Antituberculosis Activity of a Naturally Occurring Flavonoid

Article pubs.acs.org/jnp

Antituberculosis Activity of a Naturally Occurring Flavonoid, Isorhamnetin Hum Nath Jnawali,†,∥ Dasom Jeon,†,∥ Min-Cheol Jeong,† Eunjung Lee,† Bongwhan Jin,† Sungweon Ryoo,‡ Jungheon Yoo,§ In Duk Jung,⊥ Seung Jun Lee,⊥ Yeong-Min Park,⊥ and Yangmee Kim*,† †

Department of Bioscience and Biotechnology, Bio-Molecular Informatics Center, Konkuk University, Seoul, 143-701, South Korea Korean Institute of Tuberculosis, Osong, Cheongju, 361-954, South Korea § Quantamatrix Inc., Seoul National University, Seoul, 151-742, South Korea ⊥ Department of Immunology, Lab of Dendritic Cell Differentiation & Regulation, School of Medicine, Konkuk University, Chungju, 380-701, South Korea ‡

S Supporting Information *

ABSTRACT: Isorhamnetin (1) is a naturally occurring flavonoid having anticancer and anti-inflammatory properties. The present study demonstrated that 1 had antimycobacterial effects on Mycobacterium tuberculosis H37Rv, multi-drug- and extensively drug-resistant clinical isolates with minimum inhibitory concentrations of 158 and 316 μM, respectively. Mycobacteria mainly affect the lungs, causing an intense local inflammatory response that is critical to the pathogenesis of tuberculosis. We investigated the effects of 1 on interferon (IFN)-γ-stimulated human lung fibroblast MRC-5 cells. Isorhamnetin suppressed the release of tumor necrosis factor (TNF)-α and interleukin (IL)-12. A nontoxic dose of 1 reduced mRNA expression of TNF-α, IL-1β, IL-6, IL-12, and matrix metalloproteinase-1 in IFN-γ-stimulated cells. Isorhamnetin inhibited IFN-γ-mediated stimulation of extracellular signalregulated kinase and p38 mitogen-activated protein kinase and showed highaffinity binding to these kinases (binding constants: 4.46 × 106 M−1 and 7.6 × 106 M−1, respectively). The 4′-hydroxy group and the 3′-methoxy group of the B-ring and the 5-hydroxy group of the A-ring of 1 play key roles in these binding interactions. A mouse in vivo study of lipopolysaccharide-induced lung inflammation revealed that a nontoxic dose of 1 reduced the levels of IL-1β, IL-6, IL-12, and INF-γ in lung tissue. These data provide the first evidence that 1 could be developed as a potent antituberculosis drug.

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be infected by, and support active replication of, M. tuberculosis.2 The cooperative interaction of fibroblasts with other immune cells is an integral component of granuloma structures generated in response to M. tuberculosis infection, where it elicits immune regulatory functions to control mycobacteria infection.1 It has been reported that M. tuberculosis can actively upregulate matrix metalloproteinase (MMP)-1 in a manner that is dependent on signal transducer and activator of transcription 3, p38 mitogenactivated protein kinase (MAPK), and nuclear factor (NF)-κB in human lung fibroblast MRC-5 cells.3 The period between 1950 and 1970 was a turning point in the battle against TB; most of the current anti-TB drugs were discovered, and new therapeutic regimens made TB a curable disease.4 The initial optimism of the TB-control community began to wane when drug-resistant M. tuberculosis strains emerged. M. tuberculosis strains classified as multi-drug-resistant (MDR) are resistant to two of the most potent first-line anti-TB

uberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, which is transmitted through inhalation of aerosolized droplets. This bacterium primarily affects the lung, where it infects and activates macrophages, fibroblasts, and dendritic cells and is characterized by extensive focal inflammation and development of granulomas. TB is therefore a chronic inflammatory condition in which regulatory and pro-inflammatory processes occur either mutually or stagewise that contribute to the establishment and progression of disease. On the other hand, cytokines (interferons, interleukins, tumor necrosis factor) and cells (macrophages, fibroblasts, regulatory T cells, type 1 helper lymphocytes) are the major host components that are involved in TB inflammation. These components in turn exhibit dual features; either they foster or repress the local inflammatory events. The granuloma that forms following M. tuberculosis infection is surrounded by a peripheral mantle of fibroblasts. It has been assumed that the main function of these fibroblasts is to contain and isolate mycobacteria by secreting collagen at the periphery of the granuloma.1 Fibroblasts are the most abundant cell type within connective tissue that can © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 17, 2015

