Journal of Ethnopharmacology 148 (2013) 940–945

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Aqueous ethanol extract of the fruit of Xylopia aethiopica (Annonaceae)exhibits anti-anaphylactic and anti-inflammatory actions in mice

David D. Obiri n, Newman OsafoDepartment of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science andTechnology, Kumasi, Ghana

a r t i c l e i n f o

Article history:Received 7 March 2013Received in revised form6 May 2013Accepted 23 May 2013Available online 6 June 2013

Keywords:Xylopia aethiopicaMast cellsAllergyAnaphylaxisAnti-inflammatory

41/$ - see front matter & 2013 Elsevier Irelanx.doi.org/10.1016/j.jep.2013.05.047

esponding author. Tel.: +233 3220 60372, +233 3220 6063684.ail addresses: ddobiri.pharm@knust.edu.gh,i@yahoo.com (D.D. Obiri).

a b s t r a c t

Ethnopharmacological relevance: Xylopia aethiopica has been traditionally used in the form of the driedfruit decoction to treat bronchitis, asthma, arthritis and rheumatism in Ghana, Nigeria and Cameroon.Aim of the study is to evaluate the anti-anaphylactic and anti-inflammatory effects of a 70% aqueousethanol extract of the fruits of Xylopia aethiopica.Materials and methods: Systemic anaphylaxis was induced by the injection of either compound 48/80 orlipopolysaccharide, LPS and survival rates of mice monitored for 1 h or 7 days respectively while IgE-mediated anaphylaxis in a local allergic reaction was studied in the pinnal inflammation model in mice.Clonidine-induced catalepsy in mice was used to evaluate the indirect antihistamine effect of Xylopiaaethiopica, XAE. The effects of XAE assessed on the maximal and total oedema responses in thecarrageenan-induced paw oedema in mice was used to evaluate the anti-inflammatory action of theextract.Results: Administered at 30, 100, 300 and 1000 mg kg−1 p.o., XAE dose dependently suppressedcompound 48/80-induced mouse systemic anaphylactic shock and offered 63% protection to miceagainst LPS-induced endotoxic shock at a dose of 300 mg kg−1. In addition, the extract (30–300 mg kg−1)in a dose dependent manner significantly inhibited by 23–62% the mouse pinnal inflammation.Clonidine-induced catalepsy in mice was significantly suppressed in a dose and time dependent mannerwhen administered both prophylactically and therapeutically. In the same doses, when administeredbefore the induction of the mouse carrageenan-induced paw oedema, the mean maximal swellingattained during 6 h was reduced to 41.0276.94%, 35.6174.30%, and 29.0974.90% of the inflamedcontrol response respectively and total paw swellings induced over the 6 h were also dose-dependentlyand significantly suppressed to 74.84714.84%, 63.9579.37%, and 48.13710.90% of the inflamed controlresponse respectively. Administered after the induction of the carrageenan paw oedema the meanmaximal swelling attained during 6 h was suppressed to 49.8473.95%, 43.6271.01%, and 35.9771.34%of the inflamed control response respectively while the total paw swellings induced over the 6 h werealso dose-dependently and significantly suppressed at 100 and 300 mg kg−1 to 72.3974.38% and60.8173.25% of the inflamed control response respectively.Conclusion: These findings suggest that XAE inhibits mast cell-dependent immediate allergic reactionsand exhibit anti-inflammatory actions through the inhibition of histamine release from mast cells viastabilizing the cell membrane. Our results contribute towards validation of the traditional use of Xylopiaaethiopica in the treatment of bronchitis, asthma, arthritis and rheumatism.

& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Anaphylaxis, the most dramatic clinical presentation of allergyis one of the common human disorders of the immune system.It presents with symptoms collectively termed immediate

d Ltd. All rights reserved.

