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(+)-Nootkatone and (+)-valencene from rhizomes of Cyperus rotundus increase survival rates in septic mice due to heme oxygenase-1 induction
Journal of Ethnopharmacology 137 (2011) 1311–1317
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(+)-Nootkatone and (+)-valencene from rhizomes of Cyperus rotundus increase survival rates in septic mice due to heme oxygenase-1 induction Konstantin Tsoyi a,1 , Hwa Jin Jang a,1 , Young Soo Lee a , Young Min Kim a , Hye Jung Kim a , Han Geuk Seo a , Jae Heun Lee a , Jong Hwan Kwak b , Dong-Ung Lee c,∗∗ , Ki Churl Chang a,d,∗ a
Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju 660-290, Republic of Korea College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea c Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea d The Research Institute of Natural Science, Gyeongsang National University, Jinju 660-701, Republic of Korea b
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Article history: Received 5 April 2011 Received in revised form 26 July 2011 Accepted 29 July 2011 Available online 6 August 2011 Keywords: Valencene Nootkatone Heme oxygenase-1 iNOS Inflammation Sepsis
a b s t r a c t Ethnopharmacological relevance: The rhizomes of Cyperus rotundus have been used as traditional folk medicine for the treatment of inflammatory diseases. However, the mechanism by which extract of rhizomes of Cyperus rotundus (ECR) elicits anti-inflammation has not been extensively investigated so far. The aim of the present study was to test whether heme oxygenase (HO)-1 induction is involved in the anti-inflammatory action of ECR. Materials and methods: Induction of HO-1 and inhibition of inducible nitric oxide synthase (iNOS)/NO production by ECR and its 12 constituents (3 monoterpenes, 5 sesquiterpenes, and 4 aromatic compounds) were investigated using RAW264.7 cells in vitro. In addition, anti-inflammatory action of ECR and its two active ingredients (nookkatone, valencene) were confirmed in sepsis animal model in vivo. Results: ECR increased HO-1 expression in a concentration-dependent manner, which was correlated with significant inhibition of iNOS/NO production in LPS-activated RAW264.7 cells. Among 12 compounds isolated from ECR, mostly sesquiterpenes induced stronger HO-1 expression than monoterpenes in macrophage cells. Nootkatone and valencene (sesquiterpenes) significantly inhibited iNOS expression and NO production in LPS-simulated RAW264.7 cells. Inhibition of iNOS expression by nootkatone, valencene, and ECR were significantly reduced in siHO-1 RNA transfected cells. Furthermore, all three showed marked inhibition of high mobility group box-1 (HMGB1) in LPS-activated macrophages and increased survival rates in cecal ligation and puncture (CLP)-induced sepsis in mice. Conclusions: Taken together, we concluded that possible anti-inflammatory mechanism of ECR is, at least, due to HO-1 induction, in which sesquiterpenes such as nootkatone and valencene play a crucial role. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes the rate-limiting step in heme degradation, leading to the generation of iron ions, biliverdin, which is then converted into bilirubin by biliverdin reductase and carbon monoxide (CO). HO-1
Abbreviations: CO, carbon monoxide; CLP, cecal ligation and puncture; ECR, methanol extract of Cyperus rotundus; HO-1, heme oxygenase-1; HMGB1, high mobility group box 1; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; NO, nitric oxide; PVDF, polyvinylidene difluoride. ∗ Corresponding author at: Department of Pharmacology School of Medicine, and Institute of Health Sciences, Gyeongsang National University, Jinju 660-751, Republic of Korea. Tel.: +82 55 772 8071; fax: +82 55 772 8079. ∗∗ Corresponding author. Tel.: +82 54 770 2224; fax: +82 55 742 9833. E-mail addresses: [email protected] (D.-U. Lee), [email protected] (K.C. Chang). 1 Both these authors contributed equally to this work. 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.07.062
is highly upregulated in mammalian tissues in response to a wide variety of conditions, including vascular injury, ischemia, oxidative stress, and immune response. In this manner, HO-1 provides cytoprotective, anti-apoptotic, and immunomodulatory effects (Ryter et al., 2006). Our group and others have recently demonstrated that HO-1/CO can play a beneficial role in an experimental model of sepsis by modulation of such proinflammatory stimulators as inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-␣), and high mobility group box1 (HMGB1) (Bani-Hani et al., 2006; Cepinkas et al., 2008; Tsoyi et al., 2009a,b, 2011). Thus, it seems plausible that development and discovery of HO-1-inducible agents from traditional herbs may have great potential for therapeutic intervention in such systemic inflammatory disorder as sepsis. The genus Cyperus includes common weeds found upland and in paddy fields in temperate to tropical regions. In Asian countries, the rhizomes of Cyperus rotundus have been used as traditional folk medicine for the treatment of stomach and bowel disorders, as well as inflammatory diseases (Gupta et al., 1971; Seo et al.,
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2001). Although mono- and sesquiterpenes are the major components of this herb, the mechanism by which methanol extract of rhizomes of Cyperus rotundus (ECR) elicits anti-inflammation has not been extensively investigated so far. Thus, we tested our hypothesis that ECR might induce HO-1 for its anti-inflammatory action. Furthermore, we wanted to identify which components are responsible for the anti-inflammatory action of ECR. To do this, activity-guided fractionation of the ECR was performed, depending on solvent polarity, and 12 constituents were isolated from the active fractions. HO-1 induction of each isolated compound was investigated using RAW264.7 cells. We found that ECR induced HO1 in a concentration-dependent manner, which was correlated with significant inhibition of iNOS/NO production in lipopolysaccharide (LPS)-activated RAW264.7 cells. Interestingly, sesquiterpenes such as nootkatone and valencene significantly inhibited not only NO production but also HMGB1 release, an important late cytokine in sepsis, in LPS-simulated RAW264.7 cells. We report here that nootkatone and valencene have a great potential for the development of therapeutic drugs in the treatment of sepsis since both increased survival rates in cecal ligation and puncture (CLP)induced sepsis in mice. 2. Materials and methods
27.1 (C-6), 37.8 (C-7), 25.9 (C-8), 44.8 (C-9), 150.7 (C-10), 143.0 (C11), 108.3 (C-12), 20.9 (C-13), 18.4 (C-14), 15.7 (C-15); EI-GC/MS m/z 204 [M]+ (35), 161 (base peak). 2.3. Cell culture RAW264.7 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in RPMI-1640 medium supplemented with 25 mM N-(2-hydroxyethyl) piperazine-N-2ethanesulphonic acid, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% heat-inactivated fetal calf serum. 2.4. Cell viability Cell viability was determined colorimetrically using the MTT assay. Cells at the exponential phase were seeded at 1 × 104 cells/well in 24-well plates. After different treatments, 20 l of 5 mg/ml MTT solution was added to each well (0.1 mg/well) and incubated for 4 h; supernatants were aspirated, and formazan crystals in each well were dissolved in 200 l dimethyl sulfoxide for 30 min at 37 ◦ C; optical density at 570 nm was read on a Microplate Reader (Bio-Rad, Hercules, CA).
2.1. Materials
2.5. Cell stimulation
Dulbecco’s modified Eagle’s medium, fetal bovine serum, and antibiotics (penicillin/streptomycin) were purchased from Gibco BRL (Rockville, MD). Anti-iNOS antibody was purchased from Transduction Laboratories (Lexington, KY). Horseradish peroxidase labeled goat anti-rabbit IgG and -actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence (ECL) Western blotting detection reagent was purchased from Amersham (Buckinghamshire, UK). All other chemicals including LPS (Escherichia coli serotype 0128:B12) were purchased from Sigma–Aldrich. (St. Louis, MO).
