http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(8): 1069–1078 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.885060

ORIGINAL ARTICLE

Analgesic activity of Eugenia jambolana leave constituent: A dikaempferol rhamnopyranoside from ethyl acetate soluble fraction Madhu Cholenahalli Lingaraju1, Shikha Anand2, Venkanna Balaganur1, Rashmi Rekha Kumari1, Amar Sunil More1, Dinesh Kumar1, Brijesh Kumar Bhadoria2, and Surendra Kumar Tandan1 Pharmaceutical Biology Downloaded from informahealthcare.com by University of Melbourne on 09/12/14 For personal use only.

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Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India and 2Division of Plant Animal Relationship, Indian Grassland Fodder Research Institute, Jhansi, Uttar Pradesh, India Abstract

Keywords

Context: Eugenia jambolana Lam. (Myrtaceae) is a medicinal plant used in folk medicine for the treatment of diabetes, inflammation, and pain. Objective: We investigated the antinociceptive effect of kaempferol-7-O-a-L-rhamnopyranoside]40 -O-40 -[kaempferol-7-O-a-L-rhamnopyranoside (EJ-01), isolated from the E. jambolana leaves. Materials and methods: EJ-01 (3, 10, and 30 mg kg1, orally) was assessed for peripheral (formalin-nociception and acetic acid-writhing) and central (hot plate and tail flick test) analgesic activity in mice and the in vitro anti-inflammatory activity (25, 50, and 100 mg mL1) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Results and discussion: EJ-01 (10 and 30 mg kg1) significantly inhibited mean writhing counts (37.74 and 36.83) in acetic acid writhing and paw licking time (55.16 and 45.66 s) in the late phase of the formalin test as compared with the respective control (60.66 and 104.33 s). EJ-01 did not show analgesic activity in central pain models. Significant reduction in the tumor necrosis factor (TNF)-a (295.48, 51.20, and 49.47 pg mL1) and interleukin (IL)-1b (59.38, 20.08, and 15.46 pg mL1) levels were observed in EJ-01-treated medium (25, 50, and 100 mg mL1) as compared with vehicle-treated control values (788.67 and 161.77 pg mL1), respectively. Significant reduction in total nitrite plus nitrate (NOx) levels (70.80 nmol) was observed in the EJ-01-treated medium (100 mg mL1) as compared with the vehicle-treated value (110.41 nmol). Conclusion: EJ-01 is a valuable analgesic constituent of E. jambolana leaves and this study supports the pharmacological basis for the use of this plant in traditional medicine for curing inflammatory pain.

Cytokines, formalin nociception, in vitro anti-inflammatory, nitric oxide

Introduction Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1benzopyran-4-one) is a flavonoid found in many edible plants (e.g., tea, broccoli, cabbage, kale, beans, endive, leek, tomato, strawberries, and grapes). Numerous preclinical studies have shown that kaempferol and some glycosides of kaempferol have wide range of pharmacological activities, including antioxidant, anti-inflammatory, analgesic, and antiallergic activities (Calderon-Montano et al., 2011). Analgesic activity has also been observed with several kaempferol glycosides and kaempferol-containing plants in numerous in vivo studies (Toker et al., 2004). Proinflammatory cytokines produced predominantly by activated macrophages are involved in the up-regulation of inflammatory reactions. There is abundant evidence that

Correspondence: S. K. Tandan, Ph.D., Principal Scientist, Division of Pharmacology & Toxicology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, Uttar Pradesh, India. Fax: +91 5812303284. E-mail: [email protected]

History Received 15 February 2013 Revised 19 December 2013 Accepted 15 January 2014 Published online 9 July 2014

certain pro-inflammatory cytokines such as interleukin (IL)-1 b, IL-6, and tumor necrosis factor (TNF)-a are involved in the process of pathological pain and IL-1b is expressed in nociceptive dorsal root ganglion (DRG) neurons (Copray et al., 2001) and can produce hyperalgesia following either intraperitoneal, intracerebroventricular, or intraplantar injection (Perkins & Kelly, 1994; Watkins et al., 1994). TNF-a is another inflammatory cytokine that plays a well-established, key role in some pain models. Intraplantar injection of TNF-a also produces mechanical (Cunha et al., 1992) and thermal hyperalgesia (Perkins & Kelly, 1994). As noted previously, proinflammatory cytokines are produced mainly by activated macrophages and are involved in pathological pain production (Copray et al., 2001). We hypothesized that the inhibition of cytokine production from macrophage would reduce the inflammatory response and have antinociceptive action in murine models of pain. Eugenia jambolana Lam. (Myrtaceae) (synonym: Syzgium cumini Lam.) is an evergreen tropical tree, native to Bangladesh, India, Nepal, Pakistan, and Indonesia and very commonly cultivated. Earlier investigations have reported

