The Phenolic Fraction of Mentha haplocalyx and Its Constituent Linarin Ameliorate Inflammatory Response through Inactivation of NF-κB and MAPKs in Lipopolysaccharide-Induced RAW264.7 Cells

Mentha haplocalyx has been widely used for its flavoring and medicinal properties and as a traditional Chinese medicine with its anti-inflammation properties. The present study was designed to investigate the anti-inflammatory effects and potential molecular mechanisms of the phenolic fraction of M. haplocalyx (MHP) and its constituent linarin in lipopolysaccharide (LPS)-induced RAW264.7 cells. The high-performance liquid chromatography coupled with linear ion trap-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap MS) was used to analyze the chemical composition of MHP. Using the enzyme-linked immunosorbent assay (ELISA) and quantitative realtime polymerase chain reaction (qRT-PCR), the expression of pro-inflammatory meditators and cytokines was measured at the transcriptional and translational levels. Western blot analysis was used to further investigate changes in the nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and Akt signaling pathways. Fourteen phenolic constituents were identified from MHP based on the data of the mass spectrometry (MS)/MS analysis. MHP and linarin decreased the production of NO, tumor necrosis factor-α (TNF-α), interlenkin-1β (IL-1β), and IL-6. The messenger ribonucleic acid (mRNA) expression levels of inducible NO synthase (iNOS), TNF-α, IL-1β, and IL-6 were also suppressed by MHP and linarin. Further investigation showed that MHP and linarin down-regulated LPS-induced phosphorylation content of NF-κB p65, inhibitor kappa B α (IκBα), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. However, MHP and linarin showed no inhibitory effect on the phosphorylated Akt. These results suggested that MHP and linarin exerted a potent inhibitory effect on pro-inflammatory meditator and cytokines production via the inactivation of NF-κB and MAPKs, and they may serve as potential modulatory agents for the prevention and treatment of inflammatory diseases.


Introduction
Inflammation is an important defense mechanism of the host against invasive stimuli [1,2]. However, excessive inflammatory responses may give rise to various pathological changes related to human diseases, including asthma, rheumatoid arthritis, atherosclerosis, and cancer [3]. Many risk factors are responsible for inflammation, such as pathogens, impaired cells and tissues, and physical and chemical stimuli [4]. Macrophages are phagocytic white blood cells that play an important role in innate immune inflammatory responses. Activated macrophages secrete various cytokines such as tumor necrosis factor-α (TNF-α), interlenkin-1β (IL-1β), interlenkin-6 (IL- 6), and other inflammatory cytokines [5], which show pleiotropic effects via activating adaptive immune responses and worsening inflammatory responses [6]. The nuclear factor kappa-B (NF-κB) signaling pathway is involved in regulating diverse gene expression during inflammatory responses [7]. When activated with extracellular stimuli, the NF-κB released from the cytoplasmic NF-κB-inhibitor kappa B (IκB) complex by the ubiquitination and degradation of IκB translocates into the nucleus [8,9], combining with specific DNA sequences and then regulating the transcription of inflammatory cytokines and mediators [10,11]. Moreover, phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), p38, and c-Jun NH 2 -terminal kinase (JNK) subfamilies could activate NF-κB and regulate inflammatory responses [12,13].
Mentha species are widely used throughout the world as flavoring agents and medicinal plants, particularly for the treatment of common fever, cold, flu, and motion sickness [14,15]. Although the genus Mentha comprises more than 20 species, only the fresh aerial part of Mentha haplocalyx Briq. is described and used as a traditional Chinese herb in Chinese pharmacopoeia. The leaves of M. haplocalyx Briq. are also used in teas, beverages, jellies, syrups, candies, and other products. Modern pharmacological studies have revealed that M. haplocalyx shows various biological activities, such as antiallergenic, antimicrobial, anti-inflammatory, antioxidant, antitumor, antiviral, gastrointestinal protective, hepatoprotective, and chemopreventive activities [16]. Other well-known Mentha species, including M. spicata, M. longifolia, M. piperita, M. pulegium, and M. arvensis, have been reported to express similar biological activities. For instance, gastrointestinal protective properties have been reported for M. longifolia, M. pulegium, and M. arvensis [17][18][19], chemopreventive activities for M. piperita and M. spicata [20,21], and antiviral effects for M. piperita, M. spicata, and M. longifolia [22,23]. Mentha species are characterized by two important classes of secondary metabolites: essential oils and phenolic compounds [24]. Numerous researchers have proved that these compounds contribute to the multiple biological activities of Mentha species [24,25].
In traditional Chinese medicine, M. haplocalyx is used frequently as a herb that releases the "exterior" in the common cold of wind-heat type, headache, red eye, measles, and thoracic and abdominal oppression [26]. Pharmacological studies have revealed that "exterior releasing herbs" are effective against viral and bacterial infections that usually induce inflammation [27]. Based on the fact that heat is related to inflammation in Chinese medicine [28] and that M. haplocalyx has cooling properties, it can be assumed that M. haplocalyx could exert an inhibitory effect on inflammation. Indeed, some studies have proved that flavonoids and phenolic acids from the aqueous extract of M. haplocalyx [29][30][31] express high anti-inflammatory activity [16,32]. In our previous work, we have optimized a method for the purification of the phenolic fraction of M. haplocalyx (MHP), mainly consisting of flavonoids and phenolic acids [33]. In the present study, our particular interest was to investigate the mechanism of the anti-inflammatory effect of M. haplocalyx extract, as well as to analyze the chemical composition of MHP by high-performance liquid chromatography coupled with linear ion trap-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap MS). We explored the mechanism of inflammatory activity by evaluating the impact of MHP on the production of pro-inflammatory meditators and the activation of NF-κB, Akt, and MAPKs in lipopolysaccharide (LPS)-induced RAW264.7 cells. Since the linarin was the main flavonoid from MHP, the mechanism of its activity was also investigated for the first time in this study.

