In vitro studies of anti-inflammatory and anticancer activities of organic solvent extracts from cultured marine microalgae

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  • ABSTRACT

    Marine microalgae are a promising source of organisms that can be cultured and targeted to isolate the broad spectrum of functional metabolites. In this study, two species of cyanobacteria, Chlorella ovalis Butcher and Nannchloropsis oculata Droop, one species of bacillariophyta, Phaeoductylum tricornutum Bohlin, and one species of Dinophyceae, Amphidinium carterae (Hulburt) were cultured and biomasses used to evaluate the proximate comical compositions. Among the determined proximate chemical compositions of the cultured marine microalgae, the highest content of crude proteins and lipids were exhibited in P. tricornutum and A. carterae, respectively. Solvent-solvent partition chromatography was subjected to fractionate each of the cultured species and separated n-hexane, chloroform, ethyl acetate, and aqueous fractions. Nitric oxide production inhibitory level (%) and cytotoxicity effect on lipo-polysaccharide?induced RAW 264.7 macrophages were performed to determine the anti-inflammatory activity. N. oculata hexane and chloroform fractions showed significantly the strongest anti-inflammatory activity at 6.25 μg mL-1 concentration. The cancer cell growth inhibition (%) was determined on three different cell lines including HL-60 (a human promyelocytic leukemia cell line), A549 (a human lung carcinoma cell line), and B16F10 (a mouse melanoma cell line), respectively. Among the extracts, C. ovalis ethyl acetate and A. carterae chloroform fractions suppressed the growth of HL-60 cells significantly at 25 and 50 μg mL-1 concentrations. Thus, the cultured marine microalgae solvent extracts may have potentiality to isolate pharmacologically active metabolites further using advance chromatographic steps. Hence, the cultured marine microalgae can be described as a good candidate for the future therapeutic uses.


  • KEYWORD

    Amphidinium carterae , anticancer , anti-inflammatory effect , Chlorella ovalis , cultured marine microalgae , Nannchloropsis oculata , Phaeoductylum tricornutum

