3.1. Analysis of the Extracts and Characterization of the Isolated Compounds
The components present in the extracts B and C are summarized in
Figure 2. In column chromatography, five visible spots were visualized and isolated. Among them, only spots 2, 3 and 4 were obtained as single compounds, glabranin (M2), pinocembrin (M3) and licoflavanone (M4) (
Figure 2), and fully characterized via NMR analysis.
Spot M2. glabranin or (S)-5,7-dihydroxy-8-(3-methylbut-2-en-1-yl)-2-phenylchroman-4-one. Amorphous white solid. clogP: 5.06285.
m/z: 324 [M
+].
1H-NMR (CDCl
3, 300 MHz) δ (ppm) 12.12 (s, 5-OH), 7.59–7.29 (m, 6H), 6.65 (m, 1H), 6.05 (s, 1H), 5.43 (dd, 1H,
J = 3.1, 12.9 Hz), 5.20 (d, 1H,
J = 7.0 Hz), 3.33 (d, 1H,
J = 7.0 Hz), 3.05 (dd, 1H,
J = 12.9, 17.1 Hz), 2.85 (dd, 1H,
J = 3.1, 17.1 Hz), 1.80 (s, 6H).
13C-NMR (CDCl
3, 75 MHz) δ (ppm) 196.2, 163.7, 162.2, 159.6, 138.7, 134.6, 128.7, 128.6, 127.9, 125.9, 125.1, 121.6, 106.3, 103.3, 96.9, 78.9, 43.2, 25.7, 21.8, 17.8. These data are in agreement with those reported [
32].
Spot M3. (S)-pinocembrin or (S)-5,7-dihydroxy-2-phenylchroman-4-one). White solid m.p.: 198–199 °C. clogP: 3.11185.
m/z: 256 [M
+]. Spectroscopic data are in agreement with those reported [
13,
20].
Spot M4. licoflavanone or (S)-5,7-dihydroxy-2-(4-hydroxy-3-(3-methylbut-2-en-1-yl)phenyl)chroman-4-one. Pale yellow needles m.p.: 134–135 °C. clogP: 4.39585.
m/z: 340 [M
+].
1H-NMR (CDCl
3, 300 MHz) δ (ppm) 12.0 (bs, 1H), 7.28–7.20 (m, 1H), 7.19–7.05 (m, 1H), 6.89 (d, 1H,
J = 8.3 Hz), 6.05–5.75 (m, 3H), 5.25 (d, 1H,
J = 3.0 Hz), 3.35 (d, 2H,
J = 7.3 Hz), 3.08 (dd, 1H,
J = 12.9, 17.0 Hz), 2.72 (dd, 1H,
J = 3.0, 17.0 Hz,) H-3), 1.75 (s, 6H).
13C-NMR (CDCl
3, 75 MHz) δ (ppm) 196.8, 164.9, 163.7 (× 2C), 155.0, 131.5 (× 2C), 128.8, 128.5, 124.4, 123.1, 116.2, 102.8, 95.1, 94.6, 82.8, 43.2, 28.1, 24.6, 18.5. Spectroscopic data are in agreement with those reported [
23,
33,
34].
3.2. Antioxidant Profile of Glycyrrhiza glabra L. Leaf Extracts and Their Flavanone-Components
In order to evaluate and compare the antioxidant power of
Glycyrrhiza glabra L. leaf extracts A, B, C were subjected to DPPH and ABTS assays. In these experiments, a similar antioxidant profile for the different extracts emerged (
Figure 3), with IC
50 values ranging from 13.49 to 18.05 µg/mL, in DPPH assay, and from 5.88 to 6.76 µg/mL, in ABTS assay (
Table 2), a result that reflects the similar total phenolic content estimated for each extract (
Table 3). Similar experiments were performed on the three isolated compounds, M2, M3 and M4, to evaluate their contribution to the remarkable antioxidant profile of the extracts. Our results highlighted that the presence of a prenyl group can contribute to the antioxidant profile of flavanones, according to previously reported studies [
35]. Indeed, as depicted in
Figure 3, the presence of the prenyl group in position 8, characterizing glabranin (M2), improved by itself the poor antioxidant power of its not prenylated analogue pinocembrin (M3). Moreover, licoflavanone (M4) showed the best antioxidant profile, suggesting that either the prenyl group position or the presence of an additional hydroxyl group on the C ring of the flavanone backbone could significantly modify the antioxidant power of these natural products.
