JOURNAL OF MEDICINAL FOOD J Med Food 17 (6) 2014, 678–685 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.2936
Anti-Inflammatory Effects of an Ethanolic Extract of Guava (Psidium guajava L.) Leaves In Vitro and In Vivo Mi Jang,1,2 Seung-Weon Jeong,1 Somi K. Cho,3 Kwang Seok Ahn,4 Jong Hyun Lee,4 Deok Chun Yang,2 and Jong-Chan Kim1 1
2
Department of Food Analysis & Standardization, Korea Food Research Institute, Seongnam, Gyeonggi-do, Korea. Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University, Gyeonggi-do, Korea. 3 Faculty of Biotechnology College of Applied Life Sciences, Jeju National University, Jeju, Korea. 4 Department of Oriental Pathology, College of Oriental Medicine, Kyung Hee University, Seoul, Korea. ABSTRACT Plant extracts have been used as a source of medicines for a wide variety of human ailments. Among the numerous traditional medicinal herbs, Psidium guajava L. (Myrtaceae), commonly known as guava, has long been used in folk medicines as a therapeutic agent for the treatment of numerous diseases in East Asian and other countries. The aim of this study was to investigate the anti-inflammatory activity of an ethanolic leaf extract of P. guajava (guava) in vitro and in vivo. Our results demonstrated that guava leaf extract (GLE) significantly inhibited lipopolysaccharide (LPS)-induced production of nitric oxide and prostaglandin E2 in a dose-dependent manner. GLE suppressed the expression and activity of both inducible nitric oxide synthase and cyclooxygenase-2 in part through the downregulation of ERK1/2 activation in RAW264.7 macrophages. Furthermore, GLE exhibited significant anti-inflammatory activity in 2 different animal models—Freund’s complete adjuvant-induced hyperalgesia in the rat and LPS-induced endotoxic shock in mice. KEY WORDS: cyclooxygenase-2 health functional food inflammation nitric oxide signaling
Among the numerous traditional medicinal herbs, Psidium guajava L. (Myrtaceae), commonly known as guava, has long been used in folk medicines as a therapeutic agent for the treatment of a number of diseases, e.g., as an antiinflammatory, for diabetes, rheumatic pain, hypertension, wounds, ulcers, and reducing fever.4 Over the last few decades, extracts of guava leaves have been heavily commercialized in Taiwan, Japan, China, and Korea, and these extracts are commonly taken as dietary supplements showing various pharmacological effects. The main constituents of guava leaf extract are a variety of polyphenolics, flavonoids, and triterpenoids.5 Although guava leaves have been shown to exert various physiological effects, little is known about the underlying pharmacological mechanisms of the ethanolic leaf extract of P. guajava (guava). In the present study, we evaluated the inhibitory effect of guava leaf extract (GLE) on inflammatory biomarkers such as NO and PGE2 production and iNOS and COX-2 expression in LPS-stimulated RAW264.7 cells. To investigate the underlying mechanisms, the involvements of mitogen activated protein kinases (MAPKs) and nuclear factor-jB (NF-jB) were examined. Moreover, we investigated the potential therapeutic effects of GLE in 2 different animal models, Freund’s complete adjuvant (FCA)-induced hyperalgesia in the rat and LPS-induced endotoxic shock in mice.
INTRODUCTION
T
he inflammatory response occurs when cells and body tissues are injured by biological, chemical, or physical stimuli such as bacteria, trauma, toxins, or heat. It is one of the most important defense mechanisms, which is aimed at removal of the injurious stimuli and initiation of the healing process. Macrophages are key players in various inflammatory diseases and in the immune response where they release proinflammatory mediators and proteins, including interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS).1 The RAW264.7 mouse macrophage cell line, when activated by lipopolysaccharide (LPS), produces proinflammatory cytokines and other inflammatory mediators, including nitric oxide (NO) and prostaglandin E2 (PGE2), which are synthesized by iNOS and COX-2, respectively.2 Plant extracts have been used as a source of medicines for a wide variety of human disorders. Herbal and natural products have recently received increased attention because of their biological and pharmacological activities.3
Manuscript received 8 May 2013. Revision accepted 21 February 2014. Address correspondence to: Jong-Chan Kim, Korea Food Research Institute, 516 Baekhyeon-dong Bundang-gu, Gyeonggi-do 463-746, Korea, E-mail: [email protected]
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MATERIALS AND METHODS Plant materials and extract preparation Plant material was obtained from the College of Applied Life Science, Jeju National University, Korea. The plant was taxonomically identified and authenticated by one author (S.K.C). The air-dried Jeju guava leaves (20 g) were extracted with 400 mL of 55% ethanol (v/v) at 47C for 4.9 h and the extracted solution was then filtered and evaporated. The extract was then freeze-dried to obtain powder and used as the GLE. Cell culture RAW264.7 macrophages were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) and were maintained in RPMI 1640 medium (L-glutamine, 25mM HEPES buffer, and sodium bicarbonate [Gibco-BRL, Grand Island, NY, USA]) containing antibiotics (100 units/mL penicillin A and 100 lg/mL streptomycin) and 10% heat-inactivated FBS (Gibco-BRL) at 37C in a humidified incubator (5% CO2 and 95% air). Measurement of cell viability The cytotoxicity of GLE was measured using the colorimetric MTT assay before the biological assay was performed.6 RAW264.7 cells were plated at a density of 1 · 104 cells per well in a 96-well plate and then treated with GLE (5, 10, 30, and 50 lg/mL). The cells were incubated for 24 h, and the medium was replaced with fresh medium containing MTT solution (Sigma-Aldrich, St. Louis, MO, USA) (2 mg/mL in PBS) for another 2 h at 37C. The optical density of the cells was measured using a microplate reader (model 680, Bio-Rad, Hercules, CA, USA) at 570 nm. Nitrite assay The cells were pretreated with the indicated concentrations of GLE for 2 h, and then were induced with a 1 lg/mL concentration of LPS for an additional 22 h. The inhibitory effect of GLE on NO production was determined with Griess reagent, as previously described.7 Measurement of PGE2 and IL-6 levels RAW264.7 macrophage cells were plated at a density of 2 · 105 cells per well in a 24-well plate. The cells were pretreated with the indicated concentrations of GLE for 2 h and then induced with 1 lg/mL LPS for an additional 22 h. The concentrations of PGE2 and IL-6 were determined using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Reverse transcription-polymerase chain reaction Total RNA was isolated using TRIzol reagent (Gibco). The concentration and integrity of RNA were determined by measuring absorbance at 260 and 280 nm and then calculating
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the 260/280 nm ratio. The forward and reverse primers for iNOS were 50 -TCT TTG ACG CTC GGA ACT GTA GCA-30 and 50 -CGT GAA GCC ATG ACC TTT CGC ATT-30 , respectively, and the forward and reverse primers for COX-2 were 50 -TTG CTG TAC AAG CAG TGG CAA AGG-30 and 50 AGG ACA AAC ACC GGA GGG AAT CTT-30 , respectively. The forward and reverse primers for GAPDH (used as a control for the total RNA content of each sample) were 50 -AAC TTT GGC ATT GTG GAA GGG CTC-30 and 50 -TGG AAG AGT GGG AGT TGC TGT TGA-30 , respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using a ONE-STEP RT-PCR PreMix kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Western blot analysis Equal amounts of lysates resolved on sodium dodecylpolyacrylamide gel electrophoresis (SDS-PAGE) were transferred to a nitrocellulose membrane. The blots were probed with primary and secondary antibodies, and then the immunoreactive bands were developed with enhanced chemiluminescence (ECL, Amersham Biosciences, Little Chalfont, United Kingdom), as previously described.7 All of the primary and secondary antibodies, including iNOS, COX-2, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38, IjB-a, p65, b-actin, and lamin B were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Animals Male Sprague-Dawley (SD) rats (weighing 200–230 g) and male BALB/c mice (weighing 20–23 g) were used for the experiments. Animals were provided standard laboratory rodent food and water ad libitum and were housed in a temperature-controlled environment (21 – 2C) on a 12-h light/12-h dark cycle. The experimental protocol was reviewed and approved by the Animal Care Committee of Wonkwang University (WKU13-01) and all experiments were conducted in accordance with following ethical guidelines of the International Association for the Study of Pain for investigations on experimental pain in animals.8 Thermal hyperalgesia assessment The experiment was performed using a slight modification of the procedure described by Hargreaves et al.9 Rats were randomly divided into 6 groups (n = 7 for each group) according to the following treatments: saline, FCA (3 mg/mL), FCA with diclofenac (30 mg/kg), and FCA with GLE (100, 200, and 400 mg/kg). All groups, except for the saline group, which was similarly injected with saline, were injected subcutaneously with 25 lL of FCA into the plantar surface of the right hind paw. Twenty-four hours after starting the experiment, paw withdrawal latency (PWL) from a beam of infrared radiation was assessed in both experimental and control groups using the plantar test (Stoelting Stereotaxic Instrument, Ugo Basile, Chicago, IL, USA). PWL was automatically measured by the apparatus using an infrared intensity of 70 W. PWL was measured 3 times separated by a
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minimum interval of 5 min, and the average PWL was calculated from these 3 measurements. Minimum and maximum cutoffs were assigned at 1 and 30 sec, respectively.
calculated with Mann-Whitney tests using SPSS software (SPSS Inc., Chicago, IL, USA). Kaplan-Meier survival analysis was performed with log-rank tests. P values £ .05 were considered statistically significant.