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DOI: 10.1021/acs.jnatprod.5b01033 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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drugs: isoniazid and rifampicin.5−7 Extensively drug-resistant (XDR) strains are resistant to either isoniazid or rifampicin, any fluoroquinolone, and at least one of three second-line anti-TB injectable drugs, i.e., capreomycin, kanamycin, or amikacin.5−7 Patients infected with these strains required special treatment with expensive and toxic second-line drugs followed by hospitalization to manage their toxic reactions and other complications, which in turn requires significant healthcare resources. Therefore, there is an urgent need to develop novel drugs with new modes of action capable of combating MDR and XDR strains of M. tuberculosis. Flavonoids are naturally occurring polyphenolic compounds containing two benzene rings linked together by a heterocyclic pyran or pyrone ring. Flavonoids, which are normal constituents of the human diet, are a large group of secondary plant metabolites comprising more than 6000 known compounds. Flavonoids are believed to act as health-promoting substances. To date, a large number of biological and pharmacological effects have been ascribed to flavonoids, including antiallergic, antiinflammatory, antioxidant, antitumor, antiviral, and antimicrobial activities.8,9 These potentially beneficial properties have increased interest in the role of nutrients in health and disease. Research into natural products with the potential to combat M. tuberculosis has intensified, facilitated by the development of easier, faster, and safer screening techniques. Previous studies demonstrated that several plant constituents were active against M. tuberculosis. For example, quercetin and luteolin showed weak antimycobacterial activities.10 Isorhamnetin (1; 3′-methoxy-3,4′,5,7-tetrahydroxyflavone) is a naturally occurring O-methylated flavonol that is abundant in apples, blackberries, cherries, and pears.11 Isorhamnetin (1) is also present in medicinal herbs and plants, such as the sea buckthorn (Hippophae rhamnoides L.) and water dropwort (Oenanthe javanica), which are frequently used in traditional medicines for the prevention and treatment of a range of diseases.12 It has been reported that 1 carries many biological properties, including anti-inflammatory, anticarcinogenic, and antioxidant activities.13−15 Flavonoids including 1 prevent the oxidation of low-density lipoprotein cholesterol, thereby inhibiting the formation of atherosclerotic plaques in the arterial wall. They stimulate the enzymes that are involved in the detoxification of carcenogenic substances and inhibit the inflammation associated with the local production of free radicals.16 The anti-inflammatory effects of 1 and their underlying mechanisms were previously evaluated in lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages.17 Previously, it has also been reported that isorhamnetin inhibits MAP (mitogen-activated protein)/ERK kinase (MEK) 1 and PI3K in squamous cell carcinoma skin cancer.18 Dietary intake of such phytochemicals may inhibit or delay pathological conditions and diseases. The effects of 1 on M. tuberculosis have not been reported to date. The present study investigated this for the first time. We also examined the effects of 1 on interferon (IFN)-γ-stimulated human lung fibroblast MRC-5 cells in vitro and the effect of 1 in a mouse model of LPS-induced lung inflammation in vivo. In order to understand the structure−activity relationships, the antituberculosis activity of 1 was compared with those of its isomers, tamarixetin (2; 4′-methoxy-3,3′,5,7-tetrahydroxyflavone) and quercetin (3; 3,3′,4′,5,7-pentahydroxyflavone). Furthermore, we used fluorescence quenching and molecular docking studies to investigate the interactions of 1 with extracellular signalregulated kinase (ERK)1 and p38 MAPK.

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RESULTS AND DISCUSSION Anti-TB Activities. We determined the effects of 1 and 2 on the M. tuberculosis H37Rv strain and on clinical isolates from patients with MDR and XDR M. tuberculosis. Isorhamnetin (1) inhibited H37Rv strain growth with an MIC90 value (the lowest concentration to produce 90% inhibition of M. tuberculosis growth) of 158 μM (Figure 1A). The MIC90 values for the MDR

Figure 1. Effects of isorhamnetin (1) on growth of the M. tuberculosisP887 (H37Rv; panel A), M. tuberculosis-M22 (MDR; panel B), and M. tuberculosis-X24 (XDR; panel C) strains. The red square indicates the MIC90 for the relevant strain.

and XDR isolates were 316 μM (Figure 1B and C). The MIC90 value of control compound rifampicin was 0.61 μM in the H37Rv strain (M. tuberculosis-P887 strain) (Supporting Information, Figure S1), but it did not show antimycobacterial activity against MDR and XDR M. tuberculosis strains due to resistance against B



