Antitumor and antioxidant activity of Polyalthia longifolia stem bark ethanol extract.

In the present study, the ethanol extract of stem bark of Polyalthia longifolia Benth. and Hook (Annonaceae) was screened for its in vitro and in vivo antitumor activity. In vitro cytotoxicity of P. longifolia extract was assessed in murine cancer cells and in human cancer cells by Trypan blue exclusion assay and MTT assay, respectively. P. longifolia extract showed concentration-dependent cytotoxicity in Ehrlich’s ascites carcinoma (EAC) and Dalton’s ascites lymphoma (DLA) cells with IC50 values of 45.77 and 52.52 µg/mL, respectively. In the MTT assay, the IC50 values of P. longifolia extract against HeLa and MCF-7 cells were 25.24 and 50.49 µg/mL, respectively. In vivo antitumor activity against Ehrlich’s ascites tumor and Dalton’s solid tumor models was assessed by administering 50 and 100 mg/kg of P. longifolia extract, i.p., for 7 consecutive days. P. longifolia extract, at a dose of 100 mg/kg, significantly enhanced mean survival time (MST) and marginally improved hematological parameters when compared to EAC control mice. And the same dose significantly reduced the tumor volume as compared to control DLA inoculated mice. Positive control, cisplatin (3.5 mg/kg, i.p., single dose), significantly enhanced MST and improved hematological parameters when compared to EAC and significantly reduced the tumor volume when compared to DLA control. In vitro antioxidant potential of P. longifolia extract was also determined owing to the role of reactive oxygen species in tumor initiation and progression. P. longifolia extract scavenged DPPH radicals, reduced ferric ions and inhibited lipid peroxidation with IC50 values of 18.14, 155.41 and 73.33 µg/mL, respectively.

reported (Verma et al., 2008). But so far the in vivo antitumor and antioxidant activity of the stem bark of PL has not been reported. Hence, the present study focuses the in vitro antioxidant potency and in vitro and in vivo antitumor activity of ethanol extract of Polyalthia longifolia stem bark.

Cell lines
Dalton's lymphoma ascites (DLA) and Ehrlich's ascites carcinoma (EAC) originally obtained from Amala Cancer Research Center, Thrissur, India, were maintained and propagated as ascites tumor in Swiss albino mice by serial intra-peritoneal transplantation at the Central Animal Research Facility, Manipal University, Manipal, India. MCF-7 (human breast adenocarcinoma) and HeLa (human cervical tumor cells) cells procured from the National Center for Cell Science, Pune, India were sub-cultured every two to three days and maintained in 25 cm² tissue culture flasks (Tarsons Products, Kolkata, India) containing MEM medium supplemented with 10% fetal bovine serum (FBS) and 50 µg/mL gentamicin sulfate at 37°C in a CO 2 incubator (NuAire, Plymouth, USA) in an atmosphere of humidified 5% CO 2 in 95% air.

Animals
Eight to ten week old Swiss albino mice weighing between 25 and 30 g were selected from an inbred colony maintained under the controlled conditions of temperature (23° ± 2°C), humidity (50% ± 5%) and light (14 h and 10 h of light and dark, respectively). The animals were provided with sterile food and water ad libitum. Four animals were housed in each polypropylene cage containing paddy husk as bedding. Animal care and handling was done according to the guidelines issued by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

Plant material and extraction
The fresh stem bark of Polyalthia longifolia was collected in and around Mysore, India, in the month of December and was authenticated by Dr. M. N. Naganandhini, Department of Pharmacognosy, J.S.S. College of Pharmacy, Mysore, Karnataka. Fresh bark of Polyalthia longifolia was shade dried at room temperature and powdered. The bark powder (100 g) was extracted with 95% ethanol in a Soxhlet extractor exhaustively. The extract was then cooled, filtered and concentrated in a rotary flash evaporator. The residue was first dried over a water bath and then in a desiccator over fused calcium chloride. The percentage yield of ethanol extract was found to be 16.8% w/w.

Short-term cytotoxicity studies in DLA and EAC cells
Short-term in vitro cytotoxicity of P. longifolia extract was assessed by the Trypan blue exclusion method (Sheeja et al., 1997). In brief, Dalton's lymphoma ascites and Ehrlich ascites carcinoma (DLA/EAC) cells aspirated from the mice's peritoneal cavities were washed with PBS two to three times and one million cells were incubated with different concentration of P. longifolia extract in 1 mL of PBS for 3 h at 37°C in sterile test tubes. After incubation, 100 µL of Trypan blue dye (0.4% in PBS) was added and the total number of dead (stained) and viable (unstained) cells were counted using a hemocytometer, and percentage cytotoxicity was calculated using the formula: where T dead is the number of dead cells in the treated group, C dead is the number of dead cells in the control group and T tot is the total number of dead and live cells in the treated group.

