Antonie van Leeuwenhoek

, Volume 103, Issue 5, pp 947–961

Cyanobacteria: potential candidates for drug discovery

Authors

    • Algology SectionCSIR-National Botanical Research Institute
  • M. R. Suseela
    • Algology SectionCSIR-National Botanical Research Institute
Review Paper

DOI: 10.1007/s10482-013-9898-0

Cite this article as:
Dixit, R.B. & Suseela, M.R. Antonie van Leeuwenhoek (2013) 103: 947. doi:10.1007/s10482-013-9898-0

Abstract

Cyanobacteria are a rich source of vast array of bioactive molecules including toxins with wide pharmaceutical importance. They show varied bioactivities like antitumor, antiviral, antibacterial, antifungal, antimalarial, antimycotics, antiproliferative, cytotoxicity, immunosuppressive agents and multi-drug resistance reversers. A number of techniques are now developed and standardized for the extraction, isolation, detection and purification of cyanobacterial bioactive molecules. Some of the compounds are showing interesting results and have successfully reached to phase II and phase III of clinical trials. These compounds also serve as lead compounds for the development of synthetic analogues with improved bioactivity. Cyanobacterial bioactive molecules hold a bright and promising future in scientific research and great opportunity for drug discovery. This review mainly focuses on anticancerous, antiviral and antibacterial compounds from cyanobacteria; their clinical status; extraction and detection techniques.

Keywords

Bioactive moleculesAnticancerousAntiviralAntimicrobialClinical trialsExtraction methods

Introduction

Cyanobacteria are a group of photosynthetic prokaryotes and are among the most successful and oldest life forms present on earth (Gademann and Portmann 2008; Bajpai et al. 2010). They represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. They can be found in terrestrial, glaciers, aerial, marine, brackish and fresh water environments. Cyanobacteria are often a main component of phytoplankton in many freshwater and marine ecosystems. Cyanobacteria produce one or a range of bioactive compounds, which are potentially rich source of a vast array of products with applications in feed, food, nutritional, cosmetic, pharmaceutical and neutraceutical industries (Tan 2007). Due to their high chemical stability and water solubility, these compounds have important implications. They have a bright future in scientific research and for human welfare.

According to World Health Organisation (WHO), approximately 80 % of the world population depends on traditional remedies for their primary health care needs. Since, thousands of years natural products have been found to be used for treating various diseases and they form a major milestone for modern therapeutics. In the microbial world, especially cyanobacteria are prolific producers of secondary metabolites, many of which show various biological activities or bioactivity. Gerwick et al. (2008) found that secondary metabolites are mostly isolated from the members of oscillatoriales (49 %), followed by nostocales (26 %), chroococcales (16 %), pleurocapsales (6 %) and stigonematales (4 %). Cyanobacteria such as Anabaena, Nostoc, Microcystis, Lyngbya, Oscillatoria, Phormidium and Spirulina produce variety of high value compounds such as carotenoids, fatty acids, lipopeptides, polysaccharides and other bioactive compounds. Apoptogenic activity was more abundant in the genera Anabaena and Microcystis compared to Nostoc, Phormidium, Planktothrix, and Pseudoanabaena (Oftedal et al. 2011). Interestingly synthesis of these biomolecules remains an enigma and unresolved puzzle to the scientific world.

A majority of these biomolecules are peptides and are synthesized by large multimodular nonribosomal polypeptide (NRPS) or mixed polyketide (PKS)-NRPS enzymatic systems (Schwarzer et al. 2003). In aquatic environments, these metabolites usually remain within the microbial cells and are released in substantial amounts on cell lysis (Chorus and Bartram 1999). Richard E. Moore (1970s to early 2000s), revealed that the marine cyanobacteria are an exceptionally rich source of “secondary metabolites” (Cardellina and Moore 2010). Cyanobacterial secondary metabolites includes different compounds like cytotoxic (41 %), antitumor (13 %), antiviral (4 %), antimicrobial (12 %) and other compounds (18 %) include antimalarial, antimycotics, multi-drug resistance reversers, antifeedant, herbicides and immunosuppressive agents (Burja et al. 2001). Thus, cyanobacteria continue to be explored and their metabolites are now evaluated in number of biological areas and they are becoming an exceptional source of leading compounds for drug discovery (Singh et al. 2005, 2011; Nunnery et al. 2010; Bajpai et al. 2010). Anticancerous, antiviral and antibacterial bioactive molecules produced by various cyanobacteria are listed in Table 1. These natural products not only serve directly as drugs, but also being used as template for the discovery and synthesis of new drugs.
Table 1

Bioactive molecules produced by various cyanobacteria

Bioactive molecules

Cyanobacteria

Bioactivity

References

Symplocamide A

Symploca sp.