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Figure 1. Structural formula of EJ-01.

the antioxidant (Banerjee et al., 2005), anti-inflammatory (Muruganandan et al., 2001, 2002), nitric oxide (NO) scavenging activity (Jagetia & Baliga, 2004) from different parts of E. jambolana, while the anti-inflammatory and analgesic activities have been reported from the bark of E. jambolana (Hegde et al., 2011); the leaves are still poorly studied and its pharmacological potential and phytochemical constituents are required to be explored. The leaves are rich in flavonoids like acylated flavonol glycosides (Mahmoud et al., 2001), quercetin, myricetin, myricitin, and myricetin 3-O-4-acetyl-L-rhamnopyranoside (Timbola et al., 2002). Recently, we isolated a pure compound, a dikaempferol from E. jambolana leaves and its structure was identified on the basis of chemical reactions and spectral analysis (NMR) as [kaempferol-7-O-a-L-rhamnopyranoside]-40 -O-40 [kaempferol-7-O-a-L-rhamnopyranoside]. This compound is abbreviated as EJ-01 (Figure 1). Therefore, considering the therapeutic properties attributed to this plant, flavonoid, and the anti-inflammatory effect demonstrated by previous studies and involvement of macrophage-derived cytokines in pain production, we investigated the antinociceptive action of the EJ-01, isolated from the leaves of this plant, on several models of pain in mice, and possible involvement of pro-inflammatory cytokines by employing LPS-stimulated murine macrophages.

Materials and methods Chemicals, drugs, and kits Dulbecco’s modified Eagle’s minimum essential medium (DMEM) medium, penicillin, streptomycin, and fetal calf serum (FCS) were purchased from Hyclone (Logan, UT). Lipopolysaccharide (LPS), dexamethasone, morphine hydrochloride, dimethyl sulfoxide (DMSO), and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Aldrich, St. Louis, MO. Etoricoxib was obtained from Ranbaxy Research Lab (Gurgaon, India). Mouse TNF-a and IL-1b ELISA kits were

purchased from eBiosciences, San Diego, CA. Formalin was procured from Qualigens Fine Chemicals, GlaxoSmithKline Pharmaceuticals Ltd, Mumbai, India. Acetic acid was purchased from Sisco Research Laboratories Pvt. Ltd, Mumbai, India. Dipotassium hydrogen phosphate and potassium dihydrogen orthophosphate were purchased from Himedia, Mumbai, India. N-Naphthylethylenediamine, sulfanilamide, and all other chemicals were purchased from Sigma (St Louis, MO). Isolation of the compound The E. jambolana leaves were collected from the plant natural forest area of Central Research Farm of Indian Grassland fodder Research Institute (IGFRI), Jhansi (UP) in August, 2011, and the identity of the plant was confirmed by A. Singh, senior scientist, Grassland Silvipasture Management Division, IGFRI, Jhansi (UP), deposited at herbarium (Voucher no. IGFRI – 38101). The dried and powdered leaves (6 kg) were extracted exhaustively with alcohol by percolation method at room temperature. The combined extract was concentrated under vacuum at 40  C and extractive was segregated into water soluble and insoluble fractions. The water insoluble fraction was subsequently partitioned with hexane, benzene, ethyl acetate, acetone, and methanol. The concentrated ethyl acetate soluble fraction (55.9 g) was chromtographed in two sets simultaneously on Si-gel (60–120 mesh) columns (100  2.5 cm, 264 g each) and eluted with dichloromethane and methanol in different ratios with increasing polarity. The eluents collected from dichloromethane:methanol (9:1) yielded a yellow crystalline, thin layer chromatography (TLC) homogeneous substance with a melting point, 290  C. It responded to all the tests of phenolic nature, cherry red with Shinoda test, and positive Molisch’s test for sugar. The compound C42H38O19 (M + H, 847, FABMS) was degraded into aglycone and glycone (sugar) part by acid hydrolysis, which were identified as kaempferol and L -rhamnose, respectively, by co-comparison with the

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authentic sample. Finally, the diglycoside nature of the molecule was established by spectral data of UV, 1H, 13C and Hetero Multiple Bond Coherence NMR and compound is named as [kaempferol-7-O-a-L-rhamnopyranoside]-40 -O-40 [kaempferol-7-O-a-L-rhamnopyranoside]. It is first report of kaempferol as diflavoinoidal rhamnoside from E. jambolana leaves.

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Animals Throughout this study, male Swiss albino mice (18–25 g) were used except in the acetic acid writhing test, where the female mice were used. Healthy mice were procured from Laboratory Animal Resource Section of the Institute. These animals were kept in polypropylene cages in ambient environment (room temperature 24 ± 2  C; relative humidity 60–70%; 12 h light–dark cycle) and maintained on a balanced ration obtained from the Feed Technology Unit of the Institute, offered fresh drinking water ad libitum daily. The housing conditions and experimental protocols were duly approved by the Animal Ethics Committee of the Indian Veterinary Research Institute. The mice were acclimatized for 1 week before use.