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inducing the expression of a wide variety of genes that encode pro-inflammatory mediators and cytokines, including inducible nitric oxide synthase, cyclooxygenase-2, nicotinamide adenine dinucleotide phosphate phagocyte oxidase, IL-1β, and IL-12.21,22 M. tuberculosis infection is characterized by the formation of granulomas, usually in the lungs and lymph nodes.23 The tuberculous granuloma is an organized structure of immune cells that forms in response to persistent TB infection and consists of macrophages, neutrophils, fibroblasts, and lymphocytes.24,25 Fibroblasts are the most abundant cell type within connective tissue, which can be infected by, and support active replication of, M. tuberculosis.26 The interaction between M. tuberculosis and cells of both the innate and adaptive immune systems results in the secretion of chemokines and cytokines, the most important being TNF-α, IL-1β, IL-6, IL-12, and IFN-γ. TNF-α is a prototype pro-inflammatory cytokine that plays a key role in granuloma formation,27 induces macrophage activation, and has immunoregulatory properties.28 MMPs are a family of zinc- and calcium-dependent endopeptidases that are emerging as central mediators of tissue destruction in tuberculosis.29 MMPs can degrade most of the extracellular matrix (ECM) components.30 MMP-1 is an interstitial collagenase that can destroy the strongest fibrillar components of the ECM (type I collagen).29,31 We determined the influence of 1 on IFN-γ-stimulated MRC-5 cells and investigated the signaling pathways downstream of IFN-γ by monitoring levels of mRNA for IL-1β, IL-6, IL-12, TNF-α, and MMP-1 using reverse transcription-polymerase chain reaction (RT-PCR). As shown in Figure 2A−F, isorhamnetin (1) reduced the mRNA levels of IL-1β, IL-6, IL-12, TNF-α, and MMP-1 by 39%, 69%, 76%, 56%, and 41%, respectively, as compared to the levels present in cells stimulated with IFN-γ alone. These data indicated that 1 may reduce IFN-γ-induced lung damage. Effects of Isorhamnetin (1) on IFN-γ-Induced Proteins. We assessed whether 1 (20 μM) inhibited the IFN-γ-induced phosphorylation of MAPK family members (ERK, JNK, and p38 MAPK) in MRC-5 cells. Figure 3A, C, and D show that 1 significantly suppressed the activation of ERK and p38 MAPK, as shown by their reduced phosphorylation, as compared with cells stimulated by IFN-γ alone. In contrast, 1 did not significantly affect the levels of phosphorylated JNK (Figure 3A and B). These results indicate that 1 decreased IFN-γ-induced activation of ERK and p38 MAPK in IFN-γ-stimulated MRC-5 cells. Measurement of IL-12 and TNF-α in INF-γ-Stimulated MRC-5 Cells. The levels of TNF-α were 37.5% and 43.75% lower in INF-γ-stimulated MRC-5 cells treated with 5 and 10 μM 1, respectively (Figure 4A), as compared to those exposed to INF-γ only. The levels of IL-12 were 45% and 50% lower in the presence of 5 and 10 μM 1, respectively, as compared with cells that were not treated with 1 (Figure 4B). Isorhamnetin (1) Inhibits LPS-Induced Production of Pro-inflammatory Cytokines in Mouse Lung Tissue. We employed a mouse model of LPS-induced lung inflammation to investigate the effects of 1 on inflammation in vivo. The levels of the pro-inflammatory cytokines, IL-12, IL-1β, IL-6, and INF-γ were lower in lung homogenates from mice that received 1 prior to LPS injection (Figure 5A−D), as compared to vehicle-treated animals. Intact IL-1-mediated signals are essential components of the host defense to mycobacteria.32 IL-6, which has both pro- and anti-inflammatory properties,33 is produced early during mycobacterial infection and at the site of infection.34 IL-12 is a key player in host defense against M. tuberculosis. IL-12 has a crucial role in the induction of IFN-γ production.35

these strains. Medicinal plants and foods provide a promising natural source of anti-TB drugs, and the in vitro activities of several secondary metabolites have already been recognized. A flavonoid that is able to inhibit the growth of H37Rv, MDR, and XDR M. tuberculosis strains, such as 1, would be of extremely high value in the clinic, particularly in cases of MDR (e.g, M. tuberculosis-M22) and XDR (e.g., M. tuberculosis-X24) TB. Isorhamnetin (1) is therefore a new prototype molecule that exerts a relevant biological effect against the mycobacteria responsible for XDR and MDR-TB, a pandemic that is currently increasing and that represents a serious global health problem. Luteolin and quercetin were previously reported to have MICs of 827.28 μM (236.8 μg/mL) and >827.17 μM (>250 μg/mL), respectively, against M. tuberculosis H37Rv.10 We also determined the MIC of an isoform of 1, tamarixetin (2), against M. tuberculosis H37Rv, MDR, and XDR strains (Table 1) in order Table 1. Inhibitory Effects of Isorhamnetin (1) and Tamarixetin (2) on Different Strains of M. tuberculosis MIC90 (μM) name of strain

strain type

isorhamnetin (1)

tamarixetin (2)