3 24 4573543 (mobile);

hypersensitivity reactions that could potentially be life threaten-ing. This acute allergic response is mediated by mast cells andbasophils (Nigovic and Lee, 2005; Yu et al., 2006). Mast cellsexpress among other surface receptors the high affinity receptorfor immunoglobulin E, IgE (FcεRI) which is critical for mast celldevelopment and function (Kinet, 1999). In the absence of anantigen, the FcεRI binds to IgE. Subsequent cross-linking of theIgE-bound FcεRI by a cognate antigen triggers a cascade ofsignalling pathways resulting in three downstream responses;degranulation, release of arachidonic acid and its subsequent

D.D. Obiri, N. Osafo / Journal of Ethnopharmacology 148 (2013) 940–945 941

metabolism and finally the release of an array of cytokines. Analternative pathway of activation of the mast cell which is a non-immunologic mechanism is the peptidergic pathway. This isachieved by polycationic compounds such as compound 48/80referred to as the basic secretagogues of mast cells (Langunoffet al., 1983). Stimulation of mast cells with IgE or compound 48/80triggers the activation of signal transduction pathways, which leadto pro-inflammatory mediator release notably histamine (Readand Lenney, 1972). All of these mediators contribute to both acuteanaphylaxis and delayed chronic inflammation (Kemp and Lockey,2002; Kraft and Kinet, 2007; Rivera and Gilfillan, 2006; Beaven,2009). The life threatening side effects of orthodox drugs incurrent use for treatment of inflammation and allergic conditionshave stimulated the search for new regimens especially from plantsources. In addition, glucocorticoids which are the most potentanti-inflammatory drugs are ineffective in anaphylaxis (Choo et al.,2012) or effective only when combined with a phosphataseinhibitor (Obiri et al., 2012). One of such plants for which thedried fruit decoction is used to treat bronchitis, asthma, arthritisand rheumatism in Ghana, Nigerian and Cameroon is Xylopiaaethiopica (Burkill, 1985).

Xylopia aethiopica [Dunal] A. Rich (Annonaceae) is commonlyknown as “African guinea pepper” or “Ethiopian pepper”. It is atropical evergreen tree that grows as high as 20 m. The seeds arearomatic and contain bitter principles. Among the secondarymetabolites are alkaloids, glycosides, saponins, tannins, sterols,carbohydrates, protein and free fatty acids, mucilages and acidiccompounds (Burkill, 1985).

In the current study, we show that aqueous ethanol extract ofXylopia aethiopica has inhibitory effects on experimental immedi-ate allergic reactions and also exhibit anti-inflammatory actionsin mice.

2. Materials and methods

2.1. Materials

2.1.1. Preparation of plant extractSamples of the dried fruits of Xylopia aethiopica [Dunal] A. Rich

were purchased from a commercial herb market in Kumasi inMarch, 2012. The identity was confirmed as the fruit of Xylopiaaethiopica [Dunal] A. Rich by anatomical observation and directcomparison with the authentic specimens stored in the Herbariumin the Department of Herbal Medicine, KNUST, Kumasi. A voucherspecimen (No. FP/09/77) has been deposited in the same depart-ment. 2.7 kg of the dried fruit was ground using a heavy dutyblender (37BL85 (240CB6), WARING Commercial, USA) andextracted with 70% v/v aqueous ethanol (5 L) by maceration for24 h. The ethanol filtrate was concentrated under reduced pres-sure at 45 1C by a vacuum rotary evaporator (R-210, BUCHI,Switzerland) and further dried in an oven (Gallenkamp OMT,SANYO, Japan) to yield a solid mass of weight 167 g. The driedextract, XAE was freshly emulsified with Tween-80 and preparedwith saline (0.9% w/v) before use.

2.2. Animals

C57BL/6 and ICR mice (25–30 g) of both sexes purchased fromNoguchi Memorial Institute for Medical Research, University ofGhana, Accra, Ghana were kept in the Animal House of theDepartment of Pharmacology, College of Health Sciences, KNUST,Kumasi, Ghana. For 2 weeks, the animals were allowed toacclimatize to the laboratory conditions (temperature 2372 1Cwith a 12 hour light–dark cycle), while having free access tocommercial pellet diet (GAFCO, Ghana) and water ad libitum.

