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Anti-inflammatory and analgesic effects of human placenta extract

This article was downloaded by: [University of Arizona]On: 05 January 2013, At: 13:14Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Natural Product Research: Formerly
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Anti-inflammatory and analgesic effects
of human placenta extract
Kwan-Hoo Lee a , Tae-Hoon Kim a , Woo-Cheol Lee a , Sang Hoon
Kim b , Sung Youl Lee b & Sun-Mee Lee aa School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, Koreab Business Development, Green Cross Corporation, Yongin,Gyeonggi-do, Korea To cite this article: Kwan-Hoo Lee , Tae-Hoon Kim , Woo-Cheol Lee , Sang Hoon Kim , Sung Youl Lee
& Sun-Mee Lee (2011): Anti-inflammatory and analgesic effects of human placenta extract, Natural
Product Research: Formerly Natural Product Letters, 25:11, 1090-1100
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Natural Product ResearchVol. 25, No. 11, July 2011, 1090–1100 SHORT COMMUNICATION Anti-inflammatory and analgesic effects of human placenta extract Kwan-Hoo Leea, Tae-Hoon Kima, Woo-Cheol Leea, Sang Hoon Kimb,Sung Youl Leeb and Sun-Mee Leea* aSchool of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, Korea; bBusinessDevelopment, Green Cross Corporation, Yongin, Gyeonggi-do, Korea (Received 16 March 2010; final version received 21 April 2010) In this study, we investigated the effects of human placenta extract (HPE,Laennec inj.) on pro-inflammatory cytokines and mediators secreted fromlipopolysaccharide-stimulated RAW264.7 macrophages. We found thatHPE significantly inhibited the production of nitric oxide, tumour necrosisfactor- and cyclooxygenase-2. We studied the anti-inflammatory andanalgesic potential of HPE in murine models of inflammation/inflamma-tory pain. Rats were assigned to six groups and were administered eithersaline or HPE (0.33, 1, 3 and 6 mL kg1) intraperitoneally. Diclofenac wasused as a positive control. HPE attenuated the swelling of the rat's hindpaw. The vascular permeability induced by acetic acid was significantlyreduced by HPE. HPE reduced the formation of granuloma in carrageenanair pouch and hind paw oedema in complete Freund's adjuvant-inducedchronic arthritis in rats. HPE attenuated writhing episodes. An increase inhot-plate latency was observed in mice receiving HPE. HPE also increasedthe pain threshold in the Randall–Selitto test. In the tail-flick assay, HPEprolonged the reaction time of rats to radiant heat stimulation. Theseresults suggest that HPE has potent anti-inflammatory and anti-nociceptiveactivities.
Keywords: acute inflammation; analgesic; chronic inflammation; granu-loma; human placenta extract; pro-inflammatory cytokines Inflammation is a beneficial host response to a variety of stimuli and local injury Downloaded by [University of Arizona] at 13:14 05 January 2013 (Sobota, Szwed, Kasza, Bugno, & Kordula, 2000). The inflammatory reaction can betriggered by physical or chemical trauma, invading organisms and antigen–antibodyreactions, and is often exacerbated by the resultant swelling or oedema of tissues,pain or even cell damage (Yam, Asmawi, & Basir, 2008). Hence, the use of anti-inflammatory agents may be helpful in the therapeutic treatment of pathologiesassociated with the inflammatory reaction. However, the side effects of the currentlyavailable anti-inflammatory drugs pose major complications in their clinical use. Forinstance, some non-steroidal anti-inflammatory drugs may cause gastric ulcerationand renal damage (Perry, 1998). Therefore, anti-inflammatory drugs with less severe *Corresponding author. Email: ISSN 1478–6419 print/ISSN 1478–6427 onlineß 2011 Taylor & FrancisDOI: 10.1080/14786419.2010.489050 Natural Product Research side effects must be developed. There is recent interest in natural products, and manynatural products are attractive as source materials for the development of newmedicines.
