Licocalchone-C Extracted from Glycyrrhiza Glabra Inhibits Lipopolysaccharide-Interferon-γ Inflammation by Improving Antioxidant Conditions and Regulating Inducible Nitric Oxide Synthase Expression

The genus Glycyrrhiza consists of about 30 species, amoung these, G. glabra is the source of several phenolic compounds, known as flavonoids, such as licoagrodin, licoagrochalcones, licoagroaurone and licochalcone C, kanzonol Y, glyinflanin B and glycyrdione A, which have shown various pharmacological activities, including antitumor, antiparasitic, antileishmanial, anti-ulcer and antioxidative effects. Among these compounds, licochalcone C was isolated but its biology has not been fully examined. In our study we reproduced an inflammatory state by treating THP-1 (human myelomonocytic leukaemia) cells with pro-inflammatory stimuli, such as LPS and IFN-γ and we investigated the possible antioxidant activity of licochalcone C at a concentration of 50 μM. Our results show that treatment with licochalcone C attenuates the LPS-IFN-γ-induced inflammatory response by significantly decreasing the expression and activity of iNOS via NFκB (nuclear factor kappa-B), by influencing extracellular O2− production, and by modulating the antioxidant network activity of SOD (superoxide dismutase), CAT (catalase) and GPx (glutathione peroxidase) activity. Based on these results we hypothesize that Licochalcone C has antioxidant properties since it reduces the production of superoxide radicals and consequently reduces the activity of iNOS.

lower cytotoxicity [18,19]. In this study, we isolated a natural compound, licochalcone C, from G. glabra and we conducted a series of experiments using human acute monocytic leukemia cell line (THP-1) that mimic the cellular inflammation model to explore potential mechanisms for the activity of the phenolic compounds. We investigated the putative antioxidant effects of licochalcone C evaluating both the enzymatic activity of the antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidise, GPx) that iNOS modulation via activation of NFkB transcription.

Cytotoxicities of Licochalcone C
In this study, we isolated licochalcone C ( Figure 1) and examined its effects on LPS (10 µg/mL) + IFN-γ (20 ng/mL)-induced inflammation in a THP-1 cell line model. First, we evaluated the cytotoxicity of licochalcone C in THP-1 cells by an MTT assay before and after 24 hr of incubation, respectively, and it was observed that this phenolic compounds did not affect cell viability between the before and after 24 h of incubation (Table 1). For all further experiments, only a non toxic concentration of 50 µM licochalcone C were used. Table 1. Licochalcone C concentration-effect curve on cell viability.

Antioxidant Activity of Licochalcone C
Many phenolic compounds present in medicinal plants possess antioxidant and anti-inflammatory activities and previous reports have evidenced that the constituents of Glycirrhyza were effective in preventing microsomal lipid peroxidation induced by Fe (III)-ADP/NADPH and licochalcone B and D exhibited potent antioxidative and superoxide scavenging activities [20].
ROS are a major constituent of inflammation that can affect normal cellular function and have pathogenic consequences. The burst of activated, oxygen-derived free-radical species is responsible for peroxidation of cell membranes, resulting in tissue edema, and protein and enzyme degradation. They can also compromise cellular repair mechanisms, cause premature aging, and trigger apoptotic processes [21]. Quenching ROS production can decrease inflammation and subsequent tissue damage. The first line of cellular antioxidant defence consist of free radical scavenging enzymes, such as SOD, CAT and GPx. Thus, to eliminate free radicals, these cellular antioxidants play an important role in maintaining a redox equilibrium under normal physiological conditions but act in first line defence against excessive production of free radicals [22]. SOD [23]. Initially, extracellular superoxide radical levels were determined spectrophotometrically ( Figure 2). In THP-1 cells treated with proinflammatory cytokines there is a marked production of such radicals, highlighting the fact that under similar conditions elevated levels of oxidative stress demonstrated by cells coincubated with cytokines, the dismutase reaction with SOD is extremely elevated ( Figure 3A).
This increase in SOD activity involves an accumulation of H 2 O 2, not adequately metabolized from CAT and GPx, with consequent intracellular accumulation of toxic radicals that, as also shown in other studies, may lead to the activation of inducible enzymes, whose catalytic product can reversibly inhibit antioxidant enzymes such as CAT and GPx ( Figures 3B, 3C) [24.25]. Treatment with licochalcone C shows a significant reduction in the production of O 2 − and as shown in Figure 2, appears to be comparable to baseline values. Taken together, these results strongly suggest that licochacone C has the ability to decrease oxidative stress produced in vitro on THP-1 cells through the activation with proinflammatory cytokines, elucidating the antioxidant profile of this natural compound.

