Pharmacognosy Magazine

: 2017  |  Volume : 13  |  Issue : 51  |  Page : 723--725

Screening of norharmane from seven cyanobacteria by high-performance liquid chromatography

Tunay Karan1, Ramazan Erenler2,  
1 Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Gaziosmanpasa University, Tokat, Turkey
2 Department of Chemistry, Faculty of Arts and Sciences, Gaziosmanpasa University, Tokat, Turkey

Correspondence Address:
Ramazan Erenler
Department of Chemistry, Faculty of Arts and Sciences, Gaziosmanpasa University, 60240-Tokat


Background: Cyanobacteria, including pharmaceutically and medicinally valuable compounds attract the great attention lately. Norharmane (9H-pyrido (3,4-b) indole found in some cyanobacteria revealed a great number of biological effects. Objective: Seven cyanobacteria were isolated and identified from Yesilirmak River and Gaziosmanpasa University Campus to determine the norharmane content. Materials and Methods: Cyanobacteria collected from Tokat, Turkey were isolated and identified by morphologically. Norharmane (9H-pyrido [3,4-b] indole) quantities were presented for seven cyanobacteria, Chroococcus minutus (Kütz.) Nägeli, Geitlerinema carotinosum (Geitler) Anagnostidis, Nostoc linckia Bornet ex Bornet and Flahault, Anabaena oryzae F. E. Fritsch, Oscillatoria limnetica Lemmermann, Phormidium sp. Kützing ex Gomont, and Cylindrospermum sp. Kutzing ex E. Bornet and C. Flahault by high-performance liquid chromatography. Results: The norharmane amount indicated for cyanobacterial culture media altered in a species-dependent kind in the range of 0.81–10.87 μ g/g. C. minutus produced the most norharmane among the investigated cyanobacteria as 10.87 μ g/g. Conclusion: Cyanobacteria could be an important source of norharmane as well as pharmaceutically valuable compounds. Abbreviations used: HPLC: High performance liquid chromatograph.

How to cite this article:
Karan T, Erenler R. Screening of norharmane from seven cyanobacteria by high-performance liquid chromatography.Phcog Mag 2017;13:723-725

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Karan T, Erenler R. Screening of norharmane from seven cyanobacteria by high-performance liquid chromatography. Phcog Mag [serial online] 2017 [cited 2022 Nov 29 ];13:723-725
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Seven cyanobacteria were isolated and identified from Yesilirmak RiverQuantitative analysis of norharmane was executed on isolated cyanobacteriaFour cyanobecteria species included the norharmaneChroococcus minutus contained the most norharmane (10.87 μg/g).


Secondary metabolites are natural products that have an important property in industrial and biomedical applications. Primer metabolites mediate synthesis of the required macromolecules, whereas the secondary metabolites are synthesized from metabolic intermediates that result from primary metabolism or from their final products.[1] Cyanobacterial metabolites may chemically be in the structure of peptides, alkaloids, indole alkaloids, polyketides, and terpenes, and many of these compounds exhibit a large variety of pharmaceutical properties.[2] Norharmane named as (9H-pyrido (3,4-b) indole, β- carboline class, is an alkaloid that has a heterocyclic amine structure derived from tryptophan amino acid. More than 140 different types of β-carbolines have been reported from plants and animals up to now.[3] Norharmane was also isolated from Streptomyces and marine dinoflagellates.[3] It is proto-toxic, including degeneration of nigrostriatal nerves in Parkinson's disease.[3] Several pharmacological effects of norharmane such as inhibition of monoamine oxidase, indoleamine 2,3 dioxygenase, nitric oxide synthase, and acetylcholinesterase enzymes were reported.[4],[5],[6] Norharmane has several pharmacological properties that affect the induction of apoptotic cell death in human neuroblastoma SH-SY5Y cells and the increase in insulin secretion from human Langerhans islets.[7]

In this study, the quantitative analysis of norharmane was investigated in Chroococcus minutus, Geitlerinema carotinosum, Nostoc linckia, Anabaena oryzae, Oscillatoria limnetica, Phormidium sp. and Cylindrospermum sp. which were collected and isolated from different location of Yesilirmak River and Gaziosmanpasa University Campus. Experimental results showed that C. minutus included the most norharmane (10.87 μg/g) among the investigated cyanobacteria.

