Stable, water extractable isothiocyanates from Moringa oleifera leaves attenuate inflammation in vitro

Abstract

Moringa (Moringa oleifera Lam.) is an edible plant used as both a food and medicine throughout the tropics. A moringa concentrate (MC), made by extracting fresh leaves with water, utilized naturally occurring myrosinase to convert four moringa glucosinolates into moringa isothiocyanates. Optimum conditions maximizing MC yield, 4-[(α-L-rhamnosyloxy)benzyl]isothiocyanate, and 4-[(4'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate content were established (1:5 fresh leaf weight to water ratio at room temperature). The optimized MC contained 1.66% isothiocyanates and 3.82% total polyphenols. 4-[(4'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate exhibited 80% stability at 37°C for 30 days. MC, and both of the isothiocyanates described above significantly decreased gene expression and production of inflammatory markers in RAW macrophages. Specifically, both attenuated expression of iNOS and IL-1β and production of nitric oxide and TNFα at 1 and 5 μM. These results suggest a potential for stable and concentrated moringa isothiocyanates, delivered in MC as a food-grade product, to alleviate low-grade inflammation associated with chronic diseases.

Keywords: 4-[(4′-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate; 4-[(α-L-rhamnosyloxy)benzyl]isothiocyanate; Chronic inflammation; Isothiocyanates; Moringa oleifera; Moringaceae.

Fig. 1

Chemical structures of moringa glucosinolates (GLSs) 1–4 and isothiocyanates (ITCs) 5–8. Molecular masses: 1 = 570, monoacetylated 2–4 = 612, 5 = 353, monoacetylated 6–8 = 311.

Fig. 2

Effect of dilution factor and temperature on concentration of 5 and 8 and percent yield of MC. A. Effect of dilution ratio of fresh leaves (g):H₂O (mL) on ITC concentration (mg of ITC/100 mg of MC). B. Effect of dilution ratio on MC percent yield (mg of MC/100 mg of fresh leaves). All extractions in A and B were performed at room temperature (22 °C). C. Effect of temperature on ITC concentration (mg of ITC/100 mg of MC). D. Effect of temperature on MC percent yield (mg of MC/100 mg of fresh leaves). All extractions in C and D were performed in a dilution ratio of 1:5. Graphs represent the mean ± SEM (n = 3). Comparisons were made by a 1-way ANOVA followed by Tukey’s posthoc test. Significant differences (p < 0.05) between sample sets are signified by letters; different letters indicate significant difference between sample sets, while the same letter or absence of a letter indicates no difference.

Fig. 3

Mass chromatogram of GLSs and ITCs after extraction at 22 °C (A) and 100 °C (B). m/z ions for 1 (570) [M-H]⁻, 2, 3, 4 (612) [M-H]⁻, 5 (370) [M (311) + C₂H₄O₂ (60)-H]⁻ and 6, 7, 8 (420) [M (311) + C₂H₄O₂ (60)-H]⁻ were selected for in both figures. A. MC prepared at 22 °C (0.1 mg MC injected) showing the presence of: a (5), b (overlapping peaks of 6 & 7) and c (8) and the absence of any ion trace for GLSs. B. MC prepared at 100 °C (0.6 mg MC injected) showing the presence of d (1), e (overlapping peaks of 2 & 3), and f (4), and the absence of any ion trace for ITCs.

Fig. 4

Effect of storage of MC at 25 °C and 37 °C on ITC stability and photograph of purified 8 at 10X magnification (insert). Each bar represents the mean ± SEM (n = 3).

Fig. 5

Anti-inflammatory effects of MC, 5 and 8 on LPS-induced iNOS, IL-1β IL-6 and TNFα gene expression in RAW 264.7 macrophage cells. Cells were pretreated for 2 h with MC, 5, or 8 and then induced with LPS for 6 h. Values show relative gene expression compared to vehicle with LPS control determined by comparative ΔΔC_t analysis. A: Effect of MC on iNOS and IL-1β B: Effect of 5 and 8 on iNOS and IL-1β C: Effect of MC on IL-6 and TNF±. D: Effect of 5 and 8 on IL-6 and TNFα. Each bar represents the mean ± SEM (n = 4), except for TNFα in D where n = 2. *** = p < 0.001, ** = p < 0.01, * = p < 0.05. Comparisons to controls were made by Dunnett’s test for iNOS measurements or Wilcoxon’s test in all other experiments.

Fig. 6

Anti-inflammatory effect of MC, 5, or 8 on LPS-induced NO and TNFα protein production in RAW 264.7 macrophage cells. Cells were pretreated for 2 h with MC or ITCs and then induced with LPS for 6 h. Values show relative fold change in production of NO or TNFα protein measured by the Greiss method and ELISA, respectively. A: Effect of MC on NO and TNF± protein production. B: Effect of ITCs on NO and TNF± protein production. Each bar represents the mean ± SEM (n = 3). *** = p < 0.001, ** = p < 0.01, * = p < 0.05. Comparisons to vehicle with LPS controls were made by a Dunnett’s test.