Antiplasmodial activity of extracts of 25 cyanobacterial species from coastal regions of Tamil Nadu

Figures & data

Table 1. Antimalarial properties of marine cyanobacterial secondary metabolites.

Organism	Isolated metabolites	Antimalarial activity against	IC50	Reference
Calothrix strains	Calothrixin A	P. falciparum	58 ± 8 nM	Rickards et al. (1999)
	Calothrixin B		180 ± 44 nM
Schizothrix sp.	Crude	P. falciparum W2	1 μg mL^−1	Linington et al. (2009)
	Gallinamide A.		8.4 μM
L. majuscula	Lagunamides A	P. falciparum NF54	0.19 μM	Tripathi et al. (2010a)
	Lagunamides B		0.91 μM
	Malyngolide dimer	P. falciparum W2	19 μM	Gutierrez et al. (2010)
	Carmabin A		4.3 μM	McPhail et al. (2007)
	Dragomabin		6.0 μM
	Dragonamide A		7.7 μM
	Lagunamide C	P. falciparum	0.29 μM	Tripathi et al. (2011)
Oscillatoria sp.	Venturamides A	P. falciparum W2	8.2 μM	Linington et al. (2007)
	Venturamides B		5.6 μM
Oscillatoria nigro-viridis	Viridamides A	P. falciparum	5.8 μM	Simmons et al. (2008)
Blennothrix cantharidosmum	Tumonoic acid I	P. falciparum	2 μM	Clark et al. (2008)
Phormidium	Hierridin B+	P. falciparum D6	5.2 μg mL^−1	Papendorf et al. (1998)
Ectocarpi	2,4-Dimethoxy-6-heptadecyl-phenol	P. falciparum W2	3.7 μg mL^−1
Symploca sp.	Symplostatin 4	P. falciparum D10	0.7 ± 0.2 mM	Stolze et al. (2012)

Figure 1. Sampling stations along the coastal regions of Tamil Nadu.

Table 2. Morphological features of cyanobacterial species and their antiplasmodial activity against Pf3D7.