Anticancer

Linington et al. (2008)

Symplostatin

Symploca sp.

Anticancer

Luesch et al. (2002b)

Apratoxins

Lyngbya majuscula, L. sordid, L. bouilloni

Anticancer

Luesch et al. (2001a), Gutierrez et al. (2008), Matthew et al. (2008), Tidgewell et al. (2010)

Aplysiatoxin

Lyngbya majuscula

Anticancer

Mynderse et al. (1977)

Lyngbyabellin B

Lyngbya majuscula

Anticancer

Luesch et al. (2000)

Acutiphycin

Oscillatoria acutissima

Anticancer

Barchi et al. (1984)

Dragonamide C, D

Lyngbya polychroa

Anticancer

Gunasekera et al. (2008)

Cryptophycins

Nostoc sp.

Anticancer

Moore et al. (1996)

Arenastatin A

Dysidea arenaria

Anticancer

Moore et al. (1996)

Borophycin

Nostoc linckia, N. spongiaeforme

Anticancer

Hemscheidt et al. (1994)

Homodolastatin 16

Lyngbya majuscula

Anticancer

Davies-Coleman et al. (2003)

Curacin A

Lyngbya majuscula

Anticancer

Simmons et al. (2005)

Tjipanazoles

Tolypothrix tjipanasensis

Anticancer

Bonjouklian et al. (1991)

Pitipeptolides A, B

Lyngbya majuscula

Anticancer

Luesch et al. (2001c)

Aurilide

Lyngbya majuscula

Anticancer

Han et al. (2006)

Carmabin A, B

Lyngbya majuscula

Anticancer

McPhail et al. (2007)

Calothrixins A, B

Calothrix sp.

Anticancer

Rickards et al. (1999)

Dolastatins

Lyngbya sp., Symploca sp.

Anticancer

Fennell et al. (2003)

Biselyngbyaside

Lyngbya sp.

Anticancer

Teruya et al. (2009b)

Ankaraholide A

Geitlerinema sp.

Anticancer

Andrianasolo et al. (2005)

Malyngamide 3

Lyngbya majuscula

Anticancer

Gunasekera et al. (2011)

Cocosamides B

Lyngbya majuscula

Anticancer

Gunasekera et al. (2011)

Bisebromoamide

Lyngbya sp.

Anticancer

Teruya et al. (2009a)

Symplocamide A

Symploca sp.

Anticancer

Linington et al. (2008)

Veraguamides

Symploca cf. hydnoides

Anticancer

Salvador et al. (2011)

Largazole

Symploca sp.

Anticancer

Taori et al. (2008)

C-phycocyanin

Aphanizomenon flos-aquae

Anticancer

Tokuda et al. (1996)

Diacylglycerols

Aphanizomenon flos-aquae

Anticancer

Tokuda et al. (1996)

Caylobolide

Phormidium sp.

Anticancer

Salvador et al. (2010)

Coibamide

Leptolyngbya sp.

Anticancer

Medina et al. (2008)

Hoiamide

Association of Lyngbya majuscula and Phormidium gracile

Anticancer

Choi et al. (2010)

Isomalyngamide

Lyngbya majuscula

Anticancer

Chang et al. (2011)

Jamaicamides

Lyngbya majuscula

Anticancer

Edwards et al. (2004)

Kalkitoxin

Lyngbya majuscula

Anticancer

White et al. (2004)

Palauamide

Lyngbya sp.

Anticancer

Zou et al. (2005)

Tasiamide

Symploca sp.

Anticancer

Williams et al. (2003a)

Tasipeptins

Symploca sp.

Anticancer

Williams et al. (2003b)

Wewakpeptins

Lyngbya semiplena

Anticancer

Han et al. (2005)

Lagunamide

Lyngbya majuscula

Anticancer

Tripathi et al. (2011)

Majusculamide

Lyngbya majuscula

Anticancer

Pettit et al. (2008)

Malevamide

Symploca hydnoides

Anticancer

Horgen et al. (2002)

Obyanamide

Lyngbya confervoides

Anticancer

Williams et al. (2002)

Palmyramide

Lyngbya majuscula

Anticancer

Taniguchi et al. (2010)

Ulongapeptin

Lyngbya sp.