Human placenta extract (HPE, Laennec inj.) has been used as a traditional therapeutic agent in many Asian countries for treatment of diseases or replenishmentof vital essence (Yeom et al., 2003). The therapeutic effects of HPE on chronic liverdiseases and inflammatory skin diseases such as psoriasis have also been reported(Banerjee, Bishayee, & Chatterjee, 1995). Recently, HPE was shown to improvemenopausal symptoms and fatigue in middle-aged women (Kong et al., 2008) and toalleviate the arthritic symptoms in adjuvant-induced arthritic rats (AIA; Yeom et al.,2003). In addition, HPE has been shown to possess anti-oxidative activities thatcounteract the adverse consequences of oxidative stress, which interferes with normalmetabolism and physiological balance by damaging biological molecules includingproteins, lipids and DNA (Halliwell, 1997). HPE promotes Interleukin-8 (IL-8)expression through activation of Jun NH2-terminal kinase/stress-activated proteinkinase (JNK/SAPK) and the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells and activating protein-1 (NF-B and AP-1) in a humanmonocytic cell line (Kang et al., 2007). Although HPE has been widely applied invarious clinical settings, limited information is available on the precise mechanismsof its anti-inflammatory properties and related therapeutic efficacy.
Therefore, in this study, we examined the anti-inflammatory and anti-nociceptive properties of HPE in several experimental models.
2.1. Cell viability The cytotoxic effects of HPE are shown in Supplementary Figure S1 (online only).
No cytotoxic effect was observed upto a concentration of 3.6 mL1.
2.2. Effects of HPE on LPS-induced nitrite and cytokine release in RAW 264.7 cell NO, produced by macrophages, increased from 0.64 to 34.71 mmol after LPSexposure. This change was significantly inhibited by HPE of 1.8 and 3.6 mL1(Supplementary Figure S2a – online only). We also investigated the effect of HPE on Downloaded by [University of Arizona] at 13:14 05 January 2013 the production of TNF-, IL-1 and IL-6. As shown in Supplementary Figure S2(b),the levels of TNF-, IL-1 and IL-6 were significantly increased by LPS. HPE dose-dependently inhibited the increase in the level of TNF-. HPE also inhibited theincreases in the level of IL-1 and IL-6 at the concentration of 3.6 mL1.
2.3. Effects of HPE on LPS-induced protein expression of iNOS and COX-2 in The levels of iNOS and COX-2 protein expression in LPS-treated group wereapproximately five- and four-folds higher than the control level, respectively.
The increases in iNOS and COX-2 protein expression were significantly suppressedby HPE in a concentration-dependent manner (Supplementary Figure S3 – onlineonly).
K.-H. Lee et al.
2.4. Effects of HPE on carrageenan-induced oedema in the hind paw Subplantar injection of carrageenan in rats resulted in a time-dependent increase inpaw volume in the vehicle-treated control group (Supplementary Figure S4 – onlineonly); this increase was observed at 1 h and was maximal at 6 h after injection ofcarrageenan injection. HPE at a dose of 0.33 mL kg1 did not affect the paw volumecompared with the vehicle-treated control group. However, at doses of 1, 3 and6 mL kg1, HPE significantly reduced this increase. Diclofenac, used as a positivecontrol, also showed a significant anti-inflammatory effect.
2.5. Effects of HPE on acetic acid-induced vascular permeability As shown in Supplementary Figure S5 (online only), dye leakage induced by aceticacid was 31.6  2.6 mg 10 g1 body weight in the vehicle-treated control group. Noapparent changes in dye leakage were observed at HPE doses of 0.33, 1 and3 mL kg1. However, 6 mL kg1 of HPE significantly reduced the dye leakage intothe peritoneal cavity.
2.6. Effects of HPE on carrageenan-induced air pouch In the vehicle-treated control animals, the volume of exudates was 6.3  0.8 mL andthe granuloma formation was 2.0  0.2 g. There were no significant changes in thevolume of exudates among any of the experimental groups. The granulomaformation was significantly attenuated by 1, 3 and 6 mL kg1 of HPE (Supplementary Table S1 – online only).
2.7. Effects of HPE on AIA In the vehicle-treated control group, the peak paw volume was observed on day 8;paw volume then decreased until day 20. Until day 5 after HPE treatment, there wereno considerable changes compared with the vehicle-treated control group and pawvolume markedly decreased from day 18 to day 20 at doses of 0.33, 1 and 3 mL kg1of HPE. At a dose of 6 mL kg1, HPE significantly decreased the paw volume atday 20 (Supplementary Figure S6 – online only).
Downloaded by [University of Arizona] at 13:14 05 January 2013 2.8. Effects of HPE on acetic acid-induced writhing response The number of writhings was 18.0  1.6 in the vehicle-treated control group.
Treatment of animals with HPE at doses of 0.33, 1 and 3 mL kg1 significantlydecreased abdominal writhing produced by acetic acid. Aminopyrine used as apositive control also significantly reduced the writhing counts (SupplementaryTable S2 – online only).