Analgesic studies Peripheral analgesic activity Formalin-induced hypernociception in mice Antinociceptive activity of flavonoid, EJ-01, was assessed by the formalin-induced hypernociception model in male Swiss albino mice as described previously (Hunskaar et al., 1985). Mice were divided into five groups of six animals each. Group I animals were treated with vehicle orally and served as the vehicle control. Groups II, III, and IV animals were treated with EJ-01 at 3, 10, and 30 mg kg1, respectively orally. Group V animals served as the positive control and they were administered standard reference anti-inflammatory drug etoricoxib (10 mg kg1; orally) (Wani et al., 2012). One hour after vehicle or drug administration, each mouse was injected subplantarly with a single dose of 20 mL of 2.5% formalin solution (0.92% formaldehyde) made up in saline. The animals were immediately placed in a glass cylinder of 20 cm in diameter and the time spent in licking the injected paw was recorded. Animals were observed from 0 to 5 min (neurogenic phase) and 15 to 30 min (inflammatory phase) and the time spent licking the injected paw was recorded with a chronometer and considered as an indicative of nociception. The time spent in paw licking in drugtreated groups was compared with that of the vehicle control group. Acetic acid-induced abdominal constriction test (writhing test) in mice Analgesic activity of flavonoid EJ-01 was assessed by the acetic acid-induced abdominal constriction test (writhing test) in female mice (Witkin et al., 1961). Female Swiss albino mice were divided into five groups of six animals each. Group I animals were administered vehicle and served as the vehicle control. Groups II, III, and IV animals were administered EJ-01 at 3, 10, and 30 mg kg1 orally. Group V

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animals were administered etoricoxib (10 mg kg1) orally. One hour after vehicle and drug administration, each mouse was injected with 3% (v/v) acetic acid, 300 mg kg1 intraperitoneally. The animals were immediately placed in a glass cylinder, 20 cm in diameter and the number of stretches or writhes (arching of the back, elongation of the body and extension of the forelimbs) were counted cumulatively over a period of 20 min. The number of writhings in drug-treated groups was compared with that of the vehicle control group. Central analgesic activity Hot plate test in mice Central analgesic activity of EJ-01 was assessed by the hot plate test using Eddy’s hot plate (Eddy & Leimbach, 1953). Overnight fasted male Swiss albino mice were placed individually on a thermostatically controlled heated metal plate (Ugo Basil, Italy) within a restraining perspex cylinder and the reaction time of each mouse was recorded. The temperature of the hot plate was maintained at 55 ± 0.5  C. The reaction time was considered as the time elapsed between placing of mouse on the hot plate and appearance of signs of acute discomfort characterized by flicking or licking of the hind paw, forepaw or jumping in an attempt to escape from the pain. The cut-off time of 20 s was used to prevent tissue damage. The mice showing initial reaction time of 10 s or less were selected for this study and were divided into five groups of six animals each. Group I animals were administered vehicle and served as the vehicle control. Groups II, III, and IV animals were administered EJ-01 at 3, 10, and 30 mg kg1 orally. Group V animals served as a positive control and they were administered the standard reference central analgesic drug morphine hydrochloride (10 mg kg1; intraperitoneally) (Bilkei-Gorzo et al., 2010). One hour after vehicle or drug administration, each mouse was subjected to hot plate test as described above. The reaction time in drug-treated groups was compared with that of the vehicle control group. Tail-flick test in mice Central analgesic activity of flavonoid EJ-01 was assessed by using the tail-flick test (D’Amour & Smith, 1941). Male Swiss albino mice were divided into five groups of six animals each. Before administration of any drug or vehicle, the baseline withdrawal latency (seconds) was recorded twice with 20 min intervals between readings for each mouse. The intensity of the light beam was focused on animal tails 2–2.5 cm from the tip. The intensity of heat stimulus was set in such a fashion that it has to elicit a tail flick within 10–12 s and the cut-off time of 20 s was used to prevent tissue damage. After this, Group I animals were administered vehicle and served as the vehicle control. Groups II, III, and IV animals were administered EJ-01 at 3, 10, and 30 mg kg1, respectively, orally. Group V animals were administered morphine hydrochloride at 10 mg kg1 intraperitoneally. One hour after vehicle or drug administration, each mouse was subjected to tail-flick test as described above. The reaction time in the drug-treated groups was compared with that of the vehicle control group.

