Gas chromatography-mass spectrometry analysis , antimicrobial , anticancer and antioxidant activities of n-hexane and methylene chloride extracts of Senna italica

1 Chemistry Department, Faculty of Science, Ain Shams University, El-Khalifa El-Mamoun, 11566 Abbassia, Cairo, Egypt. 2 Medicinal Chemistry Department, Theodor Bilharz Research Institute, Kornaish El-Nile, 12411 Warrak El-Hadar, Imbaba (P.O. 30), Giza, Egypt. 3 Microbial Chemistry Department, Genetic Engineering and Biotechnology Division, National Research Center, El Behoos Street 33, Dokki-Giza, Egypt. 4 Chemistry Department, Faculty of Science, Mansoura University, Mansoura-35516, Mansoura, Egypt.

From the biological activity point of view, different parts of the plants were reported for their vital biological activities i.e., antioxidant (Jothi et al., 2015), cytotoxicity (Kuete et al., 2013), antibacterial (Masoko et al., 2010;Dabai et al., 2012), and and antiproliferative (Masoko et al., 2010).Since, the microbial resistances against antibiotics is a great challenge and rapidly increase, so scientists search for a new trend aiming to identify naturally occurring antimicrobial agents from medicinal plants as alternative to the current antibiotics (Cowan, 1999;Ghareeb et al., 2015).Cancer is the rapid and uncontrolled cells growth leading to death, and must be fixed by chemotherapy (Ghareeb et al., 2013).The plant derived naturally occurring compounds are considered as good chemotherapeutic anticancer agents (Schwartsmann et al., 2002).Therefore, the current study aims to investigate the chemical constituents of Senna italica aerial parts as well as the evaluation of their in vitro antimicrobial, anticancer, and antioxidant activities.

Plant material
The aerial parts of Senna italica were collected from Alkharga Oasis, Alwady Algaded, Egyptduring March, 2015.The plant was identified by Prof. Dr. Ibrahim A. Mashaly, Professor of Plant Ecology and Flora, Botany Department, Faculty of Science, Mansoura University.

Extraction
The air dried powdered aerial parts of S. italica (500 g) were soaked in a mixture of organic solvents composed CH 2 Cl 2 /MeOH (1:1) for 72 hat room temperature and after filtration, the solvent was evaporated using rotatory evaporator, then the resulting crude extract was undergo further fractionation processusing different organic solvents i.e.,n-hexane, methylene chloride, ethyl acetate and n-butanol.

Antimicrobial activity
The antimicrobial activity was evaluated by filter paper disc methods (Murray et al., 1998;Sardari et al., 1998).Stock cultures of the test organisms were obtained from the microbiological Laboratory, Faculty of Medicine, Mansoura University.Bacteria test microbes used were Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumonia, E. coli, Bacillus subtilis and Erwenia carotovora.Whereas the fungus used was; Candida albicans.

Anticancer activity (MTT assay)
The anticancer activity was evaluated according to the reported procedure (Mauceri et al., 1998), using four human tumor cell lines namely; hepatocellular carcinoma (HePG-2), mammary gland breast cancer (MCF-7), Human prostate cancer (PC3), and Epitheliod Carcinoma (Hela).The cell lines were obtained from ATCC via Holding company for biological products and vaccines (VACSERA), Cairo, Egypt.Briefly, MTT assay is a colorimetric technique is based on the change of the yellow tetrazolium bromide (MTT) to a purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells.The cells were cultured in RPMI-1640 medium with 10% fetal bovine serum.Antibiotics added were 100 units/ml penicillin and 100µg/ml streptomycin at 37°C in a 5% CO 2 incubator.The cells were seeds in a 96-well plate at a density of 1.0x104 cells/wellat 37 °C for 48 hrs under 5%CO 2 .After incubation the cells were treated with different concentration of compounds and incubated for 24 hr.After 24 h of drug treatment, 20 µl of MTT solution at 5mg/ml was added and incubated for 4 hr.Dimethyl sulfoxide (DMSO) in volume of 100 µl is added into each well to dissolve the purple formazan formed.The colorimetric assay is measured and recorded at absorbance of 570 nm using a plate reader (EXL 800, USA).The relative cell viability in percentage was calculated as (A570 of treated samples/A570 of untreated sample) X 100.

Antioxidant activity (ABTS assay)
The antioxidant activity was evaluated via ABTS method (El- Gazzar et al., 2009).Briefly,2 ml of ABTS solution (60 mM) was added to 3 M MnO 2 solution (25 mg/ml) all prepared in phosphate buffer (pH 7, 0.1 M).Then, the mixturewasn, centrifuged, filtered, and the absorbance (A control ) of the resulting green-blue solution (ABTS radical solution) was adjusted at ca. 0.5 at 734 nm.Then, 50 ml of (2 mM) solution of the test sample in highly grade spectroscopic methanol/phosphate buffer (1:1) was added.The absorbance (A test )was measured and the decrease in color strength was expressed as % inhibition.The % inhibition for each sample is calculated according to the following equation:% Inhibition = A control -A test /A control x 100.Ascorbic acid was used as standard, and the blank sample contain methanol/phosphate buffer (1:1) instead of sample in absence of ABTS.While the negative control sample containmethanol/phosphate buffer (1:1).