Anticancer

Williams et al. (2003c)

Grassypeptolide

Lyngbya majuscula

Anti-proliferative

Kwan et al. (2008)

Nostoflan

Nostoc flagelliforme

Antiviral

Kanekiyo et al. (2005)

Ichthyopeptins

Microcystis ichthyoblabe

Antiviral

Zainuddin et al. (2007)

Bauerines A–C

Dichotrix baueriana

Anti-HSV-2

Larsen et al. (1994)

Sulfolipids

Lyngbya lagerhimii, Phormidium tenue

Anti-HIV

Gustafson et al. (1989)

Calcium spirulan

Spirulina platensis

Anti-HIV

Hayashi et al. (1996)

Cyanovirin

Nostoc ellipsosporum

Anti-HIV

Dey et al. (2000)

Scytovirin

Scytonema varium

Anti-HIV

Xiong et al. (2006)

Sulfoglycolipid

Scytonema sp.

Anti-HIV

Loya et al. (1998)

Ambiguines

Fischerella sp.

Antibacterial

Raveh and Carmeli (2007)

Bastadin

Anabaena basta

Antibacterial

Miao et al. (1990)

Bis-(χ-butyrolactones)

Anabena variabilis

Antibacterial

Ma and Led (2000)

Hapalindole

Nostoc CCC537, Fischerella sp.

Antibacterial

Asthana et al. (2009)

Abietane diterpenes

Microcoleous lacustris

Antibacterial

Gutiérrez et al. (2008)

Nostocine A

Nostoc spongiaeforme

Antibacterial

Hirata et al. (2003)

Noscomin

Nostoc commune

Antibacterial

Jaki et al. (2000)

Didehydromirabazole

Scytonema mirabile

Antibacterial

Stewart et al. (1988)

Tolyporphin

Tolypothrix nodosa

Antibacterial

Prinsep et al. (1992)

Muscoride

Nostoc muscorum

Antibacterial

Nagatsu et al. (1995)

Ambiguine

Fischerella ambigua

Antibacterial

Raveh and Carmeli (2007)

Anticancerous compounds

A large number of cyanobacterial bioactive compounds are found to target tubulin or actin filaments in eukaryotic cells, making them an attractive source of anticancer agents (Jordan and Wilson 1998). The small anticancerous peptide, Dolastatin 10 and Dolastatin 12 was isolated from Symploca sp. and Leptolyngbya sp. (Kalemkerian et al. 1999; Catassi et al. 2006). Curacin A shows antiproliferative activity that has been isolated from cyanobacterium Lyngbya majuscula (Nagle et al. 1995). It is also artificially synthesized because of its pharmacological importance (Muir et al. 2002). Cryptophycin isolated from Nostoc shows dose-dependent inhibition of L1210 leukemia cell line (Smith et al. 1994). Rickards et al. (1999) isolated two compounds calothrixin A and B from the organic extracts of Calothrix strains and found that it inhibited the growth of human HeLa cancer cells.

Apratoxin A from L. majuscula (Luesch et al. 2001a), Apratoxin B–C from Lyngbya sp. (Luesch et al. 2002a), Apratoxin D from L. majuscula and Lyngbya sordid (Gutierrez et al. 2008), Apratoxin E from Lyngbya bouilloni (Matthew et al. 2008) and Apratoxins F–G from L. bouilloni (Tidgewell et al. 2010) showed cytotoxicity to various cancer cell lines i.e. U2OS osteosarcoma, HT29 colon adenocarcinoma, HeLa cervical carcinoma, KB oral epidermoid cancer, LoVo colon cancer, H-460 lung cancer and HCT-116 colorectal cancer cells lines. Luesch et al. (2000) isolated Lyngbyabellin B from L. majuscula, it shows cytotoxic against KB and LoVo cells lines. Likewise, symplocamide A, isolated from Symploca sp. showed potent cytotoxicity to lung cancer cells and neuroblastoma cells (Linington et al. 2008).