All animals were euthanised at the end of each experiment. Eachanimal was therefore used only once. The animals were humanelyhandled throughout the experiment in accordance with interna-tionally accepted principles for laboratory animal use and care(EEC Directive of 1986: 86/609 EEC). Additionally all animalexperiments were approved by the Department of Pharmacology,KNUST Ethics Committee.

2.3. Chemicals and reagents

Compound 48/80 (C2313), λ-Carrageenan, Evans Blue, Aspirinand Dexamethasone were purchased from Sigma-Aldrich (St Louis,USA). Clonidine was purchased from Boehringer Ingelheim Inc,(USA). Chlorpheniramine was bought from DWD PharmaceuticalsLtd (India). Bovine Serum Albumin, BSA was purchased from thePAA Laboratories (Germany). Phosphate Buffered Saline, BPS waspurchased from Gibco (Karlsruhe, Germany).

2.4. Microorganism

Escherichia coli (strain: ATCC 25922) was a kind donation fromthe Department of Pharmaceutical Microbiology, KNUST, Kumasi.

2.5. Methods

2.5.1. Compound 48/80-induced systemic anaphylaxisCompound 48/80-induced systemic anaphylactic reaction was

examined as previously described (Kim et al., 2005). 8–10 weekold C57BL/6 mice (25–30 g) were given an i.p. injection of8 mg kg−1 of the mast cell degranulator compound 48/80. Eithervehicle or XAE 30, 100, 300 and 1000 mg kg−1 was given orally 1 hbefore compound 48/80 injection. Mortality was monitored for 1 hafter induction of anaphylactic reaction.

2.5.2. Lipopolysaccharide (LPS)-induced allergyLPS-induced systemic anaphylaxis was examined as previously

described (Lowry, 2005). Male C57BL/6 mice (12–14 week old)received i.p. injection of lipopolysaccharide, LPS (Escherichia coli,2�10 10 cells/ml dissolved in PBS). Vehicle, dexamethasone10 mg kg−1 or XAE 30, 100 and 300 mg kg−1 was given orally fortwo consecutive days before initiation of the allergic reaction.Survival rate of the animals was monitored for 7 days afterchallenge.

2.5.3. Pinnal inflammation (passive cutaneous anaphylaxis, PCA)The pinnal inflammation model used in this study was as

previously described (Church et al., 1974). Briefly, 8–10 week oldICR mice (25–30 g) were immunised with 0.1 ml of a solution ofbovine serum albumin, BSA (0.05 mg ml−1) subcutaneously on Day0. 14 days later immunisation was repeated with 0.1 ml of asolution of BSA (0.02 mg ml−1). The mice were kept for a furtherperiod of seven days, anaesthetized with ether and 0.2 mL of a 1%solution of Evans Blue dye injected into the tail vein. Promptlyafter this, and while still under anaesthesia, the mice were laidsupine and each pinna was spread out and inoculated with BSA(0.1 mg ml−1) using a 21 gauge hypodermic needle. After 30 minthe mice were sacrificed and their ears cut off, spread out and thearea of the reaction was measured by circumscribing the area ofextravasation of the blue dye and matching it with the best fit ofstandard circles. The area of the reaction was taken as the squareof the diameter (mm) of the circle of best fit. For treatments, XAE30, 100 and 300 mg kg−1, dexamethasone 10 mg kg−1 and aspirin100 mg kg−1, were given orally 1 h before challenge with theantigen (BSA). Unlike XAE or reference drugs, vehicle (saline,0.1 ml) was injected subcutaneously just before the challenge.