P887 M22 M23 X24 X59

H37Rv MDR MDR XDR XDR

158 316 316 316 316

632 632 632 >632 >632

to investigate structure−activity relationships. These data showed that 2 had less potent antimycobacterial activity than 1 against the H37Rv strain (632 μM) as well as against the MDR (632 μM) and XDR isolates (>632 μM). Structure−Activity Relationships. We investigated the relationship between compound structure and effects on M. tuberculosis. The structures of three flavonols, isorhamnetin (1), tamarixetin (2), and quercetin (3), share double bonds between C2 and C3 in ring C, along with 3-OH in ring C and the 5,7-diOH groups in ring A. However, ring B shows considerable variation in these compounds, with 3′,4′-diOH groups in quercetin (3), 3′-OCH3 and 4′-OH in isorhamnetin (1), and 3′-OH and 4′-OCH3 in tamarixetin (2). Isorhamnetin (1) showed higher antituberculosis activity (Table 1). The hydrophobicity (log P) of a molecule can significantly affect its antitubercular activity because the mycobacterial cell wall is lipid-rich and highly impermeable to hydrophilic compounds. In contrast, hydrophobic compounds may penetrate the cell membrane, enter the cytoplasm, and exert toxic effects.19 Isorhamnetin (1) and tamarixetin (2) have higher log P values (1.856) than quercetin (3) (1.630) and lower polar surface areas (PSA) than 3 (116.45 and 127.45, respectively). PSA is defined as the surface sum of all polar atoms.20 Although 1 and 2 had similar hydrophobicity and PSA values, 1 showed greater antituberculosis activity compared to 2 (Table 1). This may be due to strong interactions between the 3′-OCH3 group and the targeted protein, leading to more stability and potency. On the basis of our data and other reported data relating to 3 (MIC > 827.17 μM (250 μg/mL),10 we suggest that the presence of 3′-OCH3 in ring B increases antimycobacterial activity. Future studies should be conducted to investigate the interactions between this and targeted proteins in more detail. Effects of Isorhamnetin (1) on mRNA Expression of Inflammatory Cytokines in IFN-γ-Stimulated MRC-5 Cells. IFN-γ produces pro-inflammatory effects in macrophages by C



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Figure 2. Effects of isorhamnetin (1) on mRNA expression of inflammatory cytokines in IFN-γ-stimulated MRC-5 cells. MRC-5 cells were stimulated without (negative control) or with 20 ng/mL IFN-γ in the presence or absence of 1 for 3 h. Competitive RT-PCR was performed as described previously.40,41 Targets were amplified from cDNA by PCR using the primers listed in the Experimental Section. (A) mRNA expression of IL-1β, IL-6, IL-12, TNF-α, MMP-1, and β-actin in IFN-γ-stimulated MRC-5 cells. (B−F) Densitometry of the RT-PCR results from IFN-γ-stimulated MRC-5 cells. Each bar graph indicates the relative expression, as compared with β-actin mRNA. Relative expression was quantified using ImageJ, and the data represent the mean ± standard deviation. **P < 0.005; ***P < 0.001 for the comparison with cells exposed to IFN-γ only.

The protective role of IFN-γ in TB is well established,36 primarily in the context of antigen-specific T-cell immunity.37 Lung macrophages have also been reported to produce IFN-γ in M. tuberculosis-infected mice.38 The in vitro and in vivo data generated by the present study provide further evidence that 1 produces potent anti-TB and anti-inflammatory effects. Blood Tests in Vivo. To know whether 1 shows toxic effects or not, blood tests in vivo were carried out to determine serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) levels (Figure 6). These analyses indicated that 1 did not have cytotoxicity in vivo. No visible clinical signs of toxicity, such as irritability, twisting, breathing, weight loss, or death, were observed. Cytotoxicity against Mammalian Cells in Vitro. We determined in vitro cytotoxicity effect of 1 in three different mammalian cells. As shown in Supporting Information Figure S2A, at its MIC (158 μM) against M. tuberculosis-P887 strain, cell

survival rates of noncancerous human keratinocyte (HaCaT) cells, mouse embryonic fibroblast NIH3T3 cells, and MRC-5 cells were 90%, 78%, and 54%, respectively, while at its MIC (316 μM) against MDR and XDR strains, cell survival rates were 71%, 63%, and 47%, respectively. Furthermore, even at 400 μM, 1 showed 66% and 58% cell survival of HaCaT cells and NIH3T3 cells, respectively. These data indicated that 1 showed an applicable level of toxicity as a candidate anti-TB and antiinflammatory agent. On the other hand, rifampicin showed 55%, 62%, and 53% survival rates of HaCaT cells, NIH3T3 cells, and MRC-5 cells, respectively, at 16 μM concentration (Supporting Information, Figure S2B). Spectrofluorophotometric Determination of Isorhamnetin Binding Affinity for ERK1 and p38 MAPK. We examined the interactions of 1 with ERK1 and p38 MAPK using fluorescence quenching in order to determine the binding constant, K. Figure 7A and B show fluorescence titration curves D