Survival study Mice were divided into 3 groups that received saline, LPS (9 mg/kg), and LPS + GLE (n = 15 per group). GLE was administered orally through an esophageal catheter at a dose of 400 mg/kg twice (at 24 h and 2 h) before LPS injection, and then the mice were monitored for 72 h and survival recorded. Whole blood samples were collected from the retro-orbital plexus after LPS injection at the 3 h mark and sera were prepared to measure TNF-a and IL-6 (R&D Systems, Minneapolis, MN, USA). Liquid chromatography/tandem mass spectrometry analysis The content of 11 selected phenolic compounds was investigated by liquid chromatography (LC)/tandem mass spectrometry (MS/MS), and standard compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ultrahigh performance liquid chromatography (UPLC; Waters Inc., Milford, MA, USA), coupled with electrospray ionization (ESI)–MS/MS (Quattro Premier XE), was used for analysis. The compounds were separated using a Kinetex C18 (100 mm · 2.1 mm · 2.6 lm) column from Phenomenex (Phenomenex Inc., Torrance, CA, USA). A mass spectrometry system equipped with ESI was operated in negative ion mode. For each compound, the optimum conditions of MRM were determined in infusion mode (Table 1). Statistical analysis The results are expressed as the mean – SD, and analysis of variance (ANOVA) with Duncan’s multiple-range test was used for multiple comparisons. Survival curves were
RESULTS Cytotoxic effects of GLE in RAW264.7 cells To investigate the effect of GLE on cell viability, RAW264.7 cells were treated with various concentrations (5, 10, 30, and 50 lg/mL) of GLE for 24 h, and cytotoxicity was measured by the MTT assay. We found that GLE had no effect on cell viability (data not shown), and the doses of GLE listed above were used in all subsequent experiments. GLE inhibits NO production by suppressing iNOS expression in LPS-stimulated RAW264.7 cells The effects of GLE on LPS-induced NO production in RAW264.7 cells were investigated by measuring the amount of nitrite released into the culture medium using the Griess reaction. As shown in Figure 1A, GLE significantly inhibited the production of LPS-induced NO in a dosedependent manner, with > 65% inhibition at a concentration of 50 lg/mL. As shown in Figure 1B, un-stimulated cells expressed no detectable levels of iNOS protein; however, in response to LPS (1 lg/mL), iNOS expression markedly increased. Pretreatment with GLE decreased the levels of iNOS protein expression in LPS-stimulated cells in a dose-dependent manner (Fig. 1B). LPS treatment of cells upregulated iNOS mRNA, whereas pretreatment with GLE inhibited LPSmediated iNOS mRNA upregulation in a dose-dependent manner (Fig. 1C). These results indicate that GLE inhibited NO production by suppressing iNOS gene expression in LPS-stimulated RAW264.7 cells.
Table 1. Liquid Chromatography and Mass Spectrometry Characteristics and Contents of Selected Bioactive Compounds in Guava Leaf Extract Compound-specific MS/MS parametersa Compounds Gallic acid (+)-Catechin Chlorogenic acid Caffeic acid Rutin hydrate Luteolin 7-glucoside q-Coumaric acid Myricetin Quercetin Luteolin Kaempferol a
RT (min)
MRM (m/z)
DP (V)
CE (V)
Contentsb (lg/g)
1.19 2.39 2.42 2.67 2.88 2.97 3.06 3.32 3.67 3.67 3.99
169 > 125 289 > 109 353 > 191 179 > 135 609 > 300 447 > 284 163 > 119 317 > 151 301 > 151 285 > 133 285 > 117
20 35 20 40 40 40 30 40 40 50 30
13 25 13 11 40 40 20 25 25 40 40
213.97 – 6.24 4456.49 – 43.27 55.86 – 2.16 4.54 – 0.25 0.27 – 0.01 1.91 – 0.02 1.26 – 0.09 1.70 – 0.01 15.65 – 2.98 0.23 – 0.02 0.66 – 0.02
Common parameters for all compounds: Mobile phase: eluent A, formic acid in water (0.1%, v/v); eluent B, formic acid in acetonitrile (0.1%, v/v); A linear gradient (0–1 min, 10% B; 1–5 min, 50% B; 5–8 min, 100% B; 8–10 min, 10% B); flow rate was 0.25 mL/min. MRM scan mode operated under the following conditions: capillary voltage 3.20 kV, cone voltage 30 V, source temperature 120C, desolvation temperature 300C and collision gas flow 0.22 mL/min, negative polarity. b Values are mean – SD of triplicate determinations. RT, retention time; MRM, multiple reaction monitoring; DP, declustering potential; CE, collision energy.
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FIG. 1. GLE-mediated inhibition of LPS-induced NO and iNOS expression in RAW264.7 macrophages. (A) RAW264.7 cells were pretreated with different concentrations of GLE for 2 h, and then stimulated with LPS (1 lg/mL) for 22 h. The concentrations of nitrite in the conditioned media of sample-treated cells were calculated from the values measured compared to those of standard concentrations of sodium nitrite dissolved in RPMI 1640. Three independent experiments were performed, and the data are presented as the means – SD. abcdeValues with different letters are significantly different from cells treated with LPS alone (P < .05). (B) RAW264.7 cells were pretreated with different concentrations of GLE for 2 h, and then stimulated with LPS (1 lg/mL) for another 22 h. Cell lysates were obtained, and iNOS protein levels were analyzed via Western blotting. Representative results of 4 independent experiments are shown. b-Actin expression was used as an internal control for Western blotting. (C) RAW264.7 cells were pretreated with the indicated concentrations of GLE for 2 h, and then incubated with LPS (1 lg/mL) for 22 h. Total RNA was isolated, and levels of iNOS mRNA were measured by RT-PCR. GAPDH levels were measured as a control for the cDNA content of each sample. The results shown are representative of the three independent experiments. GLE, guava leaf extract; LPS, lipopolysaccharide; NO, nitric oxide; iNOS, inducible nitric oxide synthase.
GLE inhibits PGE2 production by suppressing COX-2 expression in LPS-stimulated RAW264.7 cells
Suppression of LPS-induced EPK1/2 phosphorylation by GLE
Pretreatment of cells with GLE significantly inhibited LPS-induced PGE2 production in a concentration-dependent manner (Fig. 2A). At a GLE concentration of 50 lg/mL, PGE2 was reduced to the basal level (Fig. 2A). As shown in Figure 2B, unstimulated RAW264.7 cells did not express detectable COX-2 protein; however, COX-2 expression was markedly increased in response to LPS (1 lg/mL), whereas pretreatment with GLE suppressed LPS-activated COX-2 expression in a dose-dependent manner (Fig. 2B). We also attempted to determine whether the expression of COX-2 mRNA paralleled its protein levels. The results showed that in LPS-treated cells, COX-2 mRNA was upregulated, but pretreatment with GLE inhibited the LPS-mediated COX-2 mRNA increase in a dose-dependent manner (Fig. 2C).
To investigate whether inhibition of NO production by GLE was mediated by downregulation of MAPKs and NF-jB activation in RAW264.7 cells, we measured the phosphorylation of MAPKs (ERK, p38, and JNK), p65, and IjB-a in LPS-stimulated macrophages. As shown in Figure 4A, LPS treatment induced phosphorylation of ERK1/2, p38, and JNK. Pretreatment with GLE suppressed LPS-induced phosphorylation of ERK1/2 in a dosedependent manner, but did not suppress p38 or JNK phosphorylation. In addition, GLE did not affect p65 at Ser 276 or IjB-a, which is positively correlated with the extent of NF-jB activation (Fig. 4B). These results suggest that GLE inhibits LPS-induced NO production in part through suppression of the ERK1/2 signaling pathway in RAW264.7 cells.
