Bioassay-guided Isolation of New Antitumor Agent from Ficus faveolata (Wall. ex Miq.)

Ficus (Fig genus) is one of the largest and most important genus of the family Moraceae (mulberry). It consists of more than 800 species with habitats in lowland rainforest of tropical region [1]. Ficus species have been used in both Ayurvedic and Traditional Chinese Medicine (TCM); however the medicinal uses of these species are widely found and originated in Middle East [2]. The different parts (roots, stem, leave, fruits and latex) of Ficus spp. have shown anti-diabetic, anticancer and anti-inflammatory activities [3]. The phytochemical investigation of these species resulted into the isolation of different classes of bioactive secondary metabolites such as phenanthroindolizidine alkaloids, coumarins, multiple flavonoids, triterpenoids, different triacylglycerols and a number of volatile compounds [3-6].


Introduction
Ficus (Fig genus) is one of the largest and most important genus of the family Moraceae (mulberry). It consists of more than 800 species with habitats in lowland rainforest of tropical region [1]. Ficus species have been used in both Ayurvedic and Traditional Chinese Medicine (TCM); however the medicinal uses of these species are widely found and originated in Middle East [2]. The different parts (roots, stem, leave, fruits and latex) of Ficus spp. have shown anti-diabetic, anticancer and anti-inflammatory activities [3]. The phytochemical investigation of these species resulted into the isolation of different classes of bioactive secondary metabolites such as phenanthroindolizidine alkaloids, coumarins, multiple flavonoids, triterpenoids, different triacylglycerols and a number of volatile compounds [3][4][5][6].
The Ficus foveolata is a scandent climber shrub distributed in most of the Asia [7]. In Pakistan this plant is widely distributed in northern regions (Khyber Pukhtoonkhwa) of Pakistan, where it is locally called 'baat anzar' and has wide uses in folk medicines. According to our survey the powdered stem of Ficus foveolata is mixed with other local medicinal plants for the treatment of a cancer type locally called 'nasoor' which convinced us for future phytochemical investigation of this plant. Furthermore the Ficus foveolata has also been reported to be used in traditional medicines in the rest of the world for different ailment and disorders. For example, in Nepal its bark is used as lactating agent for milk secretion [8] and in Thailand, people use it as a tonic in a number of ways [9]. Literature survey showed only two recently published articles on the phytochemical constituents of this plant which reported the isolation of (1E,22E)-1,22-docosanediol diferulate [10] and two new eudesmane-type sesquiterpenes [11]. In the present study, we explored the selective anti-tumor potential of crude methanolic fractions and pure compound 3α-hydroxylean-12en-29,19α-olide (1), along with isolation and characterization studies of compound 1.
The phytochemical study of Ficus foveolata was under taken with small scale extraction of stem (300 g) for cytotoxic screening and dereplication purpose. The crude methanolic extract was partitioned into different fractions of hexane, dichloromethane and methanol. All the five fractions FA, FB, FC, FD and FE were screened for their antiproliferative effect by using the disk diffusion assay (In vitro). Initial stimulus for further research on dichloromethane fraction (FB) was its impressive activity against twelve different cancer cell lines (assayed at U.S. Josephine Ford Cancer Center). The LC-MS profile of the FB fraction was developed for dereplication purpose which exhibited few peaks with one major at m/z 454. The known data was dereplicated by comparing the UV and MS data with reported compounds in Dictionary of Natural Product (DNP). A number of hits were observed in DNP for the major peak at m/z 454 however due to interesting 1 H NMR spectrum the peak was selected for purification and characterization. Large scale extraction of Ficus foveolata stem (13 kg) resulted into the isolation of 3α-hydroxylean-12-en-29,19α-olide; Ficusonolide (1, C 30 H 46 O 3 , m/z 454) (Figure 1).

