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This study investigated the effects of the methanol extracts of Morinda citrifolia containing numerous anthraquinone and iridoid on phospholipase A2 (PLA2) isozyme. PLA2 activity was measured using various PLA2 substrates, including 10-pyrene phosphatidylcholine, 1-palmitoyl-2-[14C]arachidonyl phosphatidylcholine ([14C]AA-PC), and [3H]arachidonic acid (AA). The methanol extracts suppressed melittin-induced [3H]AA release in a concentration-dependent manner in RAW 264.7 cells, and inhibited cPLA2/sPLA2-induced hydrolysis of [14C]AA-PC in a concentration- and time-dependent manner. A Dixon plot showed that the inhibition by methanol extracts on cPLA2 and sPLA2 appeared to be competitive with inhibition constants (Ki) of 3.7µg/ml and 12.6µg/ml, respectively. These data suggest that methanol extracts of Morinda citrifolia inhibits both Ca2+-dependent PLA2 such as, cPLA2 and sPLA2. Therefore, Morinda citrifolia may possess anti-inflammatory activity secondary to Ca2+-dependent PLA2 inhibition.
Phospholipase A2 (PLA2) plays an important role in inflammatory responses through hydrolysis of cell membrane phospholipids. This leads to arachidonic acid and lysophospholipid production . PLA2 isozymes are classified according to their nucleotide sequences and are classified into three types secretory PLA2 (sPLA2), cytosolic Ca2+-dependent PLA2 (cPLA2), and Ca2+-independent PLA2 (iPLA2) based on cellular activity and functions . sPLA2 has a low molecular mass (~18 kDa) and can be activated at millimolar concentrations of Ca2+. sPLA2 is secreted by inflammatory cells upon their activation and by damaged tissues in inflammatory diseases . cPLA2 is located in the cytosol, has a high molecular mass (~85 kDa), and can be activated at the micromolar concentration of intracellular Ca2+ . iPLA2 can be detected in both cytosolic and membrane fractions with a molecular mass ranging from 29 to 85 kDa, and does not require Ca2+ for activation .
Morinda citrifolia (Rubiaceae) is widely distributed across Polynesia and contains numerous anthraquinone and iridoids, such as asperuloside, asperulosidic acid and deacetylasperulosidic acid . It has been traditionally used as a folk medicine for the treatment of many disease, including arthritis, diabetes, hypertension, heart disease, neoplasia, and atherosclerosis . Although Morinda citrifolia has a wide range of biological activities, there is no evidence of inhibitory effects on PLA2 isozymes. We measured the effects of the methanol extracts of Morinda citrifolia composing major biological active components on PLA2 isozyme activity in the present study.
PLA2 from honey bee venom, arachidonyl trifluoromethyl ketone (AACOCF3), bromoenol lactone (BEL) and melittin, was purchased from the Sigma Chemical Co. (St. Louis, Mo, USA). 10-Pyrene phosphatidylcholine (10-pyrene PC) was purchased from Molecular Probes (Leiden, Netherlands), [3H]arachidonic acid ([3H]AA) was obtained from NEN (Boston, USA), 1-palmitoyl-2-[14C]arachidonyl phosphatidylcholine ([14C]AA-PC) was obtained from Perkin Elmer (Boston, USA), and 1-palmitoyl-2-arachidonyl phosphatidylcholine was obtained from Avanti Polar Lipid (Alabaster, USA).
The RAW 264.7 murine macrophage cell line was obtained from the Korean Cell Line Bank (Seoul, Korea) and were cultured in DMEM supplemented with 10% heat-inactivated FBS and antibiotic-antifungal mix (100 IU/ml penicillin G, 100 µg/ml of streptomycin and 0.25 µg/ml of amphotericin B) at 37 with 5% CO2.
Morinda citrifolia fruit powder was purchased from the NP Nutra Co. (Hawaii). Dried Morinda citrifolia fruit powder (800 g) was extracted with 70% methanol at 40. It has been previously reported that the methanol extracts contained a numerous antharquinone and iridoids, such as asperuloside, asperulosidic acid and deacetylasperulosidic acid [6,8].
RAW 264.7 cells were harvested with Krebs buffer (137 mM NaCl, 2.7 mM KCl, 0.4 mM Na2HPO4, 0.5 mM MgCl2, 10 mM HEPES (pH 7.4), 1.8 mM CaCl2, and 5 mM glucose). Cells were labeled with [3H]AA (0.4 µCi/ml) at 37 for 2 h and were washed with Krebs buffer containing 0.5 mg/ml bovine serum albumin to trap the liberated [3H]AA. AA release was induced by 0.5 µM melittin either with or without the methanol extracts for 30 min. Both pellets and supernatants were transferred to liquid scintillation vials after centrifugation. Radioactivity was measured with a liquid scintillation counter , and the percentage of [3H]AA release was calculated as supernatants/(supernatants+pellets)×100.
