A

Ma

b

a

ARR2AA

KZZZAZC

1

d2mls(tn

0d

Journal of Ethnopharmacology 141 (2012) 57– 60

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

journa l h o me page: www.elsev ier .com/ locate / je thpharm

ntiulcer principle from Zingiber montanum

ohammad Al-Amina, Gazi Nurun Nahar Sultanab, Chowdhury Faiz Hossaina,∗,1

Department of Pharmacy, East West University, 43 Mohakhali C/A, Dhaka 1212, BangladeshCentre for Advanced Research in Sciences (CARS), University of Dhaka, Dhaka 1000, Bangladesh

r t i c l e i n f o

rticle history:eceived 4 July 2011eceived in revised form0 September 2011ccepted 27 January 2012vailable online 17 February 2012

eywords:ingiber montanumingiber purpureumingiber cassumunarntiulcer activityerumboneyclic sesquiterpene

a b s t r a c t

Ethnopharmacological relevance: Rhizome of Zingiber montanum has been extensively used as a folkmedicine to ameliorate peptic ulcer at northern part of Bangladesh.Aim of the study: To identify the antiulcer principle of the MeOH extract of the rhizome of Zingibermontanum by an ex vivo bioassay guided chromatographic separation and purification, and structureelucidation of the purified compound by spectroscopic methods.Materials and methods: Dried powder of Zingiber montanum rhizomes was extracted with MeOH. Theantiulcer activity of the crude extract and its chromatographic fractions were evaluated by the inhibitionof 1 N HCl induced gastric lesions in Swiss albino mice. The pure compound was purified from the activefraction by crystallization with hexanes. Structure of the pure compound was elucidated by spectroscopicmethods. The antiulcer activity of the pure compound was evaluated by the inhibition of 1 N HCl, 95%ethanol and indomethacin induced gastric lesions in mice.Results: The MeOH extract of Zingiber montanum showed 61.97% and 83.10% inhibition of the 1 N HClinduced gastric lesions at doses of 200 mg/kg and 400 mg/kg, respectively, in mice. Chromatographicseparation on silica gel of the extract was yielded seven fractions and the fraction 2 was found to havemost potent antiulcer activity in mice. This fraction showed 77.46% inhibition of the 1 N HCl inducedgastric lesions at a dose of 40 mg/kg in mice. Crystallization of the fraction yielded 1 (zerumbone, 180 mg).It showed statistically 45.77% and 92.25% inhibition of 1 N HCl induced gastric lesions in mice at doses of20 mg/kg and 40 mg/kg, respectively. It also showed 29.07% and 45.35% inhibition of 95% ethanol induced

gastric mucosal damage, and 64.76% and 72.38% inhibition of indomethacin induced gastric lesions inmice at doses of 20 mg/kg and 40 mg/kg, respectively.Conclusion: Zerumbone (1) showed potent cytoprotective effect against necrotizing agent (HCl) and non-steroidal anti-inflammatory drug (indomethacin) induced gastric ulceration. It also exhibited moderatecytoprotective effect against noxious agent (EtOH) induced gastric lesions. It can be considered as apromising new antiulcer natural drug lead.

© 2012 Elsevier Ireland Ltd. All rights reserved.

. Introduction

As part of our continuing search for new drug leads from tra-itional medicine (Taufiq-Ur-Rahman et al., 2005; Hossain et al.,005), we investigated a Bangladeshi medicinal plant, Zingiberontanum (J. Koenig) Link ex A. Dietr. (family: Zingiberaceae,

ocal name: Banada). There are several reports on Zingiber cas-umunar Roxb and Zingiber purpureum Roscoe in the literature

Masuda and Jitoe, 1994; Bandara et al., 2005), but according tohe Integrated Taxonomic Information System (itis.gov), these twoames are registered as synonym of the officially accepted name of

∗ Corresponding author. Tel.: +880 2 9882308x311; fax: +880 2 8812336.E-mail address: cfhossain@yahoo.com (C.F. Hossain).

