Chiang Mai J. Sci. 2013; 40(3) 321

Chiang Mai J. Sci. 2013; 40(3) : 321-331http://it.science.cmu.ac.th/ejournal/Contributed Paper

Anti-inflammatory Activity of Ethanol Extract from theLeaves of Pseuderanthemum palatiferum (Nees) Radlk.Taddaow Khumpook [a], Siriwadee Chomdej [a,c], Supap Saenphet [a], DoungpornAmornlerdpison [b] and Kanokporn Saenphet*[a][a] Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.[b] Faculty of Fisheries Technology and Aquatic Resources, Maejo University, Chiang Mai 20290, Thailand.[c] Materials Science Research Center, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.*Author for correspondence; e-mail: k_saenphet@yahoo.com

Received: 10 June 2012Accepted: 16 August 2012

ABSTRACTPseuderanthemum palatiferum (Nees) Radlk. is one of most frequently used

medical plants in Thailand for treating a variety of symptoms and several inflammatorydiseases. This study aimed to ascertain the anti-inflammatory property of ethanol extractfrom the leaves of P. palatiferum. The anti-inflammatory activity against acuteinflammation was assessed by the ethyl phenylpropiolate (EPP) induced ear edema testand cotton pellet induced granuloma model was used for chronic inflammation in albinorats. It was found that the extract showed significant anti-inflammatory activities againstboth acute and chronic inflammation as compared to the controls (P <0.05). The extractat low dose used in acute inflammatory test (1 mg/ear) exerted comparable anti-acuteinflammatory activity to phenylbutazone (PHBZ), a standard drug, whereas the extractat all doses used in chronic inflammatory test (250, 500 and 750 mg/kgBW) significantlyreduced transudative weight and granuloma formation. The extract exhibited its chronicanti-inflammatory effect in a dose dependent manner and the highest dose used hadhigher capacity than diclofenac, a standard NSAID. Due to the decrease inmalondialdehyde (MDA) and nitric oxide (NO) levels as well as the increase in superoxidedismutase (SOD) level detected in chronic inflammation-induced rats, antioxidation wassuggested as the mechanism underlying the anti-inflammatory capacity of ethanol extractfrom the leaves of P. palatiferum.

Keywords: anti-inflammation, ethanol extract, Pseuderanthemum palatiferum (Nees) Radlk.

1. INTRODUCTIONInflammation is the physiological

responses to tissue injury caused by all kindsof aggressions. Although the inflammatorycascade could help eliminate the offendingirritant and restore damaged tissues to ahealthy state, untreated or inappropriately

treated inflammation often causes harm todifferent aspects of the body and thoughtto be one of the leading causes of chronicdisease [1,2]. The effective treatment for bothacute and chronic inflammation is, thus,needed. A wide variety of anti-inflammatory

322 Chiang Mai J. Sci. 2013; 40(3)

drugs are available in pharmaceutical market,but those with no or few side effects arevery expensive. During the past decade,the interest in medicinal plants and herbalproducts has rapidly increased and the plantwith anti-inflammatory property has beenone of the most focused research subjects.Plant species having anti-inflammatoryactivity have been reported in a large bodyof literature. Those plants included: Curcumalonga Linn. [3,4], Pluchea indica Linn. [5,6],Turnera ulmifolia [7,8] and Zingiberofficinale Roscoe [9, 10]. Nevertheless,discovery of new anti-inflammatory drugsfrom plants is still challenged.

Pseuderanthemum palatiferum (Acanthaceae),known as “Hoan Ngoc” is a medicinal plantnative to Vietnam. This plant species hasbeen introduced to Thailand about 20 yearsago and it has been very popular amongThai people. It is a bush with height of 1-2 m.The leaves are in whorls, green and glossy.The flowers are pink or purple. The localVietnamese have used it to treat a wide rangeof maladies, such as diarrhea, sore throat,cancer, gastric ulcer, arthritis, as well asinflamed wound [11]. Although P. palatiferumhas been used for treating a number ofinflammatory diseases, so far, its anti-inflammatory capacity has not beenpublished based upon scientific studies.The phytochemical analysis showed that theleaves of P. palatiferum contain severaltypes of antioxidants, such as flavonoid,apigenin, triterpenoids, saponin, β-sitosterol,stigmasterol, kaempferol, salicylic acid, totalphenols and ascorbic acid [12,13]. Sincechronic inflammation exerts its cellularside effects mainly through excessiveproduction of free radicals and depletionof antioxidants [14,15], such antioxidant-rich plant like P. palatiferum may have thepotential to control inflammation in thebody through its potent antioxidant