 Materials and Methods

Cyanobacteria collection and isolation

Algae samples, taken from the pelagic regions of Yesilirmak River and Gaziosmanpasa University Campus between October and November 2014 in Tokat province and surrounding areas were collected and kept in 1 l plastic containers and tromped by filter paper (GF/C filter paper, Whatman). The samples were incubated for 2 weeks at 12/12 h (light/dark) in an enrichment medium (F2) at an average of 2465 lux at 26°C.[8] Cyanobacteria were identified and subjected to mechanical isolation under an inverted microscope through a micropipette and microinjector. C. minutus (40° 17 40.19'' N, 36° 19 28.81'' E), G. carotinosum (49° 19' 49.12'' N, 36° 34' 2.06'' E), N. linckia (40° 19' 45.655'' N, 36° 33' 45.06''E), A. oryzae (40° 19' 43.77'' N, 36°28' 22.26'' E), O. limnetica (40° 19' 51.27'' N, 36° 23' 4.69'' E), Phormidium sp.(40°19' 50.85'' N, 36° 28' 37.46'' E) and Cylindrospermum sp.(40° 20' 4.39'' N, 36° 28' 37.46'' E) were isolated. After mechanical isolation, they were streaked by 1.5% agarized medium.[9]


Bristol, Blue Green Algae (BG-11) and BG-11° (without NaNO3) which were the suitable media for cyanobacteria growth were used for cultivation of isolated cyanobacteria. An appropriate nutrient media (235 ml) and 10% of each cyanobacterium was inoculated into 250 ml flasks at 26°C, 2465 lux and kept to develop for about 2 weeks.[10]


The cultures were centrifuged at 4000 rpm for 15 min. The pellets were washed with distillated water for twice then lyophilized and were weighed on a precision scale. Each sample (0.50 mg) was placed in 1 ml glass tube and dissolved in methanol-chloroform (1/1) then vortexed for 1 min, and placed in an ultrasonic bath for 10 min.[11] After that, it was vortexed for another 1 min and filtered with a polytetrafluoroethylene syringe (Chrom Tech, 0.45 μm 13 mm).

High-performance liquid chromatography Analyses

The each extract sample (20 μl) was injected into the column. Quantitative analysis was executed by high-performance liquid chromatography (HPLC) (Shimadzo, Japan) with C18 120A (Thermo, 4.6 mm × 150 mm, 3 μm particle size) reverse phase column. Norharmane metabolite was detected at 247 nm wavelength with photodiode array detector. The flow rate was adjusted to 1 ml/min using a gradient system of A, water with 0.1% formic acid and B, acetonitrile. The gradient program was fixed as follows: 0–14 min, 100% A; 14–29 min, 80% A, 29–32 min, 60% A, 32–34 min, 0% A.[12] The amount of norharmane was calculated by the calibration curve using the Gauss method.

 Results and Discussion

Cyanobacteria and systematic

The needs for the life of cyanobacteria are mainly water, light, carbon dioxide, and simple inorganic compounds.[13] However, cyanobacteria can grow rapidly under certain environmental conditions.[14] Cyanobacteria were artificially developed in cultural media. While the C. minutus, G. carotinosum were cultivated in Bristol medium, O. limnetica, Phormidium sp. MBIC10025 were developed in BG11 medium and other cyanobacteria, Nostoc sp., A. oryzae and Cylindrospermum sp. CENA33 were cultivated in BG-11° [Table 1].{Table 1}

Quantitative analysis of norharmane

It was found out that four purified cyanobacteria, C. minutus, N. linckia, A. oryzae and G. carotinosum included norharmane, whereas the other three, O. limnetica, Phormidium sp., Cylindrospermum sp. did not. The retention time of norharmane was observed at 10th min in HPLC chromatogram [Figure 1]. Interestingly, the cyanobacteria species excreted norharmane belonged to the Nostocales order and Nostocaceae family [Table 1].{Figure 1}

Norharmane quantity was calculated according to the Gauss method by plotting the calibration curve over the absorbance value of the standard at 247 nm wavelength [Table 2].{Table 2}