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  • [Fig. 1.] Extraction approaches of cultured marine microalgae samples using solvent-solvent partition chromatography.
    Extraction approaches of cultured marine microalgae samples using solvent-solvent partition chromatography.
  • [Table 1.] Proximate chemical compositions of marine microalgae sample crude dry weight basis
    Proximate chemical compositions of marine microalgae sample crude dry weight basis
  • [Fig. 2.] Inhibitory effect of cultured marine microalga Chlorella ovalis solvent extracts by solvent-solvent partition chromatography on lipo-polysaccharide (LPS)?induced nitric oxide (NO) production (%) (A) and cell viability (%) (B) in RAW 264.7 macrophages. The incubation of extracts (COH, C. ovalis hexane fraction; COC, C. ovalis chloroform fraction; COE, C. ovalis ethyl acetate fraction; COA, C. ovalis aqueous fraction) with cells in response to LPS (1 μg mL-1) for 24 h, the NO levels in the medium was measured. CON, negative control (no LPS treated); LPS, positive control (LPS 1 μg mL-1 treated). Concentration of sample treated 25 μg mL-1 + LPS and 50 μg mL-1 + LPS, respectively. Values are mean ± standard deviation of three determinations. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.
    Inhibitory effect of cultured marine microalga Chlorella ovalis solvent extracts by solvent-solvent partition chromatography on lipo-polysaccharide (LPS)?induced nitric oxide (NO) production (%) (A) and cell viability (%) (B) in RAW 264.7 macrophages. The incubation of extracts (COH, C. ovalis hexane fraction; COC, C. ovalis chloroform fraction; COE, C. ovalis ethyl acetate fraction; COA, C. ovalis aqueous fraction) with cells in response to LPS (1 μg mL-1) for 24 h, the NO levels in the medium was measured. CON, negative control (no LPS treated); LPS, positive control (LPS 1 μg mL-1 treated). Concentration of sample treated 25 μg mL-1 + LPS and 50 μg mL-1 + LPS, respectively. Values are mean ± standard deviation of three determinations. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.
  • [Fig. 3.] Inhibitory effect of cultured marine microalga Nannchloropsis oculata solvent extracts by solvent-solvent partition chromatography on lipo-polysaccharide (LPS)?induced nitric oxide (NO) production (%) (A) and cell viability (%) (B) in RAW 264.7 macrophages. The incubation of extracts (NOH, N. oculata hexane fraction; NOC, N. oculata chloroform fraction; NOE, N. oculata ethyl acetate fraction; NOA, N. oculata aqueous fraction) with cells in response to LPS (1 μg mL-1) for 24 h, the NO levels in the medium was measured. CON, negative control (no LPS treated); LPS, positive control (LPS 1 μg mL-1 treated). Concentration of sample treated 6.25, 12.5, 25, and 50 μg mL-1, and add LPS, respectively. Values are mean ± standard deviation of three determinations. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.
    Inhibitory effect of cultured marine microalga Nannchloropsis oculata solvent extracts by solvent-solvent partition chromatography on lipo-polysaccharide (LPS)?induced nitric oxide (NO) production (%) (A) and cell viability (%) (B) in RAW 264.7 macrophages. The incubation of extracts (NOH, N. oculata hexane fraction; NOC, N. oculata chloroform fraction; NOE, N. oculata ethyl acetate fraction; NOA, N. oculata aqueous fraction) with cells in response to LPS (1 μg mL-1) for 24 h, the NO levels in the medium was measured. CON, negative control (no LPS treated); LPS, positive control (LPS 1 μg mL-1 treated). Concentration of sample treated 6.25, 12.5, 25, and 50 μg mL-1, and add LPS, respectively. Values are mean ± standard deviation of three determinations. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.
  • [Fig. 4.] Inhibitory effect of cultured marine microalga Amphidinium carterae solvent extracts by solvent-solvent partition chromatography on lipo-polysaccharide (LPS)?induced nitric oxide (NO) production (%) (A) and cell viability (%) (B) in RAW 264.7 macrophages. The incubation of extracts (ACH, A. carterae hexane fraction; ACC, A. carterae chloroform fraction; ACE, A. carterae ethyl acetate fraction; ACA, A. carterae aqueous fraction) with cells in response to LPS (1 μg mL-1) for 24 h, the NO levels in the medium was measured. CON, negative control (no LPS treated); LPS, positive control (LPS 1 μg mL-1 treated). Concentration of sample treated 25 μg mL-1 + LPS and 50 μg mL-1+ LPS, respectively. Values are mean ± standard deviation of three determinations. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.