RAW264.7 cells were plated at a density of 1 × 107 cells/100 mm dish. Cells were rinsed simultaneously with fresh medium and stimulated with LPS (1 g/ml) in the presence or absence of different concentrations of ECR or its components. For detection of iNOS/NO, cells were incubated for 8 h after LPS administration.
2.2. Extraction and isolation of rhizomes of Cyperus rotundus Rhizomes of Cyperus rotundus were purchased from Kyung Dong market place in Seoul, Korea. A voucher specimen was deposited at the Korea Food and Drug Administration (DKH-02561), South Korea. The extraction, fractionation, and isolation methods of test materials used in this experiment were followed as reported previously (Howard et al., 1995; Seo et al., 2011). The purities of isolated compounds were determined by GC–MS (HP 6890 series) to be valencene 90.6%. -Pinene 98.1%, limonene 97.2%, 4-cymene 99.0%, (+)-nootkatone 98.6%, 1,8-cineole 98%, caryophyllene oxide 89.5%, ␣-cyperone 97.4%, -selinene 95.8%, coumarin 99.0%, p-coumaric acid 92.5%, and ellagic acid 93.8%. The analytical data of representative of two essential oils are listed below: (+)-Nootkatone: C15 H22 O; Colorless oil; [˛]20 D + 195.5 (c = 0.01, CHCl3 ); 1 H NMR (500 MHz, CDCl3 ) ı: 0.97 (3H, d, J = 6.4 Hz, H-14), 1.12 (3H, s, H-15), 1.20–2.60 (methylene + methine), 1.74 (3H, s, H-13), 4.73 (2H, br s, H-12), 5.77 (1H, s, H-1); 13 C NMR (125 MHz, CDCl3 ) ı: 124.7 (C-1), 199.6 (C-2), 42.1 (C-3), 40.3 (C-4), 39.3 (C-5), 33.0 (C-6), 40.4 (C-7), 31.6 (C-8), 43.9 (C-9), 170.5 (C-10), 149.1 (C11), 109.2 (C-12), 20.8 (C-13), 14.9 (C-14), 16.8 (C-15); EI-GC/MS m/z: 218 [M]+ (18), 147 (base peak). (+)-Valencene: C15 H24 ; Pale brown oil; [˛]20 D + 79.2◦ (c = 0.5, CHCl3 ); 1 H NMR (200 MHz, CDCl3 ) ı: 0.87 (3H, d, J = 6.0 Hz, H-14), 0.95 (3H, s, H-15), 1.20–2.60 (methylene + methine), 1.71 (3H, s, H-13), 4.68 (2H, s, H-12), 5.33 (1H, br s, H-1); 13 C NMR (50 MHz, CDCl3 ) ı: 120.2 (C-1), 120.9 (C-2), 40.9 (C-3), 33.1 (C-4), 32.7 (C-5),
2.6. Assay for nitrite production Nitric oxide was measured as its stable oxidative metabolite, nitrite, as previously described (Kang et al., 1999). At the end of the incubation, 100 l of the culture medium was mixed with an equal volume of Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid). Light absorbance was measured at 550 nm, and the nitrite concentration was determined using a curve calibrated on sodium nitrite standards. 2.7. Western blot analysis Cells were harvested and lysed with buffer containing 0.5% SDS, 1% NP-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris–Cl (pH 7.5), and protease inhibitors. The protein concentration of each sample was determined using a BCA protein assay kit (Pierce, Rockford, IL). For the detection of iNOS, 20 g of the total protein was electrophoresed on an 8% polyacrylamide gel; 10% polyacrylamide gel was used for the detection of HO-1. Gels were transferred to polyvinylidene difluoride (PVDF) membranes by semidry electrophoretic transfer at 15 V for 60–75 min. PVDF membranes were blocked overnight at 4 ◦ C in 5% bovine serum albumin (BSA). Cells were incubated with primary antibodies diluted 1:500 in Trisbuffered saline/Tween 20 (TBS-T) containing 5% BSA for 2 h, and then incubated with the secondary antibody at room temperature for 1 h. Anti-rabbit IgG was used as the secondary antibody (1:5000 dilution in TBST containing 1% BSA). Signals were detected by ECL (Amersham, Piscataway, NJ). Scanning densitometry was performed using an Image Master® VDS (Pharmacia Biotech Inc., San Francisco, CA).
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2.8. HMGB1 analysis HMGB1 analysis has been described previously (Tsoyi et al., 2009b). Culture medium samples were briefly centrifuged to remove cellular debris. The same volumes of samples were then concentrated 40-fold with Amicon Ultra-4-10000 NMWL (Millipore). Centrifugation conditions were fixed angle (35◦ ) and 7500 g for 20 min at 4 ◦ C. Concentrated samples were then mixed with 2× loading dye and subjected to immunoblotting. 2.9. siRNA technique HO-1 small interference RNA (siRNA) was purchased from Invitrogen (Carlsbad, CA). The sequence of mouse HO-1 siRNA (5 –3 ) is as follows: UUACAUGGCAUAAAUUCCCACUGCC. siRNA was transfected into RAW264.7 cells using transfection reagent SuperFect® from QIAGEN, according to the manufacturer’s protocol. Cells were incubated with 100 nM HO-1 siRNA for 48 h in serum- and antibiotic-free media. Cells were then incubated for 12 h in media containing antibiotics and FBS. Following incubation, cells were treated as described above. 2.10. Animal model of sepsis To induce sepsis, ICR mice were anesthetized with ketamine (30 mg/kg) and xylazine (6 mg/kg). Next, a 2-cm midline incision was performed to allow exposure of the cecum with adjoining intestine. The cecum was tightly ligated with a 3.0-silk suture at its base, below the ileo-cecal valve, and was punctured twice with an 18-gauge needle (top and bottom). The cecum was then gently squeezed to extrude a small amount of feces from the perforation sites and returned to the peritoneal cavity. The laparotomy site was then stitched with 4.0 silk. In sham control animals, the cecum was exposed but not ligated or punctured and then returned to the abdominal cavity. Mice were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1996) and were treated ethically. The protocol was approved in advance by the Animal Research Committee (GLA-100929-m0094) of the Gyeongsang National University. 2.11. Survival rates Mice were subjected to CLP and treated right after surgery with following treatments at +12, +24, +48 h after the onset of sepsis. Survival was monitored daily, up to two weeks.
Fig. 1. Effect of ECR on HO-1 induction and iNOS/NO expression in LPS-stimulated RAW264.7 cells. (A) Cell viability of ECR was evaluated by MTT assay. (B) Cells were treated with ECR (5, 10, 25, and 50 g/ml) for 8 h for the detection of HO-1, and (C) stimulated with LPS (1 g/ml) for 8 h for iNOS/NO detection. Data are presented as mean ± SD of three independent experiments. Significance compared to control, **P < 0.01; significance compared to LPS, † P < 0.05, †† P < 0.01.
