The characterisation of bioactive compounds from an Egyptian Leptolyngbya sp. strain

Abstract

An investigation into the bioactive metabolites from a benthic, mat-forming strain dominating a polluted wastewater canal in Egypt was conducted. Phytochemical screening revealed the presence of saponins, flavonoids and alkaloids; vitamin C was also found at high concentrations. The isolate was investigated as a source of antimicrobial compounds. The lipophilic fraction was extracted using chloroform/methanol and bioassays for antimicrobial compounds were performed using strains of pathogenic bacteria. The fraction that showed the highest bioactivity was purified and its structure elucidated using UV, FTIR, proton-NMR and GC-MS. The compound’s molecular weight was 220 and it was identified as butylated hydroxytoluene which has both antimicrobial and antioxidant activities. On the ecological front, this compound, and the other metabolites detected, seem to enable the isolate to dominate its niche and protect it from adverse conditions. On the commercial front, this compound is used as a food additive and was recently discovered in different cyanobacteria, and can be used as a lead compound for both drug and food industries to substitute for the expensive and hazardous synthetic analogs. Therefore, this strain can be considered as a potential source of bioactive compounds that should be further explored.

Introduction

Cyanobacteria are oxygenic phototrophs that represent a rich source of food (Herrero et al. 2006), fuel (Chisti 2007) and bioactive compounds (Skulberg 2000). Their ability to tolerate the harshest growth conditions is mainly a result of their remarkable metabolic activities that enable them to dominate their habitats and out-compete other microorganisms (Whitton and Potts 2000). Although cyanobacteria are considered as a prolific source of secondary metabolites with a wide spectrum of bioactive effects (Thajuddin and Subramanian 2005), they are still not thoroughly investigated and have been poorly exploited (Ehrenreich et al. 2005; Barrios-Llerena et al. 2007). Extracts from cyanobacteria from India proved to be active against multidrug-resistant Mycobacterium tuberculosis, the causative agent of tuberculosis (Rao et al. 2007). Similarly, Chauhan et al. (2010) showed that extracts from Anabaena possessed antimicrobial activity against several pathogenic bacteria, and new antibacterial agents were obtained from those extracts. In order to allow for more new bioactive compounds to be discovered, new taxa from unusual habitats need to be screened for their bioactive metabolites (Skulberg 2000).

Despite the fact that several studies have focused on the characterisation of bioactive compounds from different cyanobacterial strains from different geographical locations (Falch et al. 1995; Doan et al. 2000; Ghasemi et al. 2004; Asthana et al. 2006), antimicrobial compounds derived from Egyptian cyanobacterial flora have rarely been characterised, except for some limited efforts such as those by Issa (1999), El-Sheekh et al. (2006), El Semary et al. (2009) and El Semary and Abd El Naby (2010).

To begin to address this deficiency, the current project examined the antimicrobial activity of a mat-forming, benthic Leptolyngbya sp. strain isolated from a wastewater canal called "Teraat El Khashaab" in Helwan, Egypt. This canal is being used as a sink for industrial, domestic and agricultural wastewater and is polluted with heavy metals (Abd El-Hady 2007). The isolate attracted attention as it dominated the benthos of this canal with massive mat formations. Also, it was observed to grow on a solid medium, with no obvious bacterial contamination, i.e. no apparent bacterial colonies were observed to grow on the solid medium on which this cyanobacterium was grown. Therefore, this study aimed at investigating the bioactive compounds produced by this benthic cyanobacterium.

Materials and methods

Culture conditions

Cultures of the Leptolyngbya sp. strain, originally isolated from a wastewater canal, Teraat El Khashaab, Helwan (29º50′N, 31º20′E, Egypt), were kept in Oscillatoria medium (Feuillade 1994) as both liquid and solid (1.5% agarose, w:v) cultures. Liquid cultures were grown without agitation to allow mat formation at 35°C and incident radiation of 14 μmol photon m⁻² s⁻¹ (15:9 h, light/dark cycle).