MTT assay in cultured human cancer cells
The effect of P. longifolia extract on growth of cancer cells (MCF-7 and HeLa) was assessed by MTT assay (Mossman, 1983). In brief, exponentially growing cells (1 × 10 4 cells/well) were plated in 96-well plates and allowed to adhere for 24 h prior to extract addition. The extract was dissolved in 0.1% DMSO then diluted with the medium and filtered using 0.22 µ syringe filters. The cells were then exposed to different concentrations of extract (5-200 µg/mL) for 48 h. The cells in the control wells received medium containing the same volume of DMSO (0.1%). After the incubation, 100 µL of MTT reagent (1 mg/mL in PBS) was added and cells were incubated for an additional 4 h. The formazan produced by the viable cells was solubilized by addition of 100 µL DMSO. The suspension was placed on a micro-vibrator for 5 min and absorbance was recorded at 540 nm by the ELISA reader (BIOTEK-ELx800

Acute toxicity
Median lethal dose for P. longifolia extract was determined by following the standard Organization of Economic Co-operation and Development (OECD) guidelines (2001). In brief, Swiss albino mice deprived of food for 18 h, were administered various doses of P. longifolia extract ranging from 500 to 2000 mg/kg. Animals were observed for any symptoms of toxicity continuously for 4 h then after 24 h and finally the number of survivors was recorded after 72 h.

Survival study
The tumor induction and propagation was carried out according to the method described by Jagetia and Baliga (2003). The known numbers of viable EAC cells (2.5 × 10 6 cells/mouse) were injected intraperitoneally into each mouse in an aseptic condition and the day of tumor inoculation was considered as day zero. Twenty-four hours after tumor inoculation the tumor-bearing animals were randomly divided into desired groups of six each and treated with test compound or vehicle.
The P. longifolia extract was administered for seven days consecutively starting from day 1 of tumor inoculation. Cisplatin, a single dose of 3.5 mg/kg, i.p., was injected on day 1 which served as standard drug. Every third day animals were weighed to assess the tumor growth. The animals were monitored daily for 45 days and mortality was recorded to calculate the MST (mean survival time). The percentage increase in life span (ILS) was calculated by the formula: Whole blood count Different sets of animal were used to assess hematological parameters. The experimental design was the same as described in the survival study. Blood was withdrawn on 8th day from retro-orbital plexus of mice. The total white blood cells (WBC), red blood cells (RBC) and haemoglobin content were determined using standard methods (Mukherjee, 1990).

Antitumor activity in DLA-induced solid tumor model in mice
Antitumor activity of P. longifolia extract was determined in DLA-induced solid tumor model as per the method of Rajesh Kumar et al., 2002. In brief, DLA cells (1 × 10 6 cells per mouse) were inoculated subcutaneously into the hind limb of mice. After 24 h mice were randomized and divided into four groups of six each. Group I served as control and received 0.5% CMC, i.p. Group II received 3.5 mg/kg cisplatin, i.p., single dose on the first day. Groups III and IV received P. longifolia extract 50 and 100 mg/kg, i.p., respectively, for seven consecutive days. The diameter of the tumor was measured at five-day intervals for a period of 30 days and tumor volume was calculated using the standard formula V 4 3 r r 1 2 2 = / π where r 1 and r 2 represent the radii of the tumor at two different planes.

Effect of P. longifolia extract on in vitro antioxidant activities
DPPH radical scavenging activity DPPH scavenging activity of P. longifolia extract was determined by incubating equal volumes of different concentrations of P. longifolia extract with 100 µM DPPH in methanol at room temperature. Absorbance was recorded at 517 nm after 20 min using methanol as blank and percentage DPPH radical scavenging was calculated using the formula:

Antiradical activity
Control absorbance Sample abso orbance Control absorbance 100 The experiment was carried out in triplicate and ascorbic acid was used as standard (Sreejayan & Rao, 1996).