2.12. Statistics Data are expressed as the mean ± SD of results obtained from the number of replicate treatments. Differences between data sets were assessed by one-way analysis of variance followed by Newman–Keuls tests. P < 0.05 was accepted as statistically significant. 3. Results 3.1. Effects of ERC on HO-1 induction in macrophages The MTT assay was utilized to select the optimal concentration ranges of ECR in RAW264.7 cells. As shown in Fig. 1A, cytotoxicity of ECR was not shown up to dose 50 g/ml. Next, we asked whether or not ECR possesses HO-1 induction. Fig. 1B shows that ECR concentration-dependently increased HO-1 induction in RAW264.7 cells. To see ECR inhibits iNOS and NO, the cells were then stimulated with LPS in the presence or absence of ECR at
doses of 5, 10, 25, and 50 g/ml. As shown in Fig. 1C, ECR significantly reduced iNOS protein levels and NO production in culture medium in LPS-stimulated macrophages, suggesting that ECR has potent anti-inflammatory activities. 3.2. Sesquiterpenes are active anti-inflammatory components of ECR After confirming that ECR possesses HO-1 induction, we further fractionated ECR according to solvent polarity in order to determine which fractions are responsible for anti-inflammatory action (HO-1 induction) of ECR (Fig. 2). As described in the Section 2, we isolated 12 compounds from hexane and methylene chloride fractions; each component was subjected to test for MTT and HO-1 induction (Fig. 2A–C). Among the tested compounds ellagic acid and nootkatone showed slight but significant cytotoxicity at 100 M whereas -selinene and ␣-cyperone possessed strong cytotoxicity
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Fig. 2. Effect of each fraction of ECR and individual identified components on HO-1 induction in RAW264.7 cells. Cells were incubated with twelve identified components of ECR. Cell viability was evaluated by MTT assay. After incubation cells were subjected to Western blotting for HO-1 detection. Results are representative of three independent experiments.
from 50 M therefore the latter two compounds were removed from the following experiments. (+)-Nootkatone and (+)-valencene from the hexane fraction induced HO-1 expression most significantly (Fig. 2D and E), and followed by ␣-cyperone and -selinene (Fig. 2F). 3.3. Inhibition of LPS-induced iNOS expression by nootkatone and valencene is mediated through HO-1 induction As the hexane fraction strongly induced HO-1 expression (data not shown), we wanted to confirm that this fraction is responsible for inhibition of iNOS expression and NO production when macrophages were activated with LPS. As shown in Fig. 3, LPS significantly increased iNOS expression and NO production in RAW264.7 cells. Although limonene and carophyllene oxide increased HO-1 expression (Fig. 2D), they did not effectively inhibit iNOS and NO production (Fig. 3A); however, nootkatone and valencene demon-
strated a significant inhibitory effect on expression of iNOS and NO production in LPS-stimulated macrophages (Fig. 3B). The dose (100 M) was excluded due to increased cytotoxicity of two components (ellagic acid and nootkatone) (Fig. 2B). Moreover, other sesquiterpenes such as -selinene and ␣-cyperone also inhibited iNOS/NO expression (Fig. 3C). Thus, we selected nootkatone and valencene as representative HO-1 inducers from ECR and investigated further using the following experiments. We used siHO-1 transfected cells to further confirm that induction of HO-1 can be accounted for by the action shown by nootkatone and valencene. Fig. 4A shows the efficiency (about 70%) of siHO-1 transfection by Western blotting. ECR, nootkatone, and valencene strongly inhibited LPS-induced iNOS expression in scramble RNA transfected cells. However, this effect was completely reversed by the application of siHO-1 RNA (Fig. 4B–D). Thus, we concluded that the anti-inflammatory effects of ECR, nootkatone, and valencene are mediated via HO-1 upregulation.
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Fig. 4. Inhibitory effect on iNOS expression of ECR, nootkatone, and valencene is mediated by HO-1 induction in LPS-stimulated RAW264.7 cells. Cells were transfected with scramble siRNA (ssiRNA) or siHO-1. Then iNOS expression was evaluated with (A) ECR, (B) nootkatone (C), and valencene (D). For transfection efficiency, HO1 expression was tested in A. An image analyzer was used for quantification of band intensities, and the ratio of iNOS or HO-1 to -actin is indicated below each lane. Results are representative of three independent experiments.
3.4. ECR, nootkatone, and valencene inhibit HMGB1 release in LPS-activated macrophages and increase survival rate in septic mice Our group (Tsoyi et al., 2009b) and others (Takamiya et al., 2009) have previously described HO-1 as a potent anti-inflammatory molecule in sepsis, a systemic inflammatory disorder. Keep in mind that HO-1 upregulation inhibits release of HMGB1, an important and critical cytokine for sepsis-induced death in LPS-activated macrophages; we were interested in whether or not ECR, nootkatone and valencene can inhibit release of HMGB1 in activated macrophages. As shown in Fig. 5A, release of HMGB1 was highly Fig. 3. Effects of each identified components on iNOS/NO production in LPSactivated RAW264.7 cells. Cells were treated for 1 h with each components then stimulated with LPS (1 g/ml) for 8 h. Following incubation, cells were harvested for iNOS detection; culture medium was analyzed for NO production by Griess reagent. An image analyzer was used for the quantification of band intensities,
and the ratio of iNOS to -actin is indicated below each lane. Data are presented as mean ± SD of three independent experiments. Significance compared to control, *P < 0.05, **P < 0.01; significance compared to LPS, †† P < 0.01.
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Fig. 5. ECR, nootkatone and valencene decrease LPS-stimulated HMGB1 release in macrophages and improve survival in septic animals. After incubation for 24 h, culture media was collected and subjected to HMGB1 (A) analysis. Mice were subjected to CLP and treated right after surgery with following treatments at +12, +24, +48, +72 and +96 h after the onset of sepsis. Survival was monitored daily, up to two weeks (B). The Kaplan–Meier program was utilized to compare the differences in mortality rates between groups. Significance compared to DMSO, *P < 0.05.
expressed in LPS-treated macrophages; however, ECR, nootkatone, and valencene greatly abolished this effect. Importantly, administration of ECR, nootkatone and valencene increased survival rates in CLP-induced sepsis in mice (Fig. 5B). 4. Discussion In this study, we investigated whether anti-inflammatory action of ECR and its components are related with HO-1 induction. We have found that ECR greatly inhibited iNOS/NO and HMGB1 expression in LPS-induced macrophages, and that this effect is mediated by HO-1 induction. Moreover, after screening of ECR components, it has also been determined that mostly sesquiterpenes, such as nootkatone, valencene, ␣-cyperone, and -selinene, did induce HO-1, suggesting that these components are, at least, responsible for anti-inflammatory properties of ECR. Although the antioxidant effect of ECR has been proposed to reduce NO production in RAW264.7 cells (Seo et al., 2001), neither the mechanism of action of ECR nor individual component of ECR for anti-inflammatory action has not been investigated. We found for the first time that ECR induces HO-1 expression in RAW264.7 cells, which may be accounted for antioxidant action suggested by Seo et al. (2001). Thus, it can be highly speculated that HO-1 induction by ECR could be, at least, rationalized for long-time use of this herb in oriental countries for the treatment of inflammatory disorders. Because HO-1 is well documented for anti-oxidant, antiinflammation, and anti-apoptosis, it can be explained why this herb reduced the production of reactive oxygen species in activatedmacrophages (Seo et al., 2001). The most interesting finding is