Table 1Effect of Xylopia aethiopica extract on compound 48/80-induced systemic anaphy-laxis. 8–10 week old C57BL/6 mice (25–30 g) were pretreated for 1 h with XAE 30–1000 mg kg−1 (n¼10). Compound 48/80 was injected (8 mg kg−1, i.p) and mortalitymonitored for 1 h after induction of anaphylactic shock. The experimental resultswere analysed using the Log-rank (Mantel Cox) test and the survival rates weresignificant (P≤0.001) showing a significant trend (P≤0.0008).

Treatment Deaths/events Median survival Survival proportion (%)

Normal saline 10 16 0.00XAE (mg kg−1)30 6 23 40.00100 6 33 40.00300 5 48 50.001000 0 – 100.00

20

40

60

80

100

120ControlDEX 10 mg kg-1

XAE 30 mg kg-1

XAE 100 mg kg-1

XAE 300 mg kg-1

Perc

ent s

urvi

val

D.D. Obiri, N. Osafo / Journal of Ethnopharmacology 148 (2013) 940–945942

Percentage inhibition of the inflammatory reaction wasexpressed as:

% inhibition¼ 100 1−At

A0

� �

where Ao and At are the area of extravasation of the blue dye in thepinna of the saline control and drug or extract treated micerespectively.

2.5.4. Clonidine-induced catalepsyThe indirect antihistaminic activity of the extract was demon-

strated in clonidine-induced catalepsy employing the bar test asdescribed earlier (Ferre et al., 1990). Clonidine (1 mg � kg−1, s.c.)was administered to 8–10 week old ICR mice (25–30 g) and theirforepaws placed on a horizontal bar (1 cm in diameter, 3 cm abovethe table). In the preventive (prophylactic) protocol, vehicle(5 ml kg−1), XAE (30, 100 and 300 mg kg−1), and chlorpheniramine(10 mg kg−1) was given orally for 2 consecutive days ending30 min before clonidine injection. In the curative (therapeutic)protocol drug treatment commenced 1 h after induction of cata-lepsy. The time required to remove the paws from the bar wasnoted for each animal. The duration of catalepsy was measured at30 min intervals up to 3 h after administration of clonidine.

2.5.5. Carrageenan-induced paw oedemaPaw oedema was induced by a method earlier described

(Winter et al., 1962). Briefly, a 1% carrageenan suspension innormal saline was injected (0.05 ml, s.c.) into the sub plantartissue of the right hind paw of 8–10 week old ICR mice (25–30 g).Oedema was then monitored at 1 h intervals over 6 h as percen-tage increase in paw thickness with an electronic calliper (Z22855,Milomex Ltd, Bedfordshire, UK). Total oedema induced during the6 h was measured as area under the time course curves (AUC).Drug effects were evaluated by comparing the maximal and totaloedema responses attained during 6 h in drug-treated groups withthe corresponding values attained in drug vehicle-treatedinflamed control groups. In the preventive (prophylactic) protocol,drug vehicle, XAE 30, 100 and 300 mg kg−1, and aspirin100 mg kg−1, was given orally 1 h before the induction of theoedema while in the curative (therapeutic) protocol, treatmentswere done 1 h post oedema induction.

2.6. Statistical analysis

All data are presented as the Mean7S.E.M (n¼5 or a positiveinteger). Compound 48/80-induced systemic anaphylaxis andLPS-induced septic shock data were analysed using Log-rank(Mantel Cox) test and the data presented as median survival andsurvival proportions. In the clonidine-induced catalepsy, the dataanalysis was carried out using Two-way analysis of variance(ANOVA) followed by Bonferonni's post hoc tests. Pinnal inflam-mation and carrageenan-induced paw oedema data were analysedusing One-way ANOVA followed by Newman-Keuls' post hoc test.All graphs were plotted using GraphPad Prism for WindowsVersion 5.00 (GraphPad, San Diego, CA).