Ferric ion reduction activity
Electron donating capability was evaluated by ferric chloride reduction method. Reaction mixture contained 1 mL of phosphate buffer (pH 7.4), 100 µM Fe 3+ and different concentrations of extract in 0.5 mL of PBS. After 3 min incubation, EDTA (100 M) and orthophenanthroline (300 M) were added, reaction was allowed for 10 min at room temperature and absorbance was recorded at 510 nm. Ascorbic acid was used as standard which is equivalent to 100% reduction of ferric ions, comparative reduction of Fe 3+ by P. longifolia extract was calculated (Kunchandy & Rao, 1987).

Anti-lipid peroxidation
Albino rats (180-200 g) of either sex were used for the study. Animals were anesthetized and perfused transcardially with ice-cold saline after which the brain was collected. The isolated tissue was weighed and 10% homogenate was prepared in 150 mM KCl. Inhibition of lipid peroxidation was determined in rat brain homogenate. The reaction mixture contained 0.1 mL FeCl 3 (1 mM), 0.1 mL ascorbic acid (1mM), 0.1mL of KCl (1.5 M), 0.1 mL of various concentrations of P. longifolia extract and 0.3 mL of brain homogenate (10%) in a final volume of 1 mL. After 20 min of incubation at room temperature, the reaction was stopped by addition of 1 mL of 15% TBA, 0.38% TCA and 0.05% BHT solution.
Absorbance of supernatant was recorded at 532 nm after heating at 80°C for 15 min and centrifugation at 1000 rpm (Rajakumar & Rao, 1994). Anti-lipid peroxidation activity was calculated by the formula where C is the absorbance of the control and S is the absorbance of the sample. Each experiment was carried out in triplicate and results were expressed as percentage anti-lipid peroxidation activity ± SEM.

Statistical analysis
Data obtained were expressed as mean ± SEM of indicated number of animals. Statistical analysis was carried out using one way ANOVA with Tukey's post hoc test (GraphPad Prism version 4.03 for Windows, GraphPad Software, California, USA). A value of p < 0.05 was considered to be significant. Graphs were prepared by OriginLab Origin Pro 8.0 (OriginLab Software, Northampton, MA). The daily survival was determined by Kaplan Meir's equation.

Results
In vitro cytotoxic activity P. longifolia extract exhibited dose-dependent cell death in both EAC and DLA cells with an IC 50 value of 45.77 and 52.52 µg/mL in EAC and DLA, respectively (Table 1).

Effect on cultured human cancer cells
P. longifolia extract inhibited the proliferation in both the tested human cancer cell lines in a dose-dependent manner. However, the growth inhibition of HeLa cells was greater because of shorter doubling time as compared to MCF-7cells. The IC 50 (concentration required to inhibit 50% of cell growth) value of P. longifolia extract in HeLa and MCF-7 cells was observed to be 25.24 and 50.49 µg/mL, respectively ( Table 2). The percentage of DMSO (0.1%) used in the experiment did not affect the growth of the cells.

Toxicological study (median lethal dose)
In the toxicity study, no mortality occurred within 72 h under the tested doses. The P. longifolia extract was found safe up to 2000 mg/kg. On the basis of toxicological data, therapeutic doses were selected.

Effect on mean survival time
In the EAC vehicle control group (Group 1), the average life span of animals was found to be 17.21 ± 0.93 days. The average life-span of P. longifolia extract treated animals at both the doses (50 and 100 mg/ kg) was 18.33 ± 0.65 and 22.28 ± 0.88 days. But only the higher dose was found significant (p < 0.05) when compared with control. The average life span of cisplatin treated mice was found to be 33.02 ± 1.03 days (Figure 1).

Effect on body weight changes
Substantial gain in body weight was observed in vehicle-treated mice with maximum gain of 16.35% on day 12. Cisplatin administration significantly reduced the weight gain as compared to control on all the tested days whereas the reduction in the weight gain was significant on days 9 and 12 as compared to control with P. longifolia extract treatment at both the tested doses ( Figure 2).

Effect on hematological parameters
In the EAC vehicle control, the reduction in total RBC, hemoglobin (Hb) content and increase in total WBC count were significant when compared with normal mice. Cisplatin (3.5 mg/kg i.p., single dose) significantly normalized the EAC-induced hematological changes. P. longifolia extract at both the doses (50 and 100 mg/kg) significantly reversed the elevated WBC count. However, the increase in RBC and Hb content was not significant (Figure 3).

Effect on DLA-induced solid tumor
The tumor volume of vehicle-treated animals on day 30 after tumor inoculation was found to be 9.9 cc. The tumor volume was reduced to 8.67 and 6.93 cc by P. longifolia extract administration at the dose of 50 and 100 mg/kg, respectively. Cisplatin significantly reduced the tumor volume to 3.51 cc (Figure 4). The percentage reduction in the tumor volume of P. longifolia extract-treated animals was found to be 12.68% and 30.21%, whereas cisplatin reduced the tumor volume by 64.55%.