that, after screening of each components of ECR, the increased expression of HO-1 was largely carried out by sesquiterpenes, which resulted in significant inhibition of iNOS/NO in LPS-activated macrophage cells. This finding indicates that sesquiterpenes, in particular nootkatone and valencene, are most likely active ingredients for the anti-inflammatory action of ECR. The present study clearly demonstrated that nootkatone and valencene increased HO-1 induction (relative protein levels were more than 4-fold compared to control) and significantly inhibited iNOS/NO/HMGB1 in LPS-activated RAW264.7 cells. Importantly, administration of ECR, nootkatone and valencene significantly increased survival rates in CLP-septic mice. Although limonene (monoterpenes) is the major and abundant essential oil component of Cyperus rotundus, minor components, such as nootkatone and valencene, may be more important for the anti-inflammatory effect of this plant. Indeed, a number of studies of the pharmacological effects of nootkatone have already been published. For example, nootkatone inhibited the activity of acetylcholinesterase and of human cytochrome P450 monooxygenases (Miyazawa et al., 1997; Tassaneeyakul et al., 2000). Recently, it has been documented that stimulating effects of nootkatone on an adenosine-monophosphate-activated protein kinase (AMPK), which is a well known energy-homeostatic and anti-inflammatory protein (Murase et al., 2010). In this way, we added more pharmacological information about (+)-nootkatone, which inhibited HMGB1 release through induction of HO-1 in LPSactivated RAW264.7 cells. In addition, we for the first time, report that (+)-valencene also significantly induced HO-1 in macrophages. To the best of our knowledge, there is no information on the antiinflammatory action of (+)-valencene. Thus, further study should be performed in order to determine the effect of (+)-valencene on different pathological disorders with possible application for therapeutic development. Growing lines of evidence suggest a central role for HMGB1 release from macrophages in the pathogenesis of sepsis (Wang et al., 1999; Yang et al., 2004). Recently, we demonstrated that HO-1 induction by pharmacological inducers improves survival in mice challenged with lethal endotoxemia or CLP model (Tsoyi et al., 2009b). And this beneficial effect was mostly mediated by decreased plasma HMGB1 levels in septic animals. In accordance with this result, another group has also documented that HO1 deficient animals represent higher levels of HMGB1 in blood than the wild type during endotoxemia. Moreover, the treatment of HO-1−/− animals with HMGB1 neutralizing antibodies resulted in significantly improved survival in comparison with untreated mice during LPS challenge (Takamiya et al., 2009). Thus, two independent studies have suggested a critical role for HO-1 in the regulation of HMGB1 in experimental sepsis models. Another interesting point is that several reports have suggested that HMGB1 release in macrophages is mostly dictated by NO production (Jiang and Pisetsky, 2006; Kim et al., 2009). Here, we also demonstrated that ECR, nootkatone, and valencene attenuated HMGB1 release in LPS-treated macrophages which was mediated through HO1 induction and iNOS/NO inhibition. The present finding further supports our recent report that induction of HO-1 increases survival of sepsis in animals and reduced HMGB1 release in vitro and in vivo (Tsoyi et al., 2009b, 2011) and stresses the importance of anti-inflammatory action of HO-1. Thus, the present study (ECR, nootkatone, valencene) provides scientific rationale why this herb has been traditionally used for the treatment of inflammatory disorders (Gupta et al., 1971; Seo et al., 2001) and also gives new information about the phamacological action of nootkatone and valencene, active ingredients of ECR. In addition, we recently reported that ECR and nootkatone significantly inhibited collagen-, thrombin-, and/or arachdonic acid-induced platelet aggregation (Seo et al., 2011), which may also contribute to a beneficial role in increasing survival rate in septic
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conditions in the current study. Although it remains to be determined whether anti-platelet action of ECR and nootkatone also involves HO-1 activation in platelets, anti-platelet therapy has been increasingly recognized as one of the important therapies in critical care medicine field (Knoebl, 2010). Indeed, platelets play a central role at the interface between thrombosis and inflammation (Li et al., 2011). Interestingly enough, sesquiterpenes are a large class of terpenoid compounds and common constituents of plant essential oils. Nootkatone, particularly, is also an important constituent of the flavor of grape fruits and valencene is the main constituent of orange (Citrus sinensis) peel oil. Although our intention was to identify which compounds show anti-inflammatory action from ECR, the result of the present study is more than that. Since nootkatone and valencene are natural flavors and fragrances, these essential oils have been widely used in the food, chemical, cosmetic and pharmaceutical industries. In summary, we demonstrated that HO-1 induction is at least responsible for the anti-inflammatory effect of ECR. In addition, we identified that nootkatone and valencene are strong inducers of HO-1. Although monoterpenes such as limonene induced HO-1, this induction was far behind compared to nootkatone or valencene. We concluded that the molecular mechanism for anti-inflammatory action of ECR is closely associated with HO-1 induction. Furthermore, nootkatone and valencene, its minor components but strong inducers of HO-1, may be developed as potential drugs for the treatment of systemic inflammatory disorders such as sepsis. Conflict of interest Potential conflicts do not exist. Acknowledgments This work was supported by the National Center for Standardization of Herbal Medicines funded by Korea Food and Drug Administration (2010). References Bani-Hani, M.G., Greenstein, D., Mann, B.E., Green, C.J., Motterlini, R., 2006. Carbon monoxide-releasing molecule (CORM-3) attenuates lipopolysaccharideand interferon-gamma-induced inflammation in microglia. Pharmacological Reports 58, 132–144. Cepinkas, G., Katada, K., Bihari, A., Potter, R.F., 2008. Carbon monoxide liberated from carbon monoxide-releasing molecule CORM-2 attenuates inflammation in the liver of septic mice. American Journal of Physiology, Gastrointestinal Liver and Physiology 294, G184–G191. Gupta, M.B., Palit, T.K., Singh, N., Bhargava, K.P., 1971. Pharmacological studies to isolate the active constituents from Cyperus rotundus possessing antiinflammatory, anti-pyretic and analgesic activities. Indian Journal of Medical Research 59, 76–82.
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Howard, J., Williams, H.J., Sattler, I., Moyna, G., Scott, A.I., Bell, A.A., Vinson, S.B., 1995. Diversity in cyclic sesquiterpene production by Gossypium hirsutum. Phytochemistry 40, 1633–1636. Jiang, W., Pisetsky, D.S., 2006. The role of IFN-alpha and nitric oxide in the release of HMGB1 by RAW264.7 cells stimulated with polyinosinic-polycytidylic acid or lipopolysaccharide. Journal of Immunology 177, 3337–3343. Kang, Y.J., Koo, E.B., Lee, Y.S., Yun-Choi, H.S., Chang, K.C., 1999. Prevention of the expression of inducible nitric oxide synthase by a novel positive inotropic agent YS 49, in rat vascular smooth muscle and RAW 264.7 macrophages. British Journal of Pharmacology 128, 357–364. Kim, J.H., Kim, S.J., Lee, I.S., Lee, M.S., Uematsu, S., Akira, S., Oh, K.I., 2009. Bacterial endotoxin induces the release of high mobility group box 1 via IFN-beta signaling pathway. Journal of Immunology 182, 2458–2466. Knoebl, P., 2010. Blood coagulation disorders in septic patients. Wiener Medizinische Wochenschrift 160, 129–138. Li, Z., Yang, F., Dunn, S., Gross, A.K., Smyth, S.S., 2011. Platelets as immune mediators: their role in host defense responses and sepsis. Thrombosis Research 127, 184–188. Murase, T., Misawa, K., Haramizu, S., Minegishi, Y., Hase, T., 2010. Nootkatone, a characteristic constituent of grapefruit, stimulates energy metabolism and prevents diet-induced obesity by activating AMPK. American Journal of Physiology, Endocrinology and Metabolism 299, 266–275. Miyazawa, M., Watanabe, H., Kameoka, H., 1997. Inhibition of acetylcholinesterase activity by monoterpenoids with a p-menthane skeleton. Journal of Agricultural and Food Chemistry 45, 677–679. Ryter, S.W., Alam, J., Choi, A.M., 2006. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiological Reviews 86, 583–650. Seo, E.J., Lee, D.U., Kwak, J.H., Lee, S.M., Kim, Y.S., Jung, Y.S., 2011. Antiplatelet effects of Cyperus rotundus and its component (+)-nootkatone. Journal of Ethnopharmacology 135, 48–54. Seo, W.G., Pae, H.O., Oh, G.S., Chai, K.Y., Kwon, T.O., Yun, Y.G., Kim, N.Y., Chung, H.T., 2001. Inhibitory effect of methanol extract of Cyperus rotundus rhizomes on nitric oxide and superoxide productions by murine macrophage cell line, RAW 264.7 cells. Journal of Ethnopharmacology 76, 59–64. Takamiya, R., Hung, C.C., Hall, S.R., Fukunaga, K., Nagaishi, T., Maeno, T., Owen, C., Macias, A.A., Fredenburgh, L.E., Ishizaka, A., Blumberg, R.S., Baron, R.M., Perrella, M.A., 2009. High mobility group box 1 contributes to lethality of endotoxemia in heme oxygenase-1 deficient mice. American Journal of Respiratory Cell Molecular Biology 41, 129–135. Tassaneeyakul, W., Guo, L.