This article was downloaded by: [Muhammad Arman]On: 28 August 2011, At: 04:21Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Natural Product ResearchPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/gnpl20

LC-ESI-MS characterisation ofphytoalexins induced in chickpea andpea tissues in response to a bioticelicitor of Hypnea musciformis (redalgae)Muhammad Arman aa PCSIR Laboratories Complex Karachi, Sharah-e-Dr SalimuzzamanSiddiqui, Off University Road, Karachi -75280, Pakistan

Available online: 22 Aug 2011

To cite this article: Muhammad Arman (2011): LC-ESI-MS characterisation of phytoalexins inducedin chickpea and pea tissues in response to a biotic elicitor of Hypnea musciformis (red algae),Natural Product Research, 25:14, 1352-1360

To link to this article: http://dx.doi.org/10.1080/14786419.2011.553952

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching and private study purposes. Anysubstantial or systematic reproduction, re-distribution, re-selling, loan, sub-licensing,systematic supply or distribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representationthat the contents will be complete or accurate or up to date. The accuracy of anyinstructions, formulae and drug doses should be independently verified with primarysources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material.

Natural Product ResearchVol. 25, No. 14, August 2011, 1352–1360

LC-ESI-MS characterisation of phytoalexins induced in chickpea

and pea tissues in response to a biotic elicitor of Hypnea musciformis(red algae)

Muhammad Arman*

PCSIR Laboratories Complex Karachi, Sharah-e-Dr Salimuzzaman Siddiqui,Off University Road, Karachi -75280, Pakistan

(Received 4 March 2010; final version received 10 January 2011)

A simple extraction procedure and HPLC method was developed to analysethe major and minor components of induced phytoalexins of elicited tissues(seeds) of chickpeas (Cicer arietinum L.) and peas (Pisum sativum L.)treated with a biotic elicitor (k-carrageenan) of Hypnea musciformis(red algae) from the Karachi coast. The level and timing of the inducedphytoalexin production were estimated on the basis of various elicitordilutions and as a function of time; the results are presented and discussed.A LC-ESI-MS/MS technique has been employed for the detection andcharacterisation of the induced phytochemical components (flavonoids andtheir glyco-conjugates). Nine flavonoids were identified from chickpeas:naringin, naringin malonate, liquiritigenin, naringenin, biochanin A,daidzein, formononetin, maackiain and medicarpin, while five flavonoidswere identified from peas: afrormosin, anhydropisatin, pisatin, pseudo-baptigenin and maackiain. These compounds play a vital role as phyto-alexins because of their antimicrobial activity.

Keywords: elicitor; induced secondary metabolites; phytoalexins; polysac-charides; Hypnea musciformis; flavonoids; LC-ESI-MS/MS

1. Introduction

As a result of infection or stress, plants exhibit some natural resistance responses.Amongst these, induced browning and phytoalexin production have especiallygained attention (Castoria, Maria, Anna, & Marisa, 1995). Phytoalexins are lowmolecular weight, biologically active compounds produced by plants as a defensivereaction to various exogenous stimuli, particularly fungal invasion, their role indisease resistance has been deduced from their antifungal activity (Arnold & Merlin,1990). Some of the reported phytoalexins in leguminosae species are alkaloids,coumarins and especially isoflavonoid derivatives (Grayer & Kokubun, 2001).The biosynthesis of isoflavonoid phytoalexins has been intensively studied and theirmetabolic regulation is known to be different according to the inductor agent, plantcell type and type of elicitors (Smith & Banks, 1986). It is reported thathypersensitive responses produced in plants after microbial attack may be triggered

*Email: aspcsir@gmail.com

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2011 Taylor & Francis

DOI: 10.1080/14786419.2011.553952

http://www.informaworld.com

Dow

nloa

ded

by [

Muh

amm

ad A

rman

] at

04:

21 2

8 A

ugus

t 201

1

by elicitors that are the compounds isolated from cell wall, culture filtrate andcytoplasm of parasitic and non parasitic plant pathogens. Elicitors are diverse innature and are usually polysaccharides, proteins and fatty acids (Rao, Sarada, &Ravishankar, 1996).

Chickpea (Cicer arietinum L.) and peas (Pisum sativum L.) belonging to thefamily leguminosae are important crop plants and have both economical andnutritional importance. Due to high protein contents, they are consumed widely inmany countries as a meat substitute for poor people. The antimicrobial activities ofthese two plants are due to the presence of their phytoalexins i.e. pisatin andmaackiain (Ho, Lin, Labbe, & Shetty, 2003). A relationship between phytoalexinaccumulation and defense against pathogenic microorganisms in peas(Pisum sativum), alfalfa (Medicago sativa), barrel medic (Medicago truncatula) andchickpea (Cicer arietinum) is recently reported in literature (Liu et al., 2006).

It is documented that in most cases elicitor activity is associated withpolysaccharides fraction of various elicitor preparations when tested in chickpeaand other plant tissues (Melotto & Lebavitch, 1994). In the present study, theseaweed (Hypnea musciformis, red algae) polysaccharides were evaluated as anelicitor or inducer of plant defense responses in terms of phytoalexin production.A simple HPLC separation method was developed to analyse the major and minorcomponents of the mixture of induced secondary metabolites ‘phytoalexins’ and theindividual components were identified by LC-Electro Spray Mass Spectrometrictechnique with positive and negative ionisation modes.

