ANTILISTERIAL POTENTIAL OF IMPERATORIN AND LIMONIN FROM PONCIRUS TRIFOLIATA RAFIN

ANTILISTERIAL POTENTIAL OF IMPERATORIN AND LIMONINFROM PONCIRUS TRIFOLIATA RAFINjfbc_528 1..7

ATIQUR RAHMAN1, MINKYUN NA2 and SUN CHUL KANG3,4

1Department of Applied Chemistry and Chemical Technology, Islamic University, Kushtia, Bangladesh2College of Pharmacy, Yeungnam University, Gyeongsan-si, Gyeongbuk, Korea3Department of Biotechnology, Daegu University, Kyoungsan, Kyoungbook 712-714, Korea

4Corresponding author. TEL:+82-53-850-6553; Fax: +82-53-850-6559;EMAIL: [email protected]

Accepted for Publication September 30, 2010

doi:10.1111/j.1745-4514.2010.00528.x

ABSTRACT

To discover natural compounds with antilisterial potential, we investigated an ethylacetate extract of the seeds of Poncirus trifoliata Rafin., and isolated imperatorin andlimonin. The structures were determined on the basis of spectroscopic analysis. Thecompounds were tested for antibacterial activity against some Listeria monocytoge-nes strains and found to possess potential antilisterial activity against L. monocytoge-nes American Type Culture Collection (ATCC 19116, 19111, 19166, 19118 and15313) with minimum inhibitory concentration (MIC) values ranging from 15.62to 62.5 mg/mL. The scanning electron microscopic studies also demonstrated thatimperatorin and limonin caused morphological changes of L. monocytogenes ATCC19116 at the MIC value, along with the potential effect on cell viabilities of the testedbacteria.

PRACTICAL APPLICATIONS

The use of natural compounds of P. trifoliata as antilisterial agents will be suitable forapplications on the food industry as natural preservatives or flavoring to controlfoodborne pathogens. They can be used as growth inhibitors of L. monocytogenes,the most representative foodborne pathogen. The main reason for their suitability isthat usually, plant-derived natural compounds are considered as nonphytotoxic andpotentially effective against microorganisms. However, they can be toxic or harmfulto humans when they are inappropriately used. Caution is generally required ifnatural compounds are to be taken internally or used on food commodities.

INTRODUCTION

Microbial activity is a primary mode of deterioration of manyfoods and is often responsible for the loss of quality andsafety. Concern over pathogenic and spoilage microorgan-isms in foods is increasing due to the increase in outbreaks offoodborne diseases. Listeria monocytogenes is an importantfoodborne pathogen due to the severity of infection with ahigh mortality rate. This gram-positive bacterium is respon-sible for the severe foodborne illness, listeriosis. Most reportsassociate listeriosis with the consumption of contaminatedready-to-eat foods such as dairy products, processed or curedmeat and poultry, salads, seafood and uncooked eggs (Garciaet al. 2004). There is therefore still a need for new methodsof reducing or eliminating foodborne pathogens to ensure

public health. A range of synthetic antimicrobial agents havebeen used to inhibit the growth of foodborne pathogens infoods, although concerns about the safety of these chemicalshave increased consumer demand for naturally processedfood. Hence, there has been recent interest in testing naturalproducts, including plant-derived compounds, for antibacte-rial properties as these may be used as natural preservativesin foods (Nair et al. 2005).

Poncirus trifoliata Rafin. (Rutaceae), known as trifoliataorange or Korean bitter orange, is a deciduous shrub, a nativeof northern China and Korea, and closely related to the Citrustrees. The premature fruits of P. trifoliata have been widelyused as a traditional remedy for gastritis, dyspepsia, digestiveulcers, dysentery, inflammation and allergy. A scientific inves-tigation on the health-maintaining properties of trifoliata

Journal of Food Biochemistry ISSN 0145-8884

1Journal of Food Biochemistry •• (2011) ••–•• © 2011 Wiley Periodicals, Inc.