Analysis of the Chemical Composition of the Phenolic Fraction of Mentha haplocalyx by HPLC-LTQ-Orbitrap MS
Structural elucidation of the MHP constituents was performed according to the obtained chromatographic retention times and mass spectrometry (MS)/MS data by comparison with the data of available reference compounds including rosmarinic acid, linarin, or literature data. As a result, 14 phenolic compounds were identified in the MHP sample. The total ion chromatogram (TIC) profile of MHP is shown in Figure 1 and the data for compound identification are summarized in Table 1.

Analysis of the Chemical Composition of the Phenolic Fraction of Mentha haplocalyx by HPLC-LTQ-Orbitrap MS
Structural elucidation of the MHP constituents was performed according to the obtained chromatographic retention times and mass spectrometry (MS)/MS data by comparison with the data of available reference compounds including rosmarinic acid, linarin, or literature data. As a result, 14 phenolic compounds were identified in the MHP sample. The total ion chromatogram (TIC) profile of MHP is shown in Figure 1 and the data for compound identification are summarized in Table 1.

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on NO Production and iNOS Expression Level in LPS-Induced RAW264.7 Cells
The toxicity of drugs could have an influence on the proliferation and function of cells, misleading the judgement of their efficacy. Thus, we firstly evaluated the cell cytotoxicity of MHP and linarin. The RAW264.7 cells were treated with increased concentrations of MHP and linarin for 24 h and cell cytotoxicity was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 2, MHP and linarin did not exhibit toxic effects on RAW264.7 cells at 0-200 μg/mL and 0-20 μM, respectively.

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on NO Production and iNOS Expression Level in LPS-Induced RAW264.7 Cells
The toxicity of drugs could have an influence on the proliferation and function of cells, misleading the judgement of their efficacy. Thus, we firstly evaluated the cell cytotoxicity of MHP and linarin. The RAW264.7 cells were treated with increased concentrations of MHP and linarin for 24 h and cell cytotoxicity was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 2, MHP and linarin did not exhibit toxic effects on RAW264.7 cells at 0-200 µg/mL and 0-20 µM, respectively.

Analysis of the Chemical Composition of the Phenolic Fraction of Mentha haplocalyx by HPLC-LTQ-Orbitrap MS
Structural elucidation of the MHP constituents was performed according to the obtained chromatographic retention times and mass spectrometry (MS)/MS data by comparison with the data of available reference compounds including rosmarinic acid, linarin, or literature data. As a result, 14 phenolic compounds were identified in the MHP sample. The total ion chromatogram (TIC) profile of MHP is shown in Figure 1 and the data for compound identification are summarized in Table 1.