0 30 60 90 120 150 1800

Time after challenge (h)

Fig. 1. Effect of Xylopia aethiopica extract on lipopolysaccharide (LPS)-inducedallergy in mice. Male C57BL/6 mice (12–14 week old) received injection oflipopolysaccharide, LPS (Escherichia coli, 2�1010 cells/ml dissolved in PBS, i.p).Vehicle, dexamethasone 10 mg kg−1 or XAE 30–300 mg kg−1 was given orally fortwo consecutive days before initiation of the allergic reaction. Survival rate of themice was monitored for 7 days. Data was analysed using Log-rank (Mantel Cox)test. (n¼10), Survival curves were significant (P≤0.001) with significant trend(P≤0.0275).

3. Results

3.1. Effect of Xylopia aethiopica extract, XAE on compound48/80-induced systemic anaphylaxis

Intraperitoneal administration of compound 48/80 inducedfatal anaphylactic shock in mice. When the mice were pre-treated orally 1 h with XAE before compound 48/80 administra-tion there was significant anti-anaphylactic effects as compared to

control on mortality. The extract (30–1000 mg kg−1) offered 40–100% survival proportions in mice. With increasing doses of XAE,there was a corresponding increase in the median survival of themice. Behavioural observation showed a delayed or totally inhib-ited tremors induced by compound 48/80 (Table 1).

3.2. Effect of Xylopia aethiopica extract, XAE on LPS-induced allergy

Death following an allergen challenge is an irrefutable evidenceof anaphylactic shock. Intraperitoneal administration of LPS(Escherichia coli) caused significant mortality among the vehicle-treated mice starting with 50% mortality in 24 h and increasing to90% in 72 h and 100% mortality after 96 h. The dexamethasone(10 mg kg−1)-treated group exhibited maximum protection againstendotoxic shock induced with the LPS presenting a survivalproportion of 77.78%. The extract, XAE (30 100 and 300 mg kg−1)showed a dose dependent protection against endotoxic shock by25.40%, 40.50% and 63.49% respectively. Increasing doses of XAE,presented a corresponding increase in the median survival of themice (Fig. 1).

Table 2Effect of Xylopia aethiopica extract on pinnal inflammation in mice. 8–10 week oldICR mice (20–30 g) were sensitised twice as described earlier and treated withaspirin 100 mg kg−1, dexamethasone 10 mg kg−1 and XAE 30–300 mg kg−1, p.o. 1 hafter drug administration the mice were anaesthetized, injected i.v with Evans Bluedye and immediately challenged with BSA by inoculation into the pinna. Vehicle(saline, 0.1 ml) was injected subcutaneously just before the challenge. 30 minafterwards mice were euthanized and ears cut off. The reaction area was measuredby circumscribing the area of extravasation of the dye and matching it with best fitstandard circle. Data represent Means of 10 ears7S.E.M (n¼5). Significancebetween saline group and drug or extract group denoted by *P≤0.001.

Treatment Mean reactionarea(mm2)

% inhibition of extravasationof Evans Blue dye

Normal saline(0.1 ml p.o.)

37.822570.00 –

Dexamethasone(10 mg kg−1 p.o.)

9.7470.59 72.0070.59*

Aspirin(100 mg kg−1 p.o.)

10.0870.28 71.0270.28*

XAE (mg kg−1, p.o.)30 26.7571.52 23.0671.52*100 20.5571.21 40.9171.21*300 12.9370.83 62.8370.83*

0

10

20

30

40

**

***

***

*** *****

30 60 90 120 150 180Time (min)

ControlChlorpheniramineXAE 30 mg kg-1

XAE 100 mg kg-1

XAE 300 mg kg-1

* ***

Mea

n du

ratio

n of

cat

alep

sis (

s)0

10

20

30

40

30 60 90 120 150 180Time (min)

***

***

***

**

*****

ControlChlorpheniramineXAE 30 mg kg-1

XAE 100 mg kg-1

XAE 300 mg kg-1

**

Mea

n du

ratio

n of

cat

alep

sis (

s)