Inhibition of IL-6 secretion by GLE in LPS-stimulated RAW264.7 cells
Effect of GLE on inflammatory hyperalgesia
IL-6 is produced in response to infection, burns, or other tissue damage leading to inflammation. IL-6 levels are indicative of the progression of inflammation. To confirm that GLE inhibited proinflammatory cytokines, IL-6 concentration was measured in culture supernatants by ELISA. As shown in Figure 3, pretreatment with GLE significantly inhibited IL-6 production in LPS-treated cells in a dosedependent manner.
FCA injection caused a substantial increase in hyperalgesia, which was obvious in the first hour, and lasted for more than 24 h. As shown in Figure 5, injection of FCA caused a dramatic decrease in PWL at 24 h. Administration of GLE significantly alleviated thermal withdrawal latency in a dose-dependent manner, compared with thermal withdrawal latency in the saline group (Fig. 5).
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FIG. 2. GLE-mediated inhibition of LPS-induced PGE2 production and COX-2 expression in RAW264.7 macrophages. (A) RAW264.7 cells were pretreated with different concentrations of GLE for 2 h, and then stimulated with LPS (1 lg/mL) for 22 h. Culture media were collected to measure PGE2 concentration using the mouse PGE2 ELISA kit. Three independent experiments were performed, and the data are presented as means – SD. abcd Values with different letters are significantly different from cells treated with LPS alone (P < .05). (B) RAW264.7 cells were pretreated with GLE for 2 h, and then stimulated with LPS for 22 h. Cell lysates were obtained, and COX-2 protein levels were analyzed via Western blotting. Representative results of three independent experiments are shown. b-Actin expression was used as an internal control for Western blotting. (C) RAW264.7 cells were pretreated with the indicated concentrations of GLE for 2 h, and then incubated with LPS (1 lg/mL) for 22 h. Total RNA was isolated, and COX-2 mRNA levels were measured by RT-PCR. GAPDH mRNA levels were measured as a control for the cDNA content of each sample. The results shown are representative of the 3 independent experiments. PGE2, prostaglandin E2; COX-2, cyclooxygenase-2.
Effect of GLE on the survival of mice with endotoxic shock To determine the protective effect of GLE against LPSinduced shock in mice, we monitored the effect of GLE on the mortality of mice with lethal endotoxemia. The survival
rates of the mice after endotoxin injection are shown in Figure 6A. We found that the LPS-injected mice all died in the first 35 h after LPS injection, whereas mice administered GLE (400 mg/kg) had a 67% survival rate after 72 h (Fig. 6A). These results suggest that GLE has a protective effect against septic shock in mice. We also examined the effect of GLE on the serum levels of the inflammatory cytokines, TNF-a and IL-6, in LPS-challenged mice. As shown in Figure 6B and C, serum levels of TNF-a and IL-6 were significantly elevated 3 h after LPS injection. GLE (400 mg/kg) significantly decreased TNF-a and IL-6 levels in mice with LPSinduced endotoxic shock. UPLC-ESI-MS/MS identification of bioactive compounds Guava leaves have been reported to contain numerous polyphenolic compounds and other chemical compounds that have been shown to exhibit various pharmacological effects, including anti-inflammatory and antioxidant activities.4 In this study, 11 compounds were detected and identified, including phenolic acid and flavonoids (Table 1). GLE shows the highest catechin content, followed by gallic acid, chlorogenic acid, and quercetin in decreasing order, as shown in Table 1.
FIG. 3. GLE-mediated inhibition of LPS-induced IL-6 production in RAW264.7 macrophages. Cells were pretreated with GLE (5, 10, 30, or 50 lg/mL) for 2 h, and then stimulated with LPS (1 lg/mL) for 22 h. The amount of IL-6 released was determined with a mouse IL-6 ELISA kit. Three independent experiments were performed, and the data presented are the means – SD. abcdValues with different letters are significantly different from cells treated with LPS alone (P < .05).
DISCUSSION The objective of this study was to elucidate the anti-inflammatory potential of GLE through RAW264.7 macrophage and animal experiments. We conclusively demonstrated that GLE suppressed iNOS mRNA and COX-2
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FIG. 5. Effect of different doses of GLE on paw withdrawal latency (PWL) 24 h after FCA injection. GLE was administered 24 h after FCAinduced hyperalgesia. Data are expressed as the mean – SD (n = 7). ab Values with different letters were significantly different from the saline group treated with FCA (P < .05). FCA, Freund’s complete adjuvant.
FIG. 4. Effects of GLE on activation of (A) ERK, JNK, and p38 and (B) IjB-a and translocation of p65 in LPS-stimulated RAW264.7 cells. (A) Cells were preincubated with GLE (5, 10, 30, or 50 lg/mL) for 2 h, and then stimulated with LPS (1 lg/mL) for 15 min. Wholecell extracts were prepared, resolved by SDS-PAGE, and electrotransferred to a PVDF membrane, and then Western blot analyses using phosphospecific anti-p38, anti-ERK, and anti-JNK antibodies were performed. The same membranes were reblotted with anti-p38, anti-ERK, and anti-JNK antibodies. The results shown are representative of 3 independent experiments. (B) Cells were preincubated with GLE (5, 10, 30, or 50 lg/mL) for 2 h, and then stimulated with LPS (1 lg/mL) for 15 min. Equal amounts of protein were analyzed using antibodies specific for IjB-a and p65. The results shown are representative of three independent experiments.
mRNA and protein expression in LPS-induced RAW264.7 cells by suppressing ERK1/2 phosphorylation. In addition, GLE had a protective effect on FCA-induced inflammatory hyperalgesia in rats and inhibited the LPS-mediated cytokine release and decreased the mortality rate in LPS-challenged mice. NO, which is synthesized by iNOS, is a well-known proinflammatory mediator that is involved in various physiological and pathological processes. Recently, suppression of NO production has been emphasized as a new pharmacological strategy for the treatment of inflammationrelated diseases.10 Here, we found that GLE inhibited NO production in LPS-induced RAW264.7 cells by suppressing iNOS gene expression. These data indicate that GLE is a an anti-inflammatory agent because the excess NO produced by iNOS mediates both acute and chronic inflammation.11 It has been reported that iNOS inhibitors attenuate osteoarthritis,12 periodontitis,13 septic shock,14 and other chronic
inflammatory diseases. Inhibition of iNOS activity is a significant therapeutic target for the treatment of many pathological processes. COX-2 is an enzyme that generates prostaglandins, which is induced by proinflammatory cytokines and other activators, such as LPS, resulting in the release of a large amount of PGE2 at inflammation sites.15 Therefore, identification of COX-2 inhibitors is considered to be a promising approach to protecting against inflammation and tumorigenesis. We found that GLE significantly inhibited LPS-stimulated PGE2 production and COX-2 protein and RNA expression in a dose-dependent manner, indicating that the actions of GLE occur at the level of transcription. Recent studies have found that signaling pathways of MAPKs play critical roles in the regulation of inflammatory response and in coordinating the induction of many genes encoding inflammatory mediators.16 The 3 major MAPK pathways consist of a highly conserved family of protein kinases such as ERK, JNK, and p38. These kinases regulate immune responses, including proinflammatory cytokine production, mitosis, differentiation, and cell survival/ apoptosis.17,18 In this study we found that pretreatment with GLE suppressed LPS-induced phosphorylation of ERK1/2, but not of p38 or JNK. The ERK signaling module, which was the first MAPK cascade to be characterized, is a vital mediator of cellular fates, including growth, proliferation, and survival.19 ERK plays a critical role in COX-2 expression for LPS-treated RAW264.7 cells and peritoneal macrophages.20 It has been reported that the ERK1/2 inhibitor, PD98059, and the p38 inhibitor, SB203580, suppressed LPS-induced NO production.21 We also found that GLE did not affect the phosphorylation of p65 at Ser 276 or IjB-a, which is positively correlated with the extent of NF-jB activation. NF-jB regulates the expression of various genes
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FIG. 6. Treatment with GLE protected against lethal endotoxemia and inhibited the production of IL-6 and TNF-a in LPS-injected mice. (A) Mice were injected intraperitoneally (i.p.) with LPS (9 mg/kg). GLE was administered orally at a dose of 400 mg/kg twice (at 24 h and 2 h) before LPS injection, and then the mice were monitored for 72 h to determine their survival rate (n = 15). The difference in the survival curves was significant, as calculated by log-rank test (P = .0004). GLE (400 mg/kg) was administered orally through an esophageal catheter at 24 h and 2 h before the LPS was injected. Serum was collected 3 h after LPS injection and TNF-a (B) and IL-6 (C) levels were determined by ELISA kit. Data are shown as the mean values – SD (n = 7). abValues with different letters are significantly different compared to treatment with LPS alone (P < .05).
