Preliminary phytochemical and antibacterial screening of Sesuvium portulacastrum in the United Arab Emirates

Collection and validation of samples

The leaves and stems of S. portulacastrum were collected from the gardens of Ras Al Khaimah Medical and Health Sciences University (RAKMHSU) in July 2010. Plants were cross-identified by their vernacular names and later validated at the Faculty of Food and Agriculture, United Arab Emirates University, Al Ain - UAE by Dr. Adil Ismail El Awad.

Processing of samples of leaves and stems

Two hundred and fifty grams of withered leaves and stems of S. portulacastrum were splashed with tap water to eliminate dust. They were left in the shade to dry for 15-20 days. The dried material was sliced into small fragments and ground to fine powder using mortar and pestle. The powder passed through a sieve of pore size 0.5 mm, which was extracted at room temperature thrice with ethanol, distilled water and dichloromethane for 48 h on an orbital shaker to make the extracts.[910] Finally, the extracts were concentrated using a rota-evaporator (R 215 Buchi Instrument, Switzerland) at a reduced pressure and at < 40°C.

Phytochemical analysis

The presence of phytochemicals in the three extracts such as alkaloids, saponins, tannins (5% ferric chloride), terpenoids (2, 4-dintrophenyl hydrazine) and steroids (Liebermann-Burchard test) were evaluated according to the methods described by Edeoga et al.[11]

Preparation of media and nutrient agar

Media for assessing the antimicrobial activity was prepared by dissolving 8 g of nutrient broth (Merck, UK) in 1:l of freshly distilled or completely demineralized water. Agar was prepared by dissolving 5 g of Bacto-agar (Difco laboratories, UK) in 200 ml of distilled water with continuous stirring and heating until clear solution appears. Both the media and agar were sterilized by autoclaving at 121°C for 15 min. Then they were left to cool down at 50-55°C.

Staphylococcus aureus and Escherichia coli were obtained from clinical isolates and were supplied by microbiology department in Ras Al Khaimah Medical and Health Sciences University, College Of medical Sciences. The bacteria were grown in nutrient broth at 37^° C and maintained on nutrient agar at 4^° C.[12]

Determination of the antimicrobial activity

Standard methods were used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the whole extracts. Strains were cultured overnight at 37°C in nutrient broth. Concentrations of the whole extracts were 25, 50, and 100 μg/ml, respectively. In addition broth containing bacteria only without extract and broth containing the extract only without bacteria served as control. The test was carried out by weighing 25, 50, 100 μg/ ml, respectively of each extract dissolved or suspended in 1 ml of the solvent. Sterile agar media weighing 99 ml were added under aseptic conditions to 150 ml flat bottom sterile Petri dishes together with 1 ml of plant extract at different concentrations. These plates were allowed to solidify on a level surface. A loop full of inoculums suspensions (bacteria) was taken and streaked on a radial pattern on the agar containing the plant extracts, a triplicate agar plate was used in order to obtain accurate results, standard and control plates were treated in the same way without the plant extract (standard antibiotics were penicillin, vancomycin and cefotaxime). Results were taken from plates having the expected appearance of colonies; otherwise the plates were discarded. Subcultures of these were used to determine MBC. Two susceptibility endpoints were recorded for each isolate. The MIC is defined as the lowest concentration of compounds at which the microorganisms tested did not demonstrate the visible growth. The MBC is defined as the lowest concentration yielding negative subculture or only one colony.[13]

Determination of antimicrobial susceptibility using agar well diffusion assay

The extracts were tested for antimicrobial activity using the agar well diffusion method. Circular wells (6 mm in diameter) were cut in the agar culture media and filled with 25, 50, and 100 μg/ml extract.[14]

Gas chromatography/mass spectrometry (GC/MS)

GC-MS analysis was carried out on an Shimadzu 2010 QB gas chromatography with a MSD detector equipped with HP-5 fused silica capillary column (30m×0.25mm×25μm film thickness). The ethanolic plant extract was injected via an all- glass injector working with split mode, with the Heleium as the carrier gas with a flow rate of 1 ml/min. Temperature program: injected temp 200° C, ion source 200° C, interphase 200° C. Column temperature was raised to 45° C (3min hold at 45° C, 4° C/min), then gradually increased to 150° C (3min hold at 150° C, 4° C/min) then raised to 250° C and a 15 min hold. A split ratio of 1:5 was used. Identification of each individual constituent of the volatile compound was achieved by comparing the retention times with those of authentic compounds or the spectral data obtained from the Wiley Library and National Institute of Standards and Technologies library, and with data published in the literature.

Statistical analysis

Analysis of data was performed by using SPSS (version 18). Results are expressed as mean ± S.D. Statistical differences were determined by Student-Newman-Keul test for multiple comparisons after ANOVA (Freund, 1981).

Phytochemical analysis

Phytochemical screening of the ethanolic extract of the leaves and stems of S. portulacastrum showed the presence of steroids. While the aqueous extract was positive toward the presence of alkaloids, saponins, tannins and terpenoids. However, the dichloromethane extract was negative to all chemical tests done, as shown in Table 1. Presence of tested secondary metabolites in the leaves and stems of S. portulacastrum will be promising for further studies on the plant as a potential study area for other researchers. The phytoconstituents detected in the plant materials could be responsible for the antimicrobial activity though their exact mode of action which is poorly understood up till now.

Table 1

Phytochemical screening of ethanol, aqueous and dichloromethane extract of the leaves and stems of S. portulacastrum

GC-MS study

The possible phytoconstituents were further confirmed through the use of GC-MS, which suggested the presence of the following phytochemicals: 22, 23-dihydrostigmasterol,[15] benzoic acid 3, 4, 5-trihydroxy-(gallic acid),[16] (2R, 3R)- (- )- epicatechin and capsaicin,[1718] as shown in Table 2 and Figure 1.

Table 2

Phytocomponents suggested in the ethanolic extract of leaves and stems of S. portulacastrum By GC-MS

Table 3

Phytocomponents and its biological activities obtained through the GC/MS study of S. portulacastrum

Figure 1

Chromatogram obtained from GC-MS with the ethanolic extracts of the leaves and stems of S. portulacastrum

Microbiological study

The ethanolic extract of the plant showed varying degree of antibacterial activities against the test bacterial species [Tables 4 and 5]. The antibacterial activities of the ethanol extract were compared with three standard antibiotics (penicillin G, cefotaxime and vancomycin) and showed a broad spectrum activity against gram negative and gram positive bacteria. The ethanolic extract of S. portulacastrum obtained MIC and MBC of 50 μg/ml against Staphlyococcus aureus and E. coli. The inhibition zone for the ethanolic extract was positive in different concentrations 25, 50 and 100 μg/ ml against Staphlyococcus aureus and E. coli as shown in Tables 4 and 5, Figure 2.

Table 4

Minimum inhibitory concentration of the plant extracts against Staphlyococcus aureus and E. coli

Table 5

Effect of the plant extract on the growth of bacteria

Figure 2

Determination of antimicrobial susceptibility using agar well diffusion assay for ethanolic extract 50 μg/ml against Staphylococcus aureus (staph) and E. coli (E.C.)