2.9. Effects of HPE on Randall–Selitto assay The pain threshold of inflamed rat paw was steady at all test times in the vehicle-treated control group. HPE at doses of 0.33 and 1 mL kg1 did not increase the pain Natural Product Research threshold. However, at doses of 3 and 6 mL kg1, HPE caused a significant increasein nociceptive threshold compared with the vehicle-treated control group(Supplementary Figure S7 – online only).
2.10. Effects of HPE on tail-flick test In the vehicle-treated group, the reaction time remained low throughout theexperiment. At a dose of 0.33 mL kg1, HPE significantly increased the reaction timeat 5, 30 and 60 min; at a dose of 1 mL kg1, HPE significantly increased the reactiontime only at 5 min. At doses of 3 mL kg1 and 6 mL kg1, HPE significantlyincreased the nociceptive threshold (Supplementary Figure S8 – online only).
2.11. Effects of HPE on hot-plate test (Supplementary Table S3 – online only). At doses of 0.33, 1 and 3 mL kg1, HPEexhibited anti-nociceptive effects by increasing the latency time to 25.2, 23.6 and25.0 s, respectively, which differed significantly from the vehicle-treated controlgroup.
In this study, the anti-inflammatory and anti-nociceptive activities of HPE wereinvestigated using in vitro cell culture and in vivo animal models.
LPS-stimulated RAW264.7 mouse macrophage cell has been used as an inflammatory cellular model to study the effect of anti-inflammatory drugs andherbs (Kim et al., 2003). Activation of macrophages plays an important role inthe initiation and propagation of inflammatory responses by production ofpro-inflammatory cytokines such as TNF-, IL-1 and IL-6, as well as inflammatorymediators, including NO, which is synthesised by iNOS, and COX-2 (Chan &Riches, 2001). So these key inflammation cytokines and mediators were evaluated inLPS-stimulated RAW264.7 cells to make the anti-inflammatory effects of HPE clear.
HPE showed no cytotoxicity against murine macrophage, but displayed inhibitoryeffects on LPS-induced NO, TNF- and COX-2 production. Thus, our results Downloaded by [University of Arizona] at 13:14 05 January 2013 suggest that HPE has anti-inflammatory activity related with its inhibition of NO,TNF- and COX-2 production.
The carrageenan-induced rat paw oedema is a suitable test for evaluating anti-inflammatory drugs and has frequently been used to assess the anti-oedematouseffects. Oedema development in the paw of the rat after carrageenan injection is abiphasic event (Vinegar, Schreiber, & Hugo, 1969). The initial phase observed duringthe first hour is attributed to the release of histamine and serotonin and the secondphase of oedema is due to the release of prostaglandin (Di Rosa, Giroud, &Willoughby, 1971). In the vehicle-treated control group, the paw volume of ratsignificantly increased after injection of carrageenan, indicating acute inflammatorydamage. In contrast, treatment with HPE markedly attenuated the swelling of rathind paw. These results suggest that HPE may have potential clinical applications fortreating inflammatory disorders.
K.-H. Lee et al.
Increased vascular permeability is one of the essential features of the acute inflammatory response. Mediators of inflammation such as histamine, prostaglan-dins and leukotrienes are released following stimulation (Hseu et al., 2005). Thisleads to dilation of arterioles and venules and to increased vascular permeability(H. Vogel & W. Vogel, 1997); as a result, fluid and plasma protein are extravasatedto the abdominal cavity and oedemas form. HPE reduced the intensity of theperitoneal inflammation produced by acetic acid, indicating that it may have theability to inhibit the permeability of small blood vessels during acute inflammation.
In vivo anti-inflammatory effects of HPE were also tested in animal models of subchronic inflammation. This model was characterised by exudate volume,leukocyte accumulation, granuloma, vascular permeability and protein clearance upto 7 days after injection of carrageenan (Hambleton & Miller, 1989). HPEsignificantly inhibited granuloma formation. This result reflects the ability of HPEto inhibit the proliferation phase of the inflammation process.
We investigated whether the HPE was able to exert anti-inflammatory effects in a chronic inflammatory disease model, i.e. the rat AIA model. HPE significantlyinhibited the development of chronic joint swelling induced by the adjuvant.
Injection of complete Freund's adjuvant into the rat's hind paw induces inflamma-tion at the primary lesion; this inflammation peaks after 3–5 days. Secondary lesionsoccur after injection, i.e. a decrease in weight and reduced immune response.