Fig. 2. Effect of Xylopia aethiopica extract on clonidine-induced catalepsy. 8–10week old ICR mice (25–30 g) received clonidine 1 mg � kg−1, s.c. and their forepawsplaced on a horizontal bar (1 cm in diameter, 3 cm above the table). (A) In thepreventive (prophylactic) protocol, vehicle (5 ml kg−1), XAE (30, 100 and300 mg kg−1), and chlorpheniramine (10 mg kg−1) was given orally for 2 consecu-

D.D. Obiri, N. Osafo / Journal of Ethnopharmacology 148 (2013) 940–945 943

3.3. Effect of Xylopia aethiopica extract, XAE on pinnal inflammation(PCA)

Antigen-induced pinnal inflammation in bovine serum albumin-sensitised mice was suppressed by dexamethasone (10 mg kg−1) andaspirin (100 mg kg−1) by 72.0070.59% and 71.0270.28% respec-tively. Xylopia aethiopica extract (30–300 mg kg−1) in a dose depen-dent manner inhibited the extravasation of the Evans Blue dye by23.0671.52%–62.8370.83% (Table 2).

tive days ending 30 min before clonidine injection. (B) In the curative (therapeutic)protocol drug treatment commenced 1 h after induction of catalepsy. The durationof catalepsy was measured at 30 min intervals up to 3 h after administration ofclonidine. Values are Mean7 S.E.M. (n¼5). Significance between vehicle and drug/extract treated mice denoted by *** P≤0.001, ** P≤0.01 and * P≤0.05.

3.4. Effect of Xylopia aethiopica extract, XAE on clonidine-inducedcatalepsy

Maximum catalepsy was observed in all the groups 90 minafter the administration of clonidine (1 mg kg−1, s.c). Mice treatedwith chlorpheniramine (10 mg kg−1, p.o.) and XAE (30, 100 and300 mg kg−1, p.o.) showed significant inhibition of clonidine-induced catalepsy in time dependent manner in both the pre-ventive (Fig.2A) and curative (Fig.2B) protocols.

3.5. Effect of Xylopia aethiopica extract, XAE on carrageenan-induced paw oedema

Xylopia aethiopica (30, 100, 300 mg kg−1) when administeredbefore (preventive) the induction of the carrageenan paw oedemacaused the mean maximal swelling attained during the 6 h to bereduced to 41.0276.94%, 35.6174.30%, and 29.0974.90% of theinflamed control response respectively (Fig. 3A). The total pawswellings induced over the 6 h (measured as the area under the timecourse curve, AUC) were also dose-dependently and significantlysuppressed to 74.84714.84%, 63.9579.37%, and 48.13710.90% ofthe inflamed control response respectively (Fig. 3B).

Xylopia aethiopica administered in the same doses after theinduction of the carrageenan paw oedema (curative) also sup-pressed the mean maximal swelling attained during the 6 h to49.8473.95%, 43.6271.01%, and 35.9771.34% of the inflamedcontrol response respectively (Fig. 3C). The total paw swellingsinduced over the 6 h were also dose-dependently and significantlysuppressed at 100 and 300 mg kg−1 to 72.3974.38% and 60.8173.246% of the inflamed control response respectively (Fig. 3D).

4. Discussion

Anaphylactic shock is an extreme and fatal allergic reaction,and also a component of the inflammatory response. A drugcapable of suppressing it is endowed with a very potent anti-allergic property. In the present study we demonstrate the anti-anaphylactic and anti-inflammatory actions of the hydro-alcoholicextract of the fruits of Xylopia aethiopica.