orange fruit has revealed its anti-inflammatory, antibacterialand anti-anaphylactic activities (Kim et al. 1999). In Korea,the fruit extracts of P. trifoliata are used as an over-the-counter drug for the treatment of gastrointestinal disorders(Lee et al. 2005). A recent in vitro study conducted by Yi et al.(2004) suggested that the poncirus fruit is a potent antileuke-mic agent due to promoting apoptosis of cancer cells. Fla-vonoids, coumarin and triterpenoids have been reported asconstituents of this species (Kim et al. 2007; Xu et al. 2008),and have been demonstrated to have biological activities thatinclude anti-inflammatory and cytoprotective activities (Kimet al. 2007; Zhou et al. 2007). However, there is no reportavailable in the literature on the constituents of the seedsof P. trifoliata and the antilisterial property. Hence, our efforthas been made to investigate the active compounds frombioassay-guided fractionation of ethyl acetate seed extract ofP. trifoliata with an antilisterial potential.

In this study, we isolated a limonoid, limonin and a furano-coumarin, imperatorin, from an ethyl acetate extracts of theseeds of P. trifoliate, and their structures were established onthe basis of spectroscopic analysis.

The antibacterial potential of the compounds were evalu-ated for controlling the growth of some Listeria strains withemphasis for the potential use of the natural compounds asalternative antibacterial agent.

MATERIALS AND METHODS

General

Melting points were recorded on a Gallenkamp apparatus(Shanghai, China). Optical rotations were measured using aJASCO DIP-1000 (Tokyo, Japan) automatic digital polarim-eter. IR spectra (KBr) and ultraviolet (UV) spectra (MeOH)were obtained from Shimadzu DR-8001 IR (Kyoto, Japan)and LKB 4053 UV spectrophotometer (Cambridge,England), respectively. Nuclear magnetic resonance (NMR)spectra were measured in CDCl3 on Brucker WH 250 MHzinstrument (Darmstadt, Germany) with tetramethylsilane asan internal standard. Purity of the compounds was checkedby thin layer chromatography (TLC) and high performanceliquid chromatography.

Plant Material

The fruits of P. trifoliata were collected from the local area ofKyoungsan, Republic of Korea, in September and October2007. The plant was identified by Prof. Sun Chul Kang and avoucher specimen number (PTR-0993) has been deposited inthe herbarium of College of Engineering, Department of Bio-technology, Daegu University, Republic of Korea. The seedswere separated from fruits, washed with distilled water and airdried at room temperature for 5 days.

Extraction and Isolation

Dried seeds of the plant (2.5 kg) were milled into powder.Ethyl acetate was added to it and kept at room temperature for10 days. The clear solution was collected by filtration and thesolvent was evaporated under reduced pressure. The residue(7 g) was subjected to column (diameter 1.3 cm, length51 cm) chromatography over silica gel (230–400 mesh,Merck, Germany) and was eluted successively with hexane,increasing amount of ethyl acetate in hexane and finally withmethanol. Twelve fractions were collected separately andmonitored by TLC. All the fractions were tested for antiliste-rial activity. Of the fractions tested, fraction 5 and 8 werefound to be active against the microorganisms and wereselected for purification. The fraction 5 (Rf = 0.51) elutedwith hexane : ethyl acetate (1:1) and fraction 8 (Rf = 0.63)eluted with hexane : ethyl acetate (1:9) were further purifiedby preparative TLC over silica gel GF254 using solvents hexane-ethyl acetate (1:1) and hexane-ethyl acetate (1:4) to givecompounds imperatorin (216 mg) and limonin (112 mg),respectively.

Imperatorin

The purified compound was crystallized from n-hexane-methanol to give white crystal (200 mg), mp 102–103C; UVumax (log e) 219, 248, 302 nm; IR umax (KBr) 1721, 1586, 1147,835/cm (Shults et al. 2003). 1H NMR (CDCl3, 250 MHz) d:7.76 (1H, d, J = 9.6 Hz, H-4), 7.69 (1H, d, J = 2.0 Hz, H-2′),7.35 (1H, s, H-5), 6.81 (1H, d, J = 2.0 Hz, H-3′), 6.37 (1H, d,J = 9.6 Hz, H-3), 5.61 (1H, t, J = 7.3 Hz, H-2″), 5.01 (2H, d,J = 7.3 Hz, H-1″), 1.74, 1.72 (3H each, s, Me ¥ 2).