cells. However, we cannot exclude the possible inhibition of other transcription factors. Thus, these results suggest that GLE partially modulates biomarkers via blocking ERK1/2 signaling pathway in cells. In addition to the in vitro study, we also demonstrated the inhibitory effect of GLE in 2 different experimental models of inflammation. Freund’s complete adjuvant (Mycobacterium butycirium) has commonly been used to produce inflammation in the hind paw of rats23 and is generally used to induce arthritis in animal models.24,25 FCA-induced inflammatory hyperalgesia in the rat is a well-established model for the evaluation of the analgesic activity of drugs.26 It has been shown out that FCA-injected rats have elevated serum IL-6, severe hyperalgesia, and edema during the first week after intervention.27 Our results show that injection of FCA caused a dramatic decrease in PWL at 24 h. Administration of GLE significantly alleviated thermal withdrawal latency in a dosedependent manner, compared with thermal withdrawal latency in the saline control group. Intraplantar injection of FCA into the hind paw of rats induces a persistent localized inflammatory state that is associated with thermal hyperalgesia and increased excitability of spinal cord dorsal horn neurons.23,28 Septic shock is a systemic response to serious infection that is generally caused by Gram-negative bacterial endotoxins. LPS can induce large amounts of NO and PGE2 release as well as proinflammatory cytokines in immuneactivated macrophages and at inflammation sites. Excess production of these immune mediators may result in the clinical syndrome of sepsis.29,30 The presence of endotoxins in experimental animals leads to pathophysiologic changes that are similar to septic shock syndrome in humans, and lethal endotoxemia has been extensively used as an experimental model of Gram-negative septic shock.31 In the present study, we found that GLE exhibited dosedependent inhibitory activity in FCA-induced PWL, and significantly improved the survival rate of mice with lethal endotoxemia. These results showed that GLE has very strong anti-inflammatory activity in vitro as well as in vivo, and could relieve LPS-induced systemic inflammation. In conclusion, this study provides evidence that GLE inhibits the secretion of inflammatory mediators, such as NO and PGE2, in LPS-stimulated macrophages. GLE also suppressed LPS-induced iNOS and COX-2 expression through suppression of the ERK1/2 MAPK signaling pathway. Furthermore, we also demonstrated significant anti-inflammatory effects of GLE in vivo. Therefore, GLE may have the potential to prevent inflammatory diseases. Further investigation is needed to identify the active constituents in Psidium guajava L. that possess these biological activities. AUTHOR DISCLOSURE STATEMENT No competing financial interests exist.
that encode proinflammatory cytokines, adhesion molecules, chemokines, growth factors, and chemoattractants, such as COX-2, and iNOS.22 These results suggest that GLE inhibits LPS-induced NO production, in part, through suppression of the ERK1/2 signaling pathway in RAW264.7
REFERENCES 1. Grip O, Janciauskiene S, Lindgren S: Macrophages in inflammatory bowel disease. Curr Drug Targets Inflamm Allergy 2003; 2:155–160.
ANTI-INFLAMMATORY EFFECTS OF P. GUAJAVA L. 2. Kim SS, Oh OJ, Min HY, et al.: Eugenol suppresses cyclooxygenase-2 expression in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells. Life Sci 2003;73:337–348. 3. Amirghofran Z: Herbal medicines for immunosuppression. Iran J Allergy Asthma Immunol 2012;11:111–119. 4. Gutie´rrez RM, Mitchell S, Solis RV: Psidium guajava: a review of its traditional uses, phytochemistry and pharmacology. J Ethnopharmacol 2008;117:1–27. 5. Peng RY, Hsieh CL, Chen KC: Review on the medicinal use of Psidium gujava L. In Phytopharmacology and Therapeutic Values II, Vol. 20. (Govil JN, ed.) Studium Press, Houston, 2008, pp. 215–248. 6. Denizot F, Lang R: Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986; 89:271–277. 7. Kim JY, Kim HJ, Kim SM, et al.: Methylene chloride fraction of the leaves of Thuja orientalis inhibits in vitro inflammatory biomarkers by blocking NF-jB and p38 MAPK signaling and protects mice from lethal endotoxemia. J Ethnopharmacol 2011;133:687–695. 8. Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109–110. 9. Hargreaves K, Dubner R, Brown F, Flores C, Joris J: A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988;32:77–88. 10. Tinker AC, Wallace AV: Selective inhibitors of inducible nitric oxide synthase: potential agents for the treatment of inflammatory diseases? Curr Top Med Chem 2006;6:77–92. 11. Szabo´ C: Alterations in nitric oxide production in various forms of circulatory shock. New Horizons 1995;3:2–32. 12. Pelletier JP, Jovanovic D, Fernandes JC, et al.: Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase. Arthritis Rheum 1998;411:1275–1286. 13. Lohinai Z, Benedek P, Fehe´r E, et al.: Protective effects of mercaptoethylguanidine, a selective inhibitor of inducible nitric oxide synthase, in ligature-induced periodontitis in the rat. Br J Pharmacol 1998;123:353–360. 14. Levy B, Valtier M, De Chillou C, Bollaert PE, Cane D, Mallie JP: Beneficial effects of L-canavanine, a selective inhibitor of inducible nitric oxide synthase, on lactate metabolism and muscle high energy phosphates during endotoxic shock in rats. Shock 1999;11:98–103. 15. Yoon WJ, Ham YM, Yoo BS, Moon JY, Koh J, Hyun CG: Oenothera laciniata inhibits lipopolysaccharide induced production of nitric oxide, prostaglandin E2, and proinflammatory cytokines in RAW264.7 macrophages. J Biosci Bioeng 2009; 107:429–438. 16. Zhang W, Liu HT: MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res 2002;12:9–18.
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17. Pearson G, Robinson F, Beers Gibson T, et al.: Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 2001;22:153–183. 18. Ajizian SJ, English BK, Meals EA: Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-gamma. J Infect Dis 1999;179:939–944. 19. Boulton TG, Nye SH, Robbins DJ, et al.: ERKs: a family of proteinserine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 1991;65:663–675. 20. Raymond B, Crepin VF, Collins JW, Frankel G: The WxxxE effector EspT triggers expression of immune mediators in an Erk/ JNK and NF-jB-dependent manner. Cell Microbiol 2011;13: 1881–1893. 21. Sung MJ, Davaatseren M, Kim W, et al.: Vitisin A suppresses LPS-induced NO production by inhibiting ERK, p38, and NFkappaB activation in RAW264.7 cells. Int Immunopharmacol 2009;9:319–323. 22. Barnes PJ, Karin M: Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. New Engl J Med 1997;336:1066–1071. 23. Hylden JLK, Nahin RL, Traub RJ, Dubner R: Expansion of receptive fields of spinal lamina I projection neurons in rats with unilateral adjuvant-induced inflammation: the contribution of dorsal horn mechanisms. Pain 1989;37:229–243. 24. Kim HK, Son KH, Chang HW, Kang SS, Kim HP: Inhibition of rat adjuvant-induced arthritis by ginkgetin, a biflavone from Ginkgo biloba leaves. Planta Med 1999;65:465–467. 25. Philippe L, Gegout-Pottie P, Guingamp C, et al.: Relations between functional, inflammatory, and degenerative parameters during adjuvant arthritis in rats. Am J Physiol 1997;273:R1550–R1556. 26. Kumar VL, Chaudhary P, Ramos MV, Mohan M, Matos MP: Protective effect of proteins derived from the latex of Calotropis procera against inflammatory hyperalgesia in monoarthritic rats. Phytother Res 2011;25:1336–1341. 27. Zaringhalam J, Manaheji H, Mghsoodi N, Farokhi B, Mirzaiee V: Spinal mu-opioid receptor expression and hyperalgesia with dexamethasone in chronic adjuvant-induced arthritis in rats. Clin Exp Pharmacol Physiol 2008;35:1309–1315. 28. Stein C, Millan MJ, Herz A: Unilateral inflammation of the hindpaw in rats as a model of prolonged noxious stimulation: alterations in behavior and nociceptive thresholds. Pharmacol Biochem Behav 1988;31:445–451. 29. Kubes P, McCafferty DM: Nitric oxide and intestinal inflammation. Am J Med 2000;109:150–158. 30. Guha M, Mackman N: LPS induction of gene expression in human monocytes. Cell Signal 2001;13:85–94. 31. Danner RL, Elin RJ, Hosseini JM, Wesley RA, Reilly JM, Parillo JE: Endotoxemia in human septic shock. Chest 1991;99:169–175.