Malyngamide 3 and cocosamides B are recently isolated from L. majuscula and it showed weak cytotoxicity against MCF7 breast cancer and HT-29 colon cancer cells (Gunasekera et al. 2011). Oftedal et al. (2010) have screened several cyanobacteria for the acute myeloid leukemia (AML), which is the second most common form of leukemia. They have found that the aqueous cyanobacterial extract is most effective in causing apoptosis to AML cell lines. The combination of a moderate concentration of the anticancer drug daunorubicin with cyanobacterial extract induced a synergistic apoptotic response in AML cells. It can be concluded that these cyanobacterial apoptogens have the ability to greatly improve the therapeutic index of daunorubicin (Oftedal et al. 2010). Also, there was no correlation between mouse toxicity and induction of apoptosis neither in T cell lymphoma nor in AML-cells (Oftedal et al. 2011).

Bisebromoamide, a new cell toxin that inhibits cancer cell lines, was obtained from an Okinawan collection of Lyngbya sp. (Teruya et al. 2009a). It inhibits the phosphorylation of extracellular signal-related protein kinase (ERK). Veraguamides from cyanobacterium Symplocacf. hydnoides showed moderate to weak cytotoxic activity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cell lines (Salvador et al. 2011). Taori et al. (2008) reported that largazole isolated from Symploca sp. shows cytotoxicity against transformed mammary epithelial cell lines (MDA-MB-231). The molecular target for largazole is found to be histone deacetylases (HDACs), and it is categorized as a class I HDAC inhibitor.

Some workers have also reported the anticancerous activity of photosynthetic pigment. Li et al. (2010) reported anti-tumor activities of C-phycocyanin (C-PC) mediated photodynamic therapy in MCF-7 breast cell lines. Aphanizomenon flos-aquae extract containing a high concentration of phycocyanin inhibited the in vitro growth of tumor cell lines, Phormidium tenui contain several diacylglycerols that inhibit chemically induced tumors in mice (Tokuda et al. 1996). Recently, Gantar et al. (2012) reported that C-PC in combination with lower dose (10 % of typical dose) of anticancer drug topotecan can kill cancer cells at higher rate than used alone at full dose.

Cyanobacteria cyclopeptides as a lead compound for cancer treatment

Cyanobacterial cyclopeptides, microcystins (MCs) and nodularins at high concentration are considered to be toxic to humans (Carmichael et al. 2001; Funari and Testai 2008). MCs are the most common cyanobacterial toxin prevalent in the water bodies. They can be ingested through contaminated drinking water (Oberholster et al. 2004), fish (Poste et al. 2011) or sea foods (Mulvenna et al. 2012). From a pharmacological point of view, MCs are stable hydrophilic cyclic heptapeptides causes cellular damage following uptake via organic anion transporting polypeptides (OATP) (Sainis et al. 2010; Fischera et al. 2005). Their intracellular biological effects involve inhibition of catalytic subunits of protein phosphatase 1 (PP1) and PP2, glutathione depletion and generation of reactive oxygen species (ROS) (Amado and Monserrat 2010). However, there are certain OATPs which are prominently expressed in cancer tissue as compared to normal tissue, qualifying MCs as potential candidates for cancer drug development (Sainis et al. 2010; Monks et al. 2007).

In the era of targeted cancer therapy, cyanobacterial toxins comprise a rich source of natural cytotoxic compounds with a potential to target cancers expressing specific uptake transporters (Sainis et al. 2010). Furthermore, a high proportion of these natural products target eukaryotic cytoskeleton, such as tubulin and actin microfilaments, making them an attractive source of anticancer drugs (Tan 2010).

Antiviral compounds

The global spread of deadly viral diseases like HIV/AIDS and avian influenza (H5N1 virus) etc. have showed fatal consequences. Food and Drug Administration (FDA) approved anti-HIV treatment highly active antiretroviral therapy (HAART), is effective in controlling the progression of HIV infections but found to be toxic (Luescher-Mattli 2003). Thus, novel drugs are now urgently required to combat deadliest diseases. Antiviral compound isolated form cyanobacteria are usually found to show bioactivity by blocking viral absorption or penetration and inhibiting replication stages of progeny viruses after penetration into cells. The protection of human lymphoblastoid T cells from the cytopathic effect of HIV infection with the extract of Lyngbya lagerheimeii and Phormidium tenue has been reported by Gustafson et al. (1989). A new class of HIV inhibitors called sulfonic acid, containing glycolipid, was isolated from the extract of cyanobacteria and the compounds were found to be active against the HIV virus. Cyanovirin-N (CVN), a peptide isolated from cyanobacteria, inactivates the strains of HIV virus and inhibits cell to cell and virus to cell fusion (Yang et al. 1997). In vitro and in vivo antiviral tests suggested that the anti-HIV effect of CVN is stronger than a well-known targeted (viral entry) antibody (2G12) and another microbicide, PRO2000 (Xiong et al. 2010).