It appears that bacterial peptidoglycan and muramyl dipeptide are responsible forthe induction of these symptoms (Crofford, 1993). In tests of short duration (such asthose involving carrageenan-induced oedema in rats) as well as in tests of longerduration (such as those based on complete Freund's AIA in rats), HPE exhibitedsignificant anti-inflammatory effects. These results indicate that HPE is effective forthe treatment of not only acute but also chronic inflammation.
HPE exhibited analgesic activity when assessed in abdominal constriction, hot- plate, Randall–Selitto and tail-flick tests. Acetic acid causes algesia due to theliberation of endogenous substances (Berkenkopf & Weichman, 1988). This methodhas been associated with prostanoids in general, e.g. increased levels of PGE2 andPGF2, in peritoneal fluids (Deraedt, Jouquey, Delevallee, & Flahaut, 1980) as wellas lipoxygenase products (Dhara, Suba, Sen, Pal, & Chaudhuri, 2000). In the pawpressure test developed by Randall and Selitto, half of the oedematous response toyeast results from direct capillary changes produced by released 5-HT (Stone, Downloaded by [University of Arizona] at 13:14 05 January 2013 Wenger, Ludden, Starvorski, & Ross, 1961). Inflammation and strong hyperalgesiadevelop 1–3 h after yeast is injected (Winter & Flataker, 1965). HPE exertedsignificant inhibitory effects on the writhing response and increased the painthreshold, signifying peripherally mediated activities.
To assess the central mechanism of HPE in producing acute analgesia, the hot-plate and tail-flick methods were employed. In these methods, pain is mediatedthrough a central mechanism. These methods can be further distinguished bythe response to pain stimuli conducted through different pathways. The tail-flickresponse is mediated by a spinal reflex to nociceptive action, while thehot-plate response involves higher brain centres and is considered a supraspinallyorganised response (Chapman et al., 1985). HPE significantly increased the reactiontime in the hot-plate test and the withdrawal latency time in the tail-flick test, therebyindicating its central action in producing an analgesic effect.
Natural Product Research Overall, HPE appears to have potent anti-inflammatory and analgesic activities, indicating that HPE could have therapeutic applications in various diseasesassociated with inflammation.
Dulbecco's modified Eagle's medium (DMEM), Dulbecco's phosphate buffer saline(PBS), penicillin/streptomycin (1000 U mL1, 1000 mg mL1, respectively) and foetalbovine serum (FBS) were obtained from Gibco BRL, Life Technologies (GrandIsland, NY, USA). Lipopolysaccharide (LPS; Escherichia coli serotype 0127:B8),3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT), and all theother materials required for culturing of cells were purchased from Sigma ChemicalCompany (St Louis, MO, USA). Type IV lambda carrageenan, Brewer's yeast,complete Freund's adjuvant, diclofenac and Evans blue were supplied by SigmaChemical Co. (USA). All the other chemicals used in this study were of reagent gradeand were locally and commercially available.
4.2. Preparation and composition of HPE We used Laennec, the hydrolysate of human placenta commercially marketed underthe trade name HPE, supplied by Green Cross Japan Bio Products Ltd. (Korea).
Human placentas collected upon full-term delivery were immediately placed in ice.
They were tested for human immunodeficiency virus and hepatitis B and C virusesand then cut into pieces and extracted with water using enzymatic molecularseparation and chemical hydrolysis. Extraction and sterilisation were performedunder the quality control guidelines. The final products of HPE were stored in 2 mLampules. Each ampule contained various amino acids including arginine (0.08%),lysine (0.1%), phenylalanine (0.08%), tyrosine (0.03%), leucine (0.12%), methionine(0.03%), valine (0.04%), alanine (0.08%), serine (0.07%) and threonine (0.06%).
Insoluble macromolecules such as polysaccharides and polynucleotides wereexcluded during the manufacturing process.
Downloaded by [University of Arizona] at 13:14 05 January 2013 4.3. Cell culture Macrophage RAW264.7 cells were obtained from American Type Culture Collection(Manassas, VA, USA). These cells were maintained at subconfluence in 95% air, 5%CO2 humidified atmosphere at 37C. The medium for routine subculture wasDMEM supplemented with 10% FBS, 2 mmol glutamine and 100 mg mL1 penicil-lin/streptomycin.
