In the present study, we demonstrate for the first time that artichoke leaf extract and artichoke compounds downregulate iNOS expression human coronary artery smooth muscle cells when administered concurrently with an inflammatory stimulus.
2.4. Artichoke Compounds Inhibit iNOS Expression
Artichoke is rich in polyphenolic compounds, with mono- and dicaffeoylquinic acids as the major chemical components [
28]. The most well-known caffeoylquinic acid derivative identified in artichoke extracts (heads and leaves), even though it is not the most abundant, is cynarin [
28]. Besides caffeoylquinic acid derivatives, other phenolics belonging to the flavonoid class such as the flavones (e.g., luteolin and its 7-
O-glucoside, cynaroside) and the anthocyanidins (e.g., cyanidin) have been identified in artichoke tissues [
28]. Therefore, we studied the effects of these four well-known artichoke compounds on iNOS expression in HCASMC.
As shown in
Figure 5, all four tested compounds (cynarin > cyanidin > luteolin ≈ cynaroside) downregulated iNOS mRNA expression in HCASMC, with cynarin being the most potent one (
Figure 5a). At the protein level, cynarin and cyanidin clearly reduced iNOS expression, whereas luteolin and cynaroside had only minor effects (
Figure 5b).
In our previous study, we have found that the flavone luteolin and its 7-
O-glucoside cynaroside enhance eNOS expression in endothelial cells whereas the caffeoylquinic acids (cynarin and chlorogenic acid) are without effect [
23]. These results, together with the data from the present study, indicate that the active constituents responsible for the artichoke effects on eNOS and iNOS are likely to be different compounds.
Figure 5.
Artichoke compounds downregulate iNOS expression. Human coronary artery smooth muscle cells were treated with the cytokine mixture (CM) or CM in combination with an artichoke leaf extract (ALE, 10 µg/mL) or the artichoke compounds (10 µM each) for 6 h (a) or 24 h (b). Human iNOS mRNA expression was analyzed with real-time RT-PCR (a). * p < 0.05, compared with CM. Protein expression of iNOS was studied with Western blot analyses (b). The blots shown are representative of three independent experiments with similar results.
Figure 5.
Artichoke compounds downregulate iNOS expression. Human coronary artery smooth muscle cells were treated with the cytokine mixture (CM) or CM in combination with an artichoke leaf extract (ALE, 10 µg/mL) or the artichoke compounds (10 µM each) for 6 h (a) or 24 h (b). Human iNOS mRNA expression was analyzed with real-time RT-PCR (a). * p < 0.05, compared with CM. Protein expression of iNOS was studied with Western blot analyses (b). The blots shown are representative of three independent experiments with similar results.
2.5. Potential Therapeutic Relevance
Artichoke is one of the world’s oldest medicinal plants. It has been known by the ancient Egyptians, and the ancient Greeks and Romans used it as a digestive aid. Long known as an herbal medicine, the dried leaves of artichoke have been used in folk medicine because of their choleretic and hepatoprotective activities [
28].
In various pharmacological test systems, artichoke leaf extracts have shown choleretic activity [
29], hepatoprotective [
30,
31] and prebiotic effects [
32,
33]. In addition, artichoke leaf extracts contain compounds with antifungal [
32] and antimicrobial activities [
34].
Recent studies indicate that artichoke leaf extracts may also have therapeutic potential for cardiovascular disease. The plant contains multiple compounds with high antioxidant properties [
35,
36]. Antioxidative effects of artichoke extracts have been shown in endothelial cells [
37,
38] and leukocytes [
37,
39]
in vitro, as well as in experimental animals
in vivo [
40,
41]. Luteolin-rich artichoke extracts and luteolin itself protect low-density lipoprotein (LDL) from oxidation
in vitro [
42]. In a rat model of streptozotocin-induced diabetes, an artichoke leaf extract reduced the plasma malondialdehyde and urinary 8-hydroxydeoxyguanosine levels, and increased erythrocyte glutathione levels [
41].