Calcium spirulan (Ca-SP), a novel sulphated polysaccharide, is an antiviral agent. This compound selectively inhibits the entry of enveloped virus (Herpes simplex, humancytomegalo virus, measles virus) into the cell (Hayashi et al. 1996). Rechter et al. (2006) have analyzed polysaccharide fractions isolated from Arthrospira platensis. These fractions containing spirulan-like molecules showed a pronounced antiviral activity against human cytomegalovirus, herpes simplex virus type 1.

Yakoot and Salem (2012) has conducted first human trial to address the effect of Spirulina platensis dried extract on virus load, liver function, health related quality of life and sexual functions in patients with chronic hepatitis C virus (HCV) infection. They found the therapeutic potential of S. platensis in chronic HCV patients, and in some cases (13 %) the viral infection is complexly nullified. Mansour et al. (2011) have found that the polysaccharides isolated from Gloeocapsa turgidus and Synechococcus cedrorum showed higher antiviral activity against rabies virus than that against herpes-1 virus. The exopolysaccharide from Aphanothece halophytica has an antiviral activity against influenza virus A (H1N1), which shows an 30 % inhibition of pneumonia in infected mice (Zheng et al. 2006).

Antibacterial compounds

Noscomin from Nostoc commune exhibited antibacterial activity against Bacillus cereus, Staphylococcus epidermidis, and Escherichia coli (Jaki et al. 2000). Nostocarboline from Nostoc was found to inhibit the growth of other cyanobacteria and green alga (Blom et al. 2006). Hirata et al. (2003) found that nostocine A isolated from Nostoc spongiaeforme exhibited growth inhibitory stronger to green algae than to cyanobacteria. Asthana et al. (2009) have isolated hapalindole (alkaloids) from Nostoc CCC537 and Fischerella sp. and found antimicrobial activity against Mycobacterium tuberculosis H37Rv, Staphylococcus aureus ATCC25923, Salmonella typhi MTCC3216, Pseudomonas aeruginosa ATCC27853, E. coli ATCC25992 and Enterobacter aerogenes MTCC2822.

Ambiguine, a hapalindole-type alkaloid from Fischerella ambigua shows antibacterial activity against M. tuberculosis and against Bacillus anthracis (Raveh and Carmeli 2007). Guo et al. (2009) have isolated 6-cyano-5-methoxy-12-methylindolo (2,3-a) carbazole from cyanobacteria and identified as a B. anthracis inhibitor. The methanolic extract of S. platensis showed broad spectrum antimicrobial activity and the inhibition recorded was maximum for S. aureus followed by E. coli, P. aeruginosa and S. typhi (Kaushik and Chauhan 2008). Because of the growing bacterial resistance against antibiotics and commercial standard the search for new active substances with antibacterial activity is urgently needed and cyanobacteria are the potential and promising candidates.

Cyanobacterial bioactive molecules under clinical trials

The drug-development process normally proceeds through various phases of clinical trials (phase 0 or pre clinical, phase I, phase II and phase III). The FDA must approve each phase before the study can continue. Drugs are first tested in laboratory animal (pre clinical phase) and in further phases healthy human are tested. In phase I, II and III the number of subjects range from 20 to 80, few dozen to 300 and several hundred to 3,000 people, respectively. If the drug successfully passes through all the phases it will usually be approved by the National Regulatory Authority (NRA) for use by the general population. There are few prominent molecules from cyanobacteria such as dolastatins, cryptophycins, curacin and their analogues which are in clinical trials as potential anticancer drugs. The structures were drawn with the help of ChemDraw (Fig. 1).
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Fig. 1

