Hexane soluble extract of Mallotus philippensis (Lam.) Muell. Arg. root possesses anti-leukaemic activity

Background Mallotus philippensis (Lam.) Muell. Arg. is a well known medicinal plant of Asia and Australia. Various compounds from different aerial parts of the plant have been reported possessing potent pharmacological, antiviral, antibacterial and cytotoxic activities. We were interested to determine the effects of some root extracts from M. philippensis on human promyelocytic leukemia HL-60 cell proliferation, cell cycle regulators and apoptosis in order to investigate its anti-leukemic potential. Results Root extract of M. philippensis was initially extracted in organic solvents, hexane, ethyl acetate, and n-butanol. The hexane extract showed highest toxicity against p53-deficient HL-60 cells (IC50 1.5 mg dry roots equivalent/ml medium) after 72 h and interestingly, inhibition of cell proliferation was preceded by the upregulation of the proto-oncogenes Cdc25A and cyclin D1 within 24 h. The hexane extract induced 18% apoptosis after 48 h of treatment. Chemical composition of the hexane extract was analyzed by GC-MS and the 90% fragments were matched with polyphenolic compounds. Conclusions The present study confirms that the hexane fraction of M. philippensis root extract possesses anti-leukemic activity in HL-60 cells. The polyphenols were the main compounds of the hexane extract that inhibited proliferation and induced apoptosis.


Background
Mallotus philippensis (Lam.) Muell. Arg (Euphorbiaceae) are shrubs or small trees which grow on mountain slopes or valleys, limestone hills or river valleys and forests at an altitude of 300-1600 m in Asia and Australia. Different parts of the plant have been used in traditional medicine. Kamala, a red powder consisting of glandular hairs from plant capsule has been used as anthelmintic and cathartic in traditional medicine [1,2] and an orange dye for silk [3]. Kamala is commonly administered in its curd form for the elimination of intestinal worms and also for skin irritation, ringworm, and freckles [4]. The fruit of the plant is purgative for animals [5].

Materials and methods
Cell culture HL-60 human promyelocytic cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum, 1% L-Glutamine and 1% Penicillin/Streptomycin (Life Technologies, Paisley, Scotland) at 37°C in a humidified atmosphere containing 5% CO2.

Collection and extraction of root powder
Mallotus philippensis was collected from Margalla Hills (Islamabad, Pakistan) and identified by one of the authors in the Herbarium of Quaid-i-Azam University Islamabad. Roots were washed, air dried and grounded. Powdered M. philippensis root (20 g) was extracted four times (each for hexane, ethyl acetate and n-butanol) with methanol (MeOH). These extracts were collected and concentrated with a Rotavapor at 40°C. The three separate concentrated MeOH extracts were dissolved in distilled water and extracted three times each with hexane, ethyl acetate (EtOAc), n-butanol (BuOH) and concentrated to complete dryness. 9.23 g, 4.00 g and 7.08 g dried hexane, EtOAc and BuOH extracts were obtained respectively.

Hoechst dye 33258 and propidium iodide double staining
Hoechst staining was performed according to method described by Grusch et al. [15]. HL-60 cells (0.1 × 10 6 /ml) were seeded in T25 cell culture flasks and exposed to increasing concentrations of M. philippensis hexane extract for 48 h. Hoechst 33258 (HO) and propidium iodide (PI, both Sigma, St Louis, MO) were added directly to the cells to final concentrations of 5 and 2 mg/ml, respectively. After incubation for 60 min at 37°C, cells were examined under fluorescence microscope (Axiovert, Zeiss) equipped with a filter and a camera. This method allows discriminating between early apoptosis, late apoptosis, and necrosis. Cells were judged according to their morphology and the integrity of their cell membranes, which could easily be observed after PI staining.