Antimicrobial activity
The antimicrobial potentials of the n-hexane and methylene chloride extracts of Senna italica were examined via disc diffusion assay, using twelve pathogenic microbial species.The results in Table 1 revealed that n-hexane extract showed weak to moderate antimicrobial effect against all the tested organisms with inhibition zones ranged from 3.8 to 7 mm.While the methylene chloride extract showed a remarkable effect against eight organisms compared to standard antibiotics i.e., Escherichia coli (18mm/ ampicillin24mm), Staphylococcus aureus (15mm/ ampicillin 24mm), Streptococcus pyogenes (12mm/ ampicillin 20mm), Candida albicans (10mm/ clotrimazole 20mm), Klebsiella pneumoniae (9.2mm/ ampicillin 25), Erwinia spp.(9mm/ streptomycin 35mm), Staphylococcus epidermis (9mm/ ampicillin 24mm), andBacillus subtilis (9mm/ kanamycin 20mm).At concentration 30 mg/ml the n-hexane extract of Senna italica leaf growing in Nigeria was tested as antibacterial agent, which showed a strong activity against five bacterial microorganisms namely; Staphylococcus aureus, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa and Streptococcus pneumoniae, with inhibition zones of11.60, 11.60, 16.0, 12.60, and 14.0 mm respectively (Dabai et al., 2012).Masoko et al (2010) reported that acetone extract of the roots of Senna italica growing in South Africa showed antibacterial activity against Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli and Staphylococcus aureus with MICs ranging from 0.08 to 0.16 mg/ml (Masoko et al., 2010).Also, another study revealed that the methanolic extract of Cassia italica leaves growing in Iraq showed a significant inhibition in growth of three pathogenic microbial starins, E.coli, Staphylococcus aureus and Candida albicans, exhibited inhibition zones of 15.35, 21.35, and 14.35 mm respectively at concentration 50 mg/ml (Al-Naimy et al., 2010).Moreover, As four et al (2015) investigated the n-hexane, ethyl acetate, aqueous and total methanol extracts from Cassia italica aerial parts growing in Saudi Arabia exhibited antimicrobial activity.The n-hexane exhibited weak effect against two organisms S. aureus (6mm) at 500 mg/ml compared to Gentamycin (27mm/ 50 mg/ml) and C. albicans (5mm)compared to Clotrimazole (22mm50 mg/ml) (Asfour et al., 2015).

ABTS free radical scavenging antioxidant activity
The in vitro free radical antioxidant activity of the nhexane and methylene chloride extracts was evaluated using ABTS assay.The results in Table 3 showed that the antioxidant activity (% inhibition) against ABTS radical was 36.5% and 86.3% respectively for the n-hexane and methylene chloride extracts, compared to ascorbic acid as standard with % inhibition of 89.2%.The results showed that methylene chloride extract had a potent antioxidant activity to scavenge ABTS radicals with nearly the same effect of ascorbic acid.There are limited previously published data about the ABTS radical scavenging activity of plant.For instance, Amutha et al (2014) studied the radical scavenging activity of ABTS of six solvent extracts of Cassia senna leaf growing in India.The inhibition percentages (%) were 92%, 90.6%, 93%, 92%, 90.8%, and 92% respectively for petroleum ether, benzene, chloroform, ethyl acetate, ethanol, and aqueous extracts (Amutha et al., 2014).Also, Jothi et al (2015) reported that the ABTS radical scavenging activity of different solvent extracts of the aerial part of Senna italica i.e., (methanol, ethanol, petroleum ether, benzene, and ethyl acetate), with inhibition percentages 49.84%, 43.18%, 36.84%,29.92%, and 40.18% at 100 μg/ml respectively for methanol, ethanol, petroleum ether, benzene, and ethyl acetate extracts (Jothi et al., 2015).

Fig. 2 :
Fig. 2: Gas ion chromatogram of the of the n-hexane extract of S. italica aerial part.

Fig. 3 :
Fig. 3: Gas ion chromatogram of the methylene chloride extract of S. italica aerial part.

Fig. 6 :
Fig. 6: Major compounds identified in the n-hexane extract

Table 2 :
Anticancer activity of n-hexane and methylene chloride extracts of S. italica against human tumor cells compared to 5-fluorouracil as standard.

Table 1 :
The inhibition zone (mm) and activity index% of n-hexane and methylene chloride extracts of S. italica compared to standard antibiotics.

Table 3 :
Antioxidant activity of n-hexane and methylene chloride extracts of S. italica using ABTS assay.

Table 4 :
The compounds identified in the n-hexane extract of S. italica aerial part by GC/MS analyses.

Table 5 :
The compounds identified in the methylene chloride extract of S. italica aerial part by GC/MS analyses.