1 Present address.

378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2012.01.046

Zingiber montanum. The plant is grown in tropical Asia and it hascreeping fleshy rhizomes. The rhizomes are very popular for thetreatment of gastric ulcer as a folk medicine at Rangpur Divi-sion of Bangladesh. Besides, it is also reported to be used in folkmedicine for several ailments such as inflammation, colic, diarrhea,vermifuge, stimulant, pain, sprains, wounds, and asthma (Khare,2007; Ozaki et al., 1991; Pithayanukul et al., 2007). Although severalpharmacological studies of this plant such as anti-inflammatory(Ozaki et al., 1991), antimicrobial, antioxidant (Habsah et al., 2000),fungitoxic (Tripathi et al., 2008), and antiallergic (Tewtrakul andSubhadhirasakul, 2007) activities have been reported, yet there isno report about its antiulcer activity.

On the basis of the use in folk medicine of Bangladesh, thepresent study was carried out to investigate the antiulcer activityof the MeOH extract of Zingiber montanum rhizomes and to identifythe active principle of extract.

5 hnoph

2

2

IOPPtocpHdUPotTr

2

RNDdodptfied

2

c(e1(Mf(t7(c

621111dJ

2

t

8 M. Al-Amin et al. / Journal of Et

. Materials and methods

.1. Drugs and chemicals

Organic solvents were obtained from Merck, Germany.ndomethacin was purchased from Opsonin Pharma, Bangladesh.meprazole and lansoprazole were purchased from Squareharma, Bangladesh. Sucralfate was purchased from Beximcoharma, Bangladesh. Hydrochloric acid and formaldehyde solu-ion were obtained from BDH Chemicals Ltd., England. Microscopicbservation was done using Carl Zeiss microscope, USA. Thin layerhromatography (TLC) analyses were made on Merck TLC platesre-coated with Si60F254 with visualization by spraying with 10%2SO4 in MeOH and heating. Open column chromatography wasone by using Silica gel 60 (70–230 mesh), Merck, Germany. TheV–visible spectrum was obtained by using a Shimadzu UV-1700harma Spec UV–VIS spectrophotometer. The IR spectrum wasbtained using a Shimadzu IR Prestige-2 FT-IR. The 1H NMR spec-rum was recorded on an ultra shield Bruker DPX 400 spectrometer.he NMR spectrum was recorded running gradients and usingesidual solvent peak (ı 7.25s) as internal reference.

.2. Plant materials and extraction

Zingiber montanum rhizomes were collected from Panchagarh,angpur Division of Bangladesh in April, 2010 and identified byational Herbarium of Bangladesh, Chiriakhana Road, Mirpur-1,haka 1216, Bangladesh, where a voucher specimen has beeneposited (accession number 35583). After collection, the rhizomesf the plant were washed with water, cut into pieces and sun-ried. The dried rhizomes were powdered by using grinder. Driedowdered rhizomes (200 g) were extracted with MeOH at roomemperature (1000 ml × 72 h × 3 times). The extracting solvent wasltered, and the filtrate was concentrated in vaccuo by a rotaryvaporator (45 ◦C) to get crude MeOH extract (10.0 g, yield: 5% fromried sample).

.3. Separation of the MeOH extract

Crude extract (9.0 g) was subjected to silica gel (35.0 g) openolumn chromatography and eluded with hexanes-ethyl acetateHex-EtOAc), ethyl acetate-methanol (EtOAc-MeOH) step gradi-nts. Following 49 fractions of 100 ml each was collected as follows:–5 (hexanes), 6–22 (Hex-EtOAc, 1:1), 23–35 (EtOAc), 36–39EtOAc-MeOH, 9:1), 40–41 (EtOAc-MeOH, 1:1), 42–45 (EtOAc-

eOH, 3:7), 46–49 (MeOH). On the basis of the analytical TLC, theseractions were pooled in seven new fractions as follows: fraction 11–5, 50 mg), fraction 2 (6–7, 3.0 g), fraction 3 (8–10, 700 mg), frac-ion 4 (11–12, 300 mg), fraction 5 (13–21, 1.0 g), fraction 6 (22–38,50 mg), and fraction 7 (39–49, 2.7 g). Antiulcer active fraction 23.0 g) was crystallized with hexanes to get colorless crystals ofompound 1 (180 mg, yield: 2.0% from dried MeOH extract).