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on NO Production and iNOS Expression Level in LPS-Induced RAW264.7 Cells
The toxicity of drugs could have an influence on the proliferation and function of cells, misleading the judgement of their efficacy. Thus, we firstly evaluated the cell cytotoxicity of MHP and linarin. The RAW264.7 cells were treated with increased concentrations of MHP and linarin for 24 h and cell cytotoxicity was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 2, MHP and linarin did not exhibit toxic effects on RAW264.7 cells at 0-200 μg/mL and 0-20 μM, respectively.   To investigate the anti-inflammatory activities of MHP and linarin, we studied the inhibitory effect of MHP and linarin on LPS-induced nitric oxide (NO) production in RAW264.7 cells. As shown in Figures 3A and 4A, the NO production level significantly increased after LPS-stimulation for 24 h in RAW264.7 cells. MHP at 50-200 µg/mL and linarin at 5-20 µM showed markedly inhibitory effects on the increase of NO in a dose-dependent manner. Subsequently, we investigated the cause of reduced NO production by measuring the expression of inducible NO synthase (iNOS) mRNA. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that both MHP and linarin were able to inhibit the expression of iNOS mRNA ( Figure 5A,B). The results suggested that MHP and linarin inhibited NO production by down-regulating the expression of iNOS mRNA. To investigate the anti-inflammatory activities of MHP and linarin, we studied the inhibitory effect of MHP and linarin on LPS-induced nitric oxide (NO) production in RAW264.7 cells. As shown in Figures  3A and 4A, the NO production level significantly increased after LPS-stimulation for 24 h in RAW264.7 cells. MHP at 50-200 μg/mL and linarin at 5-20 μM showed markedly inhibitory effects on the increase of NO in a dose-dependent manner. Subsequently, we investigated the cause of reduced NO production by measuring the expression of inducible NO synthase (iNOS) mRNA. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that both MHP and linarin were able to inhibit the expression of iNOS mRNA ( Figure 5A,B). The results suggested that MHP and linarin inhibited NO production by down-regulating the expression of iNOS mRNA.   To investigate the anti-inflammatory activities of MHP and linarin, we studied the inhibitory effect of MHP and linarin on LPS-induced nitric oxide (NO) production in RAW264.7 cells. As shown in Figures  3A and 4A, the NO production level significantly increased after LPS-stimulation for 24 h in RAW264.7 cells. MHP at 50-200 μg/mL and linarin at 5-20 μM showed markedly inhibitory effects on the increase of NO in a dose-dependent manner. Subsequently, we investigated the cause of reduced NO production by measuring the expression of inducible NO synthase (iNOS) mRNA. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that both MHP and linarin were able to inhibit the expression of iNOS mRNA ( Figure 5A,B). The results suggested that MHP and linarin inhibited NO production by down-regulating the expression of iNOS mRNA.     The mRNA expression levels of iNOS, TNF-α, IL-1β, and IL-6 were measured by quantitative real time polymerase chain reaction (qRT-PCR). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The data shown are representative of three experiments and expressed as mean ± SD. * p < 0.05 and ** p < 0.01 versus the group treated with LPS alone.

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on the Production of TNF-α, IL-1β, and IL-6 in LPS-Induced RAW264.7 Cells
To determine the effects of MHP and linarin on the production of inflammatory cytokines including TNF-α, IL-1β, and IL-6, the RAW264.7 cells were pretreated with LPS in the presence or absence of screened concentrations of MHP and linarin. The results of enzyme-linked immunosorbent assay (ELISA) showed that the amount of TNF-α, IL-1β, and IL-6 significantly increased in RAW264.7 cells induced by LPS alone. As shown in Figure 3B-D, the level of TNF-α was significantly inhibited by MHP at 100 and 200 μg/mL, while the levels of IL-1β and IL-6 were markedly suppressed by MHP at 50, 100, and 200 μg/mL. Meanwhile, linarin remarkably suppressed the secretion of TNF-α, IL-1β, and IL-6 at concentrations of 10 and 20 μM ( Figure 4B-D).

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on TNF-α, IL-1β, and IL-6 mRNA Expression in LPS-Induced RAW264.7 Cells
In order to investigate whether MHP and linarin inhibited the expression of pro-inflammatory cytokines at the transcriptional level, we examined TNF-α, IL-1β, and IL-6 mRNA expression levels in RAW264.7 cells. As expected in Figure 5A, 100 and 200 μg/mL of MHP significantly inhibited the

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on the Production of TNF-α, IL-1β, and IL-6 in LPS-Induced RAW264.7 Cells
To determine the effects of MHP and linarin on the production of inflammatory cytokines including TNF-α, IL-1β, and IL-6, the RAW264.7 cells were pretreated with LPS in the presence or absence of screened concentrations of MHP and linarin. The results of enzyme-linked immunosorbent assay (ELISA) showed that the amount of TNF-α, IL-1β, and IL-6 significantly increased in RAW264.7 cells induced by LPS alone. As shown in Figure 3B-D, the level of TNF-α was significantly inhibited by MHP at 100 and 200 μg/mL, while the levels of IL-1β and IL-6 were markedly suppressed by MHP at 50, 100, and 200 μg/mL. Meanwhile, linarin remarkably suppressed the secretion of TNF-α, IL-1β, and IL-6 at concentrations of 10 and 20 μM ( Figure 4B-D).