Structures of biomolecules which were in clinical trials

Dolastatins are the group of structurally unique peptides which were first reported from marine animal Dolabella auricularia (Pettit et al. 1987), using microalgae as diet, but later on they were isolated from the cyanobacteria Symploca (Luesch et al. 2001b). Dolastatins show anticancerous activity by inhibiting microtubule assembly and many of its analogues are in clinical trials. Till date there are sixteen dolastatin forms isolated and are simply named as dolastatin 1, 2, 3, and so on. Dolastatin 10 and 15 are found to be showing promising results. National Cancer Institute of US conducted the phase I clinical trials of dolastatin 10 and progressed further to phase II. Unfortunately, it was dropped from clinical trials, due to some toxic effects (Simmons et al. 2005). This finding results in the development of dolastatin 10 synthetic analogues like soblidotin, usually with improved pharmacological and pharmacokinetic properties. Interestingly, its synthetic analogue soblidotin, has cleared phase I and II successfully and now undergoing phase III clinical trials under the supervision of Aska Pharmaceuticals, Tokyo, Japan (Bhatnagar and Kim 2010). The antitumor activity of soblidotin, was found to be superior to existing anticancer drugs, such as paclitaxel and vincristine (Watanabe et al. 2006). The third generation dolastatin 15 analogues are cemadotin and tasidotin. Tasidotin is antitumor agent and has cleared phase I trials (Mita et al. 2006) and now undergoing phase II trials with Genzyme Corporation, Cambridge, MA (Bhatnagar and Kim 2010).

Cryptophycins are a group of cytotoxic depsipeptides, first isolated from Nostoc sp. as an antifungal compound (Schwartz et al. 1990), later it was reported to be effective against drug-resistant cancer cell lines (Smith et al. 1994). Moore group (Chaganty et al. 2004; Golakoti et al. 1994, 1995) have isolated twenty-six cryptophycins forms from Nostoc sp. GSV 224. Of the various forms, cryptophycin 52 was found to be the most successful and evaluated in phase II clinical trials for the treatment of platinum-resistant ovarian cancer (D’Agostino et al. 2006) and advanced lung cancer (Edelman et al. 2003). Unfortunately, the clinical trails were further discontinued as it causes neuropathy and pain in the patients. Magarvey et al. (2006) have analyzed cryptophycin biosynthetic pathways that have opened more avenues to create novel cryptophycin analogs (Sammet et al. 2010; Weiß et al. 2012).

Another group, curacins are unique thiazoline-containing lipopeptides that inhibits microtubule assembly and it is a potent competitive inhibitor of the binding of colchicine to tubulin (Blokhin et al. 1995). Gerwick group have isolated curacin A (Gerwick et al. 1994); curacin B and C (Yoo and Gerwick 1995); curacin D (Márquez et al. 1998) from Lyngbya majuscule, which is prevalent in different water bodies. Clinical development of curacin has been hindered due to its low water solubility and thus it is unable to produce activity during in vivo animal trails. Hence, curacin was withdrawn from pre clinical phase, but it served as a lead compound for the development of synthetic analogs which are more water soluble (Wipf et al. 2004). Isolation of all of these compounds offer great opportunity and a platform for the discovery of promising anticancer agents.

Methods used for isolation and detection of novel biomolecules

There are number of methods used for the extraction of valuable metabolites. The extraction can either be simultaneous (extra and intracellular metabolites) or sequential (intercellular metabolites only). In both types of extraction first quenching is done with liquid nitrogen to freeze the metabolic activity. In simultaneous extraction, quenching is immediately followed with extraction using a suitable solvent (butanol/acetone/hexane/chloroform/methanol/aqueous methanol/water/hexane/dichloromethane), while in sequential extraction biomass separation is done after quenching and then various fractions are collected by extracting with solvents of decreasing polarity sequentially (water, aqueous methanol, methanol, hexane) (Fig. 2). Conventional extraction methods employed are solid–liquid extraction (SLE) or liquid–liquid extraction (LLE). SLE is usually done by soxhlet apparatus, it is the process of removing a solutes from a solid (fixed phase) or matrix by using of liquid solvent (mobile phase). Whereas in LLE, both phase are in liquid phase and the separation of the solute depend on the distribution coefficient of the solute in mobile phase. Both SLE and LLE require high volumes of solvents, long extraction times and reproducibility of the results are low.
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Fig. 2

Extraction procedure commonly followed for the isolation of novel biomolecules from cyanobacteria

Recently low cost, chemical free green extraction (GE) methods are used such as supercritical fluid extraction (SFE), pressurized liquid extraction (PLE). In SFE, the dissolved solutes are separated from the raw material using certain gases (act as solvent) above critical points. Carbon dioxide (CO2) gas is commonly used solvent for the extraction of biomolecules. Some of the researchers are also using ethanol as cosolvent along with the CO2 gas. Mendiola et al. (2007) have used SFE–CO2 (220 bar, 26.7 °C) with 10 % of ethanol (cosolvent) for the extraction of antimicrobial component from S. platensis. Onofrejová et al. (2010) have used combination of pressurized-liquid with solid-phase extraction (PLE–SPE) for the isolation of bioactive phenols freshwater algae.

