Antimycobacterial Activity from Cyanobacterial Extracts and Phytochemical Screening of Methanol Extract of Hapalosiphon.

ABSTRACT

The recent increase in the number of multidrug-resistant clinical isolates of M. tuberculosis. has created an urgent need for the discovery and development of new antituberculosis leads. Natural products form one avenue in the search for new antitubercular agents. Cyanobacteria (blue-green algae) are a diverse group of photosynthetic, prokaryotic microorganisms found in fresh and marine waters. They produce a diversity of secondary metabolites having potential activity as antimicrobials, antivirals, and as other pharmacologically active compounds. In the current study, organic extracts of 10 cyanobacterial strains collected from the fields of an agricultural research institute in India were investigated for their antimycobacterial properties by an agar diffusion bioassay followed by minimum inhibitory concentration determination. Preliminary phytochemical screening methodologies were performed to identify active component of an extract from Hapalosiphon. sp. This study may be the first documentation of the activity of these species against mycobacteria.

Keywords:

• Antimycobacterial
• cyanobacteria
• Hapalosiphon. sp

Introduction

Tuberculosis (TB) mainly caused by Mycobacterium tuberculosis. (order Actinomycetales, Family Mycobacteriaceae) (NCBI taxonomy browser, 2004) is the leading killer among all infectious diseases worldwide and is responsible for more than 2 million deaths annually (Dye et al., 1999). For more than 30 years, no antitubercular agents with new mechanisms of action have been developed. The recent increase in the number of multidrug-resistant clinical isolates of M. tuberculosis. has created an urgent need for the discovery and development of new antituberculosis leads. Natural products have long being recognized as an important source of therapeutically effective medicines (Cragg et al., 1997; Strohl 2000; Copp, 2003). It is estimated that approximately 70% of antimicrobial and anticancer drugs currently approved for human use originate from natural products. More recently, marine sources have also begun to yield many novel natural products with exciting biological activity (Burja, 2001). The molecular diversity among the natural products is an important factor that outweighs synthetic chemistry.

Cyanobacteria, the blue-green algae, are an ancient and diverse group of photosynthetic microorganisms that have evolved to inhabit many different and extreme environments (i.e., in both freshwater and marine environments). Although the origin of these organisms dates back 3 billion or 4 billion years (Schopf & Walter, 1983), there has been increasing interest in these organisms during the past decade (Glombitza & Koch, 1989; Schwartz et al., 1990; Falch et al., 1995) as potential sources of new drugs. With about 2000 strains of cyanobacteria distributed all over the world, they show a remarkable ecological diversity. Their adaptability to different niches is because of their ability to produce a unique range of defensive metabolites. The advantages of these are that a sustainable supply of a desired material can be achieved, which is not always possible from a product derived from a microorganism source. Investigations by several workers revealed that cyanobacteria are a rich source of potentially useful natural products like novel antitumor, antifungal, and, to a lesser extent, antibacterial compounds (Patterson, et al., 1994; Borowitzka, 1995; Kulik, 1995; Burja, 2001; Soltani et al., 2005). Many compounds isolated from cyanobacteria such as dolastatins, curacins, and cryptophycins have shown antitumor activity (Simmons et al., 2005). Among these, dolastatin 10 and cryptiphycin 52 have entered into clinical trials for solid tumors (Jonge et al., 2005) and lung cancer, respectively (Edelman et al., 2005).

Kreitlow and Sabine (1999) evaluated the antibiotic activities of hydrophilic and lipophilic extracts of 12 strains of cyanobacteria collected from fresh and brackish water. Screening against seven different microorganisms showed that the extracts were inactive against the three Gram-negative bacteria Escherichia coli., Proteus mirabilis., and Serratia marcescens. and the yeast Candida maltosa., but inhibited the growth of at least one of the Gram-positive bacteria Micrococcus flavus., Staphylococcus aureus., and Bacillus subtilis. based on the agar diffusion method (Collins & Franzblau, 1989). Mian et al. (2003) screened the hydrophilic and lipophilic extracts of terrestrial and freshwater cyanobacteria for their antimicrobial and antifungal activities, and a majority of the extracts showed activity against Gram-positive bacteria. Soltani et al. (2005) examined aqueous, petroleum ether, and methanol extracts from 76 microalgae for antimicrobial properties against four bacteria and two fungi. Of total microalgae, 22.4% (17 cyanobacteria) exhibited antimicrobial effects. However, studies on the inhibitory potential of these organisms against mycobacteria are either scarce or nonexistent. In this study, we performed random screening of about 10 cyanobacterial strains against mycobacteria, and identified some active species. The partial purification and phytochemical screening revealed an active component from the methanol extract of Hapalosiphon. sp.