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Figure 3. MRC-5 cells were pretreated with isorhamnetin (1) for 1 h followed by IFN-γ stimulation for another 3 h. (A) Cell lysates were extracted, and protein levels of MAPKs in IFN-γ-stimulated MRC-5 cells were determined. β-Actin was used as a loading control. (B−D) The relative intensities of the phosphorylated JNK, p38 MAPK, and ERK bands were normalized to the protein levels of JNK, p38 MAPK, and ERK, respectively. Relative protein expression was quantified using ImageJ. Data represent the mean ± standard deviation. ***P < 0.001 for the comparison with cells exposed to IFN-γ only.

and Phe169 residues (Figure 8B). These findings indicated that the presence of a methoxy group at the 3′ position and hydroxy groups at the 4′ and 5 positions are important for the interaction with MAPK. The results of this molecular docking study should prove helpful in understanding the mechanism by which 1 inhibits ERK1 and p38 MAPKs.

for ERK1 and p38 MAPK with 1, respectively. These indicated that the tryptophan fluorescence of ERK1 and p38 MAPK were significantly quenched in the presence of 1. Isorhamnetin (1) bound strongly to ERK1 and p38 MAPK, with K values of 4.46 × 106 M−1 and 7.6 × 106 M−1, respectively. On the other hand, quercetin (3) bound weakly to ERK1 and p38 MAPK, with a K of 5.62212 × 105 M−1 and 9.86734 × 105 M−1, respectively, while tamaraxetin (2) has weaker binding activity to ERK1 and p38 MAPK, with a K of 6.31 × 104 M−1 and 4.64 × 104 M−1, respectively, as compared to isorhamnetin (1). Docking Simulations of Isorhamnetin (1) with ERK1 and p38 MAPKs. We carried out docking studies to investigate the interaction between 1 and the ATP-binding active sites of ERK1 and p38 MAPK. We found that 1 interacted with conserved amino acid residues, thus leading to more stability and potency. We first investigated the importance of hydrogenbonding interactions between 1 and ERK1 or p38 MAPK. In ERK1, the Lys71, Glu88, and Asp184 side chains formed a network of hydrogen bonds with the 4′-hydroxy groups of the B-ring of 1, while the 5-hydroxy group of the A-ring of 1 formed hydrogen bonds with the backbone amide of Met125 (Figure 8A). Similarly, in p38 MAPK, the Lys53, Glu88, and Asp168 side chains formed hydrogen bonds with the 4′-hydroxy groups of the B-ring of 1, while the 5-hydroxy group of the A-ring formed a hydrogen bond with the backbone amide of Met109 (Figure 8B). The A-ring of 1 formed additional hydrophobic interactions with Ile48 and Val56, and the 3′-methoxy group of the B-ring interacted with Leu173 and Phe185 in ERK1 (Figure 8A). On the other hand, Val30, Val38, and Ala51 in p38 MAPK formed a hydrophobic interaction with the A-ring of 1, and the 3′-methoxy group of the B-ring interacted with Leu167

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CONCLUSION Isorhamnetin (1) is a naturally occurring dietary flavonoid antioxidant that is present in vegetables and fruits. The present study is the first investigation of the antimycobacterial activities of 1 against the H37Rv M. tuberculosis strain and against clinical MDR and XDR isolates. A structure−activity study of isorhamnetin (1), tamarixetin (2), and quercetin (3) implied that the 3′-OCH3 group of the B-ring of 1 may play a key role in its antimycobacterial activity. Hydrophobicity is also a determinant of antimycobacterial activity because it influences membrane permeability. Although the observed antimycobacterial activities of 1 were moderate, their coadministration with standard antiTB drugs should be evaluated. Secretion of a variety of cytokines and chemokines is an essential element of the early immune response to M. tuberculosis infection. In our study, exposure of IFN-γ-stimulated MRC-5 human lung fibroblast cells to 1 reduced their mRNA levels of IL-1β, IL-6, IL-12, TNF-α, and MMP-1. Mycobacteria trigger key signaling pathways involved in cytokine responses and inflammation, including those involving MAPK and NF-kB.39 Exposure of MRC-5 cells to IFN-γ increased the expression and phosphorylation of p38 MAPK and ERK, and these effects were attenuated in the presence of 1. Computational binding studies predicted that the 4′-hydroxy group and 3′-OCH3 group of the B-ring and 5-hydroxy group of E