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In vitro studies Anti-inflammatory activity in LPS-treated murine macrophage cell line (RAW 264.7)

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Procurement, propagation, and maintenance of cell line RAW 264.7 cells were obtained from cell repository of animal cells, National Centre for Cell Science, Pune, India. Subculture: on the receipt of a 25 cm2 flask containing cell monolayer, the cells were subcultured using DMEM with 4 mM L-glutamine adjusted to contain 1.5 g L1 sodium bicarbonate and 4.5 g L1 glucose and supplemented with 10% (v/v) FCS and antibiotic (100 U mL1 penicillin, 100 mg mL1 of streptomycin). The growth conditions were 37  C and a humidified atmosphere containing 5% CO2 condition. The cells were subcultured again as soon as they reach confluency. The old medium was removed and fresh DMEM with 10% FCS was added. Then the cells were dislodged using a cell scraper and dispensed into a new flask, kept in an incubator with slightly loose cap. The volume of medium used for the subculture was according to usual ratio of medium volume to surface area, i.e., 0.2–0.5 mL cm2. Whenever needed, the number of flasks was increased by subculture and maintained by DMEM with 2% FCS, changed three times per week. Preparation of stock solution of EJ-01 EJ-01 was suspended in DMSO at a concentration of 100 mg mL1 (DMSO final concentration ¼ 0.1%). Different concentrations of EJ-01 were prepared from this stock solution in DMEM with 2% FCS, sterilized by filtration. For total nitrite plus nitrate (NOx) estimation, all concentrations were prepared in phenol red-free DMEM. Preparation of stock concentration of LPS Stock solution of LPS (Escherichia coli 055:B5, SigmaAldrich, St. Louis, MO) (200 mg mL1 in phosphate-buffered saline (PBS), pH 7.4) was prepared and then sterilized by filtration. MTT assay for cell viability The mitochondrial-dependent reduction of MTT assay was used to measure cell respiration as an indicator of cell viability (Denizot & Lang, 1986). MTT, a yellow color dye, is converted into blue color formazan crystals by living cell with active mitochondria. MTT assay was performed to know noncytotoxic concentrations of EJ-01 to be employed for further study. The different concentrations of EJ-01 (25, 50, 100, 150, and 200 mg mL1) were prepared from stock solution in DMEM with 2% FCS. After pre-conditioning, media was decanted and 100 mL of fresh media (DMEM with 2% FCS) containing different concentrations of EJ-01 was added into wells containing cells. Vehicle control cells were incubated with 0.1% DMSO in media and EJ-01 treatment groups were incubated with the above said concentration in media. After 48 h of incubation, 20 mL of MTT dye (5 mg MTT mL1 of PBS) was added in each well and incubated for 4 h in dark at 37  C and 5% CO2. Further media were removed by tilting the plate from side, DMSO (100 mL) was added to each well

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where formazan crystals are formed by viable cells. A violet blue color develops in wells. The absorbance was taken at 550 nm in an ELISA plate reader. The mean OD value of the content of the four well was used as cell viability expression as % of control. The assays were performed in triplicate. Based on the above exploratory work, three increasing noncytotoxic concentrations of EJ-01 (25, 50, and 100 mg mL1) were selected for assessing the effect on NOx, IL-1b, and TNF-a levels in LPS-stimulated macrophage cells. NOx assay For NOx assay, phenol red-free DMEM medium was used from seeding step onward. Cells were seeded in 96-well plates at a concentration of 5  104 mL1 in 10% phenol red-free DMEM medium and incubated for 12 h. After removing medium, cells of groups III, IV, and V were treated with media (phenol red-free DMEM with 2% FCS) containing 25, 50, and 100 mg mL1 of EJ-01, respectively. Cells of group VI were treated with 0.5 mg mL1 dexamethasone in the media and served as a positive control (Zhao et al., 2008). The group I was a naive control (untreated) and group II was a vehicle control treated with vehicle as mentioned above. Now cells of all groups except naı¨ve control were treated with 10 mL of stock solution of LPS. After incubation for 24 h, the supernatant was collected and stored at 20  C for estimation of NOx concentration by previously described method (Sastry et al., 2002). Results were expressed as mean ± SE of three replicates of one representative experiment. Pro-inflammatory cytokines: TNF- and IL-1 assay Cells were seeded in 96-well plates at a concentration of 5  104 in DMEM with 10% FCS and incubated for 12 h. After removing medium, cells of groups III, IV, and V were treated with media (DMEM with 2% FCS) containing 25, 50, and 100 mg mL1 of EJ-01 while cells of group VI were treated with dexamethasone at 0.5 mg mL1 in the media, respectively. The group I was a naive control and group II was a vehicle control. Macrophage cells of all groups except naı¨ve control were treated with 10 mL of stock solution of LPS. After incubation for 24 h, the cell supernatant was collected and stored at 70  C until tested for cytokines. The quantities of these cytokines were estimated by ELISA as per manufacturer’s instruction. Statistical analysis Data were expressed as mean ± S.E.M. The level of statistical significance was determined by a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using the Graph Pad Prism-4 software (GraphPad Software Inc. V2.04, San Diego, CA). Statistical differences were considered significant at ‘‘p’’ value less than 0.05.