Materials and Methods

Cyanobacterial cultures

Ten cultures of freshwater cyanobacteria belonging to Stigonometaceae, Nostocaceae, and Oscillatoraceae were selected from the herbarium of the Indian Agricultural Research Institute, New Delhi, India, and used for primary screening by zone diffusion assay against mycobacteria.

Growth conditions

The cyanobacterial cultures were grown in BG11 media in sterile conditions at temperatures between 20°C and 25°C under an illumination of 800–1000 lux.

BG11 composition

From Rippka and Herdman (1993): [Composition of the micronutrient solution from Kuhl and Lorenzen (1964)]

Table

	Stock solution (g/100 mL)	Nutrient solution (mL)
NaNO3	15	10
K2HPO4 _ 3H2O	0.4	10
MgSO4 _ 7H2O	0.75	10
CaCl2 _ 2H2O	0.36	10
Citric acid	0.06	10
Ferric ammonium citrate	0.06	10
EDTA (dinatrium-salt)	0.01	10
Na2CO3	0.2	10
Micronutrient solution		1
Distilled water		919

Added to 1000 ml of distilled water.

Table

H3BO3	61.0 mg
MnSO4 H2O	169.0 mg
ZnSO4 7H2O	287.0 mg
CuSO4 5H2O	2.5 mg
(NH4)6Mo7O24 4H2O	12.5 mg

The cultures were subjected to alternate light and dark periods of 12 h each. After growing for about 6–8 weeks, the cultures were filtered and centrifuged for 2500 × g for 15 min. The spent medium and the biomass were collected. The biomass was weighed and frozen until used for extraction.

Aqueous extraction

Aqueous extraction was performed with water. Biomass (50 mg) was placed in a test tube and 3 mL of distilled water was added to it. The mixture was kept at room temperature (25–27°C) for 30 min, followed by centrifugation at 2500 × g (T21, Sorvall centrifuge Soovall, GMI Inc., Minnesota, USA) for 6 min. The supernatant was withdrawn and stored at –80°C until further use in bioassay.

Organic extraction

Biomass (10–20 g) was obtained from the cultures after filtering and drying of the obtained material. The freeze-dried cyanobacterial biomass was extracted successively using different solvents in increasing order of polarity (i.e., n.-hexane–chloroform–methanol). The solvent extracts were concentrated using a rotary evaporator under reduced pressure at 25°C for n.-hexane and chloroform and at 40°C for methanol. The weight of the solid residue was recorded and taken as the yield of the crude extract. The extracts were resuspended in dimethyl sulfoxide (DMSO) to achieve a stock concentration of 2 mg/mL.

Thin-layer chromatography

TLC was performed with the crude methanol extracts of Hapalosiphon. sp. (order Stigonometceae) (NCBI taxonomy browser, 2004) using silica gel F₂₅₄ precoated aluminum foil with a layer thickness of 0.2 mm (1.05554, Merck, NJ, USA). Two bands of 10 µL were applied on a chromatoplate (CAMAG Linomat 5 MEP Instruments Pvt. Ptd., Australia) and run in the solvent system (chloroform and methanol, 85:15). The chromatogram was sprayed with anisaldehyde sulfuric acid reagent.

Column chromatography

The methanol extract of Hapalosiphon. sp. was subjected to column chromatography to identify the active component. Column chromatography was carried out using silica gel 60–120 mesh (Ranbaxy), and TLC was carried out using silica gel G (Ranbaxy). Biomass (50 mg) was dissolved in 1 mL of methanol and laid on top of the silica gel column. The column was eluted sequentially with a 50 mL mixture of chloroform and methanol from low to high polarity, (i.e., starting with 1 part methanol and 9 parts chloroform and increasing the concentration of methanol in each step). Methanol was added in the last step to elute all the components. Fractions were collected sequentially in labeled tubes and analyzed by TLC.

Antimycobacterial bioassay

Bioassays using the agar-well diffusion method were performed against eight different strains of mycobacteria including one M. tuberculosis. ATCC 27294 H₃₇Rv-sensitive strain, M. tuberculosis. ATCC 35801 Erdman strain, two M. tuberculosis. clinical multidrug-resistant (MDR) strains, Mycobacterium avium-.complex, Mycobacterium intracellulare. (from the Mycobacterium aviumintracellulare. complex; MAC), and one atypical mycobacteria, Mycobacterium aurum.. The cultures were incorporated in the molten Middlebrook 7H11 agar medium for mycobacteria, and 50 µL of the extracts/drugs (2 mg/mL of the extract) were added in the wells punched in the agar. Zones of inhibition were measured after 21–28 days except in case of the atypical mycobacteria, Mycobacterium aurum., where the zone diameters were read after 5–7 days.

