HPLC analysis of aqueous chamomile extract demonstrated two major peaks with retention times of 1.179 minutes (27.7%) and 1.520 minutes (63.3%) and five other minor peaks which together constitute 9% of the total flavonoids (). The two major peaks in the aqueous chamomile extract correspond to apigenin 7-O-glucoside (63.3%) and apigenin 7-O-neohespridoside (27.7%). The presence of these constituents in the aqueous chamomile extract was also confirmed by LC-MS analysis (data not shown).
Figure 1 (A) HPLC chromatogram of apigenin 7-O-glucoside and aqueous chamomile extract demonstrating apigenin 7-O-glucoside as major constituent, (B) standardization of chamomile extract with apigenin 7-O-glucoside concentration, (C) effect of chamomile on PGE (more ...)
In the process of standardization of aqueous chamomile extract for ongoing studies, we defined the doses in equivalent molar concentration corresponding to apigenin 7-O-glucoside, as it is the major constituent of aqueous chamomile extract. For this, different concentrations of apigenin 7-O-glucoside were prepared in methanol and subjected to HPLC (). The peak area (retention time 1.5 to 1.7 min) were calculated and plotted to obtain a standard curve, which corresponded to the concentration of apigenin 7-O-glucoside in the aqueous extract on the basis of peak area ().
Next we determined the effect of aqueous chamomile extract on inhibition of endogenous prostaglandin E2 (PGE2) levels in RAW 264.7 macrophages. As shown in , treatment of macrophages with chamomile caused a decrease in endogenous PGE2 levels in RAW 264.7 macrophages which was more pronounced at 20 and 40 μg/mL doses of chamomile. Chamomile exposure did not affect cell viability at the test concentration up to 40 g/mL doses as determined by MTT reduction assay (>95% cell viability; ).
In the next series of experiments, we used LPS challenge as treatment of RAW 264.7 macrophages, which causes induction of COX-2, and converts LPS-induced endogenous arachidonic acid to PGE2
(Simmons et al., 2004
). RAW macrophages treated with LPS (1 g/mL) in the presence of chamomile (5–40 g/ml) for 24 h exhibited a dose-dependent decrease in endogenous PGE2
production (). As shown in , exposure of cells to chamomile caused a time-dependent decrease in PGE2
release in the cell culture medium. The IC50
value was calculated to be 24.0 μg/mL for apigenin 7-O
-glucoside corresponding to 15.0 μM concentration of aglycone, apigenin.
Figure 2 Effect of chamomile on PGE2, COX-1 and COX-2 expression in RAW 264.7 macrophages. (A) effect of chamomile on PGE2 accumulation in cell supernatant activated with 1 μg/mL LPS in the absence and presence of chamomile (5–40 μg/mL) (more ...)
In an attempt to find the underlying mechanism leading to reduced PGE2 production and release after chamomile exposure, we first examined the influence of chamomile on LPS-induced COX-2 mRNA levels. As shown in , COX-2 mRNA levels were significantly elevated after LPS challenge to RAW 264.7 macrophages. A marked decrease in COX-2 mRNA expression was noted after treatment of cells with at 20 and 40 μg/mL doses of chamomile. Surprisingly, treatment of RAW 267.4 cells with test doses of chamomile did not cause any significant changes in the COX-1 mRNA levels. Since increased COX-2 mRNA steady state levels may lead to increased COX-2 protein, we examined the influence of chamomile on LPS-induced COX-2 protein expression. As shown in , treatment of cells at 5–40 μg/mL doses of chamomile caused a significant decrease in COX-2 protein expression, whereas COX-1 protein expression remained unchanged at these doses of chamomile.
Next we determined the efficacy of chamomile in inhibition of COX-2 activity and compared its effects with those of NS398, a selective COX-2 inhibitor. To determine this, we used RAW 264.7 macrophages in which COX-2 was induced by LPS and exogenous arachidonic acid was added as substrate. Since exogenous arachidonic acid can be utilized by either COX-1 or COX-2 to produce PGE2, COX-1 was irreversibly blocked by acetylsalicylic acid before COX-2 was induced with LPS. As shown in , chamomile was able to inhibit the conversion of exogenous arachidonic acid to PGE2 in a dose-dependent manner which corresponded to inhibition of COX-2 activity. The IC50 value determined (28.0 μg/mL for apigenin 7-O-glucoside corresponding to 17.6 μM apigenin) was very similar to that calculated for chamomile-dependent inhibition of PGE2 production in LPS-activated RAW 264.7 macrophages.
Figure 3 Effect of chamomile on COX-2 enzyme activity in LPS activated RAW 264.7 macrophages. RAW 264.7 macrophages were treated with 5–40 μg/mL doses of chamomile, in which COX-1 was irreversibly inactivated by acetylsalicylic acid and activated (more ...)
Inhibition of prostaglandin synthesis by direct interference with the cyclooxygenase enzymes is a common mechanism of non-steroidal anti-inflammatory drugs (NSAIDs). Since chamomile appeared to have actions similar to those of NSAIDs, we hypothesized that sulindac, an NSAID that inhibits the enzyme activity of COX-1 and COX-2, and NS398, a specific COX-2 inhibitor, might affect PGE2 production and release in LPS-activated RAW 264.7 macrophages in a similar way as chamomile. As shown in , sulindac and NS398 significantly inhibited endogenous PGE2 production and release in RAW cells with and without LPS treatment, similar to that observed after chamomile treatment. The decrease in PGE2 levels after chamomile treatment corresponded with the decrease in COX-2 mRNA and protein expression; however, the expression of only COX-2 protein was decreased after treatment with sulindac and NS398 treatments. Treatment of RAW 264.7 macrophages with sulindac and NS398 did not affect the mRNA COX-2 levels, demonstrating that NSAIDs action in murine macrophages is not directed towards the transcription of COX-2.
Figure 4 Comparative effects of chamomile, sulindac and NS398 on PGE2, COX-2 mRNA and protein levels in RAW 264.7 macrophages. (A) effect of chamomile (20 μg/mL), sulindac (500 μM) and NS398 (5 μM) on PGE2 accumulation in cell supernatant (more ...)