Results Peripheral analgesic activity Effects of EJ-01 on formalin-induced hind paw licking The results of oral administration of flavonoid EJ-01 on the early phase (0–5 min) of formalin-induced hind paw licking

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reduced the time spent in paw in licking at the doses of 10 and 30 mg kg1 as compared with the vehicle control. The reference drug etoricoxib at 10 mg kg1 (p50.001) also inhibited significantly the time spent in paw licking as compared with the vehicle control.

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Effects of EJ-01 on acetic acid-induced writhing in female mice The data on the effect of EJ-01 on the number of writhing movements induced by intraperitoneal administration of acetic acid in mice are summarized in Figure 2(C). EJ-01 did not significantly reduce the writhing counts at the dose of 3 mg kg1; however, significantly reduced the writhing counts at the doses 10 (p50.05) and 30 mg kg1 (p50.001) as compared with the vehicle control. The reference drug etoricoxib at 10 mg kg1 also inhibited significantly (p50.001) the writhing counts as compared with the vehicle control. Central analgesic activity Effects of EJ-01 on hot plate method in mice The reaction time following oral administration of different doses of EJ-01 at 1, 3, and 5 h of its administration are summarized in Figure 3(A)–(C), respectively. EJ-01 did not increase significantly the mean reaction time of mice in the hot plate test in 1, 3, and 5 h of its administration at 3 and 10 mg kg1 doses as compared with the vehicle control. EJ-01 at the dose of 30 mg kg1 also did not increase significantly the mean reaction time in the hot plate test at 1 and 3 h, however, increased mean reaction time at 5 h (p50.001). The reference drug morphine hydrochloride at 10 mg kg1 dose intraperitoneally increased significantly the mean reaction time of hot plate test at 1 (p50.01) and 3 h (p50.001) of its administration as compared with the vehicle control, but failed to increase significantly the mean reaction time of a hot plate test at 5 h of its administration as compared with the vehicle control. Figure 2. Mice were treated with the indicated dose of EJ-01(PO) and etoricoxib (PO) 30 min before chemical nociceptive stimuli (formalin 2.5% v/v; 20 mL; intraplantar). Observations (number of flinches/ lickings) were recorded at (0–5 min) early phase (A) and (15–30 min) late phase (B). Acetic acid writhing test (acetic acid 3% v/v; 300 mg/kg IP) observations (number of writhes) were recorded over a period of 20 min after stimuli (C). Bars (mean ± SE; six animals were kept in each group) with dissimilar superscript differ significantly (p50.05).

are summarized in Figure 2(A). In this model, EJ-01 did not significantly exhibit the analgesic activity in the early phase (0–5 min) at any of the doses employed in this study as compared with the vehicle control. The reference drug etoricoxib at 10 mg kg1 also did not significantly reduce paw-licking time in this phase as compared with the vehicle control. The results of oral administration of flavonoid, EJ-01 on the inflammatory phase (15–30 min) of formalin-induced hind paw licking are summarized in Figure 2(B). In the inflammatory phase, EJ-01 did not significantly reduce the time spent in paw licking at the dose 3 mg kg1 as compared with the vehicle control, however, significantly (p50.001)

Effects of EJ-01 on tail flick test in mice The tail flick latency data following oral administration of different doses of EJ-01 at 1, 3, and 5 h of its administration are summarized in Figure 3(D)–(F), respectively. EJ-01 did not increase significantly the tail flick latency at 1, 3, and 5 h of its administration at 3 and 10 mg kg1 doses as compared with the vehicle control. EJ-01 at 30 mg kg1 dose also did not increase significantly the tail flick latency at 1 and 3 h, however increased tail flick latency at 30 mg kg1 at 5 h (p50.001). The reference drug morphine hydrochloride at 10 mg kg1 dose intraperitoneally increased significantly the tail flick latency at 1 (p50.001) and 3 h (p50.001) of its administration as compared with the vehicle control but failed to increase significantly the tail flick latency at 5 h of its administration as compared with the vehicle control. Effects of EJ-01 on LPS-stimulated murine macrophage cell line (RAW 264.7) MTT assay. In the first place, a MTT assay for cell

viability was performed to determine three non-cytotoxic

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Figure 3. Mice were treated with the indicated dose of EJ-01(PO) and morphine (IP) 30 min before thermal nociceptive stimuli (hot plate test). Observations (jumping/hind paw flinching/hind paw licking) were recorded at 1 h (A), 3 h (B), and 5 h (C) of test. Tail flick test observations (tail withdrawal latency) were recorded at 1 h (D), 3 h (E), and 5 h (F) of test. Bars (mean ± SE; six animals were kept in each group) with dissimilar superscript differ significantly (p50.05). Table 1. Effect of EJ-01 on RAW 264.7 cells viability (MTT assay).