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on TNF-α, IL-1β, and IL-6 mRNA Expression in LPS-Induced RAW264.7 Cells
In order to investigate whether MHP and linarin inhibited the expression of pro-inflammatory cytokines at the transcriptional level, we examined TNF-α, IL-1β, and IL-6 mRNA expression levels in RAW264.7 cells. As expected in Figure 5A, 100 and 200 μg/mL of MHP significantly inhibited the  To determine the effects of MHP and linarin on the production of inflammatory cytokines including TNF-α, IL-1β, and IL-6, the RAW264.7 cells were pretreated with LPS in the presence or absence of screened concentrations of MHP and linarin. The results of enzyme-linked immunosorbent assay (ELISA) showed that the amount of TNF-α, IL-1β, and IL-6 significantly increased in RAW264.7 cells induced by LPS alone. As shown in Figure 3B  In order to investigate whether MHP and linarin inhibited the expression of pro-inflammatory cytokines at the transcriptional level, we examined TNF-α, IL-1β, and IL-6 mRNA expression levels in RAW264.7 cells. As expected in Figure 5A, 100 and 200 µg/mL of MHP significantly inhibited the LPS-induced increase of the TNF-α mRNA expression while MHP at 50, 100, and 200 µg/mL decreased the expression of IL-1β and IL-6 mRNA induced by LPS. In addition, linarin significantly repressed IL-1β and IL-6 mRNA expression at 5-20 µM and the mRNA expression level of TNF-α was significantly down-regulated by linarin at 20 µM ( Figure 5B). The activation of NF-κB in response to LPS is a vital step in inducing the expression of inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 [34]. Here, we investigated whether MHP and linarin inhibited the phosphorylation of inhibitor kappa B α (IκBα) and p65. As depicted in Figure 6, the relative content of phosphorylated (p-)IκBα and p-p65 significantly increased after LPS stimulation, which suggested that LPS stimulation triggered the phosphorylation of IκBα and p65. However, these increases were inhibited by MHP and linarin. MHP treatment significantly restrained the level of p-IκBα at 50, 100, and 200 µg/mL ( Figure 6A), and at 100 and 200 µg/mL for p-p65 ( Figure 6B). The increased levels of p-p65 and p-IκBα were also repressed by linarin at 10 and 20 µM ( Figure 6C,D). These results indicated that MHP and linarin exerted anti-inflammatory effects through inhibition of LPS-induced activation of the NF-κB signaling pathway.  Figure 5B).

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on NF-κB p65 and IκBα in LPS-Induced RAW264.7 Cells
The activation of NF-κB in response to LPS is a vital step in inducing the expression of inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 [34]. Here, we investigated whether MHP and linarin inhibited the phosphorylation of inhibitor kappa B α (IκBα) and p65. As depicted in Figure 6, the relative content of phosphorylated (p-)IκBα and p-p65 significantly increased after LPS stimulation, which suggested that LPS stimulation triggered the phosphorylation of IκBα and p65. However, these increases were inhibited by MHP and linarin. MHP treatment significantly restrained the level of p-IκBα at 50, 100, and 200 μg/mL ( Figure 6A), and at 100 and 200 μg/mL for p-p65 ( Figure 6B). The increased levels of p-p65 and p-IκBα were also repressed by linarin at 10 and 20 μM ( Figure 6C,D). These results indicated that MHP and linarin exerted anti-inflammatory effects through inhibition of LPS-induced activation of the NF-κB signaling pathway.

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on Akt in LPS-Induced RAW264.7 Cells
A previous study has reported that activated Akt, a key protein of the PI3K signaling pathway, could turn on the activation of NF-κB [12]. Thus, we investigated the inhibitory effects of MHP and linarin on LPS-induced expression of phosphorylated Akt protein in RAW264.7 cells. However, the results shown in Figure 7A,B indicate that MHP and linarin exerted no inhibitory effect on the LPS-induced increase of the phosphorylation level of Akt.

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on Akt in LPS-Induced RAW264.7 Cells
A previous study has reported that activated Akt, a key protein of the PI3K signaling pathway, could turn on the activation of NF-κB [12]. Thus, we investigated the inhibitory effects of MHP and linarin on LPS-induced expression of phosphorylated Akt protein in RAW264.7 cells. However, the results shown in Figure 7A,B indicate that MHP and linarin exerted no inhibitory effect on the LPS-induced increase of the phosphorylation level of Akt.

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on MAPKs in LPS-Induced RAW264.7 Cells
To further explore whether MHP and linarin exerted inhibitory effects on the inflammatory mediators and cytokines through inactivation of the MAPK signaling pathway, we investigated the phosphorylation levels of MAPKs including ERK1/2, JNK, and p38. As observed in Figure 8, LPS stimulation increased the relative content of p-p38, p-JNK, and p-ERK1/2. MHP treatment remarkably inhibited the phosphorylation level of JNK at 50, 100, and 200 μg/mL ( Figure 8C) and suppressed the phosphorylation level of p38 at 100 and 200 μg/mL ( Figure 8B), while at 200 μg/mL for the phosphorylated ERK ( Figure 8A). Furthermore, linarin significantly inhibited the phosphorylation of JNK at 5, 10, and 20 μM ( Figure 8F), but at 10 and 20 μM for the phosphorylation of ERK and p38 ( Figure 8D,E). Analytical data indicated that MHP and linarin exerted an anti-inflammation effect via the inhibition of the LPSinduced MAPK signaling pathway.