-Q., Fukuda, K., Ohta, T., Yamazoe, Y., 2000. Inhibition selectivity of grapefruit juice components on human cytochromes p450. Archives of Biochemistry and Biophysics 378, 356–363. Tsoyi, K., Jang, H.J., Kim, J.W., Chang, H.K., Kim, H.J., Pae, H.O., Seo, H.G., Lee, J.H., Lee, Y.S., Chung, H.T., Chang, K.C., 2011. Stimulation of alpha7 nicotinic acetylcholine receptor by nicotine attenuates inflammatory response in macrophages and improves survival in experimental model of sepsis through heme oxygenase-1 induction. Antioxidant & Redox Signalling 14, 2057–2070. Tsoyi, K., Ha, Y.M., Kim, Y.M., Lee, Y.S., Kim, H.J., Kim, H.J., Seo, H.G., Lee, J.H., Chang, K.C., 2009a. Activation of PPAR-gamma by carbon monoxide from CORM-2 leads to the inhibition of iNOS but not COX-2 expression in LPS-stimulated macrophages. Inflammation 32, 364–371. Tsoyi, K., Lee, T.Y., Lee, Y.S., Kim, H.J., Seo, H.G., Lee, J.H., Chang, K.C., 2009b. HO1 induction and CO-releasing molecule inhibit LPS-induced HMGB1 release in vitro and improve survival of mice in LPS- and CLP-induced sepsis model in vivo. Molecular Pharmacology 76, 173–182. Wang, H., Bloom, O., Zhang, M., Vishnubhakat, J.M., Ombrellino, M., Che, J., Frazer, A., Yang, H., Ivanova, S., Borovikova, L., Manogue, K.R., Faist, E., Abraham, E., Andersson, U., Molina, P.E., Abumrad, N.N., Sama, A., Tracey, K.J., 1999. HMG-1 as a late mediator of endotoxin lethality in mice. Science 285, 248–251. Yang, H., Ochani, M., Li, J., Qiang, X., Tanovic, M., Harris, H.E., Susarla, S.M., Ulloa, L., Wang, H., Roth, J., Warren, H.S., Fink, M.P., Fenton, M.J., Andersson, U., Tracey, K.J., 2004. Reversing established sepsis with antagonists of endogenous highmobility group box 1. Proceedings National Academy Sciences United States of America 101, 296–301.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
(+)-Nootkatone and (+)-valencene from rhizomes of Cyperus rotundus increase survival rates in septic mice due to heme oxygenase-1 induction Konstantin Tsoyi a,1 , Hwa Jin Jang a,1 , Young Soo Lee a , Young Min Kim a , Hye Jung Kim a , Han Geuk Seo a , Jae Heun Lee a , Jong Hwan Kwak b , Dong-Ung Lee c,∗∗ , Ki Churl Chang a,d,∗ a
Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju 660-290, Republic of Korea College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea c Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea d The Research Institute of Natural Science, Gyeongsang National University, Jinju 660-701, Republic of Korea b
a r t i c l e
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Article history: Received 5 April 2011 Received in revised form 26 July 2011 Accepted 29 July 2011 Available online 6 August 2011 Keywords: Valencene Nootkatone Heme oxygenase-1 iNOS Inflammation Sepsis
a b s t r a c t Ethnopharmacological relevance: The rhizomes of Cyperus rotundus have been used as traditional folk medicine for the treatment of inflammatory diseases. However, the mechanism by which extract of rhizomes of Cyperus rotundus (ECR) elicits anti-inflammation has not been extensively investigated so far. The aim of the present study was to test whether heme oxygenase (HO)-1 induction is involved in the anti-inflammatory action of ECR. Materials and methods: Induction of HO-1 and inhibition of inducible nitric oxide synthase (iNOS)/NO production by ECR and its 12 constituents (3 monoterpenes, 5 sesquiterpenes, and 4 aromatic compounds) were investigated using RAW264.7 cells in vitro. In addition, anti-inflammatory action of ECR and its two active ingredients (nookkatone, valencene) were confirmed in sepsis animal model in vivo. Results: ECR increased HO-1 expression in a concentration-dependent manner, which was correlated with significant inhibition of iNOS/NO production in LPS-activated RAW264.7 cells. Among 12 compounds isolated from ECR, mostly sesquiterpenes induced stronger HO-1 expression than monoterpenes in macrophage cells. Nootkatone and valencene (sesquiterpenes) significantly inhibited iNOS expression and NO production in LPS-simulated RAW264.7 cells. Inhibition of iNOS expression by nootkatone, valencene, and ECR were significantly reduced in siHO-1 RNA transfected cells. Furthermore, all three showed marked inhibition of high mobility group box-1 (HMGB1) in LPS-activated macrophages and increased survival rates in cecal ligation and puncture (CLP)-induced sepsis in mice. Conclusions: Taken together, we concluded that possible anti-inflammatory mechanism of ECR is, at least, due to HO-1 induction, in which sesquiterpenes such as nootkatone and valencene play a crucial role. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes the rate-limiting step in heme degradation, leading to the generation of iron ions, biliverdin, which is then converted into bilirubin by biliverdin reductase and carbon monoxide (CO). HO-1
Abbreviations: CO, carbon monoxide; CLP, cecal ligation and puncture; ECR, methanol extract of Cyperus rotundus; HO-1, heme oxygenase-1; HMGB1, high mobility group box 1; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; NO, nitric oxide; PVDF, polyvinylidene difluoride. ∗ Corresponding author at: Department of Pharmacology School of Medicine, and Institute of Health Sciences, Gyeongsang National University, Jinju 660-751, Republic of Korea. Tel.: +82 55 772 8071; fax: +82 55 772 8079. ∗∗ Corresponding author. Tel.: +82 54 770 2224; fax: +82 55 742 9833. E-mail addresses: [email protected] (D.-U. Lee), [email protected] (K.C. Chang). 1 Both these authors contributed equally to this work. 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.07.062
is highly upregulated in mammalian tissues in response to a wide variety of conditions, including vascular injury, ischemia, oxidative stress, and immune response. In this manner, HO-1 provides cytoprotective, anti-apoptotic, and immunomodulatory effects (Ryter et al., 2006). Our group and others have recently demonstrated that HO-1/CO can play a beneficial role in an experimental model of sepsis by modulation of such proinflammatory stimulators as inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-␣), and high mobility group box1 (HMGB1) (Bani-Hani et al., 2006; Cepinkas et al., 2008; Tsoyi et al., 2009a,b, 2011). Thus, it seems plausible that development and discovery of HO-1-inducible agents from traditional herbs may have great potential for therapeutic intervention in such systemic inflammatory disorder as sepsis. The genus Cyperus includes common weeds found upland and in paddy fields in temperate to tropical regions. In Asian countries, the rhizomes of Cyperus rotundus have been used as traditional folk medicine for the treatment of stomach and bowel disorders, as well as inflammatory diseases (Gupta et al., 1971; Seo et al.,
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2001). Although mono- and sesquiterpenes are the major components of this herb, the mechanism by which methanol extract of rhizomes of Cyperus rotundus (ECR) elicits anti-inflammation has not been extensively investigated so far. Thus, we tested our hypothesis that ECR might induce HO-1 for its anti-inflammatory action. Furthermore, we wanted to identify which components are responsible for the anti-inflammatory action of ECR. To do this, activity-guided fractionation of the ECR was performed, depending on solvent polarity, and 12 constituents were isolated from the active fractions. HO-1 induction of each isolated compound was investigated using RAW264.7 cells. We found that ECR induced HO1 in a concentration-dependent manner, which was correlated with significant inhibition of iNOS/NO production in lipopolysaccharide (LPS)-activated RAW264.7 cells. Interestingly, sesquiterpenes such as nootkatone and valencene significantly inhibited not only NO production but also HMGB1 release, an important late cytokine in sepsis, in LPS-simulated RAW264.7 cells. We report here that nootkatone and valencene have a great potential for the development of therapeutic drugs in the treatment of sepsis since both increased survival rates in cecal ligation and puncture (CLP)induced sepsis in mice. 2. Materials and methods
27.1 (C-6), 37.8 (C-7), 25.9 (C-8), 44.8 (C-9), 150.7 (C-10), 143.0 (C11), 108.3 (C-12), 20.9 (C-13), 18.4 (C-14), 15.7 (C-15); EI-GC/MS m/z 204 [M]+ (35), 161 (base peak). 2.3. Cell culture RAW264.7 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in RPMI-1640 medium supplemented with 25 mM N-(2-hydroxyethyl) piperazine-N-2ethanesulphonic acid, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% heat-inactivated fetal calf serum. 2.4. Cell viability Cell viability was determined colorimetrically using the MTT assay. Cells at the exponential phase were seeded at 1 × 104 cells/well in 24-well plates. After different treatments, 20 l of 5 mg/ml MTT solution was added to each well (0.1 mg/well) and incubated for 4 h; supernatants were aspirated, and formazan crystals in each well were dissolved in 200 l dimethyl sulfoxide for 30 min at 37 ◦ C; optical density at 570 nm was read on a Microplate Reader (Bio-Rad, Hercules, CA).