EJ-01 (mg/ml) 0 25 50 100 150 200

produced significant decrease in cell viability at 150 and 200 mg mL1 (p 5 0.001) concentrations.

Cell survival (% of control) (mean ± S.E.M.) 100.00 ± 0.00a 98.26 ± 0.90a 97.8 ± 1.27a 95.43 ± 2.24a 75.46 ± 3.70b 66.87 ± 3.72b

RAW 264.7 cells were treated with various concentrations of EJ-01 for 24 h and cell viability was measured by MTT assay. Values (mean ± SE from three separate experiments) with dissimilar superscript differ significantly (p50.05).

concentrations of EJ-01. Results of MTT cell viability assay are summarized in Table 1. EJ-01 did not produce significant decrease in cell viability at 25, 50, and 100 mg mL1 (p40.05) concentrations as compared with the control, but

Effects of EJ-01 on NOx levels in LPS-stimulated murine macrophage cell culture supernatants The results of effect of EJ-01 on NOx levels (nmol mL1, in the LPS-treated cell culture medium are summarized in Figure 4(A). EJ-01 at 25 and 50 mg mL1 concentrations did not produce significant inhibition of NOx levels in the LPS-stimulated cell culture medium as compared with the vehicle control; however, it produced significant inhibition on NOx levels in the LPS-stimulated cell culture medium at 100 mg mL1 (p50.001) concentration as compared with the vehicle control. The reference drug dexamethasone significantly inhibited the NOx levels in the LPS-treated cell culture medium at 0.5 mg mL1 (p50.001) concentration as compared with the vehicle control.

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Figure 4. RAW 264.7 cells were co-incubated with the indicated concentrations of EJ-01 and LPS (2 mg mL1) for 24 h in 10% phenol red-free DMEM medium containing 2% FCS. Naı¨ve control cells were neither treated with EJ-01 nor stimulated with LPS. Vehicle control cells were incubated with vehicle alone. The culture supernatants were analyzed for NOx (A), TNF-a (B), and IL-1b (C) by ELISA. Bars (mean ± SE from three separate experiments) with dissimilar superscript differ significantly (p50.05).

Effects of EJ-01 on pro-inflammatory cytokines: TNF- and IL-1 levels in LPS-stimulated murine macrophage cell culture supernatants The results of effect of EJ-01 on pro-inflammatory cytokine TNF-a and IL-1b levels (pg mL1) in the LPS-treated cell culture medium are summarized in Figure 4(B) and (C), respectively. EJ-01 produced significant inhibition of TNF-a and IL-1b levels in the LPS-stimulated cell culture medium at all the concentrations (25, 50, and 100 mg mL1) (p50.001) as compared with the vehicle control. The reference drug dexamethasone significantly inhibited the TNF-a and IL-1b levels in the LPS-stimulated cell culture medium at 0.5 mg mL1 (p50.001) concentration as compared with the vehicle control.

Discussion Although E. jambolana has been reported to have several biological functions, mostly related to antidiabetic activity (Ayyanara & Subash-Babu, 2012), the effects on pain and

