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Journal of Ethnopharmacology 126 (2009) 434–436

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

ast cell stabilization and antihistaminic potentialsf Curculigo orchioides rhizomes

. Venkatesha, Pulok K. Mukherjeea,∗, Satheesh Kumar Na, Neelesh K. Nemaa,. Bandyopadhyayb, Hiroyuki Fukuic, Hiroyuki Mizuguchic

School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, IndiaDepartment of Molecular Endocrinology, Indian Institute of Chemical Biology, Jadavpur, Kolkata 700032, IndiaDepartment of Molecular Pharmacology, Graduate School of Health-Biosciences, The University of Tokushima, Tokushima 770-8505, Japan

r t i c l e i n f o

rticle history:eceived 14 May 2009eceived in revised form 26 August 2009ccepted 14 September 2009vailable online 23 September 2009

eywords:urculigo orchioides

a b s t r a c t

Aim of the study: To investigate the mast cell stabilization and antihistaminic activities of the rhizomes ofCurculigo orchioides (COR). Extract of Curculigo orchioides Gaertn. (Fam. Amaryllidaceae) has been reportedto possess immunostimulant, and anti-inflammatory potentials. In Indian traditional system of medicineit is also used as anti-asthmatic and anti-inflammatory.Materials and methods: Estimation of histamine release is key parameter for evaluating any target for itsanti-allergic potential. The stabilization potential of the alcoholic extract of COR (100–400 mg/kg) againstmast cell degranulation was studied on isolated mice peritoneal mast cells. The antihistaminic activity

ast cell stabilizationystemic anaphylaxisompound 48/80

ndian traditional medicine

was performed by determining the mortality rate of mice upon exposure to compound 48/80 and effecton inhibition of histamine release upon degranulation.Results: The raised number of intact mast cells intimates that the COR stabilized the mast cell degranula-tion (60.96 ± 1.96%) and percentage antihistaminic potential of the extract (63.58 ± 1.8 inhibition at doseof 400 mg/kg) and it virtues further work towards the isolation of phytoconstituents from this plant.Conclusion: This finding provides evidence that COR inhibits mast cell-derived immediate-type allergic

egran

reactions and mast cell d

. Introduction

Mast cells are constituents of virtually all organs and tissues andre important mediators of inflammatory responses such as allergynd anaphylaxis (Church and Levi-Schaffer, 1997) in which his-amine remains the best characterized and most potent vasoactive

ediator implicated in the acute phase of immediate hypersensi-ivity upon release (Petersen et al., 1996). Mast cell degranulationan also be evoked by the compound 48/80, which is a mast cellegranulator and has been used as a direct and convenient reagento study the mechanism of anaphylaxis (Ennis et al., 1980). Thenfluence of natural products derived from plants is broadly recog-ized for their great structural diversity as well as their wide rangef pharmaceutical activities (Mukherjee, 2001).

Curculigo orchioides Gaertn. (Fam. Amaryllidaceae) is a tinyerbal plant widely distributed in India, China, Malaya, and Japan.

n Indian traditional medicine it is claimed as a medical cure foriles, asthma, jaundice, diarrhoea, colic and gonorrhoea (Kirtikar

∗ Corresponding author. Tel.: +91 33 24146046; fax: +91 33 24146046.E-mail address: naturalproductm@gmail.com (P.K. Mukherjee).

378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2009.09.019

ulation.© 2009 Elsevier Ireland Ltd. All rights reserved.

and Basu, 1935) and reported to be an immunostimulant (Bafnaand Mishra, 2006). The rhizomes of this species has been reportedto contain curculigoside, curculigoside B, curculigoside C (Fu et al.,2004), orcinol glycoside (Wu et al., 2005), 2,6-dimenthoxyl benzoicacid, curculigines A–C (Chen et al., 1999) and orchiosides A and B(Gupta et al., 2005), tannin, resin and alkaloid (Anon., 2004). Thetherapeutic attributes of plants have been inquired in the light ofrecent scientific developments throughout the world, due to theirpharmacological activities, low toxicity (Mukherjee et al., 2006).With this background, the study was designed to establish the mastcell stabilization and antihistaminic activities of alcoholic extractfrom Curculigo orchioides.