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Figure 4. Levels of (A) TNF-α and (B) IL-12 in the media of INF-γstimulated MRC-5 cells exposed to the indicated concentrations of isorhamnetin for 24 h. Each vertical bar represents the standard deviation of three independent experiments. **P < 0.005; ***P < 0.001 for the comparison with cells exposed to IFN-γ only. Figure 5. Effects of isorhamnetin (1) on in vivo expression of the proinflammatory cytokines, IL-12, IL-6, IL-1β, and INF-γ in a mouse model of LPS-induced lung inflammation. BALB/c mice were assigned to 6 experimental groups, with 3 mice per group. Animals were preadministered with 1 by ip injection. After 1 h, the mice were injected with LPS and maintained for 12 h prior to sacrifice. Lung homogenates were centrifuged, and supernatants were analyzed by ELISA. *P < 0.05; **P < 0.005 for the comparison with animals treated with LPS only.

the A-ring of 1 played key roles in its binding interactions with ERK1 and p38 MAPK, implying that these interactions may be essential for the potency of 1 as an inhibitor of ERK1 and p38 MAPK, and also 1 showed high binding affinity with p38 MAPK (7.6 × 106 M−1) and ERK1 (4.46 × 106 M−1). In conclusion, we proposed a possible functional consequence on the IFN-γ stimulated human lung fibroblast MRC-5 cells. We also found that the levels of the pro-inflammatory cytokines IL-12, IL-1β, IL-6, and INF-γ were lower in lung homogenates from mice that were administered with 1 prior to LPS injection in vivo. Furthermore, our in vivo and in vitro results did not reveal any toxic effects of 1. These data may provide a basis for the design of novel antimycobacterial drugs using the chemical structure of this compound as a prototype. Collectively, the results presented here provide insight into the application of 1 as a dietary supplement for the prevention of TB.

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EXPERIMENTAL SECTION

General Experimental Procedures. Isorhamnetin (1) and tamarixetin (2) were purchased from the Indofine Chemical Company (Hillsborough, NJ, USA) and Shanghai Xinyong Chemical Group (Shanghai, China), respectively, and purified to 99% purity using highperformance liquid chromatography. They were dissolved in dimethyl sulfoxide to produce a 10 mg/mL stock solution. Animals. Six-week-old female BALB/c mice were purchased from Orient (Daejeon, Korea). The mice were housed under pathogen-free conditions in a temperature- and humidity-controlled environment for 1 week before the experiment was initiated. All the procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Konkuk University, Korea (number KU14046). Anti-TB Activities. Stock solutions (166 μL) of the M. tuberculosis H37Rv strain and clinical isolates with MDR and XDR strains were mixed

Figure 6. Effects of isorhamnetin (1) (0, 0.01, 0.1, and 0.5 mg/kg mice) supplementation on serum levels of aspartate aminotransferase (AST), alanine transaminase (ALT), and blood urea nitrogen (BUN). The data represent the mean ± standard deviation. with 500 μL of 0.5% liquid agarose at 37 °C. Ten microliters of this mixture was loaded into the inlet well to form microfluidic agarose F



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Figure 7. Fluorescence spectra of aqueous solutions of (A) ERK1 (10 μM) and (B) p38 MAPK (10 μM) in the presence of the indicated concentrations of isorhamnetin (1) at pH 7.0. Samples were excited at 290 nm, and emission spectra were noted for light-scattering effects from 290 to 500 nm.

Figure 8. Binding model of isorhamnetin (1) with (A) ERK1 and (B) p38 MAPK, as determined by docking simulations. Red dotted lines represent hydrogen bonds between 1 and ERK1 or p38 MAPK, as appropriate.