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on MAPKs in LPS-Induced RAW264.7 Cells
To further explore whether MHP and linarin exerted inhibitory effects on the inflammatory mediators and cytokines through inactivation of the MAPK signaling pathway, we investigated the phosphorylation levels of MAPKs including ERK1/2, JNK, and p38. As observed in Figure 8, LPS stimulation increased the relative content of p-p38, p-JNK, and p-ERK1/2. MHP treatment remarkably inhibited the phosphorylation level of JNK at 50, 100, and 200 µg/mL ( Figure 8C) and suppressed the phosphorylation level of p38 at 100 and 200 µg/mL ( Figure 8B), while at 200 µg/mL for the phosphorylated ERK ( Figure 8A). Furthermore, linarin significantly inhibited the phosphorylation of JNK at 5, 10, and 20 µM ( Figure 8F), but at 10 and 20 µM for the phosphorylation of ERK and p38 ( Figure 8D,E). Analytical data indicated that MHP and linarin exerted an anti-inflammation effect via the inhibition of the LPS-induced MAPK signaling pathway.

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on Akt in LPS-Induced RAW264.7 Cells
A previous study has reported that activated Akt, a key protein of the PI3K signaling pathway, could turn on the activation of NF-κB [12]. Thus, we investigated the inhibitory effects of MHP and linarin on LPS-induced expression of phosphorylated Akt protein in RAW264.7 cells. However, the results shown in Figure 7A,B indicate that MHP and linarin exerted no inhibitory effect on the LPS-induced increase of the phosphorylation level of Akt.

Effects of the Phenolic Fraction of Mentha haplocalyx and Linarin on MAPKs in LPS-Induced RAW264.7 Cells
To further explore whether MHP and linarin exerted inhibitory effects on the inflammatory mediators and cytokines through inactivation of the MAPK signaling pathway, we investigated the phosphorylation levels of MAPKs including ERK1/2, JNK, and p38. As observed in Figure 8, LPS stimulation increased the relative content of p-p38, p-JNK, and p-ERK1/2. MHP treatment remarkably inhibited the phosphorylation level of JNK at 50, 100, and 200 μg/mL ( Figure 8C) and suppressed the phosphorylation level of p38 at 100 and 200 μg/mL ( Figure 8B), while at 200 μg/mL for the phosphorylated ERK ( Figure 8A). Furthermore, linarin significantly inhibited the phosphorylation of JNK at 5, 10, and 20 μM ( Figure 8F), but at 10 and 20 μM for the phosphorylation of ERK and p38 ( Figure 8D,E). Analytical data indicated that MHP and linarin exerted an anti-inflammation effect via the inhibition of the LPSinduced MAPK signaling pathway.

Discussion
Pharmacological investigations have shown that anti-inflammation is an essential feature of many Mentha species. M. haplocalyx ethanol extract significantly lessened the severity of airway inflammation by inhibiting the production of immunoglobulin E, IL-4, and IL-6 in bronchoalveolar lavage fluid and lung tissue [35]. Ethyl acetate and aqueous fractions of M. spicata effectively reduced the inflammation induced by carrageenan and cotton pellet [36]. The aqueous ethanol (70%) extract of M. arvensis at 100 μg/mL showed a strong inhibitory effect on IL-8 secretion [37], and the aqueous methanol (80%) extract of M. arvensis, the main constituents of which were linarin and rosmarinic acid, showed inhibitory activity against the strain Chlamydia pneumoniae CWL-029 [38]. The hexane fraction of M. longifolia, mainly containing phenolic and flavonoid compounds, was able to significantly reduce NO production, iNOS, and TNF-α mRNA expression at 0.05-0.20 mg/mL in LPS-induced J774A.1 cells [39]. The results of all the studies mentioned above suggest that Mentha species could serve as an important source of antiinflammatory agents.
In this paper, we investigated the chemical composition and anti-inflammatory activity of the phenolic fraction from M. haplocalyx. The chemical profile of the MHP sample was analyzed by HPLC-LTQ-Orbitrap MS and showed that its main constituents were phenolic acids and flavonoids, and some of them, such as protocatechuic aldehyde, caffeic acid, rosmarinic acid, salvianolic acid B, and diosmin, have been reported to display inhibitory effects on inflammation [40][41][42][43][44]. These bioactive constituents probably contribute to the anti-inflammatory activity of MHP. Flavonoid linarin is also one of the main constituents of the MHP extract. Previous research has shown that linarin from M. arvensis exhibited a selective inhibitory effect on acetylcholinesterase in a concentration-dependent manner [45]. It has also been reported that linarin possesses analgesic, antipyretic [46], and neuroprotective activities [47], and showed a protective effect against D-glactosamine/LPS-induced fulminant hepatic failure [48]. Furthermore, a tissue distribution study of linarin has revealed that it was rapidly and widely distributed in the heart, liver, spleen, lung, kidney, brain, muscle, stomach, small intestine, epididymis, pancreas, and bladder of rats after intragastric administration [49]. Since there was little data on the anti-inflammatory mechanism of linarin [50], the present study explored its involvement in the anti-inflammatory action of the MHP extract.
NO is a small diffusible molecule, which exerts a regulating effect on various biological functions including apoptosis, neurotransmission, inflammation, and blood vessel tone [51]. NO is generated from L-arginine by three types of NO synthases including neuronal NOS (nNOS), iNOS, and endothelial NOS (eNOS) [51]. Among them, iNOS is the main enzyme to catalyze NO production in

