Antimicrobial, antioxidant and antiproliferative activities of a galactose-binding seed lectin from Manilkara zapota

Abstract

A seed lectin from Manilkara zapota (MZSL) was purified using ammonium sulphate precipitation and affinity chromatography. Hemagglutination activity, neutral sugar content and physicochemical properties of the lectin were determined and toxicity was checked by brine shrimp toxicity assay. Antimicrobial, antioxidant as well as in vitro anticancer activities of MZSL were also evaluated. Our findings showed the molecular weight of MZSL to be 33.0 ± 1 kDa. Minimum hemagglutination concentration of the lectin was 15.625 μg/ml. With a neutral sugar content of 6.32 %, the lectin was fully active at a temperature range of 30-50 °C and pH 7.0-8.0 and it was mildly toxic with an LC₅₀ value of 107.93 μg/ml. The lectin demonstrated bacteriostatic activity against gram-positive bacteria in contrast to gram-negative bacteria at a concentration of 31.25 μg/ml, agglutinated Staphylococcus aureus and Shigella dysenteriae and exerted fungistatic activity against Aspergillus niger. MZSL dose-dependently reduced the formation of biofilm by E. coli. DPPH assay confirmed its antioxidant activity with an IC₅₀ value of 96.42 μg/ml. MZSL showed 21.64 % growth inhibition against Ehrlich ascites carcinoma (EAC) cells at 80 μg/ml whereas its antiproliferative potential against MCF-7 and A-549 cancer cell lines became evident with IC₅₀ values of 70.66 μg/ml and 107.64 μg/ml, respectively.

Keywords: Antibiofilm; Anticancer; Bacteriostatic; Fungistatic; Manilkara zapota; Seed lectin.

Fig. 1

(A) Crude protein sample (100 ml) was subjected to 100 % ammonium sulphate precipitation. Fractions with hemagglutination activity were collected and applied to a lactose-agarose column. After washing with TBS, fractions (2 ml/tube) were eluted (flow rate: 1 ml/min). (B) Hemagglutination activity of MZSL (C) The eluted fraction (MZSL) showed strong hemagglutination activity and SDS-PAGE of the lectin was performed in 15 % (w/v) polyacrylamide gel. Lane 1 (M): Marker proteins, Lane 2 and 3: Purified MZSL. Uncropped and unprocessed image of the polyacrylamide gel can be found as the supplementary material (Supplementary File).

Fig. 2

Consequences of increasing (A) temperature and (B) pH on the hemagglutination activity of MZSL. Error bars: Results of triplicate experiments have been presented with standard errors (SE). Data are presented as mean ± SE (n = 3).

Fig. 3

Mortality percentage of Artemia salina nauplii treated with various concentrations of MZSL after an exposure for 24 h. Error bars: Results of triplicate experiments have been presented with standard errors (SE). Data are presented as mean ± SE (n = 3). Asterisks (*P < 0.05) denote statistically significant differences compared to controls.

Fig. 4

Antimicrobial activity of MZSL. A. Inhibition of bacterial growth by MZSL. Red, green, purple and yellow columns indicate the growth reduction of Staphylococcus aureus, Escherichia coli, Shigella dysenteriae and Shigella boydii, respectively. B. Fungistatic activity of MZSL against Aspergillus niger. A and T represent the discs soaked with 100 μg of MZSL and Antifungal agent (1 % Clioquinol + 0.02 % Flumetasone Pivalate), respectively. C is the control disc with no MZSL. Error bars: Results of triplicate experiments have been presented with standard errors (SE). Data are presented as mean ± SE (n = 3). Asterisks (*P < 0.05, **p < 0.01) denote statistically significant differences compared to controls.

Fig. 5

Agglutination and antibiofilm activity MZSL. A and B. Agglutination of Staphylococcus aureus by MZSL at a concentration of 500 and 0 μg/ml, respectively. C and D. Agglutination of Shigella dysenteriae by MZSL at a concentration of 500 and 0 μg/ml, respectively. Scale bar: 50 μm. E. Antibiofilm activity of MZSL against Escherichia coli. Error bars: Results of triplicate experiments have been presented with standard errors (SE). Data are presented as mean ± SE (n = 3). Asterisks (*P < 0.05) denote statistically significant differences compared to controls.

Fig. 6

DPPH scavenging activity of MZSL. A. Green and red colored columns showed the concentration-dependent scavenging activity of vitamin C and MZSL. B. IC₅₀ values of vitamin C and MZSL. Error bars: Results of triplicate experiments have been presented with standard errors (SE). Data are presented as mean ± SE (n = 3).

Fig. 7

Antiproliferative activity of MZSL. A. Growth inhibition of EAC cells after treating with MZSL for 24 h. B. Effect of MZSL on the growth of MCF-7 (red bar) and A-549 (green bar) cell lines. Error bars: Results of triplicate experiments have been presented with standard errors (SE). Data are presented as mean ± SE (n = 3). Asterisks (*P < 0.05, **p < 0.01) denote statistically significant differences compared to controls.

figs1