Antimicrobial , antioxidant activities and cytotoxicity evaluation of Artocarpus nigrifolius C . Y . Wu from Vietnam

1 Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam. 2 Institute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam. 3 Department of Food Technology, Vinh University, 182 Le Duan, Vinh City, Nghe An Province, Vietnam. 4 Natural Products Research Unit, Department of Chemistry, Faculty of Science, Lagos State University, Badagry Express way Ojo, P. M. B. 0001, LASU Post Office, Ojo, Lagos, Nigeria.


INTRODUCTION
The genus Artocarpus (Moraceae) comprises about 60 species originated from the South-East Asia and Pacific regions.Artocarpus species are trees with white latex and comprises mainly of breadfruit and jackfruit trees and is widely used in folk medicine.The extracts and metabolites of Artocarpus particularly those from leaves, bark, stem and fruit possess several useful bioactive compounds.The flavonoid fraction from leaves of Artocarpus tonkinensis A. Chev. is a potential antioxidant agentkaemferol in the total flavonoid fraction and it has an inhibitory effect againt the lipid peroxydation of liver (Tran Luu Van Hien, Pham Thanh Ky, Trinh Hien Trung, *Corresponding author.E-mail: tran.minhhoi@yahoo.com.Tel: +84438364624.
Artocarpus nigrifolius C. Y. Wua new record for Vietnam Flora, is a straight, deciduous tree that can grow up to 15 m tall.The branchlets are brownish black and wrinkled, 1 to 2.5 mm thick, while the young buds are with short rust-colored pubescence.Mature fruiting syncarps are unknown.This paper examines the antimicrobial, anti-oxidant and cytotoxicity activities of the extracts from leaf, stem bark, twigs and root of the plant.
Up to now, no literature information on the biological activities and chemical constituents of this species.
Other chemicals were of analytical grade.

Extraction procedure
The dry and crushed leaves, stem bark, twig and roots of A. nigrifolius were separately extracted with methanol/water (95.5:5) using Soxhlet apparatus.The resultant extracts were concentrated to dryness under reduced pressure (40°C) in a rotary evaporator to yield different dried A. nigrifolius extracts namely: Stem bark (SB), Leaves (LV), Root (RT) and Twig (TW).Each dried extract was weighed and re-dissolved in DMSO for further experiments.

Antimicrobial activity assays
The antimicrobial activity of A.

Determination of antimicrobial activity
A broth microdilution method was used to determine the minimal inhibitory concentration of the extracts according to the standard Hoi et al. 1327 procedures (Basri and Fan, 2005;CLSI, 2009;Cos et al., 2005).Plant extracts re-suspended in methanol to obtain concentrations ranging from 0.5 to 128 µg/ml, was four-fold serially diluted and added to broth media in a 96-wells microtiter plate.Prediluted 100 µl of inoculums (for bacteria 5x10 5 cfu/ml and 5x10 5 cfu/ml for yeast) was added to each well.Bacterial and fungal suspensions were used as negative control, while broths containing standard drugs (Ampicilline, Streptomycine and Amphotericine) were used as positive control.The microtiter plates were incubated at 37°C for 24 h for bacteria and 28°C for 48 h for yeast.The optical density of the extract was measured at 600 nm using Genious Tecan UV-VIS spectrophotometer against the corresponding blank sample.
Where ODsample, ODblank and ODcontrol were the optical density of the extract at different concentrations, the blank sample and the control respectively.The inhibiting concentration providing 50% inhibition (IC50) was calculated from the graph of percentage inhibition against each extract concentrations (Hadacek and Greger, 2000;Cos et al., 2005).

Antioxidant activity method (DPPH assay)
The free radical scavenging activity of A. nigrifolius extracts was determined by modified methods of Liyana-Pathirama et al. ( 2005) and Thirugnanasampandan et al. (2008).Two milliliter of different concentrations (0.5 to 128 µg/ml) of each extract in methanol was added to 0.2 ml of DPPH radical solution in methanol (final concentration of DPPH was 1.0 mM).The mixture was shaken vigorously and allowed standing for 60 min in the dark.The absorbance of the resulting solutions, the blank and the control were measured at 517 nm using Genious Tecan UV-VIS spectrophotometer.Standard antioxidant compounds such as Butyl hydroxyl anisole, butyl hydroxyl toluene, Resveratrol and ascorbic acid were used as positive control.DPPH scavenging activity of the extract was calculated using the following formula: DPPH scavenging activity (%) = (ODblank -ODsample)/ ODblank) X 100% Where ODsample and ODblank were the optical density of the extract at different concentrations and the blank sample.The effective concentration providing 50% inhibition (EC50) was calculated from the graph of percentage inhibition against each extract concentrations.