Anti-Inflammatory Effects of an Ethanolic Extract of Guava (Psidium guajava L.) Leaves In Vitro and In Vivo Mi Jang,1,2 Seung-Weon Jeong,1 Somi K. Cho,3 Kwang Seok Ahn,4 Jong Hyun Lee,4 Deok Chun Yang,2 and Jong-Chan Kim1 1
2
Department of Food Analysis & Standardization, Korea Food Research Institute, Seongnam, Gyeonggi-do, Korea. Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University, Gyeonggi-do, Korea. 3 Faculty of Biotechnology College of Applied Life Sciences, Jeju National University, Jeju, Korea. 4 Department of Oriental Pathology, College of Oriental Medicine, Kyung Hee University, Seoul, Korea. ABSTRACT Plant extracts have been used as a source of medicines for a wide variety of human ailments. Among the numerous traditional medicinal herbs, Psidium guajava L. (Myrtaceae), commonly known as guava, has long been used in folk medicines as a therapeutic agent for the treatment of numerous diseases in East Asian and other countries. The aim of this study was to investigate the anti-inflammatory activity of an ethanolic leaf extract of P. guajava (guava) in vitro and in vivo. Our results demonstrated that guava leaf extract (GLE) significantly inhibited lipopolysaccharide (LPS)-induced production of nitric oxide and prostaglandin E2 in a dose-dependent manner. GLE suppressed the expression and activity of both inducible nitric oxide synthase and cyclooxygenase-2 in part through the downregulation of ERK1/2 activation in RAW264.7 macrophages. Furthermore, GLE exhibited significant anti-inflammatory activity in 2 different animal models—Freund’s complete adjuvant-induced hyperalgesia in the rat and LPS-induced endotoxic shock in mice. KEY WORDS: cyclooxygenase-2 health functional food inflammation nitric oxide signaling
Among the numerous traditional medicinal herbs, Psidium guajava L. (Myrtaceae), commonly known as guava, has long been used in folk medicines as a therapeutic agent for the treatment of a number of diseases, e.g., as an antiinflammatory, for diabetes, rheumatic pain, hypertension, wounds, ulcers, and reducing fever.4 Over the last few decades, extracts of guava leaves have been heavily commercialized in Taiwan, Japan, China, and Korea, and these extracts are commonly taken as dietary supplements showing various pharmacological effects. The main constituents of guava leaf extract are a variety of polyphenolics, flavonoids, and triterpenoids.5 Although guava leaves have been shown to exert various physiological effects, little is known about the underlying pharmacological mechanisms of the ethanolic leaf extract of P. guajava (guava). In the present study, we evaluated the inhibitory effect of guava leaf extract (GLE) on inflammatory biomarkers such as NO and PGE2 production and iNOS and COX-2 expression in LPS-stimulated RAW264.7 cells. To investigate the underlying mechanisms, the involvements of mitogen activated protein kinases (MAPKs) and nuclear factor-jB (NF-jB) were examined. Moreover, we investigated the potential therapeutic effects of GLE in 2 different animal models, Freund’s complete adjuvant (FCA)-induced hyperalgesia in the rat and LPS-induced endotoxic shock in mice.
INTRODUCTION
T
he inflammatory response occurs when cells and body tissues are injured by biological, chemical, or physical stimuli such as bacteria, trauma, toxins, or heat. It is one of the most important defense mechanisms, which is aimed at removal of the injurious stimuli and initiation of the healing process. Macrophages are key players in various inflammatory diseases and in the immune response where they release proinflammatory mediators and proteins, including interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS).1 The RAW264.7 mouse macrophage cell line, when activated by lipopolysaccharide (LPS), produces proinflammatory cytokines and other inflammatory mediators, including nitric oxide (NO) and prostaglandin E2 (PGE2), which are synthesized by iNOS and COX-2, respectively.2 Plant extracts have been used as a source of medicines for a wide variety of human disorders. Herbal and natural products have recently received increased attention because of their biological and pharmacological activities.3
Manuscript received 8 May 2013. Revision accepted 21 February 2014. Address correspondence to: Jong-Chan Kim, Korea Food Research Institute, 516 Baekhyeon-dong Bundang-gu, Gyeonggi-do 463-746, Korea, E-mail: [email protected]
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MATERIALS AND METHODS Plant materials and extract preparation Plant material was obtained from the College of Applied Life Science, Jeju National University, Korea. The plant was taxonomically identified and authenticated by one author (S.K.C). The air-dried Jeju guava leaves (20 g) were extracted with 400 mL of 55% ethanol (v/v) at 47C for 4.9 h and the extracted solution was then filtered and evaporated. The extract was then freeze-dried to obtain powder and used as the GLE. Cell culture RAW264.7 macrophages were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) and were maintained in RPMI 1640 medium (L-glutamine, 25mM HEPES buffer, and sodium bicarbonate [Gibco-BRL, Grand Island, NY, USA]) containing antibiotics (100 units/mL penicillin A and 100 lg/mL streptomycin) and 10% heat-inactivated FBS (Gibco-BRL) at 37C in a humidified incubator (5% CO2 and 95% air). Measurement of cell viability The cytotoxicity of GLE was measured using the colorimetric MTT assay before the biological assay was performed.6 RAW264.7 cells were plated at a density of 1 · 104 cells per well in a 96-well plate and then treated with GLE (5, 10, 30, and 50 lg/mL). The cells were incubated for 24 h, and the medium was replaced with fresh medium containing MTT solution (Sigma-Aldrich, St. Louis, MO, USA) (2 mg/mL in PBS) for another 2 h at 37C. The optical density of the cells was measured using a microplate reader (model 680, Bio-Rad, Hercules, CA, USA) at 570 nm. Nitrite assay The cells were pretreated with the indicated concentrations of GLE for 2 h, and then were induced with a 1 lg/mL concentration of LPS for an additional 22 h. The inhibitory effect of GLE on NO production was determined with Griess reagent, as previously described.7 Measurement of PGE2 and IL-6 levels RAW264.7 macrophage cells were plated at a density of 2 · 105 cells per well in a 24-well plate. The cells were pretreated with the indicated concentrations of GLE for 2 h and then induced with 1 lg/mL LPS for an additional 22 h. The concentrations of PGE2 and IL-6 were determined using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Reverse transcription-polymerase chain reaction Total RNA was isolated using TRIzol reagent (Gibco). The concentration and integrity of RNA were determined by measuring absorbance at 260 and 280 nm and then calculating
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the 260/280 nm ratio. The forward and reverse primers for iNOS were 50 -TCT TTG ACG CTC GGA ACT GTA GCA-30 and 50 -CGT GAA GCC ATG ACC TTT CGC ATT-30 , respectively, and the forward and reverse primers for COX-2 were 50 -TTG CTG TAC AAG CAG TGG CAA AGG-30 and 50 AGG ACA AAC ACC GGA GGG AAT CTT-30 , respectively. The forward and reverse primers for GAPDH (used as a control for the total RNA content of each sample) were 50 -AAC TTT GGC ATT GTG GAA GGG CTC-30 and 50 -TGG AAG AGT GGG AGT TGC TGT TGA-30 , respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using a ONE-STEP RT-PCR PreMix kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Western blot analysis Equal amounts of lysates resolved on sodium dodecylpolyacrylamide gel electrophoresis (SDS-PAGE) were transferred to a nitrocellulose membrane. The blots were probed with primary and secondary antibodies, and then the immunoreactive bands were developed with enhanced chemiluminescence (ECL, Amersham Biosciences, Little Chalfont, United Kingdom), as previously described.7 All of the primary and secondary antibodies, including iNOS, COX-2, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38, IjB-a, p65, b-actin, and lamin B were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Animals Male Sprague-Dawley (SD) rats (weighing 200–230 g) and male BALB/c mice (weighing 20–23 g) were used for the experiments. Animals were provided standard laboratory rodent food and water ad libitum and were housed in a temperature-controlled environment (21 – 2C) on a 12-h light/12-h dark cycle. The experimental protocol was reviewed and approved by the Animal Care Committee of Wonkwang University (WKU13-01) and all experiments were conducted in accordance with following ethical guidelines of the International Association for the Study of Pain for investigations on experimental pain in animals.8 Thermal hyperalgesia assessment The experiment was performed using a slight modification of the procedure described by Hargreaves et al.9 Rats were randomly divided into 6 groups (n = 7 for each group) according to the following treatments: saline, FCA (3 mg/mL), FCA with diclofenac (30 mg/kg), and FCA with GLE (100, 200, and 400 mg/kg). All groups, except for the saline group, which was similarly injected with saline, were injected subcutaneously with 25 lL of FCA into the plantar surface of the right hind paw. Twenty-four hours after starting the experiment, paw withdrawal latency (PWL) from a beam of infrared radiation was assessed in both experimental and control groups using the plantar test (Stoelting Stereotaxic Instrument, Ugo Basile, Chicago, IL, USA). PWL was automatically measured by the apparatus using an infrared intensity of 70 W. PWL was measured 3 times separated by a
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minimum interval of 5 min, and the average PWL was calculated from these 3 measurements. Minimum and maximum cutoffs were assigned at 1 and 30 sec, respectively.
calculated with Mann-Whitney tests using SPSS software (SPSS Inc., Chicago, IL, USA). Kaplan-Meier survival analysis was performed with log-rank tests. P values £ .05 were considered statistically significant.