The earliest event in compound 48/80 pathway of mast cellactivation involves the direct activation of the G protein Gq oractivation of pertussis toxin (Ptx)-sensitive G-proteins culminatingin the activation of phospholipase C, PLC and phospholipase A2,PLA2 (McGivney et al., 1981). Downstream of this is perturbation ofthe membrane resulting in an increase in the permeability of thelipid bilayer membrane (Tasaka et al., 1986) which may be anessential trigger for the release of mediators from mast cells. Themechanism of the anaphylactic shock induced by compound 48/80is due to the massive release of vasoactive amines such ashistamine from mast cells and basophils (Amir and English,1991; Kim et al., 1999). Our results showed that XAE pretreatmentprofoundly suppressed compound 48/80-induced systemic ana-phylaxis possibly by stabilizing the lipid bilayer membranethrough prevention of the perturbation induced by compound48/80. Consequently, degranulation of the mast cells as well asrelease of other mediators were suppressed or inhibited thusprotecting the experimental animals from anaphylactic shock.

The allergic reaction results from the release of histamine, lipidderivatives and cytokines from allergen-specific IgE-activated mast

0 1 2 3 4 5 60

10

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70

80ControlAspirin

XAE 300 mg kg-1XAE 100 mg kg-1XAE 30 mg kg-1

Time (h)

% c

hang

e in

paw

thic

ness

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Tota

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(AU

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Fig. 3. Effect of Xylopia aethiopica extract on carrageenan-induced oedema in mice. 8–10 week old ICR mice (25–30 g) were injected with 0.05 ml of a 1% carrageenansuspension in normal saline into the sub plantar tissue of the right hind paw. In the preventive (prophylactic) protocol (Left panel), drug vehicle, XAE 30, 100 and300 mg kg−1, and aspirin 100 mg kg−1, was given orally 1 h before the induction of the oedema while in the curative (therapeutic) protocol (Right panel), treatments weredone 1 h post oedema induction. Oedema was monitored at 1 h intervals over 6 h as percentage increase in paw thickness (A and C). Total oedema induced during the 6 hwas calculated as area under the time course curves, AUC (B and D). Drug effects were evaluated by comparing the maximal and total oedema responses attained during 6 hin drug-treated groups with the corresponding values attained in drug vehicle-treated inflamed control groups. Data is presented as Mean7S.E.M. (n¼5). n P≤0.05,nn P≤0.005, nnn P≤0.0005, ++ P≤0.001, +++P≤0.0001 when compared with control. ns not statistically significant P≥0.05. Arrow indicates point of extract administration in thetherapeutic protocol.

D.D. Obiri, N. Osafo / Journal of Ethnopharmacology 148 (2013) 940–945944

cells. Both in vitro and in vivo models of mast cell degranulation inwhich cells or animals have been treated with the mast celldegranulator compound 48/80 or submitted to passive cutaneousanaphylaxis have been employed to demonstrate the anti-anaphylactic activity of natural substances (Nyarko and Addy,1994;Hong et al., 2003; Kim et al., 2004; Ueda et al., 2005). In the presentwork, a true model of immuno-mediated anaphylactic shock inwhich mice collapsed to death when challenged with a specificallergen was employed as in the LPS-induced allergy test. The maleC57BL/6 mice are known to be very sensitive to LPS-induced shock(Chi et al., 2006; Zhao et al., 2006). Endotoxin is a lipopolysaccharide(LPS) present in the outer membrane of Gram-negative bacteria suchas the Escherichia coli and is involved in the pathogenesis of Gram-negative septic shock (Morrison and Ryan, 1987). Tumour necrosisfactor alpha, TNFα, a cytokine produced by macrophages duringseptic shock is one of the endogenous mediators that cause cardio-vascular injury and death (Tracey et al., 1986, 1987). The extract, XAEshowed a dose dependent protection against endotoxic shock. Withincreasing doses of XAE, there was a corresponding increase in themedian survival of the mice. Survival of up to 63% in the challengedanimals that received the highest dose of extract in the studyindicates a rapid and potent anti-allergic action consistent with theresults from the compound 48/80-induced anaphylaxis. In addition,we found that XAE-treated mice were protected from IgE-mediatedpinnal inflammation, which is one of the most important in vivo

models of anaphylaxis in a local allergic reaction (Wershil et al., 1987)mainly induced by vasoactive mediators such as histamine frommastcells. There was a dose dependent inhibition of the allergy. It isconceivable that XAE inhibits the initial phase of immediate typeallergic reactions, probably through interference with the degranula-tion mechanism. These findings suggest that XAE might be useful inthe treatment of anaphylaxis.