activity.This study was, therefore, undertaken

to evaluate the anti-inflammatory effects ofthe ethanol extract from P.palatiferum inrats using EPP-induced ear edema andcotton pellet tests. In addition, its antioxidativeactions in inflammation-induced rats werealso explored using lipid peroxidationand nitric oxide production, phenomenaassociated with oxidation stress as wellas inflammation [16,17] and superoxidedismutase, a free radical scavenging enzyme,as biomarkers. Since the liver is a uniformorgan with the highest antioxidant enzymeactivities [18], antioxidative status in thisstudy was conducted in liver samples of therats.

2. MATERIALS AND METHODS2.1 Plant Materials and Extraction

P. palatiferum were purchased from amarket in Muang District, Chiang MaiProvince on May, 2010. The plants wereidentified by a botanist and the herbariumspecimens were deposited at the QueenSirikit Botanical Garden, Chiang Mai(ID: WP2615). Fresh leaves of P. palatiferumwere washed with tap water, cut andimmersed in 80% ethanol for 24 hours.The extract was then filtered to remove theresidue and evaporated by vacuum rotaryevaporator to obtain the crude ethanolextract. The crude extract was then weighedto calculate the actual percentage yield(5.76%). Finally, the ethanol extract wasdissolved in distilled water to yield concen-trations of 50, 100 and 150 mg/ml, respec-tively and used for further experiments.

2.2 AnimalWistar rats (Rattus norvegicus) were

purchased from the National LaboratoryAnimal Center, Mahidol University,Salaya campus, Thailand. The animals were

Chiang Mai J. Sci. 2013; 40(3) 323

housed in cages and had access to tap waterand a standard diet (C.P. 082). The roomtemperature was controlled at 24-26oC ina 12 hour light/dark cycle. All proceduresinvolving the animals were conductedwith strict adherence to guidelines andprocedures reviewed and approved by theInstitutional Animal Care and Use Com-mittee of Biology Department, Faculty ofScience, Chiang Mai University.

2.3 Acute Oral Toxicity StudyFemale albino rats weighing 100 to

120 g were used for acute toxicity study.The study was carried out as per theguidelines set by OECD [19] and noadverse effects or mortality were detectedin the rat up to 2 g/kg, p.o., during the24 h observation period. Based on theresults obtained from this study, the dosefor anti-inflammatory activity in chronicinflammation was fixed to be 250, 500 and750 mg/kgBW for dose dependent study(comparable to the concentrations of 50, 100and 150 mg/ml, respectively.)

2.4 EPP-induced Ear Edema in Rats:Acute Inflammation

The method was modified from thatof Keardrit et al. (2010) [20]. The test wasperformed in male rats weighing 35 to 40 g.Ear edema was induced by topical applica-tion of ethyl phenylpropiolate (EPP)dissolved in acetone (50 mg/ml) in a volumeof 10 μl to the inner and outer surfaces ofboth ears (20 μl/ear). The test sample wastopically applied to the ear in a volume of20 μl just before the irritant. The thicknessof each ear was measured with a digitalvernier caliper at 15, 30, 60 and 120 minafter the edema induction. The edemathickness of the sample-tested group wascompared to that of the vehicle groupusing phenylbutazone (PHBZ) at a dose of

1 mg/ear as a positive control.

2.5 Cotton Pellet-induced Granulomain Rats: Chronic Inflammation

Rats of either sex weighing 180 to 210 gwere divided into five groups (n=6).For inflammatory induction, the rats weremildly anesthetized with thiopental andfour sterile cotton pellets (50mg) weresubcutaneously implanted in the dorsalregion of rats, two in the axilla and two inthe groin regions. After regaining theirnormal conditions, the inflammation-induced rats were orally administered asthe following pattern: Group I served asnegative control and received ddH2O.Group II served as positive control andreceived diclofenac 5 mg/kgBW. GroupIII- V received the extracts at doses of 250,500 and 750 mg/kgBW respectively.Additional group without inflammatoryinduction (normal control group) whichreceived only ddH2O was conducted forbiochemical analysis of antioxidative status.On the 17thday, the rats were sacrificedusing anesthetic ether, and the cottonpellets were dissected out without affectingthe surrounding granulomatous tissues.The moist pellets were weighedand then dried at 60oC for 48 h and againweighed. The weights of the cotton pelletsobtained from the extract treated groupswere compared with those of controlgroups. The granulomatous tissues werefixed in 10% buffered formalin for histo-logical assessment. Liver tissues were alsoexcised and stores in 0.9% saline at -20°Cfor biochemical analysis.