Survival study Mice were divided into 3 groups that received saline, LPS (9 mg/kg), and LPS + GLE (n = 15 per group). GLE was administered orally through an esophageal catheter at a dose of 400 mg/kg twice (at 24 h and 2 h) before LPS injection, and then the mice were monitored for 72 h and survival recorded. Whole blood samples were collected from the retro-orbital plexus after LPS injection at the 3 h mark and sera were prepared to measure TNF-a and IL-6 (R&D Systems, Minneapolis, MN, USA). Liquid chromatography/tandem mass spectrometry analysis The content of 11 selected phenolic compounds was investigated by liquid chromatography (LC)/tandem mass spectrometry (MS/MS), and standard compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ultrahigh performance liquid chromatography (UPLC; Waters Inc., Milford, MA, USA), coupled with electrospray ionization (ESI)–MS/MS (Quattro Premier XE), was used for analysis. The compounds were separated using a Kinetex C18 (100 mm · 2.1 mm · 2.6 lm) column from Phenomenex (Phenomenex Inc., Torrance, CA, USA). A mass spectrometry system equipped with ESI was operated in negative ion mode. For each compound, the optimum conditions of MRM were determined in infusion mode (Table 1). Statistical analysis The results are expressed as the mean – SD, and analysis of variance (ANOVA) with Duncan’s multiple-range test was used for multiple comparisons. Survival curves were
RESULTS Cytotoxic effects of GLE in RAW264.7 cells To investigate the effect of GLE on cell viability, RAW264.7 cells were treated with various concentrations (5, 10, 30, and 50 lg/mL) of GLE for 24 h, and cytotoxicity was measured by the MTT assay. We found that GLE had no effect on cell viability (data not shown), and the doses of GLE listed above were used in all subsequent experiments. GLE inhibits NO production by suppressing iNOS expression in LPS-stimulated RAW264.7 cells The effects of GLE on LPS-induced NO production in RAW264.7 cells were investigated by measuring the amount of nitrite released into the culture medium using the Griess reaction. As shown in Figure 1A, GLE significantly inhibited the production of LPS-induced NO in a dosedependent manner, with > 65% inhibition at a concentration of 50 lg/mL. As shown in Figure 1B, un-stimulated cells expressed no detectable levels of iNOS protein; however, in response to LPS (1 lg/mL), iNOS expression markedly increased. Pretreatment with GLE decreased the levels of iNOS protein expression in LPS-stimulated cells in a dose-dependent manner (Fig. 1B). LPS treatment of cells upregulated iNOS mRNA, whereas pretreatment with GLE inhibited LPSmediated iNOS mRNA upregulation in a dose-dependent manner (Fig. 1C). These results indicate that GLE inhibited NO production by suppressing iNOS gene expression in LPS-stimulated RAW264.7 cells.
Table 1. Liquid Chromatography and Mass Spectrometry Characteristics and Contents of Selected Bioactive Compounds in Guava Leaf Extract Compound-specific MS/MS parametersa Compounds Gallic acid (+)-Catechin Chlorogenic acid Caffeic acid Rutin hydrate Luteolin 7-glucoside q-Coumaric acid Myricetin Quercetin Luteolin Kaempferol a
RT (min)
MRM (m/z)
DP (V)
CE (V)
Contentsb (lg/g)
1.19 2.39 2.42 2.67 2.88 2.97 3.06 3.32 3.67 3.67 3.99
169 > 125 289 > 109 353 > 191 179 > 135 609 > 300 447 > 284 163 > 119 317 > 151 301 > 151 285 > 133 285 > 117
20 35 20 40 40 40 30 40 40 50 30
13 25 13 11 40 40 20 25 25 40 40
213.97 – 6.24 4456.49 – 43.27 55.86 – 2.16 4.54 – 0.25 0.27 – 0.01 1.91 – 0.02 1.26 – 0.09 1.70 – 0.01 15.65 – 2.98 0.23 – 0.02 0.66 – 0.02
Common parameters for all compounds: Mobile phase: eluent A, formic acid in water (0.1%, v/v); eluent B, formic acid in acetonitrile (0.1%, v/v); A linear gradient (0–1 min, 10% B; 1–5 min, 50% B; 5–8 min, 100% B; 8–10 min, 10% B); flow rate was 0.25 mL/min. MRM scan mode operated under the following conditions: capillary voltage 3.20 kV, cone voltage 30 V, source temperature 120C, desolvation temperature 300C and collision gas flow 0.22 mL/min, negative polarity. b Values are mean – SD of triplicate determinations. RT, retention time; MRM, multiple reaction monitoring; DP, declustering potential; CE, collision energy.
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FIG. 1. GLE-mediated inhibition of LPS-induced NO and iNOS expression in RAW264.7 macrophages. (A) RAW264.7 cells were pretreated with different concentrations of GLE for 2 h, and then stimulated with LPS (1 lg/mL) for 22 h. The concentrations of nitrite in the conditioned media of sample-treated cells were calculated from the values measured compared to those of standard concentrations of sodium nitrite dissolved in RPMI 1640. Three independent experiments were performed, and the data are presented as the means – SD. abcdeValues with different letters are significantly different from cells treated with LPS alone (P < .05). (B) RAW264.7 cells were pretreated with different concentrations of GLE for 2 h, and then stimulated with LPS (1 lg/mL) for another 22 h. Cell lysates were obtained, and iNOS protein levels were analyzed via Western blotting. Representative results of 4 independent experiments are shown. b-Actin expression was used as an internal control for Western blotting. (C) RAW264.7 cells were pretreated with the indicated concentrations of GLE for 2 h, and then incubated with LPS (1 lg/mL) for 22 h. Total RNA was isolated, and levels of iNOS mRNA were measured by RT-PCR. GAPDH levels were measured as a control for the cDNA content of each sample. The results shown are representative of the three independent experiments. GLE, guava leaf extract; LPS, lipopolysaccharide; NO, nitric oxide; iNOS, inducible nitric oxide synthase.
GLE inhibits PGE2 production by suppressing COX-2 expression in LPS-stimulated RAW264.7 cells
Suppression of LPS-induced EPK1/2 phosphorylation by GLE
Pretreatment of cells with GLE significantly inhibited LPS-induced PGE2 production in a concentration-dependent manner (Fig. 2A). At a GLE concentration of 50 lg/mL, PGE2 was reduced to the basal level (Fig. 2A). As shown in Figure 2B, unstimulated RAW264.7 cells did not express detectable COX-2 protein; however, COX-2 expression was markedly increased in response to LPS (1 lg/mL), whereas pretreatment with GLE suppressed LPS-activated COX-2 expression in a dose-dependent manner (Fig. 2B). We also attempted to determine whether the expression of COX-2 mRNA paralleled its protein levels. The results showed that in LPS-treated cells, COX-2 mRNA was upregulated, but pretreatment with GLE inhibited the LPS-mediated COX-2 mRNA increase in a dose-dependent manner (Fig. 2C).
To investigate whether inhibition of NO production by GLE was mediated by downregulation of MAPKs and NF-jB activation in RAW264.7 cells, we measured the phosphorylation of MAPKs (ERK, p38, and JNK), p65, and IjB-a in LPS-stimulated macrophages. As shown in Figure 4A, LPS treatment induced phosphorylation of ERK1/2, p38, and JNK. Pretreatment with GLE suppressed LPS-induced phosphorylation of ERK1/2 in a dosedependent manner, but did not suppress p38 or JNK phosphorylation. In addition, GLE did not affect p65 at Ser 276 or IjB-a, which is positively correlated with the extent of NF-jB activation (Fig. 4B). These results suggest that GLE inhibits LPS-induced NO production in part through suppression of the ERK1/2 signaling pathway in RAW264.7 cells.