Cyanobacteria code	Sampling site	Substrate of growth	Morphological features	Taxonomic affiliation following Desikachari (1959)	Pf3D7 IC50 (μg mL^−1)
C1	Pitchavaram	Rhizophora apiculata	Unicellular, mucilaginous, cells spherical, growing aggregate, 4.9 µm broad, sheath+	Aphanocapsa littoralis Hansgirg CNP 2022^b	74
C2	Pitchavaram	Sand	Filamentous, intercalary heterocyst, cylindrical, trichomes straight, constricted, 6.1 µm broad, sheath–	Anabaena circinalis Var. crassa Ghose CNP 2061^b	100
C3	Mandapam	Rope Scraping	Filamentous thin sheath, trichomes not constricted 2.6 µm broad, thin	Phormidium tenue (Menegh.) Gomont CNP 1009^b	100
C4	Dhanushkodi	Sea water	Filamentous, spirals tightly arranged, spiral 6.2 µm broad, sheath–	Spirulina subsalsa Orest. ex Gomont CNP 1020^b	68
C5	Pitchavaram	Rhizophora apiculata	Filamentous, trichomes not constricted 4.3 µm broad, thin	Phormidium abronema Skuja CNP 2012^b	100
C6	Mandapam	Coral Scraping	Filamentous, motile, sheath–, trichome constricted 7.3 µm broad	Oscillatoria acuta Bruhl et Biswas, orth.mut. Geitler CNP 1021^d	>100
C7	Mandapam	Stone	Filamentous, motile, sheath–, trichome slightly constricted 8.1 µm broad, end cell rounded	Oscillatoria annaevan Goor CNP 1020^b	>100
C8	Kanniyakumari	Rock Scraping	Filamentous, floating motile, trichome unconstructed 4.9 µm broad	Phormidium dimorphum Lemn. CNP 7010^d	93
C9	Kanniyakumari	Rock Scraping	Unicellular, cylindrical, trichome 5.8 µm broad, sheath–	Synechococcus pevalekii Nag. CNP 7001^b	>100
C10	Muthupet	Aegiceras corniculatum	Filamentous, intercalary heterocyst, trichomes 2.9 µm broad	Anabaena constricta (Szaffer) Geitler CNP 5007^b	35
C11	Muthupet	Avicennia marina	Filamentous false branched, trichome 9.1 µm broad	Pseudoscytonema malayense (Biswas) Elenkin CNP 5003^d	>100
C12	Dhanushkodi	Soil	Filamentous, trichome very massive thick 18.2 µm broad	Lyngbya aestuarii Liebm. ex Gomont CNP 1005^a	18
C13	Kanniyakumari	Seaweeds Scraping	Filamentous, floating movement present, sheath+, 5.1 µm broad	Phormidium corium (Ag.) Gomont CNP 7004^b	>100
C14	Thondi	Boat Scraping	Filamentous, sheath is present, trichome 3.9 µm broad	Phormidium valderianum (Delp.) Gomont CNP 4011^b	>100
C15	Dhanushkodi	Soil	Filamentous, motile, trichomes constricted 8.5 µm broad, end of trichome bent, sheath–	Oscillatoria salina Biswas CNP 1006^d	>100
C16	Pitchavaram	Excoecaria agallocha	Filamentous, basal heterocyst, trichome 9.2 µm broad, sheath+	Calothrix brevissima West, G.S.CNP 2000^d	>100
C17	Dhanushkodi	Water	Filamentous, trichome un constricted 3.5 µm broad, sheath present	Geitlerinema pseudacutissimum Geitler) Anagnostidis CNP 1019^d	>100
C18				O. boryana Bory ex Gomont BDU 141071^c	51
C19	Dhanushkodi	Scraping from solid substrate	Filamentous, sheath+, floating, trichome 5.5 µm broad, not constricted	Phormidium purpurascens (Kutz). Gomont CNP 1008^b	>100
C20	Pitchavaram	Rhizophora apiculata	Filamentous, sheath+, trichome not constricted 4.1 µm broad	Phormidium jadinianum Var. joannianum (Kutz.) Gomont CNP 2001^b	>100
C21	Thondi	Boat Scraping	Filamentous, sheath+, trichome 4 µm broad, not constricted	Phormidium purpurascens (Kutz). Gomont CNP 4005^b	>100
C22				O. boryana Bory ex Gomont BDU 91451^c	18
C23	Mandapam	Water	Filamentous, motile, end of trichome bent 16 µm broad	Oscillatoria princeps Vaucher ex Gomont CNP 1014^d	>100
C24	Thondi	Soil	Filamentous, sheath+, trichome 4.3 µm broad	Geitlerinema carotinosum (Geitler) Anagnostidis CNP 4003^b	>100
C25				O. salina Biswas BDU 141792^c	91

^aIdentified by using 16S rRNA sequencing.

^bIdentified by using classical taxonomic criteria by morphology.

^cCulture obtained from Germplasm of NFMC.

^dMorphological features given in Maruthanayagam et al. (2013).

Figure 2. Micrographs of cyanobacterial isolates at 100×. Twenty-five different strains of cyanobacteria were isolated from various coastal regions of Tamil Nadu. The monocultures of all isolated cyanobacteria were examined under light microscope (Binocular microscope inverted epifluorescence Model XTS, Italy) at the same magnification; the scale bar 50 µm applies to all panels and the genera of cyanobacteria were identified following Desikachari (1959) manual. C12 (Lyngbya aestuarii CNP 1005) and C22 O. boryana BDU 91451) showed promising antiplasmodial activity (IC₅₀ = 18 µg mL⁻¹) against Pf3D7. Table 3 indicates the names of genera for the strains in the figure.

Figure 3. Antiplasmodial activity of extracts of the marine cyanobacterial species. Concentration-dependent inhibition of blood-stage Plasmodium falciparum growth by cyanobacterial crude extracts as measured by SYBR Green fluorescence assay. Extract numbers and the corresponding color codes are indicated in the strip on the right. Standard deviation bars at each data point have been calculated from triplicate observations.

Table 3. Antiplasmodial potency, resistance and selectivity indices of potent cyanobacterial extracts.