Limonin

The purified compound was crystallized from n-hexane-methanol to give white crystal (98 mg), mp 118–120C; [a]25

D

-1.1° (c 1.21, acetone); IR umax (KBr) 3154, 2960, 1761 (lac-tones), 1719 (C = O), 1647, 1503, 1285, 1030, 875/cm (Khalilet al. 2003). 1H NMR (CDCl3, 250 MHz) d: 7.39 (2H, m, H-21,23), 6.32 (1H, br s, H-22), 5.45 (1H, s, H-17), 4.76 (1H, d,J = 13.0 Hz, H-19b), 4.44 (1H, d, J = 13.0 Hz, H-19a), 4.01(2H, br s, H-1, 15), 2.98 (1H, dd, J = 17.0, 4.0 Hz, H-2b), 2.86(1H, dd, J = 15.0, 14.0 Hz, H-6b), 2.68 (1H, dd, J = 17.0,2.0 Hz, H-2a), 2.55 (1H, dd, J = 12.0, 3.0 Hz, H-9), 2.47 (1H,dd, J = 14.0, 3.0 Hz, H-6a), 2.23 (1H, dd, J = 15.0, 3.0 Hz,H-2b), 1.27 (3H, s, H-28), 1.16 (3H, s, H-29), 1.15 (3H, s,H-18), 1.05 (3H, s, H-30).

Antilisterial Activity Screening

Five strains of L. monocytogenes ATCC 19111, 19116, 19118,19166 and 15313 were used in this study. The strains were

ANTILISTERIAL ACTIVITY OF FURANOCOUMARIN AND LIMONOID A. RAHMAN, M. NA and S.C. KANG

2 Journal of Food Biochemistry •• (2011) ••–•• © 2011 Wiley Periodicals, Inc.

obtained from the Korea Food and Drug Administration,Daegu, Republic of Korea. Active cultures for experimentaluse were prepared by transferring a loopful of cells from stockcultures to flasks and inoculated in brain heart infusion,(BHI; Difco, Detroit, MI) broth medium at 37C for 24 h.

The minimum inhibitory concentration (MIC) of thecompounds was tested by twofold serial dilution method(Chandrasekaran and Venkatesalu 2004). The test com-pounds were first dissolved in dimethyl sulphoxide (DMSO),and incorporated into BHI broth medium to obtain a concen-tration 500 mg/mL, and serially diluted to achieve 250, 125,62.5, 31.25, 15.62 and 7.81 mg/mL, respectively. The final con-centration of DMSO in the culture medium was maintainedat 0.5% (v/v). A 10 mL standardized suspension of each testedorganism (106–108 cfu/mL approximately) was transferred toeach tube. The control tubes contained only bacterial suspen-sion. All the tubes were incubated at 37C for 24 h. The lowestconcentrations of the compounds at which the tested organ-ism did not demonstrate visible growth were determined asMICs.

Effect of Imperatorin and Limonin on ViableCounts of Bacteria

L. monocytogenes ATCC 19116 was found to be the most sus-ceptible organism (the lowest MIC values) to both impera-torin and limonin, and was used for viable counts of bacteria.For viable counts, each of the tubes containing bacterial sus-pension (approximately 106–108 cfu/mL) of L. monocytogenesATCC 19116 in BHI broth medium was inoculated with theminimum inhibitory concentration of the imperatorin andlimonin in 10 mL BHI broth, and kept at 37C (Rahman andKang 2009). Samples for viable cell counts were taken out at 0,30, 60, 90, 120 and 150 min time intervals. Enumeration ofviable counts on BHI plates was monitored as follows: afterincubation, 1 mL of the resuspended culture was diluted into9 mL buffer peptone water, thereby diluting 10-fold, and1 mL of each dilution was spread on the surface of BHI agar.

The colonies were counted after 24 h of incubation at 37C.The controls were inoculated without compounds for thebacterial strain with same experimental condition as men-tioned above.

Scanning Electron Microscopic (SEM)Analysis

SEM studies were performed to determine the efficacy ofthe compounds imperatorin and limonin on morphologicalchanges of L. monocytogenes strains. Controls were preparedwithout sample. Further, to observe the morphologicalchanges, the method of SEM was modified from Kockromethod (Kockro et al. 2000). The bacterial samples werewashed gently with 50 mM phosphate buffer solution (pH7.2), fixed with 2.5 g/100 mL glutaraldehyde and 1 g/100 mLosmic acid solution. The specimen was dehydrated by usingsequential ethanol concentration ranging from 30–100%.After dehydration, the specimen was dried with CO2. Finally,the specimen was sputter-coated with gold in an ion coaterfor 2 min, followed by microscopic examinations (S-4300;Hitachi, Tokyo, Japan).