4.4. Cell viability The effect of the concentration of HPE on cell viability was assessed using a modifiedMTT assay (Mosmann, 1983). Briefly, cells (1  104 cells per well) were seeded in a96-well plate and incubated at 37C for 24 h. The cells were treated with various K.-H. Lee et al.
concentrations of HPE or a vehicle (PBS) and positive controls alone and wereincubated at 37C for additional 24 h. After incubation, 100 mL of MTT solution(5 mg mL1 in PBS) was added to each well and the plates was further incubated for3 h at 37C. Subsequently, 100 mL of dimethyl sulphoxide was added to each well tosolubilise any deposited formazon. The optical density of each well was measured at450 nm using a microplate reader (Emax, Molecular Devices, Sunnyvale, CA).
4.5. Nitrite assay Nitrite concentration in the medium was measured as an indicator of nitric oxide(NO) production according to the Griess method reported by Hishikawa, Nakaki,Suzuki, Saruta, and Kato (1992). In brief, RAW264.7 cells were plated at a density of1  105 cells mL1 in a 24-well cell culture plate with 500 mL of culture medium,incubated for 24 h, pre-treated with indomethacin (50 mmol), dexamethasone(1 mmol) or HPE (0.0018, 0.018, 0.18 and 3.6 mL1) for another 2 h, and thenchallenged with LPS (1 mg mL1) for an additional 24 h. Equal volumes of culturemedium and Griess reagent (1% sulphanilamide in 5% phosphoric acid and 0.1%naphthylethylenediamine dihydrochloride in distilled water) were mixed, and theabsorbance of the mixture at 540 nm was determined with a microplate reader(Molecular Devices, Emax, Sunnyvale, CA).
4.6. Measurement of cytokines by enzyme-linked immunoassay RAW264.7 cells were plated at a density of 1  106 cells mL1 in a six-well cell cultureplate with 2 mL of culture medium, incubated for 24 h, pre-treated with indometh-acin (50 mmol), dexamethasone (1 mmol) or HPE (0.0018, 0.018, 0.18 and 3.6 mL1)for another 2 h, and then challenged with LPS (1 mg mL1) for an additional 24 h.
Inflammatory mediators such as tumour necrosis factor- (TNF-), IL-1 and IL-6were quantified at 24 h after LPS treatment by commercial mouse enzyme-linkedimmunosorbent assay (ELISA) kits (eBioscience, San Diego, CA, USA) according tothe manufacturer's instructions.
Downloaded by [University of Arizona] at 13:14 05 January 2013 4.7. Western blot assay RAW264.7 cells were pre-treated with indomethacin (50 mmol), dexamethasone(1 mmol) or HPE (0.0018, 0.018, 0.18 and 3.6 mL1) for 2 h, and stimulated with LPS(1 mg mL1) for 24 h. Twenty micrograms of total protein extracts was separated on10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) andelectro-transferred Germany). The membranes were blocked with 5% non-fat milk for 1 h and thenincubated overnight with polyclonal antibody for inducible nitric oxide synthase(iNOS, Transduction Laboratories, San Jose, CA, USA) and cyclooxygenase-2(COX-2, Cayman, Ann Arbor, MI, USA) and monoclonal antibody for -actin(Sigma, St. Louis, MO, USA). The binding of all the antibodies was detected usingan enhanced chemiluminescence (ECL) detection system (iNtRON Biotechnology,Seoul, Korea), according to the manufacturer's instructions. The intensity of the Natural Product Research immunoreactive bands was determined using a densitometric analysis program(Image Gauge V3.12, FUJIFILM, Japan).
4.8. Treatment of animals Male Sprague-Dawley rats (150–250 g) and imprinting control region (ICR) mice(25–28 g) were obtained from DaeHan Biolink Co. (Korea) and were acclimated tothe laboratory conditions for at least 1 week. All animals were treated humanelyunder the Sungkyunkwan University Animal Care Committee guidelines. HPE wasdiluted in saline (vehicle) and administered at doses of 0.33, 1, 3 and 6 mL kg1. Sixtreatment groups (n ¼ 8–16) were examined: (1) vehicle-treated control; (2–5) HPE-treated groups; (6) positive drug (diclofenac)-treated group. This HPE dosingregimen was selected because it had been evaluated previously (Liu, Yoshii, &Kawabata, 2004).