Vitamin C

One gram of lyphilised cyanobacterial filaments was extracted in 80% methanol at room temperature. Vitamin C was detected in the extract using reverse phase HPLC (C18 Column). The mobile phase was a mixture of methanol:water (97:3), added under isocratic conditions. The flow rate was 0.5 ml/min, using UV detector at 254 nm (Jaffe 1984). The vitamin was identified by co-chromatography of authentic standards (Sigma). By comparing peaks of both the standard sample of known concentration and the sample of unknown concentration and relating this to the weight of the cyanobacterial mass from which the sample of unknown concentration was derived, we were able to calculate the amount of vitamin C.

Phytochemical screening

One gram of cyanobacterial biomass was lypholised then extracted in 80% for 2 days. The extract was used for the following qualitative tests. In the test for terpenes, 2 ml of the alcoholic extract was added to 2 ml distilled water and filtered using Whatmann filter paper number 2. One ml of 5% ferric chloride was added to the filtrate. The production of a yellowish green colour is evidence for the presence of tannins (Claus 1967). In the test for saponins, 2 ml of alcoholic extract were added to 1 ml of distilled water then filtered. The filtrate was vigorously shaken. Sapnoins are detected by their ability to develop a froth that is stable for a period of 30 min and longer (Wall et al. 1954). In the test for alkaloids, the lyphilised sample was boiled in water with 5 ml 2M HCl solution and the filtrate was treated with Mayer’s reagent which precipitates alkaloids (Scholz and Liebezeit 2006). The test for flavonoids (Wall et al. 1954) involved the dropwise addition of 1 ml of concentrated hydrochloric acid to 1 ml of algal alcoholic extract which contained a fragment of magnesium ribbon; a pinkish colour forms if flavonoids are present.

Antimicrobial screening

Extraction and column chromatography of lipophilic fractions

One gram of lyophilised cyanobacterial biomass was homogenised with a mixture of methanol: chloroform (2:1, v:v) and centrifuged at 14,000 g for 30 min (Hettich-R200 microcentrifuge, Germany). The lower layer was collected and re-extracted twice with the same solvent mixture. The lipophilic phase was collected, and the sample was applied to a silica gel G60 (Merck) column (1.5 × 25 cm) prepared from a slurry of 30 g of precipitated Silica gel G60 (Doan et al. 2000). The column was developed using the solvent system sequence in Table 1. The pathogenic bacterial strains were Bacillus subtilis HMCCBs1, Pseudomonas aeruginosa HMCCPa2, Bacillus cereus HMCCBc1 and Salmonella typhi HMCCSt1 (Helwan Microbial Culture collection). The concentrated lipophilic fractions were applied to 6-mm paper disks (Difco) and left to dry. The sensitivity of these bacterial strains to the extracted fractions was assessed by using the Disk Diffusion Susceptibility method (Bauer et al. 1966). Disks containing cholroform/methanol were left to evaporate and then used as negative controls. Disks containing the antibiotics ampicillin (6 μg/ml) and kanamycin (20 μg/ml), were used as positive controls. The bioactive fractions, the pathogens affected and the average values of three inhibition zone diameters are recorded in Table 1.

Table 1 Column fractionation of the lipophilic extracts from the cyanobacterial isolate and the antimicrobial activity of the fractions indicated by the diameter of the inhibition zone in cm

Elucidation of the structure of the bioactive compound in fraction VIII

The GC-MS spectra were recorded on a Shimadzu-QP2010-plus with library search at electron voltage 70 ev and EI ionisation mode. The UV-spectrum was recorded with a UV-VIS spectrophotometer (Shimadzu UV-160) and the FTIR spectrum was recorded on a Jasco FT/IR 4100 as a liquid sample. For proton NMR, 5 mg of the lypholised sample was dissolved in DMSO and measured at 300 MHz using microtube and NMR analyser (Oxford).

Results

Vitamin C and phytochemical screening

Vitamin C was found at a considerable concentration (10 μg/mg dry weight) using reverse phase C18-HPLC. The phytochemical screening confirmed the presence of saponins (formation of persistent froth), flavonoids (formation of pinkish colour) and alkaloids (formation of a precipitate with Mayer’s reagent), whereas tannins were absent (no formation of yellowish-green colour).

Elucidation of the chemical structure of the bioactive compound in fraction VIII

I-UV analysis

The UV spectrum of the bioactive compound showed two bands of medium intensity indicating the presence of an aromatic system. Bands were detected at 245 and 264 nm in the UV range of 200-400 nm (Pavia et al. 1996).

