Morinda citrifolia Inhibits Both Cytosolic Ca-dependent Phospholipase A(2) and Secretory Ca-dependent Phospholipase A(2)

Abstract

This study investigated the effects of the methanol extracts of Morinda citrifolia containing numerous anthraquinone and iridoid on phospholipase A(2) (PLA(2)) isozyme. PLA(2) activity was measured using various PLA(2) substrates, including 10-pyrene phosphatidylcholine, 1-palmitoyl-2-[(14)C]arachidonyl phosphatidylcholine ([(14)C]AA-PC), and [(3)H]arachidonic acid (AA). The methanol extracts suppressed melittin-induced [(3)H]AA release in a concentration-dependent manner in RAW 264.7 cells, and inhibited cPLA(2)/sPLA(2)-induced hydrolysis of [(14)C]AA-PC in a concentration- and time-dependent manner. A Dixon plot showed that the inhibition by methanol extracts on cPLA(2) and sPLA(2) appeared to be competitive with inhibition constants (K(i)) of 3.7microg/ml and 12.6microg/ml, respectively. These data suggest that methanol extracts of Morinda citrifolia inhibits both Ca(2+)-dependent PLA(2) such as, cPLA(2) and sPLA(2). Therefore, Morinda citrifolia may possess anti-inflammatory activity secondary to Ca(2+)-dependent PLA(2) inhibition.

Keywords: Arachidonic acid; Morinda citrifolia; Phospholipase A2.

Fig. 1

Effects of the methanol extracts on [³H]arachidonic acid (AA) release in RAW 264.7 cells stimulated by 0.5 µM melittin. Cells were incubated with the methanol extracts at 37℃ for 10 min and AA release was induced by 0.5 µM melittin. Results are mean±S.D. values from 4 separate experiments. ^*p<0.05 vs melittin.

Fig. 2

Effects of methanol extracts on cPLA₂ activity. cPLA₂ activity was measured using 1-palmitoyl-2-[¹⁴C]arachidonyl phosphatidylcholine ([¹⁴C]AA-PC) as a substrate by previously methods. The methanol extracts inhibited cPLA₂-induced hydrolysis of [¹⁴C]AA-PC in a concentration (A)- and time (B)-dependent manner. A Dixon plot showed that cPLA₂ inhibition by methanol extracts appeared to be competitive with an inhibition constant (K_i) of 3.7 µg/ml (C). Results are mean±S.D. values from 4 separate experiments. ^*p<0.05 vs control.

Fig. 3

Effects of methanol extracts on sPLA₂ activity. sPLA₂ activity was measured using 1-palmitoyl-2-[¹⁴C]arachidonyl phosphatidylcholine ([¹⁴C]AA-PC) as a substrate by previously described methods. The methanol extracts inhibited sPLA₂-induced hydrolysis of [¹⁴C]AA-PC in a concentration (A)- and time (B)-dependent manner. A Dixon plot showed that the inhibition of sPLA₂ by methanol extracts appeared to be competitive with an inhibition constant (K_i) of 12.6 µg/ml (C). Results are mean±S.D. values from 4 separate experiments. ^*p<0.05 vs control.

Fig. 4

Effects of methanol extracts on bee venom sPLA₂ activity with 10-pyrene phosphatidylcholine (10-Pyrene PC). 10-Pyrene PC hydrolyzed by purified sPLA₂ was inhibited by the methanol extracts in a concentration-dependent manner. Results are mean±S.D. values from 4 separate experiments. ^*p<0.05 vs control.