3.3. Flavanones from Glycyrrhiza glabra L. Leaf Extracts Affect NO Production in LPS-Stimulated RAW 264.7 Cell Line
A correlation between oxidative stress and inflammation had been already highlighted [
36], since several inflammatory stimuli, such as LPS, were able to promote many chronic inflammatory diseases by activating and regulating NO production [
37]. In this context, the anti-inflammatory potential of
Glycyrrhiza glabra L. leaf extracts and their flavanone components were investigated by monitoring their ability to modulate the production of nitric oxide (NO) in LPS-stimulated RAW 264.7 cells, murine macrophages widely used as an in vitro model to study inflammatory pathways. Firstly, we evaluated the effect of increasing concentrations of different
Glycyrrhiza glabra L. leaf extracts (ranging from 12.5 to 50 µg/mL) as well as their flavanone components (ranging from 10 to 200 µM) on LPS-stimulated RAW 264.7 cell growth, by using MTT assay. These studies displayed that the treatment with extract C did not elicit any significant reduction in cell viability. Conversely, the treatment with extracts A and B reduced cell viability only at the highest tested concentrations. A comparable inhibitory effect on NO production was also shown by using the three extracts at non-toxic concentrations. These findings are in agreement with previous literature data reporting similar anti-inflammatory properties of
Glycyrrhiza spp. and its bioactive components, tested in our same experimental model [
12]. In addition, our results highlighted how the ultrasound-assisted extraction, used in this study, represents a simple and rapid alternative to the maceration process of
Glycyrrhiza glabra L. leaves, guaranteeing biological activity preservation of the phytocomplex. On the other hand, the effects of the three isolated natural compounds were found to be significantly different from each other, both in terms of cytotoxicity and modulation of NO production. At the tested concentrations, up to 200 µM, pinocembrin showed a poor cytotoxic profile and a non-striking anti-inflammatory activity. On the contrary, the presence of the prenyl group in position 8, characteristic of glabranin, determined a drastic alteration in terms of toxicity/activity ratio, since this compound was found to be highly toxic already when used at 100 µM. Lastly, licoflavanone displayed an intermediate cytotoxicity profile between the two other natural compounds, but it exhibited a significant inhibitory activity on NO production, which was found at much lower concentrations compared to that of the other tested flavanones, reaching an IC
50 value of 37.68 µM (
Figure 4,
Table 4). On the basis of these experimental evidences and since no literature data on the pharmacological effects of licoflavanone have been reported so far, we decided to investigate the molecular mechanisms and cellular pathways underlying the promising anti-inflammatory potential of this natural compound.
3.4. Licoflavanone Exerts Anti-Inflammatory Effects by Reducing NF-kB Nuclear Translocation
NO is an inflammatory mediator generated by the catalytic activity of inducible nitroxide synthase (iNOS) enzyme, whose expression is stimulated by inflammatory stimuli such as bacterial LPS through the toll-like receptor 4 (TLR4). The induction of iNOS expression is mediated by NF-kB that is the main transcription factor involved in inflammatory response [
38]. Considering the effects exerted by licoflavanone on NO production in LPS-stimulated RAW 264.7 cells, as well as the ability of other flavanones (i.e., pinocembrin) to inhibit inflammatory signal transduction at NF-kB pathway level, we tested the ability of licoflavanone to reduce the activation of this transcription factor. In particular, after the treatment of LPS-stimulated RAW 264.7 cells, the translocation of NF-kB into the nucleus and the expression of two of its target genes, inducible nitroxide synthase (iNOS) and cyclooxygenase 2 (COX2), were monitored. The results obtained in this study highlighted the ability of this natural compound to reduce the translocation of NF-kB into the nucleus (
Figure 5) and to significantly decrease the transcription of its target genes (
Figure 6A), so excluding that the effects on NO production were due to a direct inhibitory action on iNOS. Such results highlight the significant role of licoflavanone in the inhibition of NF-kB pathway observed for
Glycyrrhiza glabra L. leaf extract, from which this precious natural product was isolated.
3.5. Licoflavanone Disrupts MAPK/NF-kB Pathway and Modulates Pro-Inflammatory Cytokines
The NF-kB pathway is interconnected with that of mitogen-activated protein kinases (MAPKs), which mediate phosphorylation processes that lead to the activation of several transcription factors, such as Activator Protein 1 (AP-1). These factors, together with NF-kB, activate the transcription of different inflammatory genes, including those encoding for the pro-inflammatory cytokines that mediate the amplification and propagation of inflammatory-mediated stress signal. On the basis of NF-kB/MAPKs crosstalk, in this work the ability of licoflavanone to inhibit the cascade of the main MAPKs such as p38, JNK and ERK1/2 was assessed. As shown in
Figure 6B, the treatment of LPS-stimulated RAW 264.7 cells with licoflavanone was able to rapidly cause a reduction of p38, JNK and ERK1/2 phosphorylation and activation. Furthermore, the disruption of the NF-kB/MAPKs signal transduction pathway, mediated by licoflavanone, was responsible for a striking reduction in mRNA levels of several pro-inflammatory cytokines, such as Tumor Necrosis Factor alpha (TNFα), Interleukin-1 beta (IL 1β) and Interleukin-6 (IL 6) (
Figure 6C). The pharmacological behavior of licoflavanone was found to be in agreement with that of other plant-derived flavanones, confirming that the ability to interfere with the MAPK/NF-kB pathway is a common feature of the different classes of flavonoids. Similar effects, indeed, although in different experimental models, had been described for several flavones, flavonols and flavanones, such as pinocembrin [
39], naringenin [
40], quercetin [
41] and luteolin [
42], for which the ability to inhibit MAPKs activation, NF-kB translocation into the nucleus, and the production of pro-inflammatory cytokines, had been reported.
However, some pharmacokinetic limits of flavanoids are their partial absorption at the gastrointestinal level, as well as that they undergo an intensive metabolic process in the liver (methylation, sulfation, and glucuronidation), which favors their elimination, determining their low bioavailability index [
43,
44,
45]. In this regard, since in scientific literature no data are reported on the bioavailability of licoflavanone, it is impossible to know if this natural product could reach, in vivo, the concentrations found to be effective in this in vitro study, therefore, future pharmacokinetic studies will be needed.