Western blotting
HL-60 cells were reinsulated for increasing time periods (from 2 to 48 h) with 1.5 mg dry roots equivalent/ml medium extract/ml. Then, cells were placed on ice, washed with ice-cold PBS (pH 7.2), centrifuged (1000 rpm, 4°C, 4 min) and the pellets lysed in 150 μl buffer containing 150 mM NaCl, 50 mM Tris pH 8.0, 1% Triton X-100, 2.5% 0.5 mM PMSF and PIC (Sigma, Schnell dorf, Germany). Debris was removed by centrifugation (12,000 rpm, 4°C, 20 min) and the supernatant collected. Then equal amount of protein was loaded onto 10% polyacrylamide gels. Proteins were electrophoresed for 2 h and then electroblotted onto PVDF membranes (Amersham, Buckinghamshire, UK) at 4°C for 1 h. To confirm equal sample loading, membranes were stained with Poinceau S. After washing with TBS (Tris base, NaCl, PH 7.6 adjust with HCl), membranes were blocked for 1 h in blocking solution containing 5% skimmed milk in TBS and 0.5% Tween 20, washed 3 times in TBS/T, and incubated by gentle rocking with primary antibodies  with a Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and cell cycle distribution was calculated with ModFit LT software (Verity Software House, Topsham, ME, USA).

Statistical analyses
To test the effect of extract triplicates of biological and technical repeats were used. The results of apoptosis and proliferation experiments were analyzed with t-test using GraphPad Prism version 4 (GraphPad Prim Sofware, Inc., San Diego, CA, USA).

Results and discussion
Inhibition of HL-60 cell proliferation by M. philippensis extracts Logarithmically growing HL-60 cells were incubated with increasing concentrations of n-hexane, EtOAc and BuOH extract for 72 h. Then, cells were counted and the inhibition of proliferation was calculated. The hexane extract showed highest toxicity against HL-60 cells (IC 50 1.5 mg dry roots equivalent/ml medium) after 72 h ( Figure 1). The inhibition of HL-60 proliferation that was observed upon treatment with hexane extract was preceded by the down regulation of the proto-oncogene cyclin D1 after 48 h. Suppression of cyclin D1 is potent mechanisms to block cancer cell growth.
Effect of hexane extract on cell cycle distribution HL-60 cells were exposed to 1.5 mg dry roots equivalent/ ml medium hexane fraction for 48 h to investigate cell cycle distribution. An accumulation of HL-60 cells in S-phase at the expense of G1-phase cells was observed, which however, was not significant (Figure 2). The increased number of S-phase cells suggested that the cell cycle became induced and this was substantiated by the increased expression of cyclin D1 and Cdc25A within 24 hours of extract treatment ( the expression of Cdc25A was still over expressed compared to control. Therefore, the expression of these two proto-oncogenes could not explain the inhibition of HL-60 proliferation and hence P value is worthless for the experiment. This implicated that other mechanisms blocked cell division. .735 minutes) have been detected upon comparison of their mass fragmentation data with already known data of polyphenols from the same genus. The mass fragmentation data of the larger peaks were about 90% related to betulin and kamalachalcone C which are polyphenols and already known from the same genus, however the exact identification of the compounds were not focused in this study.
Rottlerin (5, 7-dihydroxy-2, 2-dimethyl-6-(2, 4, 6-trihydroxy-3-methyl-5-acetylbenzyl)-8-cinnamoyl-1,2-chromine) that is also called mallotoxin, is one of the major constituents of M. philippensis. Since the main peaks of the hexane root extract were unrelated to rottlerin, other bioactive compounds were responsible for the cytotoxic effect. It has been confirmed from the present study that hexane fraction is active against leukaemic cells and it is proposed that some potent anti-carcinogenic compounds exist in M. philippensis that warrant their identification.

Conclusion
The results revealed that hexane soluble extract of Mal lotus philippensis (Lam.) Muell.Arg. root possesses antileukaemic activity, which provide some mechanistic evidence for why indigenous people of Pakistan and other Asian countries found it useful for various ailments as well as food additive.