channels surrounding the liquid medium loading well. After the gel solidified at room temperature, the bacteria became immobilized. Isorhamnetin (1), tamarixetin (2) (0−632 μM), or the positive control, rifampicin (0−19.4 μM, Sigma-Aldrich, St. Louis, MO, USA), was loaded into the liquid medium loading wells with Middlebrook 7H9 broth containing 10% oleic albumin dextrose catalase. The compounds and culture media diffused toward the microfluidic agarose channels. The 96-well format microfluidic agarose channel chip was incubated at 37 °C, and the same boundary area between the liquid medium loading well and the microfluidic agarose channel was imaged at 1, 3, 5, 7, and 9 days by inverted microscopy with a 40× lens. The images were processed to measure bacterial growth. The dark gray spots of M. tuberculosis in the time-lapse images were converted to white binary digital information by image processing. Agent susceptibility was determined by evaluating the area covered by M. tuberculosis and calculating the MIC90 value. All assays were run in triplicate. Mouse Model of LPS-Induced Lung Inflammation. BALB/c mice are widely known to express different immune responses in normal and pathological states. Therefore, we assigned BALB/c mice to 6 experimental groups, with 3 mice per group. Dosing solutions of LPS were prepared in phosphate-buffered saline (PBS). Animals were preadministered with 1 (0.01, 0.1, or 0.5 mg/kg) by intraperitoneal (ip) injection. After 1 h, the mice were ip injected with 10 mg/kg LPS and maintained for 12 h prior to sacrifice. The serum AST, ALT, and BUN levels were analyzed using total laboratory automation (Hitachi, Japan) and TBA-200FR NEO (Toshiba Medical Systems, Otawara-shi, Japan). Preparation of Lung Homogenates. Postsacrifice, the entire lung lobes were extracted and transferred to 0.5 mL of lysis buffer containing 0.5% NP-40, 1 mM EDTA, 50 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.5 mM PMSF, 10 mM NaF, and protease inhibitor cocktail

(Biobasic, Markham, ON, Canada). The lung was homogenized using a bullet blender homogenizer (Next Advance, Averill Park, NY, USA) and 1.4 mm stainless blend (Next Advance) at 4 °C. Lung homogenates were centrifuged at 13 000 rpm for 15 min at 4 °C. To quantify total lung protein, supernatants were analyzed by the Bradford assay. RT-PCR Analyses. MRC-5 cells were stimulated without (negative control) or with 20 ng/mL IFN-γ in the presence or absence of 1 for 3 h. Competitive RT-PCR was performed as described previously.40,41 Targets were amplified from cDNA by PCR using the following specific primers: hIL-1β, 5′-GAA GTA CCT GAG CTC GCC AGT GAA-3′ (sense) and 5′-AGG TTC TTC TTC AAA GAT GAA GGG-3′ (antisense); hTNF-α, 5′-AGC CGC ATC GCC GTC TCC TA-3′ (sense) and 5′-CAG CGC TGA GTC GGT CAC CC-3′ (antisense); hIL-12, 5′-CCT GCT GGT GGC TGA CGA CAA T-3′ (sense) and 5′-CTT CAG CTG CAA GTT GTT GGG T-3′ (antisense); hMMP-1, 5′-ATT CTA CTG ATA TCG GGG CTT T-3′ (sense) and 5′-ATG TCC TTG GGG TAT CCG TGT AG-3′ (antisense); hIL-6, 5′-GTG TGA AAG CAG CAA AGA G-3′ (sense) and 5′-CTC CAA AAG ACC AGT GAT G-3′ (antisense). The primers for β-actin, used as an internal control, were 5′-ATT GCC GAC AGG ATG CAG A-3′ (sense) and 5′-GAG TAC TTG CGC TCA GGA GGA-3′ (antisense). PCR was carried out by using the following conditions: 94 °C for 5 min, followed by 25 cycles of 94 °C for 1 min, 57 °C for 1 min, and 94 °C for 1 min, and a final extension step of 72 °C for 7 min. Enzyme-Linked Immunosorbent (ELISA) Assay. The levels of cytokines were evaluated in mouse lung tissue homogenates and human lung fibroblast MRC-5 cells using an ELISA kit (eBioscience, San Diego, CA, USA) according to the user manual. Briefly, each immunoplate (SPL, Korea) well was coated with a capture antibody overnight at 4 °C. The immunoplates were then washed with PBS containing 0.05% G



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Then we measured the fluorescence quantum yields of JNK1 and p38 MAPK using tryptophan emission, in the presence of increasing concentrations of compounds. Samples were excited at 290 nm, and emission spectra were recorded for light scattering effects from 290 to 500 nm. Detailed information on this method is mentioned in a previous report.43,45 Docking Studies. The ATP binding sites of ERK1 and p38 MAPK were defined using the X-ray crystallography structures of ERK1 (2ZOQ.pdb) and p38 MAPK (3S3I.pdb). Isorhamnetin (1) was docked to ERK1 and p38 MAPK using CDOCKER, a CHARMm-based molecular dynamics method for ligand docking in Discovery Studio (Accelrys Inc., San Diego, CA, USA), as previously reported.43 Statistical Analyses. At least 3 independent cell samples, tissue samples, or mice were included in each analysis. We performed statistical tests using GraphPad InStat software (version 3.05, GraphPad, San Diego, CA, USA). We considered values to be statistically significant when P < 0.05.