Compound 1: Obtained as colorless crystals from hexanes; m.p.5–67 ◦C (lit. 67.5–68.0 ◦C); UV �max (MeOH) nm (log ε): 213 (3.77),50 (3.94), 302 (2.90), 319 (2.91); IR �max (KBr) cm−1: 1263.4,386.8, 1452.4, 1700.0, 2920.2, 2941.1, 3026.3. 1H NMR ı (ppm):.05 (3H, s, 14-H/15-H), 1.19 (3H, s, 15-H/14-H), 1.52 (3H, s, 12-H),.78 (3H, s, 13-H), 1.89 (1H, d, J = 12.5 Hz, 1-Ha), 2.20–2.43 (5H, m,-Hb, 4-H2, 5-H2), 5.23 (1H, br. dd, J = 9.2, 7.2 Hz, 2-H), 5.85 (1H,, J = 16.4 Hz, 10-H), 5.96 (1H, d, J = 16.4 Hz, 9-H), 6.00 (1H, br. d,

= 11.9 Hz, 6-H).

.4. Animals

Swiss albino mice of either sex, weighing 22–28 g, obtained fromhe Animal Resource Division, International Center for Diarrheal

armacology 141 (2012) 57– 60

Disease and Research, Bangladesh (ICDDR,B), were used throughoutthe experiments. All animals were kept in standard environmentcondition, had free access to standard food (ICDDR,B formulated)and water ad libitum and fasted 18 h prior to their use.

2.5. Evaluation of antiulcer activity

2.5.1. Hydrochloric acid induced gastric mucosal membranelesions test

The experiment was performed with slight modification of thepublished methods (Karimi et al., 2004; Massignani et al., 2009).Briefly, the mice were divided into groups and each group has fivemice (n = 5). After 18 h fasting, the mice were administered vehicle(1% Tween-80 aqueous solution), reference drug in 1% Tween-80aqueous solution (omeprazole, 30 mg/kg, p.o.) as positive control,and samples in 1% Tween-80 aqueous solution (p.o.). One hourlater, each mouse received 0.2 ml of 1 N HCl orally to induce gastriclesions. The mice were killed by cervical dislocation 1 h after treat-ment with the ulcerogenic agent; the stomachs were removed andopened along the greater curvature and the stomach was excisedand inflated by injection of 2 ml of saline. The ulcerated stom-achs were fixed in 5% formalin for 30 min. After opening alongthe greater curvature, HCl induced gastric damage or ulcer wasobserved as elongated black-red lines (1–10 mm long by 0.5–4 mmwide) parallel to the long axis of the stomach in mice. The sever-ity of ulcers was classified as level I (ulcer area < 3 mm × 1.5 mm),level II (ulcer area 3 mm – 6 mm × 1.5 mm – 2.5 mm), and level III(ulcer area > 6 mm × 2.5 mm). The following parameters of ulcerswere determined: (i) quantal ulcer incidence (N ulcerated) as thenumber of mice with visible gastric lesions; (ii) lesion score as totalnumber of lesions; (iii) area total of lesion (mm2); (iv) ulcer index(UI) as 1 × (number of ulcers level I) + 2 × (number of ulcers levelII) + 3 × (number of ulcers level III); and (v) % inhibition of UI as(UIcontrol − UIsample)/UIcontrol × 100.

2.5.2. Ethanol (95%) induced gastric mucosal membrane lesionstest

The experiment was performed with slight modification of thepublished methods (Karimi et al., 2004; Massignani et al., 2009).Briefly, the mice were divided into groups and each group has fivemice (n = 5). After 18 h fasting, the mice were administered vehicle(1% Tween-80 aqueous solution), reference drug in 1% Tween-80aqueous solution (lansoprazole, 30 mg/kg, p.o.) as positive control,and 1 in 1% Tween-80 aqueous solution (20 mg/kg and 40 mg/kg,p.o.) as sample. One hour later, each mouse received 0.2 ml of 95%EtOH orally to induce gastric lesions. The mice were killed by cer-vical dislocation 1 h after treatment with the ulcerogenic agent;the stomachs were removed and opened along the greater cur-vature and the stomach was excised and inflated by injection of2 ml of saline. The ulcerated stomachs were fixed in 5% formalin for30 min. The following parameters of ulcers were determined: (i)quantal ulcer incidence (N ulcerated) as the number of mice withvisible gastric lesions; (ii) lesion score as total number of lesions;(iii) area total of lesion (ATL) (mm2); and (iv) % inhibition of ATL as(ATLcontrol − ATLsample)/ATLcontrol × 100.