    Inhibitory effect of cultured marine microalga Amphidinium carterae solvent extracts by solvent-solvent partition chromatography on lipo-polysaccharide (LPS)?induced nitric oxide (NO) production (%) (A) and cell viability (%) (B) in RAW 264.7 macrophages. The incubation of extracts (ACH, A. carterae hexane fraction; ACC, A. carterae chloroform fraction; ACE, A. carterae ethyl acetate fraction; ACA, A. carterae aqueous fraction) with cells in response to LPS (1 μg mL-1) for 24 h, the NO levels in the medium was measured. CON, negative control (no LPS treated); LPS, positive control (LPS 1 μg mL-1 treated). Concentration of sample treated 25 μg mL-1 + LPS and 50 μg mL-1+ LPS, respectively. Values are mean ± standard deviation of three determinations. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.
  • [Fig. 5.] Inhibitory effect of cultured marine microalga Phaeoductylum tricornutum solvent extracts by solvent-solvent partition chromatography on lipo-polysaccharide (LPS)-induced nitric oxide (NO) production (%) (A) and cell viability (%) (B) in RAW 264.7 macrophages. The incubation of extracts (PTH, P. tricornutum hexane fraction; PTC, P. tricornutum chloroform fraction; PTE, P. tricornutum ethyl acetate fraction; PTA, P. tricornutum aqueous fraction) with cells in response to LPS (1 μg mL-1) for 24 h, the NO levels in the medium was measured. CON, negative control (no LPS treated); LPS, positive control (LPS 1 μg mL-1 treated). Concentration of sample treated 6.25, 12.5, 25, and 50 μg mL-1, and add LPS, respectively. Values are mean ± standard deviation of three determinations. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.
    Inhibitory effect of cultured marine microalga Phaeoductylum tricornutum solvent extracts by solvent-solvent partition chromatography on lipo-polysaccharide (LPS)-induced nitric oxide (NO) production (%) (A) and cell viability (%) (B) in RAW 264.7 macrophages. The incubation of extracts (PTH, P. tricornutum hexane fraction; PTC, P. tricornutum chloroform fraction; PTE, P. tricornutum ethyl acetate fraction; PTA, P. tricornutum aqueous fraction) with cells in response to LPS (1 μg mL-1) for 24 h, the NO levels in the medium was measured. CON, negative control (no LPS treated); LPS, positive control (LPS 1 μg mL-1 treated). Concentration of sample treated 6.25, 12.5, 25, and 50 μg mL-1, and add LPS, respectively. Values are mean ± standard deviation of three determinations. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.
  • [Fig. 6.] Inhibitory effect of the growth of cancer cells against cultured marine microalga Chlorella ovalis solvent extracts by solventsolvent partition chromatography on HL-60 (A), B16F10 (B), and A549 (C) cell lines. Cells were treated with the extracts (COH, C. ovalis hexane fraction; COC, C. ovalis chloroform fraction; COE, C. ovalis ethyl acetate fraction; COA, C. ovalis aqueous fraction) at the indicated concentrations denoted as 25 and 50 μg mL-1, respectively. CON, control. After 24 h to treat the extracts cell viability was assessed by MTT assay. Values are expressed as means ± standard deviation in triplicate experiments. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.
    Inhibitory effect of the growth of cancer cells against cultured marine microalga Chlorella ovalis solvent extracts by solventsolvent partition chromatography on HL-60 (A), B16F10 (B), and A549 (C) cell lines. Cells were treated with the extracts (COH, C. ovalis hexane fraction; COC, C. ovalis chloroform fraction; COE, C. ovalis ethyl acetate fraction; COA, C. ovalis aqueous fraction) at the indicated concentrations denoted as 25 and 50 μg mL-1, respectively. CON, control. After 24 h to treat the extracts cell viability was assessed by MTT assay. Values are expressed as means ± standard deviation in triplicate experiments. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.
  • [Fig. 7.] Inhibitory effect of the growth of cancer cells against cultured marine microalga Amphidinium carterae solvent extracts by solvent-solvent partition chromatography on HL-60 (A), B16F10 (B), and A549 (C) cell lines. Cells were treated with the extracts (ACH, A. carterae hexane fraction; ACC, A. carterae chloroform fraction; ACE, A. carterae ethyl acetate fraction; ACA, A. carterae aqueous fraction) at the indicated concentrations denoted as 25 and 50 μg mL-1, respectively. CON, control. After 24 h to treat the extracts cell viability was assessed by MTT assay. Values are expressed as means ± standard deviation in triplicate experiments. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.
    Inhibitory effect of the growth of cancer cells against cultured marine microalga Amphidinium carterae solvent extracts by solvent-solvent partition chromatography on HL-60 (A), B16F10 (B), and A549 (C) cell lines. Cells were treated with the extracts (ACH, A. carterae hexane fraction; ACC, A. carterae chloroform fraction; ACE, A. carterae ethyl acetate fraction; ACA, A. carterae aqueous fraction) at the indicated concentrations denoted as 25 and 50 μg mL-1, respectively. CON, control. After 24 h to treat the extracts cell viability was assessed by MTT assay. Values are expressed as means ± standard deviation in triplicate experiments. Values with different alphabets are significantly different at p < 0.05 as analyzed by Duncan’s multiple range test.