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inflammation and their underlying mechanisms have not yet been investigated. An analgesic effect usually accompanies an anti-inflammatory effect. We, therefore, examined the analgesic effect of EJ-01 with four nociceptive animal models along with in vitro anti-inflammatory action. In this study, we have provided the first evidence showing the analgesic effect of EJ-01 in vivo. The most relevant additional findings of this work were that the EJ-01 is an inhibitor of LPS-induced TNF-a, IL-1b, and NO production in 264.7 macrophages. The methods used for investigating antinociception for peripheral and central mediated effects were the acetic acid and formalin test (chemical stimuli), while the hot plate and the tail flick test (thermal stimuli) served specifically for the central activity. In the acetic acid-induced writhing assay, a chemical stimulus is used to screen both peripheral and central analgesic activities (Panthong et al., 2007). The writhing test can predict effective analgesic doses for agents that can be used in humans (Dubinsky et al., 1987; Eaton, 2003). The mediators involved in the genesis of the nociception observed in the writhing test are the eicosanoids and sympathomimetic amines, the release of which is preceded by the release of the nociceptive cytokines TNF-a, IL-1b, and IL-8 (Duarte et al., 1988; Ribeiro et al., 2000a; Thomazzi et al., 1997). The writhing response of the mouse to an injection of noxious chemical is not a very specific nociception model, it is important to reveal a general antinociceptive effect of the compound under study (Marchioro et al., 2005). The EJ-01 at the doses of 10 and 30 mg kg1 significantly reduced the number of writhings in mice induced by acetic acid which suggested that EJ-01 had the effect to decrease visceral pain through peripheral and central mechanism. The formalin test was conducted to confirm the possible analgesic mechanism of action of the EJ-01 which can discriminate pain in central (early phase) and peripheral (late phase) components (Tjolsen et al., 1992). A drug that acts primarily on the central nervous system such as morphine can inhibit both phases (Martindale et al., 2001). Inflammatory pain is selectively inhibited by the NSAIDs because these agents reduce inflammation. In the formalin test, the early phase (0–5 min) is neurogenic phase which is also known as non-inflammatory pain and is mediated by the central effect through a direct activation of the C fibers causing substance P and bradykinin release while the late phase, also termed as inflammatory pain, is due to inflammation and mediated by the peripheral effect via prostaglandins and cytokine release (Granados-Soto et al., 2001). It is well known that intraplantar injections of TNF-a, IL-1b, IL-6, and chemokines induce hyperalgesia and nociception in the rat hind-paw (Cunha et al., 1992). Local treatment with specific antisera against these cytokines reduced the second phase of the formalin nociception which would appear to confirm that cytokines (TNF-a, IL-1b, etc.) produced peripherally are involved in this process. It is also reported that there is participation of bradykinin, cytokines, prostaglandins, and sympathetic amines in the formalin-induced orofacial nociception in rats (Chichorro et al., 2004). EJ-01 failed to reduce the licking time in the early phase of formalin test; this might indicate that it has failed to cause

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the blockade of nociceptive fiber or the release of substance P and bradykinin. Since the EJ-01 could reduce the licking time in the late phase only, it might be due to the inhibition of the inflammatory mediator. Thus, it might be concluded that the analgesic effect of EJ-01 at the late phase is due to the inhibition of the synthesis or release of inflammatory mediators such as cytokines and prostaglandins. The hot plate and the tail flick models are considered as the specific tests for the evaluation of the central pain (Marchioro et al., 2005) at the supraspinal and spinal levels (Wong et al., 1994), respectively. The EJ-01 did not considerably increase the mean reaction time to the heat stimulus in both hot plate and tail flick tests at any time period of the experiment except at 30 mg kg1 dose at 5 h of its administration when compared with the vehicle control. The hot plate method is one of the most common tests for evaluating the analgesic efficacy of drugs in rodents (Somchit et al., 2004). However, care must be taken for drugs that produce falsepositive results by modifying the behavior of the rodents (Tjolsen et al., 1991). The observed effect at 30 mg kg1 dose at 5 h in both the tests indicates a false-positive observation as we did not find antinociceptive effect in the early phase of formalin-induced pain. The central antinociceptive activity is related to activation of the endogenous inhibitory control of pain (Saade & Jabbur, 2008). In the central pain models, the antinociceptive activity was not observed probably due to the difficulty in crossing the blood–brain barrier (BBB) by the EJ-01, revealing a probable supraspinal antinociceptive effect if the compound was administered by intracerebroventricular route (Grisel & Mogil, 2000). Our result shows that EJ-01 does not possess the opioid-like drug activity as it did not show the analgesic activity in any of the central pain model (early phase of formalin-nociception, tail flick, and hot plate test) used in our study which may be due to inability of EJ-01 to cross BBB or lack of intrinsic opioid-like activity which needs to be explored by further investigation. The decreased analgesic activity of morphine in central pain models at the 5th hour of the study may be due to characteristic property of opioid agonists (e.g. morphine, fentanyl, and etorphine) which shows rapid onset with an early maximum effect, which remains for very short period after drug administration, that mediate analgesia via central mechanisms under both normal and inflammatory conditions (Aceto et al., 1997; Millan et al., 1987). Cytotoxicity was determined by the MTT assay. In this test, EJ-01 did not affect the cell viability (RAW 264.7) at 25, 50, and 100 mg mL1 concentrations as compared with the control and therefore these concentrations were employed in the present in vitro anti-inflammatory study. Among the inflammatory mediators, NO, prostaglandin E2 (PGE2), TNF-a, and IL-1b have crucial roles during autoimmune diseases, infections, pain, edema, and fever (Perkins & Kelly, 1994). Pain and the immune system influence each other, making it difficult to determine whether blocking nociception contributes for a reduction in the production of pro-inflammatory cytokines or vice-versa, with the reduction in the formation of pro-inflammatory cytokines resulting in less severe pain (Shavit et al., 2006). In murine macrophage RAW 264.7 cells, LPS induces iNOS transcription and transduction and then the NO