2.5.3. Indomethacin induced gastric mucosal membrane lesionstest

The experiment was performed with slight modification of thepublished methods (Hiruma-Lima et al., 2006; Massignani et al.,2009). The mice were divided into groups of five mice (n = 5).After 18 h fasting, the first group was given 1 mL of vehicle (1%

Tween-80 aqueous solution), reference drug in 1% Tween-80 aque-ous solution (sucralfate, 100 mg/kg, p.o.) as positive control and 1in 1% Tween-80 aqueous solution (20 mg/kg and 40 mg/kg, p.o.) assample. One hour after treatment, all mice received indomethacin

hnopharmacology 141 (2012) 57– 60 59

(ctv2fa(dbnll×

2

toS

3

ndipu0laeo4sas

mfmp2inotwp

c(t(3taaJt(J(

O

1

5

8

11

12

13

14

15

Fig. 1. Zerumbone.

M. Al-Amin et al. / Journal of Et

30 mg/kg, p.o.) to induce gastric ulcer. The animals were killed byervical dislocation 1 h after treatment with the ulcerogenic agent;he stomachs were removed and opened along the greater cur-ature and the stomach was excised and inflated by injection of

ml of saline. The ulcerated stomachs were fixed in 5% formalinor 30 min. The severity of ulcers was classified as level I (ulcerrea < 0.25 mm2), and level II (ulcer area 0.25–0.5 mm2), level IIIulcer area > 0.5 mm2). The following parameters of ulcers wereetermined: (i) quantal ulcer incidence (N ulcerated) as the num-er of mice with visible gastric lesions; (ii) lesion score as totalumber of lesions; (iii) ulcer index (UI) as 1 × (number of ulcers

evel I) + 2 × (number of ulcers level II) + 3 × (number of ulcersevel III); and (v) % inhibition of UI as (UIcontrol − UIsample)/UIcontrol

100.

.6. Statistical analyses

All data were expressed as mean ± SEM using one-sample t-est. Comparison of antiulcer activity in all groups was made usingne-way ANOVA followed by Dunnett’s multiple comparison tests.ignificance level was P < 0.05.

. Results and discussion

Intragastric application of HCl exerts a direct irritating andecrotizing topical effect on gastric mucosa and produce mucosalamage and lesions. The HCl induced gastric mucosal lesions test

s a good model to investigate a possible cytoprotective activity oflant extracts or compounds of interest and represents an acutelcer animal model (Karimi et al., 2004). Oral administration of.2 mL of a 1 N HCl induced multiple elongated reddish bands of

esions in the gastric mucosa along of the long axis of the stom-ch. Preliminary antiulcer activity test by this method of the MeOHxtract of Zingiber montanum showed 61.97% and 83.10% inhibitionf 1 N HCl induced gastric lesions in mice at doses of 200 mg/kg,00 mg/kg, respectively, and ulcer index of these inhibitions weretatistically significant (P < 0.001). The doses range for antiulcerctivity of the extract was kept far below from its published toxicitytudy in the literature (Rattanakhot et al., 2007).

The separation of the MeOH extract by silica gel column chro-atography yielded seven fractions. Fractions 2–6 were screened

or its antiulcer activity by 1 N HCl induced gastric lesions test inice at a dose of 40 mg/kg. Fractions 2 and 3 were found to be most

otent among the fractions tested. The pretreatment with fraction showed 77.46% and pretreatment with fraction 3 showed 74.65%

nhibition and ulcer index of these inhibitions was statistically sig-ificant (P < 0.001). Fractions 1 and 7 were not tested as the quantityf the fraction 1 was small (50 mg) and the TLC analysis of the frac-ion 7 showed presence of large quantity of polymerized tanninshich might give false positive activity and might be difficult tourify.