Discussion
Pharmacological investigations have shown that anti-inflammation is an essential feature of many Mentha species. M. haplocalyx ethanol extract significantly lessened the severity of airway inflammation by inhibiting the production of immunoglobulin E, IL-4, and IL-6 in bronchoalveolar lavage fluid and lung tissue [35]. Ethyl acetate and aqueous fractions of M. spicata effectively reduced the inflammation induced by carrageenan and cotton pellet [36]. The aqueous ethanol (70%) extract of M. arvensis at 100 µg/mL showed a strong inhibitory effect on IL-8 secretion [37], and the aqueous methanol (80%) extract of M. arvensis, the main constituents of which were linarin and rosmarinic acid, showed inhibitory activity against the strain Chlamydia pneumoniae CWL-029 [38]. The hexane fraction of M. longifolia, mainly containing phenolic and flavonoid compounds, was able to significantly reduce NO production, iNOS, and TNF-α mRNA expression at 0.05-0.20 mg/mL in LPS-induced J774A.1 cells [39]. The results of all the studies mentioned above suggest that Mentha species could serve as an important source of anti-inflammatory agents.
In this paper, we investigated the chemical composition and anti-inflammatory activity of the phenolic fraction from M. haplocalyx. The chemical profile of the MHP sample was analyzed by HPLC-LTQ-Orbitrap MS and showed that its main constituents were phenolic acids and flavonoids, and some of them, such as protocatechuic aldehyde, caffeic acid, rosmarinic acid, salvianolic acid B, and diosmin, have been reported to display inhibitory effects on inflammation [40][41][42][43][44]. These bioactive constituents probably contribute to the anti-inflammatory activity of MHP. Flavonoid linarin is also one of the main constituents of the MHP extract. Previous research has shown that linarin from M. arvensis exhibited a selective inhibitory effect on acetylcholinesterase in a concentration-dependent manner [45]. It has also been reported that linarin possesses analgesic, antipyretic [46], and neuroprotective activities [47], and showed a protective effect against D-glactosamine/LPS-induced fulminant hepatic failure [48]. Furthermore, a tissue distribution study of linarin has revealed that it was rapidly and widely distributed in the heart, liver, spleen, lung, kidney, brain, muscle, stomach, small intestine, epididymis, pancreas, and bladder of rats after intragastric administration [49]. Since there was little data on the anti-inflammatory mechanism of linarin [50], the present study explored its involvement in the anti-inflammatory action of the MHP extract.
NO is a small diffusible molecule, which exerts a regulating effect on various biological functions including apoptosis, neurotransmission, inflammation, and blood vessel tone [51]. NO is generated from L-arginine by three types of NO synthases including neuronal NOS (nNOS), iNOS, and endothelial NOS (eNOS) [51]. Among them, iNOS is the main enzyme to catalyze NO production in acute and chronic inflammation, and inflammatory stimuli such as LPS can cause its expression [7,52]. Our data indicated that both MHP and linarin significantly suppressed the LPS-induced NO production in RAW264.7 cells with a concentration-dependent manner via down-regulating the transcriptional level of iNOS.
Apart from NO, many pro-inflammatory cytokines including TNF-α, IL-6, and IL-1β are produced and take part in the process of inflammatory responses. TNF-α acts as a principal endogenous mediator during inflammatory responses and is also capable of stimulating the secretion of IL-6 and IL-1β, which could exert a synergistic effect with TNF-α [2,53], causing further damage to the body. Inhibiting the synthesis or release of these inflammatory cytokines could emerge as a potential therapeutic approach for inflammatory diseases. In the present study, MHP significantly blocked LPS-induced overproduction of TNF-α, IL-1β, and IL-6. Linarin attenuated TNF-α, IL-1β, and IL-6 production induced by LPS as well, which was consistent with the reported article [50]. Additionally, further investigation demonstrated that MHP and linarin treatment down-regulated the expression levels of TNF-α, IL-1β, and IL-6 mRNA. The above results indicated that MHP and linarin exerted an inhibitory effect on LPS-induced inflammation by suppressing the expression of inflammatory cytokines at the transcriptional and translational levels.