Influence of licochalcone C on iNOS signaling via NFkB
Several non-enzymatic (e.g., glutathione, flavonoids, and vitamins A, C, and E) as well as enzymatic scavengers of ROS (e.g., SOD, CAT, and GPx) prevent the accumulation of ROS. Unfortunately, these defense mechanisms are not always adequate to counteract the toxic effects of ROS, resulting in what is termed a state of oxidative stress. Phenolic compound via their antioxidant effects in protecting cellular components against ROS [26]. Antagonizing ROS production by several antioxidants abrogated the activation of NF-kB [23]. Cells that have an excessive production of free radicals lead to an activation of a series of transcription factors (e.g., AP-1, NFkB, Nrf2) associated with induction of genes coding for proteins, such as cycloxygenase (COX-2) and iNOS-2, closely involved in inflammatory responses. In our experimental model, it is possible to observe that in activated THP-1, an altered redox state leads to a high expression of the transcription factor NFkB ( Figure 4A) directly involved in the modulation of the inducible isoform of NOS [27,28].  Figure 4D), as also demonstrated by evaluating the expression of a specific marker of the main production of peroxynitrite [29,30]. Drugs that inhibit iNOS expression and/or enzyme activity, resulting in decreased NO generation, may be beneficial in treating diseases caused by an overproduction of NO [31]. The densitometric analysis showed a diminished expression and activity of iNOS in the co-stimulated cells with cytokines and licochalcone C at a concentration of 50 µM, respect to cytokines alone ( Table 2). Treatment with licochalcone C has strong antioxidant properties, since it reduces the production of ROS and restores physiological enzymatic activity in the network, it down-regulates the transcription factor NF-kB, thereby decreasing the production of a highly reactive species, such as that ONOO − . Because of its high reactivity it generates extensive lesions in the same cells that produce it.

General
Licochalcone C was extracted as described by Yoon et al. [32]. Dried roots (2 kg) from Glycyrrhiza glabra were grinded into fine powder in liquid nitrogen, with a mortar and pestle. Afterwards, two consecutive extraction with 4 L of boiling distilled water for 2.5 h were done. Fatty acids were removed from the combined water extract with 2 L of n-hexane. The defatted solution was extracted three times with 2 L of dichloromethane and then, the combined organic extract was evaporated. The residue was dissolved in chloroform and loaded onto a silica column. The elution solvent system used was a mix of n-hexane-ethyl acetate-methanol 2:1:0.1. Fractions were analyzed on TLC and those presenting large spots near the R f of pure licochalcone A (Sigma) were combined, concentrated and loaded onto a second silica column and eluted with chloroform-methanol (10:1) eluent system to obtain 15 mg of licochalcone C. Purity was checked with a HPLC system (P/U-2080, Jasco) equipped with a diode array detector (MD-2010, Jasco) using a Tracer Hypersil ODS (5 µm) C 18 column (10 × 250 mm). The compound was obtained with a purity of 98% and its structural identity was confirmed by H 1

Cell culture
Cell culture THP-1 (human myelomonocytic leukaemia) cell line (ATCC number TIB-202), were cultured at a density of 10 6 cells/mL in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 100 ng/mL streptomycin, 100 U/mL penicillin, and 2 mM L-glutamine in a 5% CO 2 air humidified atmosphere at 37 °C and passaged every 4 to 5 days. The cell viability, determined by trypan blue exclusion was >99%. Cells were seeded onto six-well tissue culture plate and incubated overnight at 37 °C in a humidified atmosphere of 5% CO 2 . Cell viability was not influenced in field exposed non-stimulated cells nor LPS-stimulated cells. More than 98% of cells were viable, as determined by trypan blue dye exclusion at the beginning of the culture, and more than 90% were viable before cells were collected. Cell were divided in: (1) control cells; (2) cells stimulated with LPS (10 µg/mL) + IFN-gamma (20 ng/mL); (3) cells stimulated with LPS (10 µg/mL) IFN-gamma (20 ng/mL) and licochalcone C (50 µM); (4) cells stimulated only with licochalcone C (50 µM) for 24 hours. The dose-effect relationship of the licochalcone C was previously determined in a set preliminary of experiments, and it was found that 50 µM was associated with the down expression of iNOS and no cellular toxicity. Cell viability was determined by trypan blue day exclusion and MTT assay (Biotium, Hayward, CA, USA).

Production of O 2
− was determined spectrophotometrically (Hewlett Packard 8452 A) by monitoring the reduction of ferricytochrome c (Type VI, Sigma) at 550 nm, as described by Pritchard [33] Ferricytochrome c (final concentration, 50 μmol/L) was added directly to the cuvette containing the cells and Dulbecco's phosphate buffered saline (DPBS) (final volume 1 mL), in the presence or absence of superoxide dismutase [(SOD), 350 U/mL], and changes in absorbance were followed for 10 minutes. Rates of O 2 − production were calculated on the basis of the molar extinction coefficient of reduced ferricytochrome c [ε = 21,000 cm −1 (mol/L) −1 ]. Cell counts were used to calculate results as nanomoles O 2 − per 10 6 cells per minute.