II-FTIR analysis

The spectrum (Table 2) was subdivided into four absorption sections mainly; 4,000–2,800, 2,800–2,000, 2,000–1,000 and 1,000-400 cm⁻¹. The identification of functional groups was performed according to guidelines detailed in Pavia et al. (1996). Absorption bands in the 4,000–2,800 cm⁻¹ region indicated the presence of –OH group that is bonded when dissolved in alcohol. To the left of the broad band a C-H aromatic group was detected. Whereas to the right of the broad band a C-H aliphatic group was detected, as well as stretching vibration due to aliphatic side substitution. Absorption bands in the 2,800–1,000 cm⁻¹ region indicated the presence of aromatic C = C as well as a CH₃ group. Absorption bands in the 1,000-400 cm⁻¹ region indicated the presence of CH of aromatic alkane and the presence of a substituted aromatic ring.

Table 2 FTIR absorption sections and the functional groups bands

II-The proton–NMR analysis

The proton-NMR spectrum showed signals indicative of: β-CH₃ group, methine group, α-C H₃ group_, OH group, CH in a benzene ring and aromatic system (Table 3).

Table 3 Proton-NMR signals and their indications

IV- Gas chromatography-mass spectrometry analysis

The mass spectrum of the compound under investigation revealed that the molecular weight of the compound was 220. A literature search confirmed the identity as butylated hydroxytoluene.

Discussion

The quenching of harmful free radicals in an environment rich in inducers of those radicals (e.g. heavy metals in industrial wastes) may explain the high concentration of vitamin C in the studied organism. Vitamin C is a powerful antioxidant and functions primarily to protect lipid membranes and structures from peroxidation caused by free radicals (Buettner 1993). Moreover, it has antimicrobial effects; for example, vitamin C from the green alga Haematococcus inhibits Helicobacter pylori infection in humans (Wang et al. 2000). This vitamin thereby provides the organism with protection that enables it to cope with the environmental stress of this unusual niche and to dominate other microorganisms that are not as protected. Meanwhile, the phytochemical screening revealed the presence of saponins, flavonoids and alkaloids and the absence of tannins. Some of these substances were detected in cyanobacterial extracts and reported to have antimicrobial effects (Kulik 1995). Similarly, it was found from an extensive phytochemical screening of microalgae that those bioactive compounds not only play a major biological role in cellular metabolism but also play a role as a defensive mechanism where they inhibit co-existing microorganisms (Scholz and Liebezeit 2006). Consistent with this, fraction VIII proved to be most effective against all bacterial pathogens tested in the antimicrobial screening.

The elucidation of the pure bioactive compound structure was performed using several chemical analyses. UV analysis showed that the compound has an aromatic ring, and FTIR confirmed this aromatic nature. The proton-NMR showed the presence of a phenolic ring as well as aliphatic substitution. Finally, the GC mass coupled with a literature search identified the compound as butylated hydroxytoluene of molecular weight 220. This compound is commonly used as a food additive due to its antioxidant activity (Sikkema et al. 1995); however, it is reported to also have antimicrobial activity (Davidson and Branden 1981). The mechanism of toxicity to microorganisms, of this and other hydrocarbons, was discussed in the comprehensive review by Sikkema et al. (1995). In their review, these authors reported that the interactions of lipophilic compounds with the cell membrane of microorganisms can cause loss of membrane integrity, leading to an increase in permeability to protons and the dissipation of the proton motive force. Both lipids and proteins of the membrane are affected. It was speculated that lipophilic compounds may also affect membrane-bound enzymes due to alteration in the hydrophobic environment around the membrane. Thus, the compound discovered herein can be used as an antimicrobial/antioxidant agent that can be easily obtained from a natural source. In line with this, Babu and Wu (2008) were able to isolate and produce this compound from naturally-occurring freshwater phytoplankton, including the filamentous cyanobacteria Oscillatoria and Cylindrospermopsis raciborskii. They also investigated some of the conditions that may trigger greater production of this substance and highlighted the possibility of substituting the chemical synthesis of this substance with natural production using those microalgae. Such clean technology is increasingly important and future research should focus on studying factors that maximise the production of this compound and other useful natural substances.