By modulating the secretion of inflammatory cytokines, NF-κB exerts a critical effect on regulating inflammatory responses to extracellular stimuli [34,54]. The major forms of NF-κB are composed of p50 and p65, which are bound to the cytoplasmic inhibitory protein IκB in the resting state [54]. Once activated by external stimuli, IκB is phosphorylated by IκB kinase (IKK) which leads to the degradation of IκBα [55], and p65 is then released and translocates to the nucleus where it may trigger the expression of multiple inflammatory genes. Among the identified constituents of MHP, protocatechuic aldehyde exerted an anti-inflammatory effect on myocardial ischemia/reperfusion injury through inhibiting the NF-κB pathway [40]. Caffeic acid exhibited the anti-inflammatory property via reducing the activation of NF-κB targeting genes [41]. Both salvianolic acid B and diosmin showed a protective effect on lung inflammation by suppressing the expression of TNF-α, IL-1β, and IL-6 through inactivation of the NF-κB pathway [42,43]. Therefore, MHP was expected to exert an anti-inflammatory effect by inhibiting the NF-κB pathway. Data in this paper showed that treatment with definite concentrations of MHP effectively blocked LPS-induced phosphorylation of IκBα and p65. Meanwhile, the increased phosphorylation levels of IκBα and p65 were also down-regulated by linarin. These suggested that MHP and linarin were able to repress the activation of the NF-κB pathway in LPS-induced RAW264.7 cells.
In addition to the NF-κB pathway, the PI3K/Akt and MAPK pathways play essential roles in regulating inflammatory cytokine production in macrophages. It has been reported that Akt and MAPKs were involved in the activation of NF-κB [56,57]. Akt is a key downsteam protein of PI3K and shows diverse roles in cellular growth, adhesion, and inflammation responses [58]. However, our finding showed that LPS-induced phosphorylated Akt was not blocked by MHP. Rosmarinic acid and linarin were two main constituents of MHP. In the previous study of the molecular mechanism of melanogenesis, rosmarinic acid did not reduce Akt phosphorylation in B16 melanoma cells [59]. The results of the present study also demonstrated that linarin displayed no significant inhibitory effect on the phosphorylated Akt. These results suggest that other signaling pathways may be involved in the anti-inflammatory mechanisms of MHP and linarin. Therefore, the impacts of MHP and linarin on MAPKs were further investigated. MAPKs including JNK, p38, and ERK are reported to be closely associated with diverse cellular processes, such as differentiation, proliferation, apoptosis, and immune responses [60]. In general, mitogen and differentiation signals are able to activate the ERK while stress stimuli activate p38 and JNK [61]. Many studies have illustrated that the up-regulation of TNF-α, IL-1β, and IL-6 is diminished by phytochemicals through the inhibition of the activation of ERK, JNK, and p38 [6,62]. In this paper, MHP and linarin down-regulated the phosphorylation levels of ERK, JNK, and p38. Taken together, these findings show that MHP and linarin could inhibit the activation of the NF-κB pathway via suppressing MAPKs activities.
In conclusion, the present study revealed that the phenolic fraction of M. haplocalyx and linarin showed potent inhibitory effects on the secretion of NO, TNF-α, IL-1β, and IL-6 and the mRNA expression of iNOS, TNF-α, IL-1β, and IL-6 in LPS-induced RAW264.7 cells. As shown in Figure 9, these effects were closely related to the inhibition of LPS-induced activation of NF-κB and MAPKs signals. The results not only clarified the anti-inflammatory molecular mechanisms of MHP and linarin, but also pointed out the potential value of MHP and linarin as anti-inflammatory agents for the prevention and treatment of inflammatory diseases.
Molecules 2017, 22,811 10 of 17 expression of iNOS, TNF-α, IL-1β, and IL-6 in LPS-induced RAW264.7 cells. As shown in Figure 9, these effects were closely related to the inhibition of LPS-induced activation of NF-κB and MAPKs signals. The results not only clarified the anti-inflammatory molecular mechanisms of MHP and linarin, but also pointed out the potential value of MHP and linarin as anti-inflammatory agents for the prevention and treatment of inflammatory diseases.