TLC analysis on silica gel of the fractions 2 and 3 showed aommon spot at Rf 0.76 in a mobile phase of hexanes–EtOAC3:1). The compound at the common spot was purified by crys-allization from fraction 2 with hexanes to yield compound 1180 mg). UV spectrum of 1 showed absorption at 213, 250, 302,19 nm which indicated the presence of a �–� conjugated sys-em in 1, the presence of carbonyl group was revealed by strongbsorption in IR spectrum at 1700 cm−1. In 1H NMR spectrum,

pair of trans olefinic protons was observed at ı 5.85 (1H, d, = 16.4 Hz) and 5.96 (1H, d, J = 16.4 Hz). The spectrum also showed

wo olefinic protons at ı 5.23 (1H, br. dd, J = 9.2, 7.2 Hz) and 6.001H, br. d, J = 11.9 Hz), six methylene protons at ı 1.89 (1H, d,

= 12.5 Hz), and 2.20–2.43 (5H, m), and four methyl groups at 1.053H, s), 1.09 (3H, s), 1.52 (3H, s), 1.78 (3H, s). Based on these

spectral data analysis, 1 was identified as zerumbone (Fig. 1),a cyclic sesquiterpene, which was previously isolated from thisplant (Ozaki et al., 1991). 1H NMR data and melting point of 1is also very consistent with that of zerumbone (Matthes et al.,1980).

It was found to be the active principal of the extract for antiul-cer activity as it showed 45.77% and 92.25% inhibition of 1 N HClinduced gastric lesions in mice at doses of 20 mg/kg, 40 mg/kg,respectively, and ulcer index of these inhibitions were statisti-cally significant (P < 0.01 and P < 0.001, respectively). Toxicity testof zerumbone showed no occurrence of death in mice over a periodof seven days at a dose of 1 g/kg in the literature (Sulaiman et al.,2010). The effects of MeOH extract of Zingiber montanum, fractions2–6 and compound 1 on HCl induced gastric mucosal membranelesions are summarized in Table 1.

As 1 showed cytoprotective and antiulcerogenic effect on HClinduced gastric ulceration, we were interested to investigate itseffect on other aggressive factors induced ulceration. EtOH is con-sidered to be a noxious compound to the stomach. Intragastricadministration of 95% EtOH is antagonistic for the cytoprotectormechanisms and produce necrotic lesions in the gastric mucosa.Bicarbonate and mucus secreted by gastric epithelium are con-sidered two of the natural cytoprotective mechanisms of gastricmucosa. EtOH (95%) reduce the secretion of bicarbonate and gas-tric mucus and make the mucus membrane more susceptible toluminal HCl attack (Marhuenda et al., 1993). In EtOH (95%) inducedgastric mucosal membrane lesions test in mice, 1 showed signifi-cant (P < 0.05) inhibition of area total of lesions of 45.35% at a dose of40 mg/kg. Although 1 showed 29.07% of area total of lesions at doseof 20 mg/kg, yet the area total of lesions inhibitions was statisticallyinsignificant (Table 2).

Clinically significant gastric ulceration is a major problem inthe use of non-steroidal anti-inflammatory drugs (NSAIDs) likeindomethacin. It causes gastric ulceration through complex mech-anism (Wallace, 2008). We also investigated the cytoprotectiveeffect of 1 against NSAIDs induced gastric ulcer. It showed 64.76%and 72.38% inhibition of indomethacin induced gastric lesions inmice at doses of 20 mg/kg, 40 mg/kg, respectively, and ulcer indexof these inhibitions were found to be significant (P < 0.001) statis-tically (Table 3).

Zerumbone (1) showed potent cytoprotective effect againstnecrotizing agent (HCl) and non-steroidal anti-inflammatory drug(indomethacin) induced gastric ulceration. It also exhibited mod-erate cytoprotective effect against noxious agent (EtOH) inducedgastric lesions. These studies revealed encouraging result for 1 as apotent cytoprotective agent against necrotizing agents and NSAIDand it can be considered as a promising new antiulcer natural druglead. Further studies are needed to elucidate the exact mecha-

nism of action involved in zerumbone (1) for this pharmacologicalaction.

60 M. Al-Amin et al. / Journal of Ethnopharmacology 141 (2012) 57– 60

Table 1Effects of different doses of MeOH extract, fractions 2–6 and zerumbone (1) on 1 N HCl induced gastric lesions in mice.