2.1. Materials
2.5. Cell stimulation
Dulbecco’s modified Eagle’s medium, fetal bovine serum, and antibiotics (penicillin/streptomycin) were purchased from Gibco BRL (Rockville, MD). Anti-iNOS antibody was purchased from Transduction Laboratories (Lexington, KY). Horseradish peroxidase labeled goat anti-rabbit IgG and -actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence (ECL) Western blotting detection reagent was purchased from Amersham (Buckinghamshire, UK). All other chemicals including LPS (Escherichia coli serotype 0128:B12) were purchased from Sigma–Aldrich. (St. Louis, MO).
RAW264.7 cells were plated at a density of 1 × 107 cells/100 mm dish. Cells were rinsed simultaneously with fresh medium and stimulated with LPS (1 g/ml) in the presence or absence of different concentrations of ECR or its components. For detection of iNOS/NO, cells were incubated for 8 h after LPS administration.
2.2. Extraction and isolation of rhizomes of Cyperus rotundus Rhizomes of Cyperus rotundus were purchased from Kyung Dong market place in Seoul, Korea. A voucher specimen was deposited at the Korea Food and Drug Administration (DKH-02561), South Korea. The extraction, fractionation, and isolation methods of test materials used in this experiment were followed as reported previously (Howard et al., 1995; Seo et al., 2011). The purities of isolated compounds were determined by GC–MS (HP 6890 series) to be valencene 90.6%. -Pinene 98.1%, limonene 97.2%, 4-cymene 99.0%, (+)-nootkatone 98.6%, 1,8-cineole 98%, caryophyllene oxide 89.5%, ␣-cyperone 97.4%, -selinene 95.8%, coumarin 99.0%, p-coumaric acid 92.5%, and ellagic acid 93.8%. The analytical data of representative of two essential oils are listed below: (+)-Nootkatone: C15 H22 O; Colorless oil; [˛]20 D + 195.5 (c = 0.01, CHCl3 ); 1 H NMR (500 MHz, CDCl3 ) ı: 0.97 (3H, d, J = 6.4 Hz, H-14), 1.12 (3H, s, H-15), 1.20–2.60 (methylene + methine), 1.74 (3H, s, H-13), 4.73 (2H, br s, H-12), 5.77 (1H, s, H-1); 13 C NMR (125 MHz, CDCl3 ) ı: 124.7 (C-1), 199.6 (C-2), 42.1 (C-3), 40.3 (C-4), 39.3 (C-5), 33.0 (C-6), 40.4 (C-7), 31.6 (C-8), 43.9 (C-9), 170.5 (C-10), 149.1 (C11), 109.2 (C-12), 20.8 (C-13), 14.9 (C-14), 16.8 (C-15); EI-GC/MS m/z: 218 [M]+ (18), 147 (base peak). (+)-Valencene: C15 H24 ; Pale brown oil; [˛]20 D + 79.2◦ (c = 0.5, CHCl3 ); 1 H NMR (200 MHz, CDCl3 ) ı: 0.87 (3H, d, J = 6.0 Hz, H-14), 0.95 (3H, s, H-15), 1.20–2.60 (methylene + methine), 1.71 (3H, s, H-13), 4.68 (2H, s, H-12), 5.33 (1H, br s, H-1); 13 C NMR (50 MHz, CDCl3 ) ı: 120.2 (C-1), 120.9 (C-2), 40.9 (C-3), 33.1 (C-4), 32.7 (C-5),
2.6. Assay for nitrite production Nitric oxide was measured as its stable oxidative metabolite, nitrite, as previously described (Kang et al., 1999). At the end of the incubation, 100 l of the culture medium was mixed with an equal volume of Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid). Light absorbance was measured at 550 nm, and the nitrite concentration was determined using a curve calibrated on sodium nitrite standards. 2.7. Western blot analysis Cells were harvested and lysed with buffer containing 0.5% SDS, 1% NP-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris–Cl (pH 7.5), and protease inhibitors. The protein concentration of each sample was determined using a BCA protein assay kit (Pierce, Rockford, IL). For the detection of iNOS, 20 g of the total protein was electrophoresed on an 8% polyacrylamide gel; 10% polyacrylamide gel was used for the detection of HO-1. Gels were transferred to polyvinylidene difluoride (PVDF) membranes by semidry electrophoretic transfer at 15 V for 60–75 min. PVDF membranes were blocked overnight at 4 ◦ C in 5% bovine serum albumin (BSA). Cells were incubated with primary antibodies diluted 1:500 in Trisbuffered saline/Tween 20 (TBS-T) containing 5% BSA for 2 h, and then incubated with the secondary antibody at room temperature for 1 h. Anti-rabbit IgG was used as the secondary antibody (1:5000 dilution in TBST containing 1% BSA). Signals were detected by ECL (Amersham, Piscataway, NJ). Scanning densitometry was performed using an Image Master® VDS (Pharmacia Biotech Inc., San Francisco, CA).
