Sterculia diversifolia bears anti-cancer and immunomodulatory activities

The present study was aimed to evaluate the methanolic extract and subsequent solvents soluble fractions of Sterculia diversifolia bark for cytotoxic, anti-cancer and immunomodulatory activities. Phytochemical investigation confirmed the presence of alkaloids, flavonoids, saponins, glycosides, etc. In the cytotoxic activity, n-hexane showed potent activity (LD 50 : 7.0 µg/mL) followed by dichloromethane fraction (LD 50 : 16.2 µg/mL). In the anti-cancer activity, dichloromethane fraction showed potent activity (IC 50 : 5.9 µg/mL) followed by ethyl acetate fraction (IC 50 : 9.5 µg/mL). While in the immunomodulatory assay, ethyl acetate fraction showed a very significant activity (IC 50 : 21.0 µg/mL) followed by dicloromethane and n-butanol fractions (IC 50 : 25.0 and 25.3 µg/mL respectively). Hence, it is clear that S. diversifolia has anti-cancer and immunomodulatory agents. Video Clip of Methodology : ROS Assay: 2 min 9 sec Full Screen Alternate

S. diversifolia is a medium size tree.The plant bears laxative, anti-bacterial, antifungal and anti-oxidant activities.Previously few fatty acids have been reported from S. diversifolia (Salem et al., 2014).The current study is aimed to investigate the methanolic extract of S. diversifolia and its subsequent solvents soluble fractions for its prospective cytotoxic, anti-cancer, and immunomodulatory activities.

Plant material
Plant material was collected from the botanical garden of Pakistan Forest Institute, University of Peshawar, Pakistan in September, 2014.Plant material was identified by Mr. Ghulam Jelani, a taxonomist at the Department of Botany, University of Peshawar.A specimen was deposited at the herbarium of the University of Peshawar under reference No. Bot.20098 (PUP).

Extraction and fractionation
The stem bark of the plant (17 kg) was air dried in the shade at ambient temperature and then crushed to powder.Dry powder was subjected to maceration using methanol for 14 days (2 × 7 days) and then filtered using Whatman No. 1 filter paper.The extract was concentrated using rotary evaporator under reduced pressure at 40°C (Ullah et al., 2016a).Methanolic extract (950 g) was mixed with 2.5 L distilled water and soaked for 24 hours, then extracted successively with n-hexane (3 × 2.5 L), dichloromethane (3 × 2.5 L), ethyl acetate (3 × 2.5 L), and n-butanol (3 × 2.5 L) to obtain their respective soluble fractions.The remaining was considered as water soluble fraction i.e. aqueous fraction.

Preliminary phytochemical screening
Methanol extract was preliminary evaluated for qualitative phytochemical analysis using the standard protocols (Kayani et al., 2007;Ullah et al., 2016b).

Brine shrimp lethality
To determine the cytotoxic potential of the methanol extract and fraction, brine shrimp lethality bioassay was performed.In this method, artificial sea water was prepared by dissolving 38 g of sea salt in double distilled water, pH 7.4 and then filtered (Meyer et al., 1982).Brine shrimp larvae were produced by placing the sea water in a small tank and then adding brine shrimp eggs and allowing it to stand for 24 hours at 25°C . During all this process, this tank was covered with aluminum foil.Stock solutions of the test sample (methanol extract and fractions) were prepared by dissolving 20 mg of the sample in 2 mL of chloroform.Then 1000, 100 and 10 μg per mL concentration of the test samples were obtained by transferring 500, 50 and 5 μL of the stock solution into vial respectively.Then three replicates were prepared for each concentration making a total of nine vials.The solvent was allowed to evaporate.Then 10 larvae were placed in each vial and volume was made to 5 mL by adding sea water.Two vials were supplemented with solvent and reference cytotoxic drug served as negative and positive control respectively.
The standard reference cytotoxic drug used was etoposide (LD50 = 7.5 μg/mL).These entire vials were incubated for 24 hours at 25-27°C.After incubation time, the number of survivals was counted.To determine the LD50, Finney computer program was used (Alves et al., 2000).

Anti-cancer (PC3) activity
Anti-cancer activity was recorded in 96-well microplate by MTT assay.Human prostate cancer cells (PC-3) were cultured in DMEM (Dulbecco's Modified Eagle's Medium), along with 5% of fetal bovine serum, 100 IU/ mL of penicillin and 100 μg/mL of streptomycin in 75 cm 2 flasks and kept in 5% CO2 incubator at 37°C.Exponentially growing cells were harvested, counted with hemocytometer and diluted using the medium.Cell culture with the concentration of 1 x 10 5 cells/mL was prepared and introduced (100 μL/well) into 96well plates.After incubation, the medium was removed and 200 μL of fresh medium was added with concentrations of test samples (1-30 μM).After 48 hours, 200 μL MTT (0.5 mg/mL) was added to each well and incubated further for 4 hours.100 μL of DMSO was added to each well.The extent of MTT reduction was calculated by measuring the absorbance at 570 nm, using a microplate reader.The cytotoxicity was measured as concentration causing 50% growth inhibition (IC50) for PC-3 cells.The percent inhibition was determined by using the following formula:

Results
The methanolic extract of S. diversifolia was screened for the presence of various phytochemicals (Table I).From the results, it was confirmed the presence of alkaloids, carbohydrates, saponins, sterols, steroids, glycosides, flavonoids, phenol's, tannins, phalbotannins, terpenoides and vitamin C.