Treatment (p.o.) Dose (mg/kg) N ulcerated Lesion score Area total of lesion (mm2) Ulcer index % inhibition

Control (dist. H2O) 10 ml/kg 5/5 12.20 ± 1.43 41.60 ± 3.44 28.40 ± 1.47 –Omeprazole 30 5/5 9.80 ± 1.02 24.40 ± 1.40* 17.60 ± 1.50* 38.03MeOH extract 200 5/5 5.80 ± 1.02** 22.80 ± 6.88** 10.80 ± 1.50*** 61.97MeOH extract 400 3/5 2.80 ± 1.24*** 9.00 ± 4.67*** 4.80 ± 2.13*** 83.10Fraction 2 40 4/5 2.60 ± 0.75*** 11.15 ± 4.29*** 6.40 ± 2.14*** 77.46Fraction 3 40 4/5 4.60 ± 1.54** 11.95 ± 4.14*** 7.20 ± 2.27*** 74.65Fraction 4 40 5/5 7.60 ± 1.21* 16.75 ± 4.93** 12.40 ± 0.93*** 56.33Fraction 5 40 5/5 12.00 ± 1.22 30.57 ± 3.15* 21.20 ± 1.66* 25.35Fraction 6 40 5/5 11.40 ± 1.08 40.40 ± 3.23 26.40 ± 2.56 7.04Zerumbone (1) 20 5/5 7.80 ± 2.13 15.55 ± 2.52*** 15.40 ± 4.59** 45.77Zerumbone (1) 40 1/5 1.00 ± 1.00*** 0.80 ± 0.80*** 2.20 ± 2.20*** 92.25

Results are mean ± SEM for five mice. Statistical comparison was performed using ANOVA followed by Dunnett’s test.* P < 0.05, when compared with control group.

** P < 0.01, when compared with control group.*** P < 0.001, when compared with control group.

Table 2Effects of different doses of zerumbone (1) on ethanol induced gastric lesions in mice.

Treatment (p.o.) Dose (mg/kg) n N ulcerated Lesion score Area total of lesion (mm2) % inhibition

Control (dist. H2O) 10 ml/kg 5 5/5 6.00 ± 0.77 17.20 ± 0.97 –Lansoprazole 30 5 5/5 3.40 ± 0.40* 10.80 ± 0.86* 37.21Zerumbone (1) 20 5 5/5 3.20 ± 0.37* 12.20 ± 2.06 29.07Zerumbone (1) 40 5 4/5 3.20 ± 0.80* 9.40 ± 2.44* 45.35

Results are mean ± SEM for five mice. Statistical comparison was performed using ANOVA followed by Dunnett’s test.* P < 0.05, when compared with control group.

Table 3Effects of different doses of zerumbone (1) on indomethacin induced gastric lesions in mice.

Treatment (p.o.) Dose (mg/kg) n N ulcerated Lesion score Ulcer index % inhibition

Control (dist. H2O) 10 ml/kg 5 5/5 12.80 ± 1.80 21.00 ± 0.89 –Sucralfate 100 5 5/5 6.80 ± 1.16* 10.00 ± 2.10** 52.38Zerumbone 20 5 4/5 6.20 ± 1.59* 7.40 ± 1.91*** 64.76Zerumbone 40 5 4/5 4.60 ± 1.50** 5.80 ± 1.96*** 72.38

Results are mean ± SEM for five mice. Statistical comparison was performed using ANOVA followed by Dunnett’s test.

A

Hs(

R

B

H

H

H

K

KM

* P < 0.05, when compared with control group.** P < 0.01, when compared with control group.

*** P < 0.001, when compared with control group.

cknowledgements

We thank Ms. Hosne Ara, the Director of Bangladesh Nationalerbarium for identification of the plant. We also acknowledge the

ervice of Bangladesh Council of Scientific and Industrial ResearchBCSIR) for running the NMR experiment.

eferences

andara, K.A.N.P., Kumar, V., Saxena, R.C., Ramdas, P.K., 2005. Bruchid (Coleoptera:Bruchidae) ovicidal phenylbutanoid from Zingiber purpureum. Journal of Eco-nomic Entomology 98, 1163–1170.

absah, M., Amran, M., Mackeen, M.M., Lajis, N.H., Kikuzaki, H., Nakatani, N.,Rahman, A.A., Ghafar Ali, A.M., 2000. Screening of Zingiberaceae extracts forantimicrobial and antioxidant activities. Journal of Ethnopharmacology 72,403–410.