RESULTS AND DISCUSSION

The bioassay-guided fractionation of ethyl acetate seedextract of P. trifoliata after column chromatography oversilica gel yielded two pure compounds, which were obtainedas white crystals with specific melting points (102–103 and118–120C, respectively). All the IR, 1H- and 13C-NMR signalsare identical to the known compounds imperatorin (Mulleret al. 2004) and limonin (Khalil et al. 2003). Thus, the struc-tures were clearly identified to be imperatorin and limonin(Fig. 1).

The result of antilisterial activity of imperatorin andlimonin is presented in Table 1. The compounds showedpotent antilisterial activity against all the tested listeria iso-lates. As shown in Table 1, the MIC value of imperatorin or

FIG. 1. CHEMICAL STRUCTURES OFIMPERATORIN AND LIMONIN ISOLATED FROMPONCIRUS TRIFOLIATA RAFIN. SEEDS

A. RAHMAN, M. NA and S.C. KANG ANTILISTERIAL ACTIVITY OF FURANOCOUMARIN AND LIMONOID


limonin was found more susceptible to L. monocytogenesATCC 19116 (15.62 mg/mL) than L. monocytogenes ATCC19111, 19166, 19118 and 15313 (31.25 ~ 62.5 mg/mL). In thisstudy, L. monocytogenes ATCC 19116 was found to be themost susceptible to both imperatorin and limonin and wassubjected to viable counts of bacteria and morphologicalobservation by SEM studies.

Based on susceptibility, further studies were carried out onL. monocytogenes ATCC 19116 to determine the effect ofimperatorin and limonin on viability of this bacteria and theresults of this study showed that both compounds had similarsensitivities on L. monocytogenes ATCC 19116. The resultsfrom the viable count assay showed that both imperatorinand limonin at MIC concentration strongly inhibited thegrowth of L. monocytogenes ATCC 19116. At 60 min exposureof the limonin and imperatorin, steep decline in colony-forming unit (cfu) numbers was observed against

L. monocytogenes ATCC 19116. The cfu numbers against thetested strain were remarkably decreased upon exposure ofthese compounds (at MIC concentration) at 90 min and thusrevealed that these compounds had potential antilisterialeffect (Fig. 2). In our future study, these compounds will betested below MIC to ascertain lower concentrations of thesecompounds be effective as preservatives if similar drop in cfuoccur.

On SEM observation, it was found that there was no anychange in the morphology of the control group strains ofL. monocytogenes (Fig. 3A1,B1), whereas treatment groupshowed partially collapse of cells or destruction in the mor-phology, swelling at endpoint with lysis of cell wall, partiallydistorted shape, pore formation (Fig. 3A2,A3,B2,B3). Thus,the compounds had detrimental effect on the morphologicaldamages of L. monocytogenes.

Food safety is a fundamental concern of both con-sumers and the food industry, especially as the number ofreported cases of food-associated infections continues toincrease and is rapidly changing. Recurring outbreaks offoodborne illness caused by Listeria monocytogenes havesustained the demand for preservation systems that limitthe proliferation of this psychotropic pathogen in refriger-ated foods. The development of suitable control strategiesfor this pathogen would benefit from the availability offunctional, effective antilisterial preservatives. Many naturalcompounds found in dietary plants, such as extracts ofherbs and fruits, possess antimicrobial activities againstfoodborne pathogens.

TABLE 1. ANTILISTERIAL ACTIVITY OF IMPERATORIN AND LIMONINFROM P. TRIFOLIATA RAFIN. SEEDS

Microorganism

MIC (mg/mL)*

Imperatorin Limonin

L. monocylogenes ATCC 19111 31.25 31.25L. monocylogenes ATCC 19116 15.62 15.62L. monocylogenes ATCC 15313 31.25 62.5L. monocylogenes ATCC 19166 31.25 62.5L. monocylogenes ATCC 19118 31.25 31.25

* MIC, minimum inhibitory concentration.