Determination of minimum inhibitory concentration by microplate Alamar blue assay

Minimum inhibitory concentration (MIC) was performed with active Hapalosiphon. and Scytonema. (order Scytonemataceae) extracts. Five cultures each of M. tuberculosis. and Mycobacterium avium-.complex were taken for the MIC assay. The cultures were adjusted to an optical density of McFarland no. 1 standard in 0.04% Tween 80 and 0.2% bovine serum albumin. Suspensions were diluted 1:25 in 7H9 GC broth, that is, Middlebrook 7H9 broth supplemented with glycerol and casitone (Difco, NJ, USA). Two hundred µL of 2X drug solution of the respective compounds was added to the wells of 96-well flat-bottom microtiter plates (Tarsons, Calcutta, India). The final drug concentration ranges were 0.03 to 8.0 µg/mL for the standard drugs rifampicin (RIF) and isoniazid (INH) and 15.6 µg/mL to 2 mg/mL for the cyanobacterial extracts. One hundred µL of M. tuberculosis. or Mycobacterium avium. inoculum was added. After incubation for about 7 days, 50 µL of a freshly prepared 1:1 mixture of Alamar blue (Accumed International, Westlake, OH, USA) reagent and 10% Tween 80 was added to the control well and incubated (Collins & Franzblau, 1997). If the well turned pink, indicating growth, the reagent mixture was added to all wells. The MIC was defined as the lowest drug concentration that prevented a color change from blue to pink.

Results and Discussion

Antimycobacterial bioassay

Our results show that the agar diffusion assay is a valuable tool for detecting the antimycobacterial effects of the different extracts of cyanobacteria. All mycobacterial strains showed very good growth in the 7H11 media, and the zones of inhibition could be clearly seen. M. tuberculosis. H37v ATCC 27294 and M. intracellulare. ATCC 35761 are good indicator strains for studying the activity of the crude or fractionated extracts. Zones of inhibition were observed with the chloroform and methanol extracts of Hapalosiphon. sp. (Table 1). Although Oscillatoria. sp. showed activity at 10 mg/mL concentration of the extract, the activity was lost at 2 mg/mL. The methanol extracts of Spirulina. sp. and Anabaena cylindrica. showed good zones of inhibition against the MAC strains. No inhibition was observed with the hexane and chloroform extracts of Scytonema. sp., but the methanol extract produced inhibition zone of 20–30 mm against the MAC strains and slight zones against the M. tuberculosis. strains.

Table 1.. Zone diffusion assay against mycobacteria (classification of cyanobacteria based on NCBI browser, 2004

		Zone of inhibition diameter (in millimeters)
Cyanobacterial strain (2 mg/mL conc.)	Extract (50 µL/well)	M. tuberculosis. H37Rv ATCC 27294	M. tuberculosis. MDR	M. avium. ATCC 49601	M. intracellulare. ATCC 35761	M. aurum.
Hapalosiphon. sp.	Hexane	—	—	—	—	—
(Order Stigonometales, Family Stigonomataceae)	Chloroform	13.81	13.26	—	—	12.84
	Methanol	16.21	16.86	16.77	14.75	16.41
	DCM-MeOH	17.27	12.60	14.06	11.42	Not tested
	Fraction (4)	17.7	16.42.	17.42	19.58	13.28
	Fraction (6)	—	—	—	19.27	—
Anabaena. sp.	Water	—	—	—	—	—
(Order Nostocales, Family Nostocaceae)	Hexane	14.14	—	12.23	12.32	—
	Chloroform	Not tested	Not tested	Not tested	Not tested	Not tested
	Methanol	20.22	sl	—	16.97	11.34
Lyngbya. sp.	Hexane	—	—	—	—	—
(Order Oscillatoriales)	Chloroform	—	—	—	—	—
	Methanol	—	—	—	—	—
Westeillopsis prolifica. Janet	Methanol	12.26	11.99	12.42	12.22	13.24
(Order Stigonematales)
Spirulina. sp.	DCM	11.47	10.77	30.06	26.50	—
(Order Nostocales, Family Nostocaceae)	Methanol	Slight	—	21.89	—	29.43
Anabaena variabilis. Kutzing	Hexane	—	—	—	—	—
(Order Nostocales, Family Nostocaceae)	Chloroform	—	—	—	—	—
	Methanol	—	—	—	—	17.20
Anabaena cylindrica. Lemmerman	Methanol	10.48	—	25.75	30.77	23.80
Aulosira fertilissima.	Hexane	—	—	—	—	—
(Order Nostocales, Family Nostocaceae)	Chloroform	—	—	—	—	—
	Methanol	—	—	—	—	—
Oscillatoria. sp.	Hexane	sl	sl	sl	sl	sl
(Order Osciillatoriales, Family Oscillatoriaceae)	Chloroform	10.42	sl	sl	sl	sl
	Methanol (10 mg/mL)	19.90	11.26	12.27	13.24	10.51
Scytonema. sp.	Hexane	sl	sl	sl	sl	sl
(Order Nostocales, Family Scytonemataceae)	Chloroform	10.42	sl	sl	sl	sl
	Methanol	19.90	11.26	21.34	30.22	15.56
Rifampicin (0.2 µg/ml)		37.18	—	25.34	23.44	40.32

Note.: S1 refers to slight zone.