Although it is still a matter of debate [
43], there is some evidence that artichoke leaf extract may lower cholesterol levels. In randomized, double-blind, placebo-controlled clinical trials, artichoke leaf extract reduced the total cholesterol levels in hypercholesterolemic adults [
44,
45].
A previous study from our laboratory shows that artichoke leaf extracts upregulate eNOS expression in human endothelial cells [
23]. Increased NO production by artichoke extracts has also been shown in porcine aortic endothelial cells [
46]. In a randomized, placebo-controlled trial, concentrated artichoke leaf juice significantly lowered blood pressure (by ≈ 3 mmHg after 12 weeks) in patients with mild hypertension [
47].
The present study demonstrates that ALE, cynarin and cyanidin inhibit iNOS expression in vascular smooth muscle cells when administered concurrently with an inflammatory stimulus. We did not analyze whether the compounds in ALE have any effect on iNOS expression without an inflammatory stimulus. The cynarin concentration in the ALE we used is not known; but it could be in similar ranges as that in methanolic extracts of artichoke (≈1.5%) [
28]. Based on this assumption, the cynarin concentration of 100 µg/mL ALE would be ≈3 µM, a concentration that is relevant to the effect of cynarin on iNOS expression. We are aware that we have only studied a few candidate compounds in the present study, and we may have missed a number of active compounds. It should also be reminded that the effects of artichoke extracts cannot be attributed to one or two single compounds. Rather, the
in vivo effect of a plant extract is more likely to result from multiple active compounds that act additively or synergistically. It is also possible that some compounds contained in ALE may have antagonistic effects, or complex and unpredictable effects worthy of future study.
The therapeutic potential of cynarin needs to be further investigated. Several issues should be considered in future studies including the bioavailability of the compound and the specificity of its action. After ingestion of artichoke extracts, cynarin and other caffeoylquinic acids are not found in human plasma. However, caffeoylquinic acid metabolites (such as caffeic acid, ferulic acid, isoferulic acid, dihydrocaffeic acid, and dihydroferulic acid) are detected in considerable concentrations [
48,
49]. It is still unknown whether the metabolites of cynarin are effective in inhibiting iNOS expression. In addition to its effect on iNOS, cynarin has antioxidative activities [
39] and immuno-suppressive effects [
50]. Recent studies indicate that cynarin may have the potential to serve as a chemosensitizing agent by reversing P-glycoprotein-mediated multidrug resistance [
31]; but it may also induce drug-drug interactions by inhibiting organic anion transporters [
51]. Therefore, animal studies are required to test the therapeutic potential and possible side effects of cynarin
in vivo.
Anthocyanidins (e.g., cyanidin) and their glycosides (
i.e., anthocyanins) are responsible for the brilliant color of fruits and flowers and are widely ingested by humans [
52]. Cyanidin and its glycosides show antioxidant, anti-inflammatory, and antimutagenic effects [
52]. Cyanidin 3-
O-β-
d-glucoside reduces cytokine-induced iNOS and cyclooxygenase-2 (COX-2) expression in intestinal cells [
53] and macrophages [
54]. Moreover, cyanidin 3-
O-β-
d-glucoside has been shown to reduce ochratoxin-induced iNOS expression
in vivo [
55]. Orally administered cyanidin 3-
O-β-
d-glucoside is metabolized to cyanidin and protocatechuic acid by intestinal microflora [
7] and these metabolites are detectable in blood and urine [
56]. Cyanidin itself has also been shown to suppresses phorbol ester-induced COX-2 and iNOS expression in human colon adenocarcinoma cell line HT-29 cells [
57] and in RAW 264.7 macrophages [
56]. In an air pouch model of inflammation in mouse skin, both cyanidin 3-
O-β-
d-glucoside and cyanidin inhibit carrageenan-induced iNOS and COX2 expression, as well as the production of inflammatory cytokines after oral application [
56]. These results are compatible with the present study and support the concept that cyanidin may have therapeutic potentials.