PLA2 activity was measured with a pyrene-labeled phosphatidylcholine in the presence of serum albumin  using a spectrophotometer. PLA2 purified from honey bee venom was used as sPLA2 . sPLA2 and the methanol extracts were vortex-mixed and incubated for 10 min at room temperature. The above enzyme was incubated with a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 2 µM 10-pyrene PC, 0.1% bovine serum albumin, and 6 mM CaCl2 for 20 min. The fluorescence was measured using excitation (345 nm) and emission (398 nm) wavelengths with a spectrophotometer (FL600, Microplate Fluorescence Reader, Bio-Tek).
RAW 264.7 cells were washed and sonicated in 10 mM Tris-HCl buffer (pH 7.4) with 100 mM NaCl, 2 mM EGTA, 100 µM leupeptin, 150 µM aprotinin, and 1 mM Na3VO4. Lysates were centrifuged at 10,000 g for 30 min at 4, and supernatants were stored at -70 and used to supply cPLA2 .
PLA2 activity was assayed by measurement of [14C]AA hydrolyzed from [14C]AA-PC. For the measurement of RAW 264.7 cell-derived cPLA2 activity, enzyme sources and methanol extracts were incubated for 30 min at room temperature in 100 mM Tris-HCl buffer (pH 8.5) containing 10 µM BEL as the iPLA2 inhibitor , and 5 mM CaCl2 and 1 mM dithiothreitol (DTT) as the sPLA2 inhibitors . The reaction mixture was incubated with 0.025 µCi [14C] AA-PC as the substrate for a given time. The cPLA2 inhibitor AACOCF3 (10 µM)  was used for the measurement of sPLA2 activity instead of DTT.
The reaction mixture was incubated at 37 for 30 min. The reaction was stopped by the addition of modified Doles reagent (n-heptane/isopropyl alcohol/1 N-H2SO4=400/390/10, 560 µl) . After centrifugation, 150 µl of upper phase was transferred to a new tube; n-heptane (800 µl) and silica gel (10 mg) were added to the tube. The mixtures were mixed and centrifuged for 2 min, and supernatants (800 µl) were mixed with scintillation solution (1.0 ml) and counted for radioactivity in a Packard Tri-carb liquid scintillation counter. The specific activity in picomoles per minute per milligram protein (pmol·min-1·mg-1) was obtained by dividing activity by the amount of enzyme protein. Protein was analyzed with a BCA (bicinchoninic acid) protein assay kit to calculate specific activity. The [14C]AA-PC hydrolysis ratio was applied to compose the Dixon plot, and the concentration of the substrate was increased by adding 1-palmitoyl-2-arachidonyl phosphatidylcholine.
The results are represented as mean±standard deviation (S.D.) values and analyzed statistically by analysis of variance (ANOVA). Differences between groups were determined with the Newman-Keul's test. Statistical significance was defined for p values less than 0.05.
The methanol extracts containing numorous anthraquinone and iridoids, such as asperuloside, asperulosidic acid and deacetylasperulosidic acid, displayed more potent inhibitory effects on melittin-induced AA release in [3H]AA-labeled RAW 264.7 cells than other fractions (data not shown). Melittin (0.5 µM) resulted in AA release by 18.3±2.0%, which was significantly decreased by the methanol extracts at 10 and 100 µg/ml to 14.9±1.8% and 12.5±0.4%, respectively (Fig. 1).
We have previously reported that the RAW 264.7 cell-derived PLA2 was significantly inhibited by 10 µM AACOCF3, but was not influenced by 1 mM DTT or 10 µM BEL. Also RAW 264.7 cell-derived PLA2 did not show any PLA2 activity in the absence of Ca2+ . These data suggest that the RAW 264.7 cell-derived PLA2 contained mainly cPLA2.
RAW 264.7 cell-derived PLA2 and PLA2-specific substrate, [14C]AA-PC were used to investigate the effects of the methanol extracts on cPLA2. The methanol extracts inhibited cPLA2 in a concentration- and time-dependent manner (Fig. 2A and 2B), and inhibited cPLA2 activity by 16.8% and 63.2% at concentrations of 10 and 100 µg/ml, respectively.