Column chromatography and TLC

The yield of the methanol extract of Hapalosiphon. sp. was 1.5%. Although good zones of inhibition were observed with Anabaena cylindrica. and Spirulina. sp., the activity was lost when the cultures were grown in large scale, probably due to batch variation. Partial purification was done with the crude extract of Hapalosiphon. sp. because it consistently showed activity against all the strains tested. TLC with the crude methanol extract of Hapalosiphon. sp. is shown in Figure 1. The Hapalosiphon. sp. was grown in a large-scale culture in 6 L of BG11 media (annexure I). The partial purification of the methanol extract with silica gel chromatography gave seven fractions. Bioassay against M. tuberculosis. cultures showed that only one fraction, no. 4, retained activity against the mycobacterial strains. The yield of the fraction was approximately 2 mg. TLC of the partially purified methanol extract of Hapalosiphon. sp. (Fig. 1) gave a major single band of R_f value 0.36 (CAMAG Reprostar).

Figure 1 TLC of Hapalosiphon. fraction 4 purified by column and Hapalosiphon. crude extract. The major band in the partially purified fraction is marked with R_f. value.

Minimum inhibitory concentration

The MIC of the methanol extracts was performed initially by the agar dilution assay, a modified method of NCCLS M24-T2 (2000), Wayne, Pa. agar proportion method (not described here), as a standard assay performed in our laboratory for antimycobacterial susceptibility testing. This was performed against a panel of 30 strains including the test strains taken for the bioassay. The MICs of the extracts were > 64 µg/mL. Because the requirement of the test substance in this method is greater, a microplate-based method, the Alamar blue assay, was used to determine the actual MICs. The MIC values for the Hapalosiphon. crude methanol extract ranged from 125 to 500 µg/mL (Table 2) for the sensitive strains of M. tuberculosis., but the MICs were ≥ 2 mg/mL for the MDR strains. Fraction no. 4, purified from column, showed activity similar to the crude extract, confirming the active principle. The MIC of Scytonema. sp. against the MAC strains showed some signs of activity.

Table 2.. Minimum inhibitory concentration (MIC) of the cyanobacterial extracts by Alamar blue assay (no inhibition was observed with DMSO/methanol).

	MIC by MABA (µg/mL)
Strain	Rifampicin (RIF)	Isoniazid (INH)	Clarithromycin	Hapalosiphon. sp. (crude)	Scytonema. sp.	Hapalosiphon. sp. fraction 4
M. tubereolosis. H37Rv ATCC 27294	0.03	0.5	Not tested	500	500	250
M. tubereolosis. H37Rv clinical	0.03	0.25	Not tested	500	500	250
M. tubereolosis. (MDR)	> 32	> 32	Not tested	2000	> 4000	2000
M. tubereolosis. (MDR)	> 32	> 32	Not tested	2000	> 4000	2000
M. tubereolosis. H37Ra	0.125	0.25	Not tested	250	250	125
M. avium. ATCC 49601	0.03	0.25	0.25	250	500	250
M. intracellulare. ATCC 35761	0.03	0.25	0.25	250	62.5	250
M. avium. ATCC 700898	0.125	Not tested	0.06	259	125	250
M. avium. clinical	0.125	Not tested	0.03	31.2	125	125
M. avium. clinical	0.125	Not tested	0.06	250	500	125

Note.: μ refers to micro, AB refers to alamar blue.

The mycobacterial bioassay established by us and the MIC by the Alamar blue assay provide a very clear indication of antimycobacterial activity in the crude and purified extract. This study is the first report of antimycobacterial activity from cyanobacteria. Two cyanobacterial strains, Hapalosiphon. sp. and Scytonema. sp. showed activity. Repeated screening with Haplosiphon. sp. showed consistency in the results and was therefore studied further to confirm the active component. Although these results do not indicate any defined antimycobacterial entity and the activity of the extracts is low, our phytochemical findings provide a basis for exploiting these organisms for a new pharmacophore on which more active analogues could be synthesized and used to meet the growing challenge of drug resistance against mycobacteria. Studies on identifying the active principle in Scytonema. sp. are also in progress.

Acknowledgment

The authors thank the Herbal Chemistry Department of Ranbaxy Research Labs for providing facilities for the chromatography studies.