iruma-Lima, C.A., Santos, L.C., Kushima, H., Pellizzon, C.H., Silveira, G.G., Vascon-celos, P.C.P., Vilegas, W., SouzaBrito, A.R.M., 2006. Qualea grandiflora, a BrazilianCerrado medicinal plant presents an important antiulcer activity. Journal ofEthnopharmacology 104, 207–210.

ossain, C.F., Kin, Y.P., Baerson, S.R., Zhang, L., Bruick, R.K., Mohammed, K.A., Agar-wal, A.K., Nagle, D.G., Zhou, Y.D., 2005. Saururus cernuus lignans-potent smallmolecule inhibitors of hypoxia-inducible factor-1. Biochemical and BiophysicalResearch Communications 333, 1026–1030.

arimi, G., Hosseinzadeh, H., Ettehad, N., 2004. Evaluation of the gastric antiulcero-genic effects of Portulaca oleracea L. extracts in mice. Phytotherapy Research 18,

484–490.

hare, C.P., 2007. Indian Medicinal Plants. Springer-Verlag, Berlin, Heidelberg.arhuenda, E., Martin, M.J., de la Lastra, C.A., 1993. Antiulcerogenic activ-

ity of aescine in different experimental models. Phytotherapy Research 7,13–16.

Massignani, J.J., Lemos, M., Maistro, E.L., Schaphauser, H.P., Jorge, R.F., Sousa, J.P.B.,Bastos, J.K., de Andrade, S.F., 2009. Antiulcerogenic activity of the essential oil ofBaccharis dracunculifolia on different experimental models in rats. PhytotherapyResearch 23, 1355–1360.

Masuda, T., Jitoe, A., 1994. Antioxidative and anti-inflammatory compounds fromtropical gingers: isolation, structure determination, and activities of cas-sumunins A, B, and C, new complex curcuminoids from Zingiber cassumunar.Journal of Agricultural and Food Chemistry 42, 1850–1860.

Matthes, H.W.D., Luu, B., Ourisson, G., 1980. Cytotoxic components of Zingiber zerum-bet, Curcuma zedoaria and C. domestica. Phytochemistry 19, 2643–2650.

Ozaki, Y., Kawahara, N., Harada, M., 1991. Anti-inflammatory effect of Zingiber cas-sumunar Roxb. and its active principles. Chemical and Pharmaceutical Bulletin39, 2353–2360.

Pithayanukul, P., Tubprasert, J., Wuthi-Udomlert, M., 2007. In vitro antimicrobialactivity of Zingiber cassumunar (Plai) oil and a 5% plai oil gel. PhytotherapyResearch 21, 164–170.

Rattanakhot, N., Toojinda, P., Temjarean, P., Sriwatanakul, K., Aramphongphan, A.,2007. P13 toxicological evaluation of commercial Thai herbal preparations. ThaiJournal of Pharmacology 29, 78–80.

Sulaiman, M.R., Perimal, E.K., Akhtar, M.N., Mohamad, A.S., Khalid, M.H., Tasrip, N.A.,Mokhtar, F., Zakaria, Z.A., Lajis, N.H., Israf, D.A., 2010. Anti-inflammatory effectof zerumbone on acute and chronic inflammation models in mice. Fitoterapia81, 855–860.

Taufiq-Ur-Rahman, M., Shilpi, J.A., Ahmed, M., Hossain, C.F., 2005. Preliminary phar-macological studies on Piper chaba stem bark. Journal of Ethnopharmacology 99,203–210.

Tewtrakul, S., Subhadhirasakul, S., 2007. Antiallergic activity of some selected plantsin the Zingiberaceae family. Journal of Ethnopharmacology 109, 535–540.

Tripathi, P., Dubey, N.K., Shukla, A.K., 2008. Use of some essential oils as post-harvestbotanical fungicides in the management of grey mould of grapes caused byBotrytis cinerea. World Journal of Microbiology and Biotechnology 24, 39–46.

Wallace, J.L., 2008. Prostaglandins, NSAIDS, and gastric mucosal protection: whydoesn’t the stomach digest itself? Physiological Reviews 88, 1547–1550.