2.6 Biochemical AnalysisThe liver tissue was homogenized with

tissue homogenizer in Tris-HCl buffer(0.1 M, pH 7.4). The homogenate wascentrifuged at 1,000 × g, for 10 minutes in

324 Chiang Mai J. Sci. 2013; 40(3)

cold centrifuge, and the supernatant wasused for estimation of malondialdehyde,superoxide dismutase and nitric oxidefallowing the methods of Wasowicz et al.,1993 [21], Marklund and Marklund, 1974[22] and Kumar et al., 2005 [23], respectively.Protein content was determined using themethod of Lowry et al., 1951 [24].

2.7 Histological AssessmentThe formalin-fixed granulomatous

tissues were dehydrated and processed forhistology examination using standardtechniques. The 6-μm-thick sections werestained with hematoxylin and eosin. Ablind histological analysis was conductedby a pathologist.

2.8 Statistical AnalysisFor statistical analysis, data were

analyzed by one-way analysis of variance(ANOVA) using SPSS statistical softwareversion 17.0 for windows. The results wereexpressed as mean ± standard deviation.A level of p value less than 0.05 wasconsidered to be significant. To determinethe relationship between cotton weightsand MDA, NO and SOD levels, a bivariatecorrelation with a Pearson correlationcoefcient (r) was used.

3. RESULTS AND DISCUSSIONThe results of this study demonstrated

that the ethanol extract of P. palatiferumpossessed anti-inflammatory activity inboth acute and chronic inflammation.Neither sign of toxicity nor mortality wasobserved when rats were treated with theextract dose up to 2,000 mg/kgBW. Theextract can therefore, be considered to berelatively safe. Based on this result, themaximum dose for anti-inflammatorystudy was set as 750 mg/kgBW.

3.1 EPP-induced Ear Edema in Rats:Acute Inflammation

Inhibition of acute inflammation byethanol extract from P. palatiferum wasclearly seen in Table 1. The extract at bothdoses used (1 and 2 mg/ear) significantlyinhibited ear edema when compared to acontrol group, but no significant differencesamong the doses was observed. During the1st h of assessment time the percentageinhibition of ear edema in rats received bothlow and high doses of the extract was lowerthan that of rats treated with phenylbutazone(1 mg/ear). Nevertheless, the extract at a doseof 1 mg/ear displayed a comparable percentof inhibition to the anti-inflammatorydrug at 2 h after topical application.Moreover, the similar time-dependent

Table 1. Effect of ethanol extract of P. palatiferum on EPP-induced ear edema in rats.

GroupsEdema thickness (μm)

15 min 30 min 60 min 120 min

% Inhibition15

min30

min60

min120min

ControlPhenylbutazoneP.palatiferum 1P. palatiferum 2

583±0.07c

477±0.03a

521±0.02b

541±0.03b

597±0.09b

486±0.05a

515±0.05a

511±0.06a

573±0.05c

437±0.03a

483±0.08ab

494±0.06b

572±0.05b

438±0.03a

445±0.02a

447±0.02a

-18.210.67.2

-18.613.714.4

-23.715.713.8

-23.422.221.9

Data represent mean ± SD (n = 6); a, b, c Means in the same column and with different superscriptsare different at P <0.05; Phenylbutazone 1 mg/ear; P. palatiferum 1 = 1 mg/ear; P. palatiferum2 = 2 mg/ear.

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manner of ear edema reduction was alsoobserved in phenylbutazone and this lowdose extract treated groups. The tentativeof better anti-edema effect observed in lowdose group might be due to its higherdegree of percutaneous penetration thanthe high dose group which vehicle solventwas more saturated with the extract. EPP-induced ear edema is a good model forevaluating the anti-inflammatory capacityof tested substances in acute inflammation.This assay is simple and provides a reliableresult with several anti-inflammatorydrugs, including phenylbutazone [25]. EPPproduces local swelling by causingvasodilatation, vascular permeability andfluid accumulation and several inflammatorymediators such as histamine, kinins,serotonin (5-HT) and prostaglandins (PGs)are found responsible for thoseinflammation signs [26]. Inhibition of thesynthesis and release of the key mediatorsof acute inflammation is the mechanismunderlying the anti-edematous effect ofphenylbutazone [27]. The results obtainedfrom this study suggested the similarmechanism for P. palatiferum extract at adose of 1 mg/ear. Due to its delay time ofaction as compared to anti-inflammatorydrug, extensive research is required forisolating the active compound (s) with fast

anti-inflammatory action.