General
Melting point was determined using Buchi 535 digital device. Optical rotation was taken on Jasco-P2000 digital polarimeter in MeOH at room temperature while the IR (KBr) data was recorded on Bruker VECTOR 22 spectrophotometer; v max in cm -1 . UV data was taken from 996 photodiode array detector connected to analytical HPLC-MS instrument. 1 H NMR (300 MHz, C 5 D 5 N) and 13

Plant material
The stem of Ficus faveolata was collected from district Buner, Khyber Pukhtoonkhwa, Pakistan during the month of July 2007. The plant was identified by taxonomist Mr. Ambara Khan and voucher specimen (Bot.15077) was deposited in the Herbarium of Department of Botany, University of Peshawar, Pakistan.

Extraction and isolation
The air dried stem (13 kg) of Ficus faveolata were repeatedly extracted (X3) with 80% MeOH/H 2 O at room temperature after every 24 hrs. The combined extract was concentrated under vacuum at 40°C, to obtain brownish thick syrup that constituted the crude aqueous methanolic extract (100 g) which was first partitioned into five fractions: FA, FB, FC, FD and FE on polarity basis. The suspended crude extract in water was defatted (X3) with petroleum ether affording fraction FA (35 g). The polarity of suspension was change to 10% MeOH/H 2 O followed by extraction with dichloromethane (DCM) to obtain DCM soluble fraction FB (11.5 g). By addition of MeOH (1.8 L) the polarity of aqueous suspension was again changed to 50% MeOH/H 2 O and extracted with DCM to obtain fraction FC (7 g). Finally the polarity of the aqueous layer was changed to approximately 70% MeOH/H 2 O which was further extracted with DCM to get DCM soluble fraction FD (10 g), while the remaining DCM insoluble phase was get concentrated under vacuum to get methanolic fraction (FE).
After all the five fractions were screened for cytotoxic activity against six cancer cell lines, the LCMS profiles of fraction FB was developed. Fraction FB (11.5 g) was subjected to HPLC (Phenomenex Luna column C 18 RP (5 µm, 150 × 4.6; 0.1 % acidic (formic acid) gradient solvent system (10-100 MeCN/H 2 O in 30 minutes) in order to purified the selected peak, which resulted into the isolation of compounds 1 (14 mg

Anti-proliferative in vitro disk diffusion assay
The Disk Diffusion Assay (DDA) was performed in Josephine Ford Cancer Center (JFCC) [12]. For determination of cytotoxic potency and solid tumor selectivity, the crude fractions (FA, FB, FC, FD and FE) and pure compound 1 (isolated from FB fraction) were screened against six and twelve cancer cell lines respectively. Selectivity is the measure of differential inhibition of solid tumor cell line against normal or leukemia cell line (200 zone units=6.5 mm) in the disk diffusion assay, while positive activity is define as zone of inhibition greater 250 zone units. After dissolution in DMSO, the samples were pipetted onto a paper disk and then applied to an agar plate which is seeded with a particular cell line. Then the agar plates were incubated for cell growth, and the samples activity was analyzed by the size of zone of inhibition (in zone units or mm) of cell growth on agar plate. For determination of IC 50 value, human tumor cells were seeded in concentration of 5 × 10 4 cells in T25 tissue culture flasks (Falcon Plastics, NJ, USA) with 5 mL media RPMI 1640 (Cellgro, Virginia) supplemented with 15% BCS (Hyclone, Utah), 5% Penicillin/Streptomycin and 5% Glutamine. After three days incubation (cells in logarithmic growth phase; 5 × 10 5 cells/ flask), test compound was added to the flasks to achieve concentrations ranging from 10 1 to 10 -5 μg/mL. The cultured flasks were then incubated for seventy-two hours, then the cells were washed, trypsinized, spun down and counted for both viable and dead cells using 0.08% trypan blue (Gibco, Maryland). The number of viable cells number was plotted as a function of concentration and the IC 50 value determined by interpolation [13]. Each point was carried out in duplicate and a standard deviation determined.   The 13 C NMR indicated the presence of ester group δ C 182.3 (C-29) and a trisubstituted double bond δ C 124.9 (C-12) and δ C 140.5 (C-13). These characteristic spectral data suggested the presence of oleane-12-en skeleton in compound 1 [13]. Furthermore the mass spectrum showed standard retro-Diel Alder fragmentation (m/z 207 and 246) (Figure 2c), indicating the presence of lactone ring at ring D and/or E [14]. The presence of one carbonyl group, one olefinic bond and six rings in compound 1 can fully satisfied eight degree of unsaturations evident in the molecular formula C 30 H 46 O 3 . All these spectral data suggested that compound 1 was olean-12-en type triterpene with a secondary hydroxyl group and lactone ring. However the position and spatial orientation of hydroxyl group and lactone ring were yet to have determined.