Cytotoxicity assay
Four human cancel cell lines were obtained from the American Type Culture Collection (Manassas, VA).They are MCF7 (breast carcinoma cell line), KB (epidermoid carcinoma cell line), LU (lung cancer cell line), HepG2 (hepatoma carcinoma cell line).They were grown in medium RPMI 1640 supplemented with 10% FBS (Fetal bovine serum), 50 IU/ml penicillin and 50 µg/ml streptomycin.All the cell lines were maintained at 37 0 C in a 5% CO2 atmosphere with 95% humidity.
The MTT assay is based on the protocol described by Mossmann (1983) and Hussain et al. (1993).The test extracts, dissolved in five concentrations, were added into the available culture cells and culture plates were incubated for 3 days.After the exposure times, the culture cells were treated with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] while OD was measured at 450 nm absorbance on microplate reader (TECAN GENIOUS).The inhibitory concentration (IC) values were calculated by formula:  IC50 is the concentration in µg/ml required for 50% inhibition of cell growth as compared to that of untreated control.

Antimicrobial activity
The data of the antimicrobial and antioxidant activities of A. nigrifolius extracts demonstrated in the Table 1, revealed a wide range of activity for A. nigrifolius extracts, with very low to higher antimicrobial action against the microorganisms tested.Moderate to strong activities were observed against B. subtilis, S. aureus and L. fermentum by leaf, stem-bark and root extracts, and while, little or no activity was seen against E. coli, P. aeruginosa, S. enterica and C. albican by all the extracts.The leaf extract displayed the best activity against B. subtilis, L. fermentum and S. aureus having MIC values between of 3.65, 4.25 and 5.49 µg/ml, respectively.When compared with standard antibiotics (Ampicilline, Penicillin/Streptomycin mixture and Amphotericine), other extracts showed a weak to moderate MIC values.The antibacterial activity showed by A. nigrifolius extracts could be attributed to the presence of some phenolic compounds including flavonoids and stilbenoids present in several species of the genus Artocarpus reported, with well-known pharmaceutical and biological activities.The crude methanolic extracts of the stem and root barks, stem and root heart wood, leaves, fruits and seeds of Artocarpus heterophyllus and their subsequent partitioning with petrol, dichloromethane, etyl acetate and butanol gave fractions that exhibited a broad spectrum antibacterial activity against Bacillus cereus, Bacillus The butanol fractions (concentration 4 mg/disc) of root bark and fruits of A. heterophyllus were found to be more active and have been examined by disc diffusion methods (Khan et al., 2003).
Bioassay  Antimicrobial testing of the three compounds indicated that Sepicanin A displayed a significant selective antibacterial activity against methicillin-resistant S. aureus (MRSA) with IC 50 and MIC values of 1.4 and 2.9 μM, respectively (Mohamed et al., 2009).
The methanolic plant extract consists of two active isoprenyl flavones artocarpin and artocarpesin were isolated from A. heterophyllus.These inhibited the growth of primary cariogenic bacteria at concentration of 3.13 to 12.5 µg/ml and also exhibited the growth inhibitory effects on plague-forming Streptococci.These findings showed that phytochemicals from A. heterophyllus would be potent compounds for the prevention of dental caries (Sato et al., 1996).
The artonin E has been isolated from the bark of Artocarpus rigida Blume.The artonin E (concentration 250 µg/disc) showed antimicrobial activity against E. coli and B. subtilis produced clear zone with a diameter of 1.2 and 0.9 cm, respectively, while the standard used the canamycin sulphate (concentration 240 µg/disc) produced clear zone with a diameter of 2.2 cm.Therefore, this compound has possibility to be used as an antibiotic.However, artonin E did not show a good antifungal activity against Rizhopus oligosporus or R. oryzae (Suhartati et al., 2008).