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2.8. HMGB1 analysis HMGB1 analysis has been described previously (Tsoyi et al., 2009b). Culture medium samples were briefly centrifuged to remove cellular debris. The same volumes of samples were then concentrated 40-fold with Amicon Ultra-4-10000 NMWL (Millipore). Centrifugation conditions were fixed angle (35◦ ) and 7500 g for 20 min at 4 ◦ C. Concentrated samples were then mixed with 2× loading dye and subjected to immunoblotting. 2.9. siRNA technique HO-1 small interference RNA (siRNA) was purchased from Invitrogen (Carlsbad, CA). The sequence of mouse HO-1 siRNA (5 –3 ) is as follows: UUACAUGGCAUAAAUUCCCACUGCC. siRNA was transfected into RAW264.7 cells using transfection reagent SuperFect® from QIAGEN, according to the manufacturer’s protocol. Cells were incubated with 100 nM HO-1 siRNA for 48 h in serum- and antibiotic-free media. Cells were then incubated for 12 h in media containing antibiotics and FBS. Following incubation, cells were treated as described above. 2.10. Animal model of sepsis To induce sepsis, ICR mice were anesthetized with ketamine (30 mg/kg) and xylazine (6 mg/kg). Next, a 2-cm midline incision was performed to allow exposure of the cecum with adjoining intestine. The cecum was tightly ligated with a 3.0-silk suture at its base, below the ileo-cecal valve, and was punctured twice with an 18-gauge needle (top and bottom). The cecum was then gently squeezed to extrude a small amount of feces from the perforation sites and returned to the peritoneal cavity. The laparotomy site was then stitched with 4.0 silk. In sham control animals, the cecum was exposed but not ligated or punctured and then returned to the abdominal cavity. Mice were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1996) and were treated ethically. The protocol was approved in advance by the Animal Research Committee (GLA-100929-m0094) of the Gyeongsang National University. 2.11. Survival rates Mice were subjected to CLP and treated right after surgery with following treatments at +12, +24, +48 h after the onset of sepsis. Survival was monitored daily, up to two weeks.
Fig. 1. Effect of ECR on HO-1 induction and iNOS/NO expression in LPS-stimulated RAW264.7 cells. (A) Cell viability of ECR was evaluated by MTT assay. (B) Cells were treated with ECR (5, 10, 25, and 50 g/ml) for 8 h for the detection of HO-1, and (C) stimulated with LPS (1 g/ml) for 8 h for iNOS/NO detection. Data are presented as mean ± SD of three independent experiments. Significance compared to control, **P < 0.01; significance compared to LPS, † P < 0.05, †† P < 0.01.
2.12. Statistics Data are expressed as the mean ± SD of results obtained from the number of replicate treatments. Differences between data sets were assessed by one-way analysis of variance followed by Newman–Keuls tests. P < 0.05 was accepted as statistically significant. 3. Results 3.1. Effects of ERC on HO-1 induction in macrophages The MTT assay was utilized to select the optimal concentration ranges of ECR in RAW264.7 cells. As shown in Fig. 1A, cytotoxicity of ECR was not shown up to dose 50 g/ml. Next, we asked whether or not ECR possesses HO-1 induction. Fig. 1B shows that ECR concentration-dependently increased HO-1 induction in RAW264.7 cells. To see ECR inhibits iNOS and NO, the cells were then stimulated with LPS in the presence or absence of ECR at
doses of 5, 10, 25, and 50 g/ml. As shown in Fig. 1C, ECR significantly reduced iNOS protein levels and NO production in culture medium in LPS-stimulated macrophages, suggesting that ECR has potent anti-inflammatory activities. 3.2. Sesquiterpenes are active anti-inflammatory components of ECR After confirming that ECR possesses HO-1 induction, we further fractionated ECR according to solvent polarity in order to determine which fractions are responsible for anti-inflammatory action (HO-1 induction) of ECR (Fig. 2). As described in the Section 2, we isolated 12 compounds from hexane and methylene chloride fractions; each component was subjected to test for MTT and HO-1 induction (Fig. 2A–C). Among the tested compounds ellagic acid and nootkatone showed slight but significant cytotoxicity at 100 M whereas -selinene and ␣-cyperone possessed strong cytotoxicity
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Fig. 2. Effect of each fraction of ECR and individual identified components on HO-1 induction in RAW264.7 cells. Cells were incubated with twelve identified components of ECR. Cell viability was evaluated by MTT assay. After incubation cells were subjected to Western blotting for HO-1 detection. Results are representative of three independent experiments.
from 50 M therefore the latter two compounds were removed from the following experiments. (+)-Nootkatone and (+)-valencene from the hexane fraction induced HO-1 expression most significantly (Fig. 2D and E), and followed by ␣-cyperone and -selinene (Fig. 2F). 3.3. Inhibition of LPS-induced iNOS expression by nootkatone and valencene is mediated through HO-1 induction As the hexane fraction strongly induced HO-1 expression (data not shown), we wanted to confirm that this fraction is responsible for inhibition of iNOS expression and NO production when macrophages were activated with LPS. As shown in Fig. 3, LPS significantly increased iNOS expression and NO production in RAW264.7 cells. Although limonene and carophyllene oxide increased HO-1 expression (Fig. 2D), they did not effectively inhibit iNOS and NO production (Fig. 3A); however, nootkatone and valencene demon-
strated a significant inhibitory effect on expression of iNOS and NO production in LPS-stimulated macrophages (Fig. 3B). The dose (100 M) was excluded due to increased cytotoxicity of two components (ellagic acid and nootkatone) (Fig. 2B). Moreover, other sesquiterpenes such as -selinene and ␣-cyperone also inhibited iNOS/NO expression (Fig. 3C). Thus, we selected nootkatone and valencene as representative HO-1 inducers from ECR and investigated further using the following experiments. We used siHO-1 transfected cells to further confirm that induction of HO-1 can be accounted for by the action shown by nootkatone and valencene. Fig. 4A shows the efficiency (about 70%) of siHO-1 transfection by Western blotting. ECR, nootkatone, and valencene strongly inhibited LPS-induced iNOS expression in scramble RNA transfected cells. However, this effect was completely reversed by the application of siHO-1 RNA (Fig. 4B–D). Thus, we concluded that the anti-inflammatory effects of ECR, nootkatone, and valencene are mediated via HO-1 upregulation.
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Fig. 4. Inhibitory effect on iNOS expression of ECR, nootkatone, and valencene is mediated by HO-1 induction in LPS-stimulated RAW264.7 cells. Cells were transfected with scramble siRNA (ssiRNA) or siHO-1. Then iNOS expression was evaluated with (A) ECR, (B) nootkatone (C), and valencene (D). For transfection efficiency, HO1 expression was tested in A. An image analyzer was used for quantification of band intensities, and the ratio of iNOS or HO-1 to -actin is indicated below each lane. Results are representative of three independent experiments.
3.4. ECR, nootkatone, and valencene inhibit HMGB1 release in LPS-activated macrophages and increase survival rate in septic mice Our group (Tsoyi et al., 2009b) and others (Takamiya et al., 2009) have previously described HO-1 as a potent anti-inflammatory molecule in sepsis, a systemic inflammatory disorder. Keep in mind that HO-1 upregulation inhibits release of HMGB1, an important and critical cytokine for sepsis-induced death in LPS-activated macrophages; we were interested in whether or not ECR, nootkatone and valencene can inhibit release of HMGB1 in activated macrophages. As shown in Fig. 5A, release of HMGB1 was highly Fig. 3. Effects of each identified components on iNOS/NO production in LPSactivated RAW264.7 cells. Cells were treated for 1 h with each components then stimulated with LPS (1 g/ml) for 8 h. Following incubation, cells were harvested for iNOS detection; culture medium was analyzed for NO production by Griess reagent. An image analyzer was used for the quantification of band intensities,
and the ratio of iNOS to -actin is indicated below each lane. Data are presented as mean ± SD of three independent experiments. Significance compared to control, *P < 0.05, **P < 0.01; significance compared to LPS, †† P < 0.01.