Reagents
Linarin and rosmarinic acid were purchased from Must Bio-technology Co. (Chengdu, China).  China). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies were obtained from Cell Signaling Technology (Danvers, CO, USA). Fluorescein-conjugated affinipure goat anti-rabbit IgG was purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). Antibodies against phospho (p)-ERK, p-p38, p-JNK, p-Akt, Akt, p-p65, and p-IκBα were obtained from Cell Signaling Technology. All other reagents were of analytical grade.

Preparation of the Phenolic Fraction of Mentha haplocalyx Extract
The sample (300 g) of M. haplocalyx was extracted three times by refluxing with 4800 mL of 30% ethanol for 1.5 h each time. Then the extracted solution was filtered and the procedure was repeated another two times. The filtered extracts were mixed together and then concentrated until there was no alcoholic odour. The concentrate was centrifuged at 3000 rpm for 30 min. The pH of the supernatant collected was adjusted to 3 by adding acetic acid. The sample solution was applied onto a column of treated HPD-400 macroporous resin (Cangzhou Baoen Chemical Co., Ltd., Cangzhou, Hebei, China). The elution conditions were as follows: 20% ethanol for two column volume, 70% ethanol for three column volume. The eluent of 70% ethanol was collected and then concentrated to dryness. MHP was obtained after the residue was dried under reduced pressure. For in vitro experiments, the phenolic fraction was dissolved in dimethyl sulfoxide with a final concentration of less than 0.1%.

HPLC-MS/MS Analysis of MHP
MHP (10 mg) was dissolved using 70% methanol in a 25 mL volumetric flask, and the solution was filtered by a 0.45 µM microporous membrane.
The operating conditions of mass spectrometry were as follows: electrospray ionization (ESI) source in positive or negative mode, capillary temperature of 350 • C; sheath gas flow, 30 arb; auxiliary gas flow, 10 arb; positive mode, 4 kV of spray voltage, 25 V of capillary voltage; 110 V of tube lens negative mode, 3 kV of spray voltage, −35 V of capillary voltage; −110 V of tube lens. The range of full-scan mass was from m/z 100 to 1000. The resolution of the Orbitrap analyzer was set at 30,000. The three most intense ions from the one-stage mass spectrometry scanning were selected for further MS 2 analysis. The other specific parameters were as follows: collision-induced dissociation (CID) of activation type, 2 amu of isolation width, 30 ms of activation time, 35% of normalized collision energy.

Cell Culture Conditions and Treatment
The RAW264.7 macrophage cell line was purchased from China Infrastructure of Cell Line Resource (Beijing, China). RAW264.7 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator with 5% CO 2 at 37 • C. The cells were treated with MHP and linarin at different concentrations and then stimulated with LPS (1 µg/mL) for the incubation time.

Cell Viability Assay
The RAW264.7 cells were seeded in 96-well plates overnight, followed by incubation with MHP (0, 12.5, 25, 50, 100, and 200 µg/mL) and linarin (0, 5, 10, 20, 40, 80, and 160 µM) for 24 h. After treatment for 24 h, the supernatant was removed, and the MTT solution (10 µL) and fresh culture medium (90 µL) were added to each well. The cells were incubated for 4 h, after which the culture medium was removed and DMSO was added to dissolve the crystals for an additional 10 min. The value of optical density was read on a microplate reader (MK3, Thermoelectric Technology Instrument Co., Ltd., Shanghai, China) at 492 nm.

Effect on NO Production
The RAW264.7 cells were seeded in 96-well plates overnight and then treated with MHP (50, 100, and 200 µg/mL) and linarin (5, 10, and 20 µM) for 1 h, followed by incubation with LPS (1 µg/mL) for an additional 24 h. Griess reagent was used to determine the content of nitric oxide in the cell culture supernatant. Briefly, the culture supernatant (50 µL) was reacted with an equal volume of Griess reagent (50 µL), and the optical density of each well was measured on the microplate reader at 570 nm.

Cytokines Measurement
For the measurement of TNF-α, IL-1β, and IL-6, the RAW264.7 cells were seeded in 24-well plates overnight and then treated with MHP (50, 100, and 200 µg/mL) and linarin (5, 10, and 20 µM) for 1 h, followed by incubation with LPS (1 µg/mL) for an additional 24 h. The content of the pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 in the supernatant were assayed using the ELISA kits according to the manufacturer's instructions.

Western Blot Analysis
The RAW264.7 cells were seeded in 100-mm dishes overnight, after which they were treated with MHP (50, 100, and 200 µg/mL) and linarin (5, 10, and 20 µM) for 4 h, and further incubated with LPS (1 µg/mL) for an additional 30 min. Subsequently, the cells were rinsed two times with cold phosphate-buffered saline (PBS), lysed using ice-cold radioimmunoprecipitation assay (RIPA) buffer supplemented with a cocktail of protease and phosphatase inhibitors, and placed on ice for 30 min. The supernatants were collected after centrifuging at 10,000 g for 15 min at 4 • C. The bicinchoninic acid (BCA) protein assay kit was used to measure protein content for each sample. Protein samples were separated on a 10% SDS-PAGE using PowerPac™ Basic Power Supply (Bio-Rad, Hercules, CA, USA), blotted onto PVDF membranes and then blocked for 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBST) solution. The membranes were then incubated at 4 • C overnight with 1:1000 dilutions of the primary antibodies. After washed with TBST solution three times for 10 min, the membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Finally, the membranes were washed with the TBST solution three times for 10 min and chemiluminescent signals of the protein bands were developed using the Super Signal TM West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.).

Statistical Analysis
The results were expressed as the means ± standard deviation (SD). Multiple group comparisons were performed using one-way ANOVA test using the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA); * p < 0.05 or ** p < 0.01 were considered statistically significant.