Antioxidant activity
DPPH is a free radical compound that has been widely used to test the free radical scavenging ability of various extracts (Nagai et al., 2005).It is considered to be a model for lipophilic radical in which a chain reaction is initiated by the lipid autoxidation.Table 1 shows the effective concentration (EC 50 ) of the scavenging activity of A. nigrifolius extracts, BHA, BHT, resveratrol and ascorbic acid on DPPH radicals at different concentrations.The standard antioxidants BHA, BHT, reveratrol and ascorbic acid demonstrated higher inhibitory activity, when compared to the extracts, which also displayed remarkable scavenging activities.The stem-bark extract showed the highest scavenging activity (EC 50 ) value of 42.08 µg/ml, while, moderate activity was displayed by the leaf (80.09 µg/ml) and root (61.05 µg/ml) extracts.The effective concentration resulting in a 50% inhibition of the DPPH radical scavenging (EC 50 ) for the extracts was less than 100 μg/ml, except for twig extract with EC 50 value greater than 128 μg/ml.It is evident that A. nigrifolius extracts, like some Artocarpus species, did show some proton-donating ability and could serve as free radical scavengers (Donsing et al., 2008;Ko et al., 1998;Iverson et al., 2010;Jayasinghe et al., 2004;Kai-Wei et al., 2009).

MTT cell proliferation assay
In vitro anticancer activity of A. nigrifolius extracts was carried out against four human cancer cell lines consisting of lung (LU), breast (MCF7), epidermoid (KB) and hepatoma (HepG2) (Table 2).Both the leaf and stem bark extracts showed moderate activity against KB (IC 50 ; 24.20 vs. 21.03μg/ml) and MCF7 (IC 50 ; 22.68 vs. 20.10μg/ml) cancer cell lines.The twig extract exhibited the most significant cytotoxicities against KB (IC 50 19.21μg/ml) and HepG2 (IC 50 10.06 μg/ml) cancer cell lines.The cytotoxicity of the various parts of A. nigrifolius extracts was found in the order: twig > stem-bark > leaves > root.Several cytotoxic principles have been isolated from the genus Artocarpus (Arung et al., 2010;Hsu et al., 2011;Jagtap and Bapat, 2010).Sahartati et al. (2001) isolated a new prenylated flavone artoindonesiani L along with four known phenolic compounds identified as artonins M and E, cycloartobiloxanthone and artonin 0 from root bark of Artocarpus rotunda (Hout) Panzer.These compounds showed significant cytotoxicity against murine P-388 leukemia cells with an LC 50 values 0.6, 7.9, 0.06, 4.6 and 0.9 µg/ml, respectively.The penylated flavonoids, artonins E and 0, artobiloxanthone and cycloartobiloxanthone were isolated from the stem bark of Artocarpus kemando showed cytotoxicity against KB (human oral epidermoid carcinoma) with an IC 50 values 3.0, 0.5, 3.5 and 2.5 µg/ml, respectively, whereas, the compounds arto artonins E and artobiloxanthone which have a catechol moiety in ring B, exhibited strong DNA strand scission activity (93 and 84% relaxation of supercoiled DNA, respectively, at 2.5 µg/ml concentration) (Seo et al., 2003).The two new isoprenylated flavones, artoindonesianins U and V have been isolated from the heartwood of Artocarpus chempeden showed strong cytotoxicity against P-388 tumor cells with IC 50 2.0 and 0.5 µg/ml, respectively (Syah et al., 2004).In another report, Ko et al. (2005) isolated new prenylated flavonoids, artelastoheterol, artelasticinol, cycloartelastoxanthone, artelastoxanthone and cycloartelastoxanthendiol from the root bark of Artocarpus elasticus.The previously known compound artonol A exhibited cytotoxic activity against the A549 human cancer cell line, with an ED 50 value of 1.1 µg/ml.
The flavonoids cycloartobiloxanthone and the xanthone artoindonesianin C compounds were active against human epidermoid carcinoma of the nasopharynx (KB) cells; the chromone artorigidusin, cycloartobiloxanthone and the xanthone artoindonesianin C showed varying toxicity to human breast cancer (BC) cells, where as the flavonoid 7-demethylartonol E, xanthone artonol B, flavonoid cycloartobiloxanthone and the xanthone artoindonesianin C compounds were active in the human small cell lung cancer (NCI-H187) cytotoxicity assay, with artonol being the most active compound (IC 50 1.26 µg/ml).The ellipticine was used as the standard drug in the test exhibited IC 50 values against these cell lines at 1.33, 1.46 and 0.39 µg/ml, respectively.All these compounds were isolated from the root bark of Artocarpus rigidus Blume subsp.rigidus (Namdaung et al., 2006).
Breadfruit (Artocarpus communis, Moraceae) is cultivated in tropical and subtropical regions as a traditional starch crop and also has potential medicinal properties.Three new geranyl chalcone derivatives including isolespeol; 5'-geranyl -2',4',4trihydroxychalcone and 3,4,2'4'-tetrahydroxy-3'geranyldihydrochalcone together with two known compounds lespeol and xanthoangelol were isolated from the leaves of A. communis and were studied for in vitro anticancer activity.The effects of geranyl chalcone derivatives on the viability of human cancer cells [including human liposarcoma cells (SW 872), human colorectal carcinoma cells (HT-29, COLO 205), human colorectal carcinoma cells (PLC5, Huh7) and human hepatoblastoma cells (HepG2, Hep3G)] were investigated.The results indicated that isolespeol showed the highest inhibitory activity with an IC 50 value of 3.8 µM in SW 872 human lip sarcoma cells.Treatment of SW 872 human lip sarcoma cells with isolespeol caused the loss of mitochondrial membrane potentiality (DeltaPsim).Western blotting revealed that isolespeol stimulated increased protein expression of Fas, FasL, and p53.The expression ratios of pro-and antiapoptotic Bcl-2 family members were also changed by isolespeol treatment to subsequently induce the activation of caspase-9 and caspase-3, which was followed by cleavage of poly (ADP-ribose) polymerase (PARP).
These results demonstrated that isolespeol induced apop-pathways (Fang et al., 2008a,b).The new oxepinoflavone, Artoindonesianin E1 from the wood of A. elasticus, along with four known prenylated flavones artocarpin, cycloartocarpin, cudraflavones A and C. The cytotoxicity of compound artoindonesianin E1 against murine leukemia P-388 cells was studied using MTT (microculture tetrazolium technique) essay; which showed moderate cytotoxicity against P-388 cells with IC 50 5.0 µg/ml (Musthapa et al., 2009).The cytotoxic effects of these compounds in human cancer cell might also have potential for anticancer application.These finding suggested that Artocarpus extracts have the potential to be developed as new chemotherapeutic agents to prevent or to inhibit the growth of tumors and cancers.