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production (Xie & Nathan, 1994). Furthermore, LPS stimulation is well known to induce NF-kB nuclear translocation (Freeman & Natanson, 2000), a transcription factor necessary for iNOS, COX-2, TNF-a, and IL-1b transcription. Therefore, RAW 264.7 cells provide an excellent model for drug screening and for subsequent evaluation of potential inhibitors of the pathway leading NO production and cytokine release by NF-kB pathway. Based on this information, efforts have been made to reveal the anti-inflammatory activities of EJ-01 on LPS-induced NO, IL-1b, and TNF-a production in murine macrophage RAW 264.7 cells. NO synthesized by iNOS plays a critical role in pain conditions with an inflammatory component and the suppression of NO production can be a very important target in the development of anti-nociceptive and anti-inflammatory agents (De Alba et al., 2006). One needs careful attention while estimating NO in biological systems (Feldman et al., 1993) as NO is rapidly oxidized to nitrite and/or nitrate. NOx content in biological samples is a good indicator of amount of NO synthesis, because quantitatively these compounds are considered to be the most stable metabolite of NO metabolism over the other metabolites (Blum et al., 2000) and measurement of NOx levels is routinely used as an index of NO production (Moshage et al., 1995). In this study, EJ-01 has been demonstrated to inhibit LPS-induced NOx production in RAW 264.7 macrophages (Figure 4A). Similar effects have been demonstrated with the other kaempferol compounds of plant origin (Aggarwal et al., 2009; Hamalainen et al., 2007). To clarify the anti-inflammatory effect of EJ-01, we analyzed the levels of TNF-a and IL-1b in LPS-stimulated macrophages. The cytokines, TNF-a and IL-1b are released by damaged tissue resident macrophages. Bradykinin, TNF-a, IL-1, and IL-8 are particularly important in eliciting the inflammatory pain. These agents liberate prostaglandins, other inflammatory pain mediators and are suggested to be correlated with pain (Burke et al., 2006). Our results are further supported by a related in vivo study in rat formalininduced nociception where TNF-a and IL-1b levels are increased in paw and reduced by ketoprofen (Ahmad et al., 2012) suggesting that EJ-01 might also produce its antinociceptive effect in a similar way in this study. IL-1b can produce hyperalgesia following either intraperitoneal, intracerebroventricular, or intraplantar injection (Perkins & Kelly, 1994; Watkins et al., 1994). Moreover, IL-1b was found to increase the production of substance P and PGE2 in a number of neuronal and glial cells (Jeanjean et al., 1995). Administrations of IL-10 and other anti-inflammatory cytokines have been demonstrated to prevent cytokine mediated inflammatory hyperalgesia (Maier et al., 1993). In the in vitro model, we noted that reduced levels of IL-1b by EJ-01 (Figure 4C) which can suggest that EJ-01 may reduce inflammatory pain. One more cytokine, TNF-a has been shown to play important role in both inflammatory and neuropathic hyperalgesia. Intraplantar injection of complete Freund’s adjuvant (CFA) in adult rats resulted in a significant elevation in the levels of TNF-a, IL-1b, and nerve growth factor (NGF) in the inflamed paw. A single injection of anti-TNF-a antiserum before the CFA significantly delayed the onset of the

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DOI: 10.3109/13880209.2014.885060

inflammatory hyperalgesia and reduced IL-1b but not NGF levels (Woolf et al., 1997). Intraplantar injection of TNF-a also produces mechanical (Cunha et al., 1992) and thermal hyperalgesia (Jeanjean et al., 1995). Apart from the anticytokine antibodies therapies, other anticytokine therapies may also provide benefit in relieving inflammation. Thalidomide and chlorpromazine are able to block TNF activity in vitro (Moreira et al., 1993) and in vivo (Aarestrup et al., 1995; Ghezzi et al., 1996). These compounds which inhibit cytokine production were also shown to be antihyperalgesic both in animals (George et al., 2000; Sommer et al., 1998) and in man (Mehl-Madrona, 1999; Peuckmann et al., 2003). It was shown that thalidomide also has an antinociceptive effect in inflammatory pain. It inhibited carrageenan and LPS-induced mechanical hyperalgesia in rats and inhibited zymosan and acetic acid induced writhing responses in mice. These effects were associated with the inhibition of TNF-a production (Ribeiro et al., 2000b) suggesting that EJ-01 might also produce its antinociceptive effect in a similar way in this study. In conclusion, the EJ-01 isolated from E. jambolana exhibited antinociceptive effect and produced a decrease in NOx, IL-1b, and TNF-a levels in stimulated RAW 264.7 cells, suggesting its anti-inflammatory effect and attenuating inflammatory pain. Our in vivo results contributed to a better knowledge of this medicinal plant and the pharmacological profile of EJ-01. The results suggested that EJ-01 is a valuable analgesic constituent of leaves of E. jambolana and support the pharmacological basis for the use this plant as traditional herbal medicine for treatment of inflammatory pain.

Declaration of interest We have no conflict of interest in the work.

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