3.2 Cotton Pellet-induced Granuloma inRats: Chronic Inflammation

Granuloma formation is the repairingphase, the last phase of chronic inflammatoryprocess, following the increase in vascularpermeability and leukocytes infiltration[28]. In this study, foreign body granulomaswere provoked in rats by subcutaneousimplantation of cotton pellets and thediminution of granuloma formation wasestimated following the administration ofthe extract. It was shown in Table 2 thatthe extract of P. palatiferum at all doses usedcould significantly reduce the wet weight ofcotton pellet (P<0.05) and it occurred in adose dependent manner. The wet weight ofcotton reflects the transudate volume whilethe dry weight correlates with the amountof granulation tissue [29]. Diclofenac, astandard NSAID, could inhibit transudationby blocking the effects of serotonin,bradykinin, histamine and prostaglandinE 1 on vascular membrane and by inhibitingthe release of these mediators [30]. It alsoreduces granuloma size by inhibiting theleukocyte infiltration and collagen fiberformation. Suppression of cytokines, suchas IL-1 and TNF, as well as growth factorsinfluence fibroblast proliferation and

Table 2. Effect of ethanol extract of P. palatiferum on cotton pellet-induced chronicinflammation in rats.

Cotton: 50 mg. Values are in milligrams. Mean ± standard deviation was computed over sixanimals/group. Percentage inhibition was compared with negative control group. a, b Means inthe same column and with different superscripts are different at P<0.05.

Treatment (mg/kgBW) Wet weight Dry weight% inhibition of

transudative% inhibition of

granulomaddH2O+cotton

Diclofenac (5) + cottonP. palatiferum (250) + cottonP. palatiferum (500) + cottonP. palatiferum (750) + cotton

261.88±19.43a

190.81±38.17b

200.87±27.69b

197.20±20.00b

157.08±29.27b

65.10±5.68a

49.03±6.26b

55.17±5.19ab

53.63±4.40b

51.22±9.83b

-27.9525.9627.0440.20

-24.6915.2517.6221.32

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mucopolysaccharides production weresuggested as the mechanisms for granulomainhibition [31, 32]. The extract may regulatevascular permeability through the samemechanism as diclofenac, eliminating orblocking vascular mediators. Nevertheless, thecapacity of P. palatiferum extract the highestdose used (750 mg/kgBW) gave the higherpercentage of transudate inhibition thandiclofenac. This potent anti-transudativeproperty together with the non-toxic result,P. palatiferum in an appropriate formulationand form might be considered as an alternativeto non-steroidal anti-inflammatory drugs.

An anti-granuloma effect was alsoachieved with the extract from P. palatiferum.The reduction of cotton dry weight foundin rats treated with the extract (Table 2)indicated the suppression of granuloma

development. The degree of granulomainhibition was in dose-dependent mannerand comparable to that of diclofenac andit was confirmed by histological results.The decrease in inflammatory cellinfiltration, fibroblast proliferation, andcollagen fiber density observed in theextract treated group were also in dose-dependent manner (Figure. 1 c, d and e).Aside from having some angiogenesis, P.palatiferum extract at the highest dose usedgave the most appreciable result inhistological confirmation showing relativelycomparable histological features todiclofenac (Figure. 1 b and e). The possiblemechanism involving anti-granulomatouseffect of the extract may contribute to thereduction of some or all key mediatingcytokines leading to the inhibition of

Figure 1. Histological section of granuloma tissue on day 17 post-surgery (a) control group(b) 5 mg/kgBW of diclofenac treated group. (c) 250 mg/kgBW of P. palatiferum treatedgroup. (d) 500 mg/kgBW of P. palatiferum treated group. (e) 750 mg/kgBW of P. palatiferumtreated group. Granulation tissue of the extracts contains comparatively more collagen, fibroblastand blood capillaries and few inflammatory cells (H & E stain 20x).

Chiang Mai J. Sci. 2013; 40(3) 327

inflammatory cell recruitment, especiallymacrophage and mast cells, the keyinitiators for granulation [33].