Effect on free radical scavenging activity
P. longifolia extract significantly scavenged DPPH radical and inhibited lipid peroxidation in rat brain homogenate with IC 50 values of 18.14 and 73.33 µg/mL, respectively. The IC 50 values of ascorbic acid and curcumin in DPPH radical scavenging and lipid peroxidation activity were 1.54 and 4.04 µg/mL, respectively. The IC 50 values of P. longifolia extract and ascorbic acid in ferric ion reduction were found to be 155.41 and 35.53 µg/mL, respectively (Table 3).

Discussion
Previously, P. longifolia extract has been reported to contain aporphine and azafluorene alkaloids (Wu & Duh, 1990), proanthocyanidine trimers, sitosterols, and clerodane diterpenes (Phadnis et al., 1988) which have antitumor activities Salatino et al., 2007;Shrivastava & Patel, 2007). Present in vivo studies revealed significant reduction in the body weights of EAC-bearing animals at a dose of 100 mg/kg. Another criterion for evaluating antitumor potential is prolongation of life span of tumor-bearing mice (Clarkson & Burchenal, 1965) with reversal of the elevated total levels of WBC (Oberling & Guerin, 1954).This parameter has also been fulfilled by P. longifolia extract as observed from the results. To further confirm antitumor activity, the effect of P. longifolia extract on solid tumor was assessed using DLA cells. Significant reduction in tumor volume was observed with P. longifolia extract treatment, which implies inhibition of DLA tumor growth. This indicates that the inhibitory effect of P. longifolia extract is not only due to its local cytotoxic effect but also due to its systemic action.
The short term in vitro cytotoxicity and antiproliferative data also supports the in vivo antitumor activity of P. longifolia extract against EAC and DLA, where P. longifolia extract caused significant cell death and inhibition of cancer cell growth.
Reactive oxygen species have multiple functions (Valko et al., 2006) and are implicated in tumor initiation and progression (Czapski & Goldstein, 1990;Sagun et al., 2006;Nishigori et al., 2004). Depleted endogenous antioxidant enzymes with enhanced free radical generation and MDA are well documented in carcinogenesis (Szatrowski & Nathan, 1991). Many tumor cells have pro-oxidant status and promote oxidative stress. This increases the surviving potential of the cancer cells by inducing mutations, activating redox signaling and stimulating pro-survival factors such as NF κB and AP-1 (Seeram et al., 2005). Antioxidants alter the intracellular redox state, thereby enhancing the effects of cytotoxic therapy. We have observed that P. longifolia extract significantly scavenged DPPH, reduced ferric ion and inhibited lipid peroxidation which proves its antioxidant activity. It was reported that plant-derived extracts containing antioxidant principles showed cytotoxicity toward tumor cells (Jiau & Larry, 1977) and antitumor activity  Figure 4. Effect of P. longifolia extract administration on solid tumor growth induced by DLA cells. DLA cells (1 × 10 6 cells) were injected s.c. into a hind limb of each mouse in an aseptic condition. After 24 h of tumor inoculation mice were treated as per the treatment schedule mentioned in the methodology, and tumor volume was measured on every fifth day. On day 30 the tumor volume and weight were recorded. All values are the mean ± SEM of six mice, a p <0.05, b p <0.01 & c p <0.001, compared to vehicle treatment. in experimental animals (Ruby et al., 1995). The cytotoxic and antitumor activity of plant-derived product is either through induction of apoptosis or inhibition of neovascularization (Ming et al., 1998). Plants with high phenol content are reported to possess effective antioxidant and antitumor properties (Lee et al., 2004) and P. longifolia extract has been found to have high phenol content from the antioxidant studies performed, hence could have antitumor activity.
A phytochemical study on the hexane extract of stem bark of PL has led to the characterization of various clerodane and ent-halimane diterpenes (Hara et al., 1995) which are active constituents that are reported to have antitumor activity (Seeram et al., 2005) and the MeOH extract of stem parts of PL have been reported to contain cytotoxic aporphine alkaloid liriodenine (Wu & Duh, 1990). The presence of any of these components may be attributed to the antitumor property of the extract. Since the present study focuses on the preliminary antitumor activity of P. longifolia extract, the tumor selective action and characterization of the active component of P. longifolia extract responsible for the activity are yet to be explored.