Cyanobacteria are the inevitable sources of natural compounds used in biotechnology for the benefit of human beings, and bioactive secondary metabolites are of great interest in medicine and agriculture. Some cyanobacterial metabolites have significant pharmaceutical potentials as antimicrobial, anticancer, antiviral, and enzyme inhibitory activities.[15],[16] Indole alkaloids, bauerins A-C were isolated from terrestrial cyanobacteria (Dichotrix baueriana GO25-5) and were revealed the activity against herpes simplex virus 2.[17] Cytotoxic antiviral indolocarbazoles were isolated from Nostoc sphaericum EX-5-1.[18]

Norharmane is the simplest example of β-carboline exhibiting the various biological and pharmacological effects.[19] The investigation of norharmane in different cyanobacteria is rather scarce, but some researches on norharmane for Nodularia harveyana, Nostoc insulare and Synechocystis sp., Anabaena cylindrica, Anabaena inaequalis, Anabaenopsis siamensi s, C. minutus, Nostoc carneum, Nostoc commune, Nodularia harveyana, and Phormidium foveolarum were mentioned.[7],[20],[21]

C. minutus showed high and rapid ability to remove nonylphenol including bioaccumulation and biodegradation. Hence, C. minutus can be used for removing of nonylphenol contaminated aqueous systems effectively by biodegradation.[22] In addition, C. minutus has the ability of decolorizing cyclic azo dyes as well.[23] Unsaturated, hydroxy, n-saturated, branched, dioic fatty acids were determined from C. minutus and major fatty acids were found as hexadecanoic (16:0), 9-hexadecenoic (16:1), hexadecadienoic (16:2), octadecanoic (18:0), and 9-octadecenoic (18:1).[24]

N. linckia can be used for selenium nanoparticles synthesis. N. linckia biomass with selenium nanoparticles may be promising agent for medicinal, pharmaceutical, and technological objective.[25]Nostoc sp. methanol extract which included the phytol, n-hexadecanoic acid, 9,12-octadecadienoic acid, 1,2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester as the major products revealed the antimicrobial activity.[26] Moreover, Nostoc sp. displayed the acetylcholinesterase inhibitory activity.[27]

In the present study, the presence of norharmane, the target pharmacological secondary metabolite in the cyanobacteria taxa was determined by HPLC. The amount of norharmane was detected in C. minutus, G. carotinosum, N. linckia and A. oryzae as 10.87, 1.92, 0.81, and 2.27 (μg/g dried algae), respectively. However, norharmane was not detected in O. limnetica, Phormidium sp. and Cylindrospermum sp. in this work.