	P. falciparum IC50 (μg mL^−1)
Cyanobacterial cultures	3D7	INDO	Resistance index (IC50 PfIndo/IC50Pf3D7)	HeLa IC50 (μg mL^−1)	Selective index (IC50 HeLa/IC50Pf3D7)
Anabaena constricta CNP 5007 (C10)*	35	>100	>2.8	>200	>5.7
L. aestuarii CNP 1005 (C12)	18	34	1.9	>200	>11.1
O. boryana BDU 141071 (C18)	51	65	1.3	>200	>4.0
O. boryana BDU 91451 (C22)	18	62	3.4	>200	>11.1
Chloroquine (CQ)	0.02	0.21	10.5	>51.5	>2500

*( ) Cyanobacterial codes.

Figure 4. Pretreatment of host red blood cells with antiplasmodial extracts to find if their action is against host red blood cells. The aim of the experiment was to judge if the extracts are selectively toxic to the malaria parasite or that they are toxic to host red blood cells leading to indirect antiplasmodial toxicity. Uninfected human red blood cells were incubated (48 h, 37 °C) with extracts of C12, C18, and C22 each at its antiplasmodial IC₁₀₀. Results shown above indicate that there was no significant effect of pre-exposure on invasion, maturation, or growth of the malaria parasite suggesting the selective action of extracts on Plasmodium falciparum with no significant toxicity against host red blood cells.

Figure 5. Morphology and molecular identification of Lyngbya aestuarii CNP 1005, the strain showing the most potent antiplasmodial action in the present studies. (A) Microscopic image of L. aestuarii CNP 1005, (B) amplification of L. aestuarii 16S rRNA, (C) amplified 16S rRNA sequence using forward primer, and (D) phylogenetic tree.

Table 4. Biological activities of compounds produced by marine cyanobacteria.

Organism	Isolated compounds	Targets	IC50	Reference
L. majuscula	Dragonamide A	L. donovani	6.5 μM	Balunas et al. (2010)
	Dragonamide E		5.1 μM
	Herbamide B		5.1 μM
Calothrix strains	Calothrixin A	HeLa	40 ±9 nM	Rickards et al. (1999)
	Calothrixin B		350 ± 82 nM
Schizothrix sp.	Gallinamide A.	L. donovani	9.3 μM	Linington et al. (2009)
		T. cruzi	16.9 μM
		Vero cells	10.4 μM
Oscillatoria sp.	Venturamides A	T. cruzi	14.6 μM	Linington et al. (2007)
		MCF-7 cells	13.1 μM
	Venturamides B	T. cruzi	15.8 μM
		MCF-7 cells	>54 μM
Oscillatoria nigro-viridis	Viridamides A	L. mexicana	1.5 μM	Simmons et al. (2008)
L. majuscula		T. cruzi	1.1 μM
	Lagunamide C	Murine leukemia cell line (P388)	24.4 nM	Tripathi et al. (2011)
		Lung carcinoma cell line (A549)	2.4 nM
		Prostate cancer cell line (PC3)	2.6 nM
		Colorectal adenocarcinoma cell line (HCT8)	2.1 nM
		Ovarian cancer cell line (SK-OV)	4.5 nM
	Isomalyngamide A	Breast cancer cell line (MCF-7)	4.6 μM	Chang et al. (2011)
		Breast cancer cell line MDA-MB-231	2.8 μM
	Cocosamides A	MCF7	29 μM	Gunasekera et al. (2011)
		HT-29 cells	30 μM
	Hantupeptin B	Leukemia MOLT-4 cell line	0.2 μM	Tripathi et al. (2010b)
		MCF-7 cancer cell lines	0.5 μM
	Pitiprolamide A	Human colorectal carcinoma cell line (HCT-116)	33 μM	Montaser et al. (2011)
	Lagunamides A	Murine leukemia cell line P388	6.4 nM	Tripathi et al. (2010a)
	Lagunamides B		20.5 nM
Lyngbya sp.	Koshikalide	HeLa S3 cells	42 μg mL^−1	Iwasaki et al. (2010)
Leptolyngbya sp.	Grassypeptolides D	HeLa	335 nM	Thornburg et al. (2011)
		Mouse neuro-2a blastoma cells	599 nM
	Grassypeptolides E	HeLa	192 nM
		Mouse neuro-2a blastoma cells	407 nM
Lyngbya sordida	Malyngamide 2	Human lung carcinoma cells (H-460)	10.1 μM	Malloy et al. (2011)

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