There are number of analytical techniques available for the detection and purification of biomolecules from cyanobacterial extract (Fig. 3). Thin layer chromatography (TLC) followed by spectrophotometric analysis is the easiest technique used for primarily identification and separation of bioactive molecules. Pelander et al. (2000) have used high performance TLC plates (HPTLC) for the separation of small cyanobacterial peptides. However, TLC/HPTLC separation is nonspecific and less sensitive. Use of high performance liquid chromatography (HPLC) for the identification and quantification has increased greatly. Nowadays more advance technique ultra performance liquid chromatography (UPLC) is available, which can be better option than HPLC. In order to get accurate identification of bioactive product, liquid chromatography is followed by mass spectrometry (LC–MS) (Harada et al. 2004). Different configurations of this approach such as fast atom bombardment (FAB-LC–MS) (Kondo et al. 1995) and electrospray ionization (ESI-LC–MS) has been developed (Barco et al. 2002; Spoof et al. 2003). Zhang et al. (2004) used LC–MS–MS with electrospray ionization. Liquid chromatography can also be coupled with quadruple time-of-flight tandem mass spectrometry (LC–QTof-MS) for cyanobacterial cyclopeptides detection (Ferranti et al. 2009). Immunoassay techniques due to their high sensitivity, specificity and operational simplicity are widely used for the characterization of biomolecules. Lindner et al. (2004) have developed the highly sensitive enzyme-linked immunosorbent assays (ELISA) for the detection and quantification of cyanobacterial cyclopeptides.
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Fig. 3

Detection and purification methods for isolation of bioactive molecules from cyanbacterial extract

Metabolites are also identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The technique requires very small amount of sample without separation or purification (Welker et al. 2002). Erhard et al. (1997) used MALDI-TOF MS for identification of secondary metabolites with intact cyanobacterial cells. Resulting mass signals which are further characterized by post source-decay fragmentation, and comparison of observed fragment spectra with theoretical ones or with those of pure reference compounds (Welker et al. 2004). In general, MALDI-TOF MS is used for the identification of peptides; however this can also be used for the identification of alkaloids (Aráoz et al. 2008). In MALDI-TOF MS, the mass fragmentation pattern of a particular analyte is distinctive, and may vary according to the ionization mode used for mass spectrometry and to the charge state of the molecule (Antoine et al. 2006; Welker et al. 2006).

Desorption electrospray ionization mass spectrometry (DESI-MS) is also an applied analytical technique for chemical profiling, characterization and quantification of low molecular-weight biomolecules (Esquenazi et al. 2009). Another technique, direct analysis in real time mass spectrometric (DART-MS) technique is very much effective in chemical profiling and fingerprinting of bioactive molecules without prior sample preparation. Singh and Verma (2012) have identified the Nostoc sp. on the basis of characteristic chemical compounds (chemical fingerprinting) using DART-MS. All these techniques are helpful for the identification and characterization of bioactive molecules.

Conclusion

Cyanobacteria are the promising sources potentially useful natural products. Microbial natural products discovery opens a new era of research. Presently, the isolation of number of natural products is increasing; however, few compounds have reached the market. Limited number of identified cyanobactererial biomolecules and analogues are in clinical trials and some of them have passed different phases of clinical trials to prove their candidature as potential drugs. In order to exploit the new opportunities available, it will be necessary to develop novel methodologies that allow the isolation and culture of microorganisms, which produce natural products unique to particular environmental conditions. Thus, there is an urgent need for extensive research in this new emerging field for drug discovery.

Acknowledgments

Authors are thankful to Director, CSIR-NBRI for all the facilities and constant encouragement. Rakhi Bajpai Dixit is grateful to Department of Science and Technology (DST, New Delhi), for providing financial assistance in the form of a project (Ref. No. SR/FT/LS-111/2010).

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© Springer Science+Business Media Dordrecht 2013