FIG. 2. EFFECT OF IMPERATORIN ANDLIMONIN (MIC CONCENTRATION) ON THEVIABILITY OF THE TESTED LISTERIAMONOCYTOGENES ATCC 19116cfu, colony-forming unit; CT, control withouttreatment.



Recently, the European Union Scientific Committee forFood has published an opinion on the levels of coumarin innatural flavoring source materials (Scientific Committee forFood 1997). Currently, the limits for coumarin are less than2 mg/kg in food and beverages with specific exceptions of10 mg/kg in “special” caramels and in alcoholic beverages(Lake 1999). As described earlier in this review based on foodintake data, with coumarin-containing foods only accountingfor 5% of total solid foodstuffs, the maximum daily intakefor a 60-kg consumer would be 1.235 mg coumarin/day or0.02 mg/kg/day (Lake 1999). On the other hand, in most ofthe studies, long-term consumption of limonoids have pro-duced no adverse effects and have been found to be safe exceptfor a few exceptions. (Jacob et al. 2000; Manners et al. 2003).Arecent study showed that as a margin of safety, a self-imposedmaximum of 3 mg/L limonin was set for the beverages to beconsidered acceptable (Breksa III et al. 2008). However, com-prehensive studies need to be conducted to ascertain thesafety levels of these compounds for using in food industry. Inthis study, the exact mechanism to inhibit the microbialgrowth has not been examined. Coumarins may cross-linkDNA and form adducts with DNA (Kleiner et al. 2002). Also,the inhibitory mechanism of limonin is not known andfurther research is needed to find mode of this antimicrobialaction.

In this study, we isolated imperatorin (a furanocoumarin)and limonin (a limonoid) from the seeds of P. trifoliata,which showed potent antibacterial activity against L. mono-cytogenes strains, the most representative foodborne patho-gens. Natural coumarins and limonoids are occurring in thefruits of Citrus sp. and plants in the family Rutaceae, whichpossess a variety of pharmacological activities. Coumarinsproduced from the shikimate pathway are one of the mostimportant plant metabolites because of their bioactivitiesassociated with the medicinal use. Antimicrobial propertiesof coumarins have been also reported (Manderfeld et al.1997; Rosselli et al. 2007). Zhao et al. (2008) isolated limoninfrom the roots of Chinese medicinal herb Dictamnus radicisCortex, which was demonstrated to possess weak antibacte-rial activity against Bacillus subtilis, Escherichia coli and Sta-phylococcus aureus. Germano et al. (2005) have reported thepresence of limonoids in Trichilia emetica Vahl., which wereresponsible for the antibacterial activity against many clini-cally isolated bacterial strains. In this study, we examined theantibacterial activities of limonin and imperatorin againstdifferent types of microorganisms namely L. monocytogenesstrains and the activities differed from previous studies(Manderfeld et al. 1997; Zhao et al. 2008). Imperatorin andlimonin have been identified from some other Citrus species(Li and Chen 2004; Roy and Saraf 2006). Besides, poncirin,

FIG. 3. EFFECT OF IMPERATORIN AND LIMONIN ON MORPHOLOGICAL CHANGES OF LISTERIA MONOCYTOGENES ATCC 19116A1, bacteria without imperatorin (control); A2 and A3, bacteria treated with imperatorin at MIC values (15.62 mg/mL) showing distorted cell, poreformation and cell lysis; B1, bacteria without limonin (control); B2 and B3, bacteria treated with limonin at MIC values (15.62 mg/mL) showingdistorted cell, pore formation and cell lysis.



coumarins, auraptine, hesperidin and naringin have beenidentified from poncirus fruits (Avula et al. 2005). However,this is the first report on the isolation of imperatorin andlimonin from the seeds of poncirus fruits and the evaluationon the antilisterial activity.

CONCLUSION

In conclusion, our finding suggests that the natural com-pounds limonin and imperatorin derived from the seeds ofP. trifoliata have a strong inhibitory effect against L. monocy-togenes strains, which provides the information that thesecompounds have potential value as an antimicrobial appli-cant in food preservation and other industries.

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ANTILISTERIAL POTENTIAL OF IMPERATORIN AND LIMONIN FROM PONCIRUS TRIFOLIATA RAFIN