Dixon plots were constructed from cPLA2-induced substrate hydrolysis rates ([14C]AA-PC of 5 and 50 nM) at various concentrations of methanol extracts to determine the inhibitory pattern on cPLA2 by methanol extracts. Fig. 2C demonstrated that the apparent Ki value of the methanol extracts on cPLA2 was 3.7 µg/ml, and such an inhibitory pattern suggests that the methanol extracts acted as a competitive inhibitor against cPLA2.
sPLA2 obtained from honey bee venom hydrolyzed 10-pyrene PC; the sPLA2-specific substrate . We used purified sPLA2 from honey bee venom to investigate the inhibitory effects of the methanol extracts on sPLA2. The methanol extracts inhibited sPLA2 in a concentration- and time-dependent manner (Fig. 3A and and3B).3B). The methanol extracts inhibited sPLA2 activity by 15.6% and 53.9% at concentrations of 10 and 100 µg/ml, respectively.
Dixon plots were constructed from sPLA2-induced hydrolysis rates of the substrate ([14C]AA-PC of 5 and 50 nM) to determine the sPLA2 inhibitory patterns by the methanol extracts. Fig. 3C demonstrated that the apparent Ki value of the methanol extracts on sPLA2 was 12.6 µg/ml, and such inhibitory patterns suggest that methanol extracts acts as a competitive inhibitor against sPLA2. Additionally, the methanol extracts inhibited bee venom sPLA2-induced 10-pyrene PC hydrolysis in a concentration-dependent manner (Fig. 4).
The methanol extracts decreased melittin-induced [3H]AA release in RAW 264.7 cells in a concentration-dependent manner in the present study. Melittin has been used as an endogenous PLA2 activator by increasing intracellular Ca2+ concentrations via receptor-operated calcium channels . Such inhibitory effects of the methanol extracts may be associated with PLA2 inhibition. However, AA release is an indirect measure of PLA2 activity since other enzymes including arachidonyl-CoA synthetase, CoA-dependent acyltransferase, and CoA-independent transacylase may result in free AA production . We measured direct effects of methanol extracts on PLA2 isozymes using [14C]AA-PC, a specific PLA2 substrate to address this issue.
RAW 264.7 cellular lysates were used as the source of cPLA2 since we did not prepare each PLA2 isozyme. RAW 264.7 cell-derived PLA2 was completely inhibited by AACOCF3 but not by DTT or BEL. RAW cell-derived PLA2 did not result in any PLA2 activity using the sPLA2-specific substrate, 10-pyrene PC. Additionally, RAW 264.7 cell-derived PLA2 showed only a low level of activity in the absence of Ca2+ . These data suggested that RAW 264.7 cell-derived PLA2 primarily contained cPLA2 rather than sPLA2 and iPLA2.
We determined the inhibitory pattern of the methanol extracts on cPLA2 with Dixon plots. The methanol extracts inhibited cPLA2-induced hydrolysis of [14C]AA-PC in a concentration- and time-dependent manner. Dixon plot was constructed to demonstrate that inhibition by the methanol extracts appeared to be competitive with an inhibition constant (Ki) of 3.7 µg/ml. It has been reported that cPLA2 is inhibited by a trifluoromethyl ketone analog of AA (AACOCF3), which presumably binds directly to the active site of cPLA2 [15,21,22]; AACOCF3 (10 µM) inhibited cPLA2 by 95.4%, and the methanol extracts (100 µg/ml) inhibited it by 63.2%. Bee venom sPLA2 was used to investigate the effects of the methanol extracts on sPLA2. The methanol extracts inhibited sPLA2-induced hydrolysis of [14C]AA-PC in a concentration- and time-dependent manner. Dixon plot demonstrated that the inhibition by methanol extracts appeared to be competitive, with an inhibition constant (Ki) of 12.6 µg/ml. These data suggest that methanol extracts of the Morinda citrifolia fruit inhibits both Ca2+-dependent PLA2 such as cPLA2 and sPLA2. Morinda citrifolia anti-inflammatory activity was observed by a locally acute inflammatory response such as, bradykinin-induced rat paw edema  and a selective inhibition effect on cyclooxygenase . These data support that the potential anti-inflammatory activity of Morinda citrifolia may be secondary to the inhibition of Ca2+-dependent PLA2.
In conclusion, the methanol extracts of the Morinda citrifolia fruit containing numerous anthraquinone and iridoids, such as asperuloside, asperulosidic acid and deacetylasperulosidic acid  decreased melittin-induced [3H]AA release in RAW 264.7 cells in a concentration-dependent manner. The methanol extracts inhibited cPLA2/sPLA2-induced hydrolysis of [14C]AA-PC in both a concentration- and time-dependent manner. A Dixon plot demonstrated that cPLA2 and sPLA2 inhibition by the methanol extracts appeared to be competitive with an inhibition constant (Ki) of 3.7 µg/ml and 12.6 µg/ml, respectively. These data suggest that methanol extracts of the Morinda citrifolia fruit inhibits both Ca2+-dependent PLA2, such as cPLA2 and sPLA2. These data supported that the anti-inflammatory activity of Morinda citrifolia can be secondary to the inhibition of Ca2+-dependent PLA2.