Inhibition of IL-6 secretion by GLE in LPS-stimulated RAW264.7 cells
Effect of GLE on inflammatory hyperalgesia
IL-6 is produced in response to infection, burns, or other tissue damage leading to inflammation. IL-6 levels are indicative of the progression of inflammation. To confirm that GLE inhibited proinflammatory cytokines, IL-6 concentration was measured in culture supernatants by ELISA. As shown in Figure 3, pretreatment with GLE significantly inhibited IL-6 production in LPS-treated cells in a dosedependent manner.
FCA injection caused a substantial increase in hyperalgesia, which was obvious in the first hour, and lasted for more than 24 h. As shown in Figure 5, injection of FCA caused a dramatic decrease in PWL at 24 h. Administration of GLE significantly alleviated thermal withdrawal latency in a dose-dependent manner, compared with thermal withdrawal latency in the saline group (Fig. 5).
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FIG. 2. GLE-mediated inhibition of LPS-induced PGE2 production and COX-2 expression in RAW264.7 macrophages. (A) RAW264.7 cells were pretreated with different concentrations of GLE for 2 h, and then stimulated with LPS (1 lg/mL) for 22 h. Culture media were collected to measure PGE2 concentration using the mouse PGE2 ELISA kit. Three independent experiments were performed, and the data are presented as means – SD. abcd Values with different letters are significantly different from cells treated with LPS alone (P < .05). (B) RAW264.7 cells were pretreated with GLE for 2 h, and then stimulated with LPS for 22 h. Cell lysates were obtained, and COX-2 protein levels were analyzed via Western blotting. Representative results of three independent experiments are shown. b-Actin expression was used as an internal control for Western blotting. (C) RAW264.7 cells were pretreated with the indicated concentrations of GLE for 2 h, and then incubated with LPS (1 lg/mL) for 22 h. Total RNA was isolated, and COX-2 mRNA levels were measured by RT-PCR. GAPDH mRNA levels were measured as a control for the cDNA content of each sample. The results shown are representative of the 3 independent experiments. PGE2, prostaglandin E2; COX-2, cyclooxygenase-2.
Effect of GLE on the survival of mice with endotoxic shock To determine the protective effect of GLE against LPSinduced shock in mice, we monitored the effect of GLE on the mortality of mice with lethal endotoxemia. The survival
rates of the mice after endotoxin injection are shown in Figure 6A. We found that the LPS-injected mice all died in the first 35 h after LPS injection, whereas mice administered GLE (400 mg/kg) had a 67% survival rate after 72 h (Fig. 6A). These results suggest that GLE has a protective effect against septic shock in mice. We also examined the effect of GLE on the serum levels of the inflammatory cytokines, TNF-a and IL-6, in LPS-challenged mice. As shown in Figure 6B and C, serum levels of TNF-a and IL-6 were significantly elevated 3 h after LPS injection. GLE (400 mg/kg) significantly decreased TNF-a and IL-6 levels in mice with LPSinduced endotoxic shock. UPLC-ESI-MS/MS identification of bioactive compounds Guava leaves have been reported to contain numerous polyphenolic compounds and other chemical compounds that have been shown to exhibit various pharmacological effects, including anti-inflammatory and antioxidant activities.4 In this study, 11 compounds were detected and identified, including phenolic acid and flavonoids (Table 1). GLE shows the highest catechin content, followed by gallic acid, chlorogenic acid, and quercetin in decreasing order, as shown in Table 1.
FIG. 3. GLE-mediated inhibition of LPS-induced IL-6 production in RAW264.7 macrophages. Cells were pretreated with GLE (5, 10, 30, or 50 lg/mL) for 2 h, and then stimulated with LPS (1 lg/mL) for 22 h. The amount of IL-6 released was determined with a mouse IL-6 ELISA kit. Three independent experiments were performed, and the data presented are the means – SD. abcdValues with different letters are significantly different from cells treated with LPS alone (P < .05).
DISCUSSION The objective of this study was to elucidate the anti-inflammatory potential of GLE through RAW264.7 macrophage and animal experiments. We conclusively demonstrated that GLE suppressed iNOS mRNA and COX-2
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FIG. 5. Effect of different doses of GLE on paw withdrawal latency (PWL) 24 h after FCA injection. GLE was administered 24 h after FCAinduced hyperalgesia. Data are expressed as the mean – SD (n = 7). ab Values with different letters were significantly different from the saline group treated with FCA (P < .05). FCA, Freund’s complete adjuvant.
FIG. 4. Effects of GLE on activation of (A) ERK, JNK, and p38 and (B) IjB-a and translocation of p65 in LPS-stimulated RAW264.7 cells. (A) Cells were preincubated with GLE (5, 10, 30, or 50 lg/mL) for 2 h, and then stimulated with LPS (1 lg/mL) for 15 min. Wholecell extracts were prepared, resolved by SDS-PAGE, and electrotransferred to a PVDF membrane, and then Western blot analyses using phosphospecific anti-p38, anti-ERK, and anti-JNK antibodies were performed. The same membranes were reblotted with anti-p38, anti-ERK, and anti-JNK antibodies. The results shown are representative of 3 independent experiments. (B) Cells were preincubated with GLE (5, 10, 30, or 50 lg/mL) for 2 h, and then stimulated with LPS (1 lg/mL) for 15 min. Equal amounts of protein were analyzed using antibodies specific for IjB-a and p65. The results shown are representative of three independent experiments.
mRNA and protein expression in LPS-induced RAW264.7 cells by suppressing ERK1/2 phosphorylation. In addition, GLE had a protective effect on FCA-induced inflammatory hyperalgesia in rats and inhibited the LPS-mediated cytokine release and decreased the mortality rate in LPS-challenged mice. NO, which is synthesized by iNOS, is a well-known proinflammatory mediator that is involved in various physiological and pathological processes. Recently, suppression of NO production has been emphasized as a new pharmacological strategy for the treatment of inflammationrelated diseases.10 Here, we found that GLE inhibited NO production in LPS-induced RAW264.7 cells by suppressing iNOS gene expression. These data indicate that GLE is a an anti-inflammatory agent because the excess NO produced by iNOS mediates both acute and chronic inflammation.11 It has been reported that iNOS inhibitors attenuate osteoarthritis,12 periodontitis,13 septic shock,14 and other chronic
inflammatory diseases. Inhibition of iNOS activity is a significant therapeutic target for the treatment of many pathological processes. COX-2 is an enzyme that generates prostaglandins, which is induced by proinflammatory cytokines and other activators, such as LPS, resulting in the release of a large amount of PGE2 at inflammation sites.15 Therefore, identification of COX-2 inhibitors is considered to be a promising approach to protecting against inflammation and tumorigenesis. We found that GLE significantly inhibited LPS-stimulated PGE2 production and COX-2 protein and RNA expression in a dose-dependent manner, indicating that the actions of GLE occur at the level of transcription. Recent studies have found that signaling pathways of MAPKs play critical roles in the regulation of inflammatory response and in coordinating the induction of many genes encoding inflammatory mediators.16 The 3 major MAPK pathways consist of a highly conserved family of protein kinases such as ERK, JNK, and p38. These kinases regulate immune responses, including proinflammatory cytokine production, mitosis, differentiation, and cell survival/ apoptosis.17,18 In this study we found that pretreatment with GLE suppressed LPS-induced phosphorylation of ERK1/2, but not of p38 or JNK. The ERK signaling module, which was the first MAPK cascade to be characterized, is a vital mediator of cellular fates, including growth, proliferation, and survival.19 ERK plays a critical role in COX-2 expression for LPS-treated RAW264.7 cells and peritoneal macrophages.20 It has been reported that the ERK1/2 inhibitor, PD98059, and the p38 inhibitor, SB203580, suppressed LPS-induced NO production.21 We also found that GLE did not affect the phosphorylation of p65 at Ser 276 or IjB-a, which is positively correlated with the extent of NF-jB activation. NF-jB regulates the expression of various genes
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FIG. 6. Treatment with GLE protected against lethal endotoxemia and inhibited the production of IL-6 and TNF-a in LPS-injected mice. (A) Mice were injected intraperitoneally (i.p.) with LPS (9 mg/kg). GLE was administered orally at a dose of 400 mg/kg twice (at 24 h and 2 h) before LPS injection, and then the mice were monitored for 72 h to determine their survival rate (n = 15). The difference in the survival curves was significant, as calculated by log-rank test (P = .0004). GLE (400 mg/kg) was administered orally through an esophageal catheter at 24 h and 2 h before the LPS was injected. Serum was collected 3 h after LPS injection and TNF-a (B) and IL-6 (C) levels were determined by ELISA kit. Data are shown as the mean values – SD (n = 7). abValues with different letters are significantly different compared to treatment with LPS alone (P < .05).