Tween 20, which was also used to wash the plates between each subsequent step. Blocking solution was added to each well and incubated for 1 h at room temperature (RT). Next, the samples of lung tissue homogenates or IFN-γ-stimulated MRC-5 cells (100 μL/well) were added and incubated for 2 h at RT. After washing, detection antibody solution was added and incubated for 1 h at RT. 3,3′,5,5′tetramethylbenzidine (TMB) substrate was added, and the blue color reaction was stopped using 1 N H2SO4. The optical density was analyzed at 450 nm using a Sunrise plate reader (TECAN, Männedorf, Switzerland). All values represent the means ± standard deviations of at least three independent experiments. Western Blotting. Proteins were isolated from IFN-γ-stimulated MRC-5 cells and analyzed as we previously reported for the RAW cells.41 Briefly, MRC-5 cells were grown at 37 °C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY, USA) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin; Invitrogen) in a humidified atmosphere containing 5% CO2. Cultured cells were incubated with isorhamnetin (1) (20 μM) for 1 h, which was followed by stimulation with 20 ng/mL INF-γ for 3 h. After incubation, cells were washed twice with PBS, detached, collected, and then centrifuged at 1000 rpm for 5 min at 4 °C. Resuspension of cell pellets was done in 100 μL of lysis buffer (1% Triton X-100, 0.1% NaN3, 1% deoxycholate), incubated for 30 min on ice, and then centrifuged at 12 000 rpm for 10 min at 4 °C. The concentration of protein in the supernatant (cytoplasmic extract) was determined by Bradford assay (Bio-Rad, Hemel Hempstead, UK). Equal amounts of protein (20 μg) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel and transferred onto a polyvinylidene fluoride microporous membrane (Millipore, Billerica, MA, USA). Blocking of membrane was performed by incubating with 5% bovine serum albumin in TBST (25 mM Tris, 3 mM KCl, 140 mM NaCl, 0.1% Tween 20) for 1 h at RT followed by overnight incubation at 4 °C with antibodies specific for phospho-ERK and ERK (1:2000; Cell Signaling Technology, Beverly, MA, USA), phosphop38 and p38 (1:2000, Cell Signaling Technology), JNK and phospho-JNK (1:1000, Cell Signaling Technology), and β-actin (1:5000, Sigma-Aldrich) and then with horseradish-peroxidase-conjugated secondary antibodies for 2 h at RT. The relative density of each protein band was quantified using ImageJ software (NIH, Bethesda, MD, USA). Cytotoxicity toward Mammalian Cells. Human lung fibroblast MRC-5 cells, mouse embryonic fibroblast NIH3T3 cells, and human keratinocyte HaCaT cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Human lung fibroblast MRC-5 cells, HaCaT cells, and NIH3T3 cells were treated with different concentrations of 1 (0−400 μM) and rifampicin (0−16 μM) for 24 h, followed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays as reported previously.42 HaCaT and NIH3T3 cells were selected to be assessed in parallel with MRC-5 cells to determine selective growth inhibition toward human lung fibroblast cells. The MTT assay has been mostly performed to determine the cytotoxic effect, and this study helped to screen the chemopreventive potential of natural products. It provides preliminary data for further in vitro and in vivo studies. Cell survival was calculated as the ratio of A570 measured in cells treated with 1 and rifampicin to A570 of untreated cells, expressed as a percentage and calculated as the average of triplicate measurements from three independent experiments. Construction, Expression, and Purification of ERK1 and p38 MAPK. We inserted human ERK1 into the pET21b expression vector (Novagen), and these plasmids were propagated in E. coli BL21. This protein was expressed in E. coli as a C-terminally His6-tagged form. Similarly, human p38 MAPK was inserted into the pET21b expression vector and expressed in E. coli as a C-terminally His6-tagged form. ERK1 and p38 MAPK were then purified, as reported previously.43,44 Fluorescence Quenching between p38 MAPK or ERK1 Proteins and Isorhamnetin (1). An RF-5301PC spectrofluorophotometer (Shimadzu, Kyoto, Japan) was used to perform fluorescence quenching experiments. Isorhamnetin (1), tamaraxetin (2), and quercetin (3) were titrated in a ratio of 1:10 (protein:inhibitor) with 10-μM ERK1 or p38 MAPK protein solution in 50 mM sodium phosphate buffer containing 100 mM NaCl at pH 8.0. The samples were kept in a 2 mL cuvette, with excitation and emission path lengths of 10 nm.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01033.

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AUTHOR INFORMATION

Corresponding Author

*Phone (Y. Kim): 822-450-3421. Fax: 822-447-5987. E-mail: [email protected]. Author Contributions ∥

H. N. Jnawali and D. Jeon contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Priority Research Centers Program (2009-0093824) and from the Basic Science Research Program (2013R1A1A2058021) through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology. This paper was written as part of Konkuk University's research support program for its faculty on sabbatical leave in 2014.

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REFERENCES

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