Cu, Zn-Superoxide Dismutase Activity
SOD activity was determined as described by Sun and Zigman [34]. The assay mixture contained 50 mM sodium carbonate buffer, pH 10, epinephrine 0.1 mM (Sigma), and tissue fraction (containing about 1-50 μg of protein) in a final volume of 2.5 mL. The inhibitory effect of SOD on the autoxidation of epinephrine, with the use of 1.25 mM KCN to discriminate the CN-insensitive MnSOD from the CN-sensitive Cu, ZnSOD was assayed spectrophotometrically at 480 nm at 25 °C. Percentage inhibition values were converted into activities by using a purified Cu, Zn bovine SOD as standard (Sigma). One unit of SOD is the amount of enzyme required to halve the rate of substrate auto-oxidation.

Catalase Activity
CAT activity was measured spectrophotometrically [35]. The decomposition of H 2 O 2 was monitored continuously at 240 nm. The assay mixture in a final volume of 3 mL contained 10 mM potassium phosphate buffer, 10 mM H 2 O 2 and 1.5-11 μg of protein of enzymatic extract. CAT units were defined as 1 μmole H 2 O 2 decomposed/min at 25 °C.

Glutathione Peroxidase Activity
Quantification of GPx activity was evaluated using the Paglia and Valentine method as modified by Di Ilio et al. [36,37]. The activity of the Se-dependent GSH peroxidase was measured with H 2 O 2 (0.25 mM) as substrate. The oxidation of NADPH was followed at 25 °C on a Hewlett Packard spectro-photometer at 340 nm. One unit was defined as 1 μmol of GSH oxidized min.

Western blot Analysis for iNOS, NF-κB and 3-nitrotyrosine
Determination of iNOS, 3-nitrotyrosine and NFkb proteins were performed in two series of protein extracts from THP-1 cell line by Western blotting. Fifty μg cytoplasmatic and 10 μg nuclear proteins, quantified by spectrophotometric assay (HP 8452A, Palo Alto, CA, USA) using the Breadford method, were separated by electrophoresis in a 7.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE; Bio-Rad, Hercules, CA, USA) and transferred at 4 °C to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) in glycine-methanol buffer. Nitrocellulose was then blocked in Tris-buffered saline (TBS)milk and incubated, overnight, with various primary antibodies: anti-human iNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal anti-3-nitrotyrosine (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-human NF-κB (Santa Cruz Biotechnology). The nitrocelluloses were then washed in TBS, incubated with a secondary antibody conjugated with alkaline phosphatase for 2 h, washed again, and developed in an alkaline buffer with nitroblue tetrazolium (NBT) as substrate (Alkaline Phosphatase Conjugate Substrate Kit, Bio-Rad, Hercules, CA, USA). Antibody anti-β-Actin (Sigma), and Actin (Sigma), were used as an internal standard. The densitometric analysis of Western blots was performed using a computerized densitometric system (Bio-Rad Gel Doc 1000, Milan, Italy).

Semi-Quantitative Reverse Transcription-Polymerase Chain Reaction for iNOS
Semi-quantitative reverse-transcribed polymerase chain reaction (RT-PCR) was used to determine mRNA levels of the inducible Nitric Oxide Synthase. Total RNA was extracted using 1 mL ULTRASPEC-RNA (Biotecx, Lab., Inc. Huston, TX, USA), as recommended by the manufacturer. RNA was dissolved in diethyl pyrocarbonate (DEPC)-treated water and quantified spectrophotometrically at 260 nm.
First-strand cDNA was generated by adding RNA (1 μg) to a mixture containing 1 mM deoxynucleoside-triphosphates (d-NTP), 1U/μL RNase inhibitor, 2.5 U/μL moloney murine leukemia virus reverse transcriptase, 2.5 μM oligo-dt, 5 mM MgCl 2 , 10x PCR buffer in a final volume of 20 μL. Reverse transcription was performed at 42 °C for 1 hr followed by heat inactivation of reverse transcriptase at 92 °C for 10 min. 18S was amplified from the same amount of RNA to correct for variation of different samples. PCR amplification was performed using a Programmable Thermal Controller (MJ Research, Inc. Waltham, MA, USA). The PCR solution contained 10 μL of first-strand cDNA, 4 μL 10x PCR buffer, 2 mM MgCl 2 . The following primer pairs were used: sense 5'-CGT AAA GAC CTC TAT GCC AA-3' and antisense 5'-AGC CAT GCC AAA TGT CTC AT-3' for iNOS, and 18S primers, sense 5'-TAC GGA GCA GCA AAT CCA C-3' and antisense 5'-GAT CAA AGG ACT GCA GCC TG-3', 2U Termophylus Acquaticus (Taq) DNA polymerase (Celbio, Milan, Italy), and water to a final volume of 50 μL. These samples were overlaid with mineral oil and subjected to 35 cycles at 95 °C for 60 sec, 60 °C for 60 sec, and to one cycle at 72 °C for 7 min for iNOS and 40 cycles at 95 °C for 60 sec, 58 °C for 60 sec, and to one cycle at 72 °C for 7 min for 18S. PCR products were run on 2% agarose gel electrophoresis and photographed after ethidium bromide staining under UV light. Bands on the gel were scanned using a computerized densitometric system (Bio-Rad Gel Doc 1000, Milan, Italy).