cells. However, we cannot exclude the possible inhibition of other transcription factors. Thus, these results suggest that GLE partially modulates biomarkers via blocking ERK1/2 signaling pathway in cells. In addition to the in vitro study, we also demonstrated the inhibitory effect of GLE in 2 different experimental models of inflammation. Freund’s complete adjuvant (Mycobacterium butycirium) has commonly been used to produce inflammation in the hind paw of rats23 and is generally used to induce arthritis in animal models.24,25 FCA-induced inflammatory hyperalgesia in the rat is a well-established model for the evaluation of the analgesic activity of drugs.26 It has been shown out that FCA-injected rats have elevated serum IL-6, severe hyperalgesia, and edema during the first week after intervention.27 Our results show that injection of FCA caused a dramatic decrease in PWL at 24 h. Administration of GLE significantly alleviated thermal withdrawal latency in a dosedependent manner, compared with thermal withdrawal latency in the saline control group. Intraplantar injection of FCA into the hind paw of rats induces a persistent localized inflammatory state that is associated with thermal hyperalgesia and increased excitability of spinal cord dorsal horn neurons.23,28 Septic shock is a systemic response to serious infection that is generally caused by Gram-negative bacterial endotoxins. LPS can induce large amounts of NO and PGE2 release as well as proinflammatory cytokines in immuneactivated macrophages and at inflammation sites. Excess production of these immune mediators may result in the clinical syndrome of sepsis.29,30 The presence of endotoxins in experimental animals leads to pathophysiologic changes that are similar to septic shock syndrome in humans, and lethal endotoxemia has been extensively used as an experimental model of Gram-negative septic shock.31 In the present study, we found that GLE exhibited dosedependent inhibitory activity in FCA-induced PWL, and significantly improved the survival rate of mice with lethal endotoxemia. These results showed that GLE has very strong anti-inflammatory activity in vitro as well as in vivo, and could relieve LPS-induced systemic inflammation. In conclusion, this study provides evidence that GLE inhibits the secretion of inflammatory mediators, such as NO and PGE2, in LPS-stimulated macrophages. GLE also suppressed LPS-induced iNOS and COX-2 expression through suppression of the ERK1/2 MAPK signaling pathway. Furthermore, we also demonstrated significant anti-inflammatory effects of GLE in vivo. Therefore, GLE may have the potential to prevent inflammatory diseases. Further investigation is needed to identify the active constituents in Psidium guajava L. that possess these biological activities. AUTHOR DISCLOSURE STATEMENT No competing financial interests exist.
that encode proinflammatory cytokines, adhesion molecules, chemokines, growth factors, and chemoattractants, such as COX-2, and iNOS.22 These results suggest that GLE inhibits LPS-induced NO production, in part, through suppression of the ERK1/2 signaling pathway in RAW264.7
REFERENCES 1. Grip O, Janciauskiene S, Lindgren S: Macrophages in inflammatory bowel disease. Curr Drug Targets Inflamm Allergy 2003; 2:155–160.
ANTI-INFLAMMATORY EFFECTS OF P. GUAJAVA L. 2. Kim SS, Oh OJ, Min HY, et al.: Eugenol suppresses cyclooxygenase-2 expression in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells. Life Sci 2003;73:337–348. 3. Amirghofran Z: Herbal medicines for immunosuppression. Iran J Allergy Asthma Immunol 2012;11:111–119. 4. Gutie´rrez RM, Mitchell S, Solis RV: Psidium guajava: a review of its traditional uses, phytochemistry and pharmacology. J Ethnopharmacol 2008;117:1–27. 5. Peng RY, Hsieh CL, Chen KC: Review on the medicinal use of Psidium gujava L. In Phytopharmacology and Therapeutic Values II, Vol. 20. (Govil JN, ed.) Studium Press, Houston, 2008, pp. 215–248. 6. Denizot F, Lang R: Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986; 89:271–277. 7. Kim JY, Kim HJ, Kim SM, et al.: Methylene chloride fraction of the leaves of Thuja orientalis inhibits in vitro inflammatory biomarkers by blocking NF-jB and p38 MAPK signaling and protects mice from lethal endotoxemia. J Ethnopharmacol 2011;133:687–695. 8. Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109–110. 9. Hargreaves K, Dubner R, Brown F, Flores C, Joris J: A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988;32:77–88. 10. Tinker AC, Wallace AV: Selective inhibitors of inducible nitric oxide synthase: potential agents for the treatment of inflammatory diseases? Curr Top Med Chem 2006;6:77–92. 11. Szabo´ C: Alterations in nitric oxide production in various forms of circulatory shock. New Horizons 1995;3:2–32. 12. Pelletier JP, Jovanovic D, Fernandes JC, et al.: Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase. Arthritis Rheum 1998;411:1275–1286. 13. Lohinai Z, Benedek P, Fehe´r E, et al.: Protective effects of mercaptoethylguanidine, a selective inhibitor of inducible nitric oxide synthase, in ligature-induced periodontitis in the rat. Br J Pharmacol 1998;123:353–360. 14. Levy B, Valtier M, De Chillou C, Bollaert PE, Cane D, Mallie JP: Beneficial effects of L-canavanine, a selective inhibitor of inducible nitric oxide synthase, on lactate metabolism and muscle high energy phosphates during endotoxic shock in rats. Shock 1999;11:98–103. 15. Yoon WJ, Ham YM, Yoo BS, Moon JY, Koh J, Hyun CG: Oenothera laciniata inhibits lipopolysaccharide induced production of nitric oxide, prostaglandin E2, and proinflammatory cytokines in RAW264.7 macrophages. J Biosci Bioeng 2009; 107:429–438. 16. Zhang W, Liu HT: MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res 2002;12:9–18.
685
17. Pearson G, Robinson F, Beers Gibson T, et al.: Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 2001;22:153–183. 18. Ajizian SJ, English BK, Meals EA: Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-gamma. J Infect Dis 1999;179:939–944. 19. Boulton TG, Nye SH, Robbins DJ, et al.: ERKs: a family of proteinserine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 1991;65:663–675. 20. Raymond B, Crepin VF, Collins JW, Frankel G: The WxxxE effector EspT triggers expression of immune mediators in an Erk/ JNK and NF-jB-dependent manner. Cell Microbiol 2011;13: 1881–1893. 21. Sung MJ, Davaatseren M, Kim W, et al.: Vitisin A suppresses LPS-induced NO production by inhibiting ERK, p38, and NFkappaB activation in RAW264.7 cells. Int Immunopharmacol 2009;9:319–323. 22. Barnes PJ, Karin M: Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. New Engl J Med 1997;336:1066–1071. 23. Hylden JLK, Nahin RL, Traub RJ, Dubner R: Expansion of receptive fields of spinal lamina I projection neurons in rats with unilateral adjuvant-induced inflammation: the contribution of dorsal horn mechanisms. Pain 1989;37:229–243. 24. Kim HK, Son KH, Chang HW, Kang SS, Kim HP: Inhibition of rat adjuvant-induced arthritis by ginkgetin, a biflavone from Ginkgo biloba leaves. Planta Med 1999;65:465–467. 25. Philippe L, Gegout-Pottie P, Guingamp C, et al.: Relations between functional, inflammatory, and degenerative parameters during adjuvant arthritis in rats. Am J Physiol 1997;273:R1550–R1556. 26. Kumar VL, Chaudhary P, Ramos MV, Mohan M, Matos MP: Protective effect of proteins derived from the latex of Calotropis procera against inflammatory hyperalgesia in monoarthritic rats. Phytother Res 2011;25:1336–1341. 27. Zaringhalam J, Manaheji H, Mghsoodi N, Farokhi B, Mirzaiee V: Spinal mu-opioid receptor expression and hyperalgesia with dexamethasone in chronic adjuvant-induced arthritis in rats. Clin Exp Pharmacol Physiol 2008;35:1309–1315. 28. Stein C, Millan MJ, Herz A: Unilateral inflammation of the hindpaw in rats as a model of prolonged noxious stimulation: alterations in behavior and nociceptive thresholds. Pharmacol Biochem Behav 1988;31:445–451. 29. Kubes P, McCafferty DM: Nitric oxide and intestinal inflammation. Am J Med 2000;109:150–158. 30. Guha M, Mackman N: LPS induction of gene expression in human monocytes. Cell Signal 2001;13:85–94. 31. Danner RL, Elin RJ, Hosseini JM, Wesley RA, Reilly JM, Parillo JE: Endotoxemia in human septic shock. Chest 1991;99:169–175.