2. Materials and methods

2.1. Chemicals and reagents

Compound 48/80 was purchased from Sigma (St. Louis, MO,U.S.A.), o-phthalaldehyde (OPT), RPMI-1640 were purchased fromHimedia Laboratories Pvt. Ltd., Mumbai. Other reagents used wereof analytical grade.

nopharmacology 126 (2009) 434–436 435

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.2. Preparation of plant extract

Rhizome of Curculigo orchioides (COR) was collected from Oota-amund, Tamilnadu, India in May 2008 and the sample wasdentified and authenticated by Botanical Survey of India [BSI],overnment of India, Shibpur, Howrah. Voucher specimen (SNPS-040) has been preserved at our university for future reference.he ethanol extract of the rhizomes of Curculigo orchioides (COR)as prepared from 1 kg of rhizome; it yielded 3.3% (w/w) of dry

xtract. The extract was dried under rotary vacuum evaporator tobtain solvent free extract. The extract was subjected into qualita-ive phytochemical tests (Mukherjee, 2002).

.3. Animals

The male Swiss albino mice (20–25 g) were acclimatized in stan-ard conditions. They were maintained on a standard pellet dietith free access to water and housed in temperature controlled

oom. The experiments were performed according to the guide-ine of the committee for the purpose of control and supervisionf experiments on animals (CPCSEA), constituted under the direc-ives of Ministry of Social Justice and Empowerment, Governmentf India.

.4. Mast cell stabilization activity

COR was administered at various doses (100–400 mg/kg, bw,.o.) to different groups of animals [n = 6]. The standard drugisodium cromoglycate (DSCG) (50 mg/kg) was administered

ntraperitoneally (due to its poor absorption through oral route)or 4 days. The toxicity profile of COR has already been reportednd it has been shown to be non-toxic up to a dose of 10 g/kgRao and Mishra, 1996). On the following day, mice were admin-stered with 5 ml of cold phosphate-buffered saline (NaCl 137 mM,aHCO3 12 mM, NaH2PO4 0.3 mM, KCl 12.7 mM, MgCl2 1.0 mM,aCl2 1.8 mM, dextrose 5.6 mM) i.p. After gentle abdominal mas-age for 10 s, the peritoneal fluid containing mast cells wereollected immediately in RPMI-1640 media (pH 7.2–7.4). Cell via-ility was checked by a trypan blue dye (0.4%) exclusion test. Thenhe cells were washed three times with RPMI-1640 media by cen-rifugation at low speed (500–600 rpm), discarding the supernatantnd resuspending the pellets of mast cells in the medium. Mast cellsrom the treated and control groups were incubated with com-ound 48/80 (1 �g/ml) at 37 ◦C for 10 min in a water bath. Thenhe cells were stained with toludine blue (1%) and the numbers ofegranulated and intact mast cells were counted from total num-er of 100 cells of each drug treated which were observed underigh power microscope (Gupta et al., 1995) and calculated by the

ormula:

ercentage of intact mast cells = total number of mast cells −total numb

.5. Compound 48/80-induced systemic anaphylaxis

Compound 48/80-induced systemic anaphylactic reaction wasnalyzed as previously described (Yi et al., 2001). Mice were givenn injection of 0.8 mg/kg, i.p. of the toxicant. COR (100–400 mg/kg)ere dissolved in carboxy methyl cellulose, a suspending agent,

nd administered orally for 4 days prior to injection of the com-ound 48/80. DSCG was used as standard. Blood sample wasbtained from the heart of each mouse for further analysis ofistamine as described later. Mortality (%) within 1 h followingdministration of the compound 48/80 injection and was repre-

number of degranulated cellsf mast cells

× 100

Fig. 1. Evaluation of the alcoholic extracts of Curculigo orchioides on mast cell sta-bilization against compound 48/80 induced degranulation. [Datum represents themean ± SEM of six independent experiments (n = 6).] ap < 0.001 compared with con-trol; bp < 0.001 compared with toxicant.

sented as:

the number of dead micetotal number of experimental mice

× 100

2.6. Histamine estimation

The blood tissues were homogenized and centrifuged at 400 × gfor 10 min and histamine content was measured in plasma by o-phthalaldehyde spectrofluorimetric method (Shore et al., 1959).This method involves extraction of histamine into n-butanol fromalkalinized perchloric acid tissue extracts; histamine returns to anaqueous solution and condensation with o-phthalaldehyde to yielda product with strong and stable fluorescence which was measuredin a spectroflourimeter. The fluorescent intensity was measured atemission wavelength of 438 nm and excitation at 353 nm.