Citrulline Synthesis
The measure of the conversion of L-arginine to L-citrulline is a standard assay method currently used to quantitative NOS activity. Briefly, 10 μL of radioactive arginine, L-(2,3,4,5-3 H) arginine monohydrochloride 64 Ci/mM, 1 μCi/μL (Amersham, Arlington Heigths, IL, USA) and 50 μL NADPH 10 mM were added to each cell homogenate samples and incubated for 30 min at room temperature. After incubation, the reactions were stopped with 400 μL of stop-buffer (50 mM HEPES, pH 5.5, 5 mM EDTA) and added of the equilibrated resin into each sample. The equilibrated resin bound unreacted arginine. After centrifugation, the radioactivity corresponding to L-( 3 H)-citrulline was measured with liquid scintillation spectrometry. Calcium was omitted from these incubations to favour the determination of the calcium independent iNOS isoform.

Statistical Analysis
The results were expressed as mean ± SD. Statistical analysis was performed using the analysis of variance (ANOVA). Probability of null hypothesis of <5% (p < 0.05) was considered statistically significant.

Conclusions
As many studies have demonstrated that the use of several compounds of plant origin show a multiplicity of pharmacological activities against a number of pathological conditions, in this study we isolated the structural isomer of licocalchone A, licocalchone C, from licorice, which in the literature was shown to exhibit various biological activities [38][39][40][41]. Traditionally, these compounds have been used in northeast Asia for the treatment of gastric and duodenal ulcers, bronchial asthma, inflammation, and other diseases. Much of the recent research on the constituents of licorice has resulted in finding the pharmacological importance of various phenolic compounds [42][43][44]. Peripheral blood monocytes may reflect the bias of the donating individual, and it is hard to assess the role of individual effects on the results of tests. PBMCs are almost impossible to standardize for use in standard tests to evaluate biological response. Because of the shortcomings of PBMCs, the THP-1 monocyte cell line has been used extensively as a model for testing the drug. The THP-1 was more sensitive than PBMCs in cytotoxic evaluations, but provided an identical ranking of relative cytotoxicities with significantly less variation. In this model, an inflammatory state has been reproduced in THP-1 with the stimulation of LPS and IFN-γ that determine activation of the iNOS gene via NF-kB, resulting in a high production of nitric oxide [23]. Our hypothesis is that treatment with the licocalchone C, resulting in a reduction of the superoxide radical, restores a physiological condition that down regulates iNOS. At the cellular level this lead to a significant reduction of NO, which influences: (1) production of peroxynitrite, as we have demonstrated in the treatment there is nitrosylation of proteins; (2) restoration of the activity of CAT and GPx. In fact, several experimental evidences suggest that in a state of oxidative stress (as in our model) there may be an interaction between NO of the heme catalase, resulting in a reversible inhibition of the enzyme [25]. Furthermore, as evidenced by Dobash et al. [24] we found a temporary inhibition of GPx probably through the interaction of NO with the thiol group of the enzyme. Therefore, our results show that the excessive NO production in vitro by endogenous iNOS in response to cytokines leads to alterations of the redox state through the down-regulation of catalase, GPX and the up-regulation of CuZn-SOD. This alteration of cellular redox may play a pivotal role in the pathophysiology associated with the induction of iNOS. Our results show for the first time that treatment with licocalchone C could attenuate LPS-IFN-γ-induced inflammatory response via the modulation of oxidative stress through decreasing O 2 − production and modulation of antioxidant network activity of SOD, CAT and GPx in a biological model. Moreover, licocalchone C, by scavenging ROS, is responsible for the inhibition of iNOS via NFkB in THP-1 cell line. Decreasing NO production, significantly prevents the formation of ONOO − . Since both the NO and NF-kB are important inflammatory mediators, they play key roles in modulating the formation of various radical species during the oxidative action of licocalchone C, and may represent a new target of action against inflammatory diseases which have in common an alteration of the redox status.