The indirect antihistaminic activity of the extract was demon-strated in a dose dependent suppression of clonidine-inducedcatalepsy. While histamine containing mast cells have been identifiedin the brain (Schwartz, 1997), different stages of catalepsy have beenshown to directly correlate with the histamine content of the brain(Chopra and Dandiya, 1975). In mice, clonidine, an α2-adrenoceptoragonist induces a dose dependent catalepsy which is inhibited byhistamine H1 receptor antagonist but not by H2 receptor antagonistand pretreatment with a precursor of histamine, L-histidine caused apotentiation of the clonidine-induced catalepsy in a dose dependentmanner (Lakdawala et al., 1980). There is evidence also to show thatclonidine releases histamine from mast cell in a similar manner to aselective degranulator such as compound 48/80 (Lakdawala et al.,1980). Both clonidine and compound 48/80 cause degranulationwithout causing any damage to the cell wall (Stanworth, 1973).Catalepsy produced by clonidine in the mouse is mediated byhistamine release from mast cells acting via H1 receptor. In thisstudy, both XAE and the antihistaminic drug chlorpheniramine

D.D. Obiri, N. Osafo / Journal of Ethnopharmacology 148 (2013) 940–945 945

(10 mg kg−1) inhibited clonidine-induced catalepsy in mice whenadministered prophylactically and therapeutically. Taken togetherwith the effect of the extract on compound 48/80-induced anaphy-laxis, XAE possibly possess antihistaminic activity and mast cellstabilizing ability in a dose dependent manner in agreement withearlier findings that extracts having antihistaminic or mast cellstabilizing effect inhibit clonidine-induced catalepsy (Dhanalakshmiet al., 2004).

In this work the acute anti-inflammatory activity of Xylopiaaethiopica fruit extract has been studied using carrageenan-inducedmouse paw oedema. Carrageenan-induced oedema is primarily avascular event initiated by a dilatation of arterioles and an eventualincrease in permeability of post capillary venules that result inexudation of inflammatory cells and fluids at the site of injury(Vinegar et al., 1987).The events in carrageenan-induced oedemahave several features in common with the early exudative phase ofthe inflammatory process and the inhibition of this acute phase ofinflammation will therefore ultimately attenuate the inflammatoryprocess. However, proven anti-inflammatory activity for a drugadministered before initiation of the inflammatory response doesnot necessarily imply an ability to act therapeutically. For example,when administered prophylactically, cyclosporin prevented the onsetof collagen-induced inflammation in rats but treatment with thedrug after the onset of disease exacerbated the condition (Kaibaraet al., 1983). Thus, the dose-dependent inhibitions of carrageenan-induced mouse paw oedema by the extract indicate the presence ofcompounds potentially capable of inhibiting the acute phase ofinflammatory processes. Therefore, though the precise mechanismof action of Xylopia aethiopica extract is yet to be unravelled, thesuppression of the carrageenan-induced swellings by the extractwhen administered either before or after the onset of the inflam-matory reaction indicates the presence of compounds probablyacting via genuine anti-inflammatory mechanisms.

5. Conclusion

Of note, we conclude that the aqueous ethanol fruit extract ofXylopia aethiopica significantly inhibits mast cell-mediated ana-phylaxis and exhibits anti-inflammatory effects in mice.

Acknowledgements

We thank the Principal Technician of the Department ofPharmacology, KNUST, Mr. Thomas Ansah, for offering us technicalassistance in carrying out this project.

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