2.7. Statistical analysis

Bonferroni’s Multiple Comparison test was carried out to com-pare between toxicant and test groups, while one-way ANOVA testwas used to make a statistical comparison between the groups.Each data was the mean and standard error of the mean (SEM) ofthe different experiments under the same conditions. Results withp < 0.001 were considered statistically significant.

3. Results and discussion

The qualitative phytochemical studies revealed the presenceof alkaloids, poly phenols, saponins, steroids and tannins in theplant extract. The desired medicinal properties of the plant may beattributed to the synergistic or individual effect of these phytocon-stituents which are well known for their anti-allergic properties.

COR inhibited percentage degranulation of mast cells up to60.96 ± 1.96 at dose of 400 mg/kg. This result was comparable toDSCG (67.46 ± 1.063 at 50 mg/kg) with significance of p < 0.001 andall the extracts tested have shown a statistically significant mast cellstabilization activity except at the lower dose (100 mg/kg) where itwas not significant (Fig. 1). It might also act by stabilization of themast cell membrane, thereby preventing its degranulation which

mimics the standard drug DSCG.

The lower rate of mortality of mice from the anaphylactic shockwas 30% at 400 mg/kg (COR) (Table 1) which was comparableto the standard (40% at 50 mg/kg). The elevation of histaminein plasma from different groups of mice was determined by

436 P. Venkatesh et al. / Journal of Ethnopha

Table 1Effect of alcoholic extracts of Curculigo orchioides on anaphylactic shock induced bycompound 48/80 in mice.

Treatment Dose (mg/kg) Compound 48/80 (mg/kg) Mortality (%)

None (saline) – – 0Toxicant – 0.8 100DSCG (std) 50 0.8 40COR 100 0.8 90COR 200 0.8 60COR 300 0.8 50COR 400 0.8 30

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spdoaiIodistmctaioo

Wu, Q., Fu, D.X., Hou, A.J., Lei, G.Q., Liu, Z.J., Chen, J.K., Zhou, T.S., 2005. Antioxida-tive phenols and phenolic glycosides from Curculigo orchioides. Chemical and

ig. 2. Effect of the alcoholic extracts of Curculigo orchioides on inhibition ofistamine release in compound 48/80 induced degranulation in mice. [Datum repre-ents the mean ± SEM of six independent experiments (n = 6).] ap < 0.001 comparedith control; bp < 0.001 compared with toxicant; cp < 0.01 compared with toxicant.

pectroflurometric method. The marked anti-allergic effect of CORre-treatment in allergen-induced systemic anaphylaxis was doseependent, i.e. 45.42 ± 2.5 and 63.58 ± 1.8 of percentage inhibitionf histamine release at 300 and 400 mg/kg, respectively (p < 0.001)nd 36.39 ± 2.2 inhibition at 200 mg/kg (p < 0.01) (Fig. 2), whichndicated that the extract directly reduced the allergic mediators.ndeed, pre-treatment with COR extract inhibited the releasef histamine efficaciously. It is particularly remarkable that theegree of inhibition furnished by COR against compound 48/80-

nduced mast cell degranulation and anaphylaxis was almostimilar to that of DSCG. The results of this study demonstrated thathe alcoholic extract of the rhizomes of Curculigo orchioides showed

arked mast cell stabilization and antihistaminic activities againstompound 48/80-induced allergic response in mice. In conclusion,

he results revealed that mast cell stabilization and antihistaminicctivities, which are closely mimicked to the standard, DSCG andt is worth isolating the phytochemical from this source for benefitf being potential anti-allergic drug, which is under progress inur laboratory.

rmacology 126 (2009) 434–436

Acknowledgements

Authors are thankful to the Department of Science and Technol-ogy [DST], Government of India and Japan Society for Promotionof Science [JSPS] F. No DST/INT/JAP/P-62/08; through India–Japancooperative science programme. Thanks are also due to theIndian Council of Medical Research, Government of India, NewDelhi: F. No – 45/29/2007/BMS/TRM, for financial support for thiswork.

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