Brine shrimp lethality (cytotoxicity)
The results for methanolic extract of S. diversifolia and various solvents soluble fractions against brine shrimp lethality assay is presented in Table II.A typical concentration dependent cytotoxic effect was observed.n-Hexane fraction showed a maximum activity with LD50 value of 7.0 μg/mL followed by dichloromethane fraction with an LD50 value of 16.2 μg/mL.The rest of the samples showed a mild to moderate cytotoxic behavior.LD50 value was 7.5 μg/mL for etoposide (standard).

Anti-cancer activity (PC 3 cell lines)
The methanolic extract of S. diversifolia and various fractions were investigated for anti-cancer activity against PC-3 cell lines (Table II).A significant anticancer potential was observed in case of dichloromethane fraction with IC50 values of 5.9 ± 0.3 μg/mL, followed by ethyl acetate (9.5 ± 0.4 μg/mL), and methanolic extract of S. diversifolia (16.4 ± 0.3 μg/mL).Rest of the samples were fairly inactive in the anticancer assay.IC50 value for the doxorubicin (standard) was 2.8 μg/mL.

Immunomodulatory activity
The methanolic extract of S. diversifolia and various fractions were evaluated for immunomodulatory activity (oxidative burst assay) (Table II).The inhibition of reactive oxygen species (ROS) was observed by calculating their IC50 values.The maximum ROS inhibition was observed for ethyl acetate fraction, followed by dichloromethane and n-butanol fractions with IC50 values of 21.0, 25.0, and 25.3 μg/mL respectively.A moderate activity was observed in case of ethyl acetate fraction with IC50 value of 95.0 μg/mL.While the metha-nolic extract of S. diversifolia, n-hexane and aqueous fractions didn't showed any significant activity.The IC50 value for ibuprofen (standard) was 11.2 ± 1.9 μg/mL.

Discussion
Plants produces pharmacologically active compounds of different chemical classes which are deposited in their specific parts.Phytohemical tests revealed the presence of alkaloids, carbohydrates, saponins, sterols, steroids, glycosides, flavonoids, phenol's, tannins, terpenoides and vitamin C, while negative result for coumarins, amino acids and proteins in methanolic extract of S. diversifolia.Presence of the compounds of the chemical classes such as alkaloids, steroids, coumarins, tannis, glycosides and tannins validates its anticancer and immunomodulatory potentials (Alam et al., 2016).
A variety of plants, their extracts, and isolated compounds have proved their use for the treatment of various cancers.Sterculiaceae family is very familiar for containing plants bearing anti-cancer potential.Brine shrimp lethality activity is an easy, convenient, and reproducible bioassay to assess the samples for their possible cytotoxic potential.Compared to positive control (etoposide LD50: 7.5 µg/mL), all the tested samples showed good brine shrimp lethality activity.Samples bearing less than 250 µg/mL, LD50 were considered significantly active (Rieser et al., 1996).
Potent cytotoxic activity indicated the samples to be further assessed for further investigation in various anti -cancer models (Meyer et al., 1982;Apu et al., 2010).
Keeping in mind the cytotoxic activity, the anti-cancer activity was performed to clarify the anti-cancer potential of methanolic extract of S. diversifolia and subsequent solvents soluble fractions.The results of anticancer activity (PC3 cell lines inhibition) showed that this plant can be recommended in the management of cancer (Alam et al., 2016).From the result it is clear that all those samples effective in cytotoxic activity were fairly active in the anti-cancer activity as well indicting the presence of a number of chemical constituents responsible for its anti-cancer potential.The main objective of cancer management with chemotherapy (anti-cancer drugs) is to kill/inhibit the neoplastic cells.There are different cells which are naturally present in the human body.These cells are part of the immune system including natural killers, cytotoxic cells and lymphokine activated cells, which are responsible to destroy abnormal and damaged cells (Alam et al., 2016).
Any agent which has got the property of cytotoxicity can be used in various pathological conditions (inflammation, AIDS, infection and cancer) (Su et al., 2009).
The ethyl acetate fraction showed good inhibitory effect against ROS followed by dichloromethane and nbutanol, which is clear from their IC50 values while the methanol extract, n-hexane and aqueous fractions possess no activity.From these results we conclude that the active chemical agent responsible for inhibition of ROS may be present in the ethyl acetate, dichloromethane and n-butanol fraction.All organisms (aerobic) produce ROS, which can easily react with different bio molecules like proteins, lipoproteins, lipids and DNA (Ullah et al., 2016c).ROS play an important role in the development of cancer.Generation of ROS is due to drugs, pesticide and other pollutants derived from tobacco cause destruction of membrane lipids, proteins and DNA (Ciolino and Levine, 1997).Various diseases including diabetes, cancer, inflammation and arthritis are associated with ROS.In living system there is a protective phenomenon which provides protection against these ROS.Anti-oxidant agents are found naturally in tissues, they work as anti-aging substances.They diminish the oxidative reaction by free radical scavenging or by chelate formation (Kourounakis et al., 1999).

Conclusion
S. diversifolia showed a very potent cytotoxic and anticancer behavior against brine shrimp and PC-3 cell lines.