3.3 Biochemical AnalysisAnother suggested mechanism for

anti-inflammatory effects of P. palatiferumprobably includes the inhibition of thegeneration of oxygen radicals. An increasein oxygen uptake initiated by activatedmacrophages in inflammatory process giverise to a variety of reactive oxygen species,including O2, nitric oxide (NO) and hydrogenH2O2 [34]. The sustained activation andphosphorylation of MAP kinases andredox-sensitive transcription factors, suchas NF-KB and AP-1 were suggested as themechanisms of enhancing inflammation[35]. The increase in gene expression ofproinflammatory mediators resulted fromalteration of nuclear histone acetylation anddeacetylation is also caused by oxidativestress [36]. Antioxidants play a role inpreventing inflammation by scavengingfree radicals [37,38], lowering lipidperoxidation and maintaining the activitystatus of various antioxidant enzymes [39].From our results, increased oxidative stress

was reflected by the high values of MDAcontent, the inflammatory biomarker andindicator for lipid peroxidation in freeradical interaction, in inflammatory-induced rats (Figure 2). The excessive NOradicals (Figure 3) also reflected the releaseof nitric oxide synthase (NOS) duringinflammatory stimulation [40].

The reduction MDA and NO byP. palatiferum was comparable in strengthto diclofenac (Figure 2 and 3) and wascorrelated with its capacity to inhibitgranuloma development (Table 2 andFigure 1). The strength of the relationshipbetween cotton dry weight to MDA leveland to NO level was indicated by thecorrelation coefficient that closed to 1(r = 0.87 and 0.85 respectively). A numberof plant species containing flavonoids andother phenolic compounds such asScutellaria baicalensis Georgi., Taxilluschinensis (DC) Danser, Sophora japonica (L.)Schott. and Mahonia fortune (Lindl.)Fedde. have been reported to exert anti-inflammatory activity by acting as thepotent free radical scavengers and inhibitingcyclooxygenase and lipooxygenase pathwaysof arachidonate metabolism [41]. Since

Figure 2. Effect of ethanol extract of P. palatiferum on MDA level in cotton pellet-inducedgranuloma in rats. Values are mean ± standard deviation. * Significantly different from control(P <0.05). (C: Cotton).

328 Chiang Mai J. Sci. 2013; 40(3)

flavonoid has been reported to be one of themajor phytochemicals in P. palatiferum,its ability to decrease MDA and NO levelwas, thus, not surprising.

The relationship between antioxidantand anti-inflammatory activities of P.palatiferum extract was further supportedby the much greater increase in SODactivity that resulted when inflammatory-induced rats were treated with the extract(Figure 4). SOD level was inversely relatedto wet weight (r = -0.89). This experimentalevidence strongly suggests that P. palatiferum

is also a rich source of enzymatic antioxidant.Besides having an ability to quench thesuperoxide free radical, preventing of theformation of plasma-derived superoxide-dependent chemotactic factor for humanneutrophils [42] was also suggested for therole of SOD in anti-inflammation process.

Since oxidative stress and inflammationare associated with the risk of severalinflammatory disorders such as atheroscle-rosis, rheumatoid arthritis, hepatitisglomerulonephritis and diabetes mellitus,the antioxidant and anti-inflammatory

Figure 4. Effect of ethanol extract of P. palatiferum on SOD level in cotton pellet-inducedgranuloma in rats. Values are mean ± standard deviation. * Significantly different from control(P <0.05). (C: Cotton).

Figure 3. Effect of ethanol extract of P. palatiferum on NO level in cotton pellet-inducedgranuloma in rats. Values are mean ± standard deviation. * Significantly different from control(P <0.05). (C: Cotton).

Chiang Mai J. Sci. 2013; 40(3) 329

activities of extract from the leaves ofP. palatiferum may shed light on potentialtherapeutic options in those diseases.

4. CONCLUSIONSIt could be concluded from our study

that ethanol extract from the leaves ofP. palatiferum possessed potent anti-inflammatory activities in both acute andchronic inflammations. Antioxidation wasproposed as the mechanism underlying itsanti-inflammatory property. Our resultsconfirm the traditional use of P. palatiferumfor treating inflammation. Further researchis needed for developing anti-inflammatoryproducts from this plant species, either asalternatives or as adjuvants to conventionalanti-inflammatory drugs.

ACKNOWLEDGEMENTSThis work was supported by a grant

from Science Achievement Scholarship ofThailand, the National Research UniversityProject under Thailand’s Office of theHigher Education Commission and Thegraduate school, Chiang Mai University.

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