Results and Discussion
The attachment of hydroxyl group at position C-3 and lactone ring on ring E (between C-17 and C-19) were made by 2D correlation spectra (COSY and HMBC) and mass fragmentation pattern (Figures  2a-2c). The connection of downfield resonating proton at δ Mass fragmentation of compound 1 indicated the presence of lactone ring in either ring D and/or E, however the exact attachment of lactone ring to ring E (between C-17 and C-19) was made by COSY and HMBC correlations (Figures 2a-2c) (Figures 2a-2c).
The relative configuration of hydroxyl group at C-3 was determined

CH3
Ring A  (Figure 2d). On the basis of spectral data and comparison with spectral data of related compounds [15], the structure of 1 was elucidated as 3α-hydroxylean-12-en-29,19α-olide (Ficusonolide), which to the best of our knowledge is a new compound.
In the preliminary screening, all the five crude fractions (FA-FE) were screened for their cytotoxicity against six cancer cell lines on different dilution ( Table 2). All the fractions exhibited variable degree of cytotoxic activity. Initial results indicated that fractions (FA and FC) did not showed reasonable cytotoxic activity compared to FB, FD and FE fractions. For further confirmation, the fractions FB, FD and FE were also evaluated on high dilution. The fraction FD was moderatively active with zone of inhibition of 750, 350, 550, 700 and 750 (200 zone units=6.5 mm) for L1210, Colon38, CFU-GM, H-116 and H-125 cell line respectively. The fraction FD were also checked on 1/4 dilution, where at low concentration the activity declined approximately at 300 zone of inhibition. The fraction FE was found to show significant activity with zone of inhibition 900 against Murine lyphocytic leukemia. At low concentrations (dilution of 1/4 and 1/16), the FE exhibited reasonable activity with zone of inhibition 800 and the activity was declined to the zone of inhibition approximately 550 at dilution of 1/64 for all cell lines. The fraction FB was found to be most significantly active among all five fractions having zone of inhibition more than 1000 for all cell lines. Interestingly the fraction FB was found consistently active even upto low dilution (1/64 dilution) exhibiting zone of inhibition more than 1000. The FB showed significant inhibition at dilution 1/256 with zone of inhibition more than 1000 against both L1210 and H-125. The activity of this fraction (FB) retain consistent at dilution of 1/4096 against H116 and H-125 with zone of inhibition 700 and 1000 respectively. The fraction FB also showed significant inhibition at very dilution 1/16384 against H-125 (Table 2). For the reason of significant inhibition of fraction FB against these cell lines especially against H116 and H-125, the fraction FB were subjected to column chromatography, as a result the compound ficusonolide (1) was identified.
Ficusonolide (1) was tested against twelve cancer cell lines (Table 3). Interestingly, Ficusonolide (1) was also found active against two H116 cells (Human colon adrenocarcinoma) and H125 cells (Human lung adrenocarcinoma) with IC 50 values 7.8 and 11.0 μg/mL respectively. The activity results of compound 1 were quite consistent with the trend observed for crude fraction FB from which leaded to the isolation of compound 1. The literature survey for the comparison and structure activity relationship did not show same activity for other lactones with similar skeleton.

Conclusions
The bioassay guided chemical investigation of the Ficus faveolata stem resulted in the isolation of a new compound 1; 3α-hydroxylean-12-en-29,19α-olide (Ficusonolide). All the crude methanolic fractions (FA, FB, FC, FD and FE) and pure compound 1 were screened for their cytotoxic activity against six and twelve cancer cell lines respectively. The selectivity and potency of the pure compound 1 were in harmony with the activity profile of the fraction FB and ethno medicinal uses of this plant. This small project on local medicinal plants has opened new vista for future research work on indigenous medicinal plants.