Screening the norhamane from algae samples, four of those comprised of the norharmane compound. Due to the consisting of bioactive compounds, cyanobacteria have a potency to be used in drug discovery and development process. Moreover, C. minutus, G. carotinosum, N. linckia and A. oryzae as could be a source of norharmane production. The isolation of bioactive secondary metabolites including norharmane from these cyanobacteria could be effective in the pharmaceutical industry.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Dos Santos M, Guaratini T, Lopes J, Colepicolo P, Lopes N. Plant cell and microalgae culture. Modern Biotechnology in Medicinal Chemistry and Industry. Kerala, India: Research Signpost; 2005.
2Gademann K, Portmann C. Secondary metabolites from cyanobacteria: Complex structures and powerful bioactivities. Curr Org Chem 2008;12:326-41.
3Nikam TD, Nitnaware KM, Ahire ML. Alkaloids Derived from Tryptophan: Harmine and Related Alkaloids. In: Ramawat KG, Mérillon J-M, editors. Natural Products: Phytochemistry, Botany and Metabolism of Alkaloids, Phenolics and Terpenes [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2013. p. 553–74.
4Connop BP, Kalisch BE, Boegman RJ, Jhamandas K, Beninger RJ. Enhancement of 7-nitro indazole-induced inhibition of brain nitric oxide synthase by norharmane. Neurosci Lett 1995;190:69-72.
5Chiarugi A, Dello Sbarba P, Paccagnini A, Donnini S, Filippi S, Moroni F, et al. Combined inhibition of indoleamine 2,3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interferon-gamma-activated macrophages. J Leukoc Biol 2000;68:260-6.
6Robinson ES, Anderson NJ, Crosby J, Nutt DJ, Hudson AL. Endogenous beta-carbolines as clonidine-displacing substances. Ann N Y Acad Sci 2003;1009:157-66.
7Volk RB. Screening of microalgae for species excreting norharmane, a manifold biologically active indole alkaloid. Microbiol Res 2008;163:307-13.
8Teneva I, Stoyanov P, Belkinova D. Production of cyanobacterial toxins from two Nostoc species (Nostocales ) and evaluation of their cytotoxicity in vitro. J BioSci Biotechnol 2012;1:33-43.
9Karan T, Dastan T, Baral I, Altuner Z. Effects of differential time applications on some cyanobacterial norharman production rates. Cumhuriyet Univ Fac Sci 2016;37:398-404.
10Galhano V, Santos H, Oliveira MM, Gomes-Laranjo J, Peixoto F. Changes in fatty acid profile and antioxidant systems in a Nostoc muscorum strain exposed to the herbicide bentazon. Process Biochem 2011;46:2152-62.
11Cheel J, Urajová P, Hájek J, Hrouzek P, Kuzma M, Bouju E, et al. Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC. Anal Bioanal Chem 2017;409:917-30.
12Erenler R, Telci I, Ulutas M, Demirtas I, Gul F, Elmastas M, et al. Chemical constituents, quantitative analysis and antioxidant activities of Echinacea purpurea (L.) Moench and Echinacea pallida (Nutt.) Nutt. J Food Biochem 2015;39:622-30.
13Hagemann M. Molecular biology of cyanobacterial salt acclimation. FEMS Microbiol Rev 2011;35:87-123.
14Bhatnagar I, Kim SK. Immense essence of excellence: Marine microbial bioactive compounds. Mar Drugs 2010;8:2673-701.
15Hirata K, Yoshitomi S, Dwi S, Iwabe O, Mahakhant A, Polchai J, et al. Bioactivities of nostocine a produced by a freshwater cyanobacterium Nostoc spongiaeforme TISTR 8169. J Biosci Bioeng 2003;95:512-7.
16Yadav S, Sinha R, Tyagi M, Kumar A. Cyanobacterial secondary metabolites. Int J Pharm Bio Sci 2011;2:144-67.
17Rastogi RP, Sinha RP. Biotechnological and industrial significance of cyanobacterial secondary metabolites. Biotechnol Adv 2009;27:521-39.
18Knübel G, Larsen LK, Moore RE, Levine IA, Patterson GM. Cytotoxic, antiviral indolocarbazoles from a blue-green alga belonging to the Nostocaceae. J Antibiot (Tokyo) 1990;43:1236-9.
19Becher PG, Beuchat J, Gademann K, Jüttner F. Nostocarboline: Isolation and synthesis of a new cholinesterase inhibitor from Nostoc 78-12A. J Nat Prod 2005;68:1793-5.
20Volk RB. Screening of microalgal culture media for the presence of algicidal compounds and isolation and identification of two bioactive metabolites, excreted by the cyanobacteria Nostoc insulare and Nodularia harveyana. J Appl Phycol 2005;17:339-47.
21Volk RB, Furkert FH. Antialgal, antibacterial and antifungal activity of two metabolites produced and excreted by cyanobacteria during growth. Microbiol Res 2006;161:180-6.
22He N, Sun X, Zhong Y, Sun K, Liu W, Duan S, et al. Removal and biodegradation of nonylphenol by four freshwater microalgae. Int J Environ Res Public Health 2016;13. pii: E1239.
23Parikh A, Madamwar D. Textile dye decolorization using cyanobacteria. Biotechnol Lett 2005;27:323-6.
24Rezanka T, Dor I, Prell A, Dembitsky VM. Fatty acid composition of six freshwater wild cyanobacterial species. Folia Microbiol (Praha) 2003;48:71-5.
25Zinicovscaia I, Rudi L, Valuta A, Cepoi L, Vergel K, Frontasyeva MV, et al. Biochemical changes in Nostoc linckia associated with selenium nanoparticles biosynthesis. Ecol Chem Eng S 2016;23:559-69.
26Devi KM, Mehta SK. Antimicrobial activities of freshwater Cyanobacterium, Nostoc sp. from Tamdil Wetland of Mizoram, India: An identification of bioactive compounds by GC-MS. Int J Pharm Sci Res 2016;7:1179.
27Zelík P, Lukesová A, Voloshko LN, Stys D, Kopecký J. Screening for acetylcholinesterase inhibitory activity in cyanobacteria of the genus Nostoc. J Enzyme Inhib Med Chem 2009;24:531-6.