4.9. Carrageenan-induced hind paw oedema The carrageenan-induced paw oedema described by Winter, Risley, and Nuss (1962)was used with slight modifications. HPE and were administered by intraperitoneal(i.p.) injection 30 min before subplantar injection of 0.1 mL of 1% sodiumcarrageenan into the rat's right hind paw. Right paw volumes were measuredimmediately and at 0.5, 1, 2, 4, 6, 8, 10 and 12 h after injection of carrageenan using awater plethysmometer from Ugo Basile (Italy). The rate of paw oedema wascalculated according to the following equation: Oedema rate ð%Þ ¼ ðB  AÞ=A  100%, where B is the volume of the paw after carrageenan injection and A is that before theinjection.
4.10. Acetic acid-induced vascular permeability After 30 min of intramuscular injection of HPE, 2.5% Evans blue solution wasintravenously injected into mice and 20 min later, 10 mL g1 body weight of 0.6% ofacetic acid solution was intraperitoneally injected. After another 20 min, mice were Downloaded by [University of Arizona] at 13:14 05 January 2013 sacrificed and the peritoneal cavity was washed with saline. The absorbance of thesupernatant of the peritoneal cavity fluid was measured at 630 nm using aspectrophotometer (Whittle, 1964).
4.11. Carrageenan-induced air pouch 8 mL of air was injected into the dorsal subcutaneous space of rats to create a pouch.
0.2% carrageenan-saline solution was injected into the pouch 24 h after the airinjection (Hambleton & Miller, 1989). Vehicle, HPE or diclofenac (5 mg kg1) wereintraperitoneally administered once-daily at the same time for 7 days. After 24 h offinal treatment, animals were sacrificed and the exudates and granuloma werecollected and measured.
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4.12. Adjuvant-induced arthritis According to the method reported by Cox and Welch (2004), animals weresubcutaneously injected in the base of the right hind paw with 1 mg of heat-killedMycobacterium butyricum in 1 mL of complete Freund's adjuvant. After 11 days ofadjuvant injection, HPE was intraperitoneally administered daily for 10 days.
Arthritis was assessed by measuring the degree of swelling in the paw with aplethysmometer. The oedema rate was measured as described above.
4.13. Acetic acid-induced writhing response HPE was intramuscularly administered 30 min before the experiment, and acetic acidin saline (0.7%, 0.1 mL 10 g1 body weight) was then injected intraperitoneally. After5 min of the IP injection of acetic acid, the number of writhings was counted for thenext 10 min (Koster, Anderson, & de Beer, 1959).
4.14. Randall–Selitto assay A 20% suspension of Brewer's yeast (0.1 mL) was injected into the plantar of thehind paw of rats. Two hours after the injection of Brewer's yeast, HPE wasintraperitoneally administered. Subsequently, the nociceptive threshold of both theinflamed and intact paws of each group was measured using an analgesymeter at 0.5,1, 2, 3 and 4 h after treatment. The nociceptive threshold was defined as the force atwhich the rat vocalised or withdrew its paw (Randall & Selitto, 1957).
4.15. Tail-flick test Tail-flick latencies were determined 5, 15, 30, 60, 90, 120 and 180 min following i.p.
injection of HPE. The heat was focussed approximately midway along the tail andthe latency time for removal of the tail from the stimulus was recorded. A 15 s cutoffwas imposed to prevent tissue damage (Minami et al., 2009).
4.16. Hot-plate test Downloaded by [University of Arizona] at 13:14 05 January 2013 For the hot-plate test, the temperature of the hot-plate was set at 55C  0.5C.
Thirty minutes after i.p. injection of HPE, the mice were placed on the hot-plate andthe latency time that elapsed until the animal jumped or licked one of its hind pawswas recorded in seconds. A cut-off time of 30 s was imposed to prevent tissue damage(Woolfe & MacDonald, 1944).
4.17. Statistical analysis Overall significance was tested by one-way analysis of variance (ANOVA).
Differences between groups at specific time points were considered significant atp 5 0.05, with the appropriate Bonferroni correction made for multiple comparisons.
All results are presented as means  standard error mean (SEM).
Natural Product Research Supplementary material Tables S1–S3 and Figures S1–S8 relating to this paper are available online.
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Am J Physiol Cell Physiol 286: C1045–C1052, 2004. First published December 18, 2003; 10.1152/ajpcell.00360.2003. Protection against hypoxia-induced blood-brain barrier disruption: changes in intracellular calcium Rachel C. Brown, Karen S. Mark, Richard D. Egleton, and Thomas P. DavisDepartment of Pharmacology, University of Arizona, Tucson, Arizona 85724

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