Conclusions
The antimicrobial activity of the methanol extracts of various parts of A. nigrifolius were studied against some strains of organisms.Among them, the leaves extract showed a promising antimicrobial potential against L. fermentum, B. subtilis and S. aureus with IC 50 values against were 4.25, 3.65 and 5.49 μg/ml, respectively.The scavenging activity of methanol extract of bark stem was strongest with EC 50 value of 42.08 μg/ml.But it was less potent than the antioxidant agent resveratrol (an antioxidant agent found in grapes fruit).
The results of cytotoxicity of A. nigrifolius C. Y. extracts (leaves, root, stem bark and branch/methanol) demonstrated week to active toxicity.This data to help selecting plant extracts with promising activity for future work.
-guided fractionation of the EtOH extract of Artocarpus sepicanus Diels leaves has led to the isolation of a new geranyl flavanone (Sepicanin A), along with the known compounds, afzelechin-3-O-α-lrhamnopyranoside and β-sitosterol glucoside.The structure of the new compound was established by UV, IR, HRESIMS, 1D and 2D NMR experiments.
Plant materials of A. nigrifolius were collected from Copia Natural Reserve, Son La province, Vietnam, in 2010.Botanical identification was performed by N. T. Hiep and the voucher (Specimen No: PVT 419) was deposited at Herbarium of the Institute of Ecology and Biological Resources (HN), Vietnam Academy of Science and Technology.

Table 1 .
Antimicrobial and antioxidant activities of A. nigrifolius extracts.