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Fig. 5. ECR, nootkatone and valencene decrease LPS-stimulated HMGB1 release in macrophages and improve survival in septic animals. After incubation for 24 h, culture media was collected and subjected to HMGB1 (A) analysis. Mice were subjected to CLP and treated right after surgery with following treatments at +12, +24, +48, +72 and +96 h after the onset of sepsis. Survival was monitored daily, up to two weeks (B). The Kaplan–Meier program was utilized to compare the differences in mortality rates between groups. Significance compared to DMSO, *P < 0.05.
expressed in LPS-treated macrophages; however, ECR, nootkatone, and valencene greatly abolished this effect. Importantly, administration of ECR, nootkatone and valencene increased survival rates in CLP-induced sepsis in mice (Fig. 5B). 4. Discussion In this study, we investigated whether anti-inflammatory action of ECR and its components are related with HO-1 induction. We have found that ECR greatly inhibited iNOS/NO and HMGB1 expression in LPS-induced macrophages, and that this effect is mediated by HO-1 induction. Moreover, after screening of ECR components, it has also been determined that mostly sesquiterpenes, such as nootkatone, valencene, ␣-cyperone, and -selinene, did induce HO-1, suggesting that these components are, at least, responsible for anti-inflammatory properties of ECR. Although the antioxidant effect of ECR has been proposed to reduce NO production in RAW264.7 cells (Seo et al., 2001), neither the mechanism of action of ECR nor individual component of ECR for anti-inflammatory action has not been investigated. We found for the first time that ECR induces HO-1 expression in RAW264.7 cells, which may be accounted for antioxidant action suggested by Seo et al. (2001). Thus, it can be highly speculated that HO-1 induction by ECR could be, at least, rationalized for long-time use of this herb in oriental countries for the treatment of inflammatory disorders. Because HO-1 is well documented for anti-oxidant, antiinflammation, and anti-apoptosis, it can be explained why this herb reduced the production of reactive oxygen species in activatedmacrophages (Seo et al., 2001). The most interesting finding is
that, after screening of each components of ECR, the increased expression of HO-1 was largely carried out by sesquiterpenes, which resulted in significant inhibition of iNOS/NO in LPS-activated macrophage cells. This finding indicates that sesquiterpenes, in particular nootkatone and valencene, are most likely active ingredients for the anti-inflammatory action of ECR. The present study clearly demonstrated that nootkatone and valencene increased HO-1 induction (relative protein levels were more than 4-fold compared to control) and significantly inhibited iNOS/NO/HMGB1 in LPS-activated RAW264.7 cells. Importantly, administration of ECR, nootkatone and valencene significantly increased survival rates in CLP-septic mice. Although limonene (monoterpenes) is the major and abundant essential oil component of Cyperus rotundus, minor components, such as nootkatone and valencene, may be more important for the anti-inflammatory effect of this plant. Indeed, a number of studies of the pharmacological effects of nootkatone have already been published. For example, nootkatone inhibited the activity of acetylcholinesterase and of human cytochrome P450 monooxygenases (Miyazawa et al., 1997; Tassaneeyakul et al., 2000). Recently, it has been documented that stimulating effects of nootkatone on an adenosine-monophosphate-activated protein kinase (AMPK), which is a well known energy-homeostatic and anti-inflammatory protein (Murase et al., 2010). In this way, we added more pharmacological information about (+)-nootkatone, which inhibited HMGB1 release through induction of HO-1 in LPSactivated RAW264.7 cells. In addition, we for the first time, report that (+)-valencene also significantly induced HO-1 in macrophages. To the best of our knowledge, there is no information on the antiinflammatory action of (+)-valencene. Thus, further study should be performed in order to determine the effect of (+)-valencene on different pathological disorders with possible application for therapeutic development. Growing lines of evidence suggest a central role for HMGB1 release from macrophages in the pathogenesis of sepsis (Wang et al., 1999; Yang et al., 2004). Recently, we demonstrated that HO-1 induction by pharmacological inducers improves survival in mice challenged with lethal endotoxemia or CLP model (Tsoyi et al., 2009b). And this beneficial effect was mostly mediated by decreased plasma HMGB1 levels in septic animals. In accordance with this result, another group has also documented that HO1 deficient animals represent higher levels of HMGB1 in blood than the wild type during endotoxemia. Moreover, the treatment of HO-1−/− animals with HMGB1 neutralizing antibodies resulted in significantly improved survival in comparison with untreated mice during LPS challenge (Takamiya et al., 2009). Thus, two independent studies have suggested a critical role for HO-1 in the regulation of HMGB1 in experimental sepsis models. Another interesting point is that several reports have suggested that HMGB1 release in macrophages is mostly dictated by NO production (Jiang and Pisetsky, 2006; Kim et al., 2009). Here, we also demonstrated that ECR, nootkatone, and valencene attenuated HMGB1 release in LPS-treated macrophages which was mediated through HO1 induction and iNOS/NO inhibition. The present finding further supports our recent report that induction of HO-1 increases survival of sepsis in animals and reduced HMGB1 release in vitro and in vivo (Tsoyi et al., 2009b, 2011) and stresses the importance of anti-inflammatory action of HO-1. Thus, the present study (ECR, nootkatone, valencene) provides scientific rationale why this herb has been traditionally used for the treatment of inflammatory disorders (Gupta et al., 1971; Seo et al., 2001) and also gives new information about the phamacological action of nootkatone and valencene, active ingredients of ECR. In addition, we recently reported that ECR and nootkatone significantly inhibited collagen-, thrombin-, and/or arachdonic acid-induced platelet aggregation (Seo et al., 2011), which may also contribute to a beneficial role in increasing survival rate in septic
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conditions in the current study. Although it remains to be determined whether anti-platelet action of ECR and nootkatone also involves HO-1 activation in platelets, anti-platelet therapy has been increasingly recognized as one of the important therapies in critical care medicine field (Knoebl, 2010). Indeed, platelets play a central role at the interface between thrombosis and inflammation (Li et al., 2011). Interestingly enough, sesquiterpenes are a large class of terpenoid compounds and common constituents of plant essential oils. Nootkatone, particularly, is also an important constituent of the flavor of grape fruits and valencene is the main constituent of orange (Citrus sinensis) peel oil. Although our intention was to identify which compounds show anti-inflammatory action from ECR, the result of the present study is more than that. Since nootkatone and valencene are natural flavors and fragrances, these essential oils have been widely used in the food, chemical, cosmetic and pharmaceutical industries. In summary, we demonstrated that HO-1 induction is at least responsible for the anti-inflammatory effect of ECR. In addition, we identified that nootkatone and valencene are strong inducers of HO-1. Although monoterpenes such as limonene induced HO-1, this induction was far behind compared to nootkatone or valencene. We concluded that the molecular mechanism for anti-inflammatory action of ECR is closely associated with HO-1 induction. Furthermore, nootkatone and valencene, its minor components but strong inducers of HO-1, may be developed as potential drugs for the treatment of systemic inflammatory disorders such as sepsis. Conflict of interest Potential conflicts do not exist. Acknowledgments This work was supported by the National Center for Standardization of Herbal Medicines funded by Korea Food and Drug Administration (2010). References Bani-Hani, M.G., Greenstein, D., Mann, B.E., Green, C.J., Motterlini, R., 2006. Carbon monoxide-releasing molecule (CORM-3) attenuates lipopolysaccharideand interferon-gamma-induced inflammation in microglia. Pharmacological Reports 58, 132–144. Cepinkas, G., Katada, K., Bihari, A., Potter, R.F., 2008. Carbon monoxide liberated from carbon monoxide-releasing molecule CORM-2 attenuates inflammation in the liver of septic mice. American Journal of Physiology, Gastrointestinal Liver and Physiology 294, G184–G191. Gupta, M.B., Palit, T.K., Singh, N., Bhargava, K.P., 1971. Pharmacological studies to isolate the active constituents from Cyperus rotundus possessing antiinflammatory, anti-pyretic and analgesic activities. Indian Journal of Medical Research 59, 76–82.
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