2. Results and discussion

In the present study alcoholic extracts of elicitor treated and control tissues ofchickpea were analysed for the estimation of ISMs (phytoalexins) with respectto various elicitor doses, 5–100 mg glu eqmL�1 and different incubation periods,6–72 h. A reproducible HPLC method was developed to analyse the major andminor components of induced phytoalexins. Typical HPLC chromatograms(Supplementary Figures S1 and S2 – online only) showed that the reproducibility ofelution time of the separated components was 2–3%. The integrated area of the peakswas assumed to be proportional to the amount of solutes (phytoalexins) present.Initial results for elicitor activity of pea tissues were not found very promising,induction of browning and estimation of inducedmetabolites (phytoalexins) in treatedtissues of peas were insignificant as compared to controls (Bi, Iqbal, Ali, Arman, &Hassan, 2008). Therefore, attempts were made to develop HPLC methods and for thecharacterisation of the components present in the pea’s chromatogram whereas theindividual components quantification was not carried out.

The data presented in Supplementary Tables S1 and S2 (online only) are a ratioof peak area/g fresh weight of treated and control samples. These results showed thatchickpea tissues responded differentially to the applied elicitor preparations andproduced a positive and definite elicitor activity in terms of phytoalexin production.100 mg elicitor concentration and 24 h incubation time were the best conditions toinduce metabolites at maximum level in the samples. Some components (marked byFT) were induced in elicitor treated samples only. Recently reported dose dependantstudies showed that 100 mgmL�1 and 1000 mgmL�1 concentrations of algal polysac-charides, � carrageenan were the effective dilutions for high induction of florescent

Natural Product Research 1353

Dow

nloa

ded

by [

Muh

amm

ad A

rman

] at

04:

21 2

8 A

ugus

t 201

1

compounds and defense related genes at 168 h and 24 h after elicitation in tobacco

leaves (Mercier et al., 2001). Cooper, Doss, Price, Peterson, and Oliver, 2005

reported that during time course studies in peas, application of a new class of insect

elicitor ‘Bruchin B’ increased the expression of isoflavone synthase gene within 8 h of

elicitor treatment followed by increase in the level of isoflavone phytoalexin pisatin

that reached the highest level at 32–64 h after elicitation.Due to the low amount of induced metabolites and non-availability of standard

materials, LC-Electro Spray Ionisation Mass Spectrometry (LC-ESI/MS) appears to

be the ideal technique for the identification of secondary metabolites (Kite et al.,

2006). In the present study, samples were run under identical conditions and

monitored simultaneously by UV and total ion current (TIC) chromatograms

(Supplementary Figures S3 and S4 – online only). UV analogue is in complete agree-

ment with most of the peaks present in TIC, however some major peaks (at RT

22.57, 8.21 and 20.61) and minor peaks (at RT 10.06 and 2.19) of these figures

appeared in TIC are not found in their UV analogs, therefore these peaks were not

characterised during this work. The retention time’s data of the identified

phytoalexins are summarised in Supplementary Table S3 (online only).Nine compounds from chickpea such as Naringin 1 (Fang et al., 2006; Zhang &

Brodbelt, 2004) Naringin malonate 2 (Liu, Ye, H. Guo, Bi, & D. Guo, 2005),

Liquiritigenin 3 (Li, Liu, & Cheng, 2007), Naringenin 4 (Zhang & Brodbelt, 2004),

Biochanin A 5 (Careri, Corradini, Elviri, & Mangia, 2007), Daidzein 6 (Careri et al.,

2007) Formononetin 7 (Kim et al., 2007) Maackiain 8 (Liu et al., 2005) and

Medicarpin 9 (Tsiri, Chinou, Halabalaki, Haralampidis, & Ganis-Spyropoulos,

2009) and five compounds from peas such as Afrormosin 10 (Carlsen et al., 2008;

Gong, Wang, Chen, Liu, & Yu, 2009), Anhydropisatin 11 (Mizuno, Tanaka,

Katsuragawa, Saito, & Iinuma, 1990;), Pisatin 12 (Mizuno et al., 1990;),

Pseudobaptigenin 13 (Wu, Wang, & Simon, 2003) have been identified by

LC-MS/MS/MS analysis. These compounds belong to the classes of flavanones,

Isoflavones, pterocarpans and their glyco conjugates (Figure 1). Maackiain was

found in both chickpea and peas tissues. Molecular ions in positive and negative

modes and the product ion spectra of these compounds were studied in order to

obtain the most specific transition (Li, Xiao, Liang, Shi, & Liu, 2004). The use of the

selected transition is necessary for the identification of these compounds.All identified phytoalexins (1–13) exhibited ionisation in both positive and

negative modes, compounds were predominantly detected as [MþH]þin positive

mode due to the high proton affinity. In negative ionisation mode all analytes

exhibited an [M�H]� ion due to loss of hydrogen and provided stable molecular ion

peaks (Liu et al., 2005). The main product ions observed for each analyte in positive

and negative ionisation modes are shown in Table S3.The MS/MS experiments were performed on each ion in positive and negative

modes, representative positive and negative ions ESI- MS and ESI-MS/MS spectra

of Naringenin are discussed and shown in Supplementary Figures S5–S8 (online

only). Literature search showed that the ions observed in positive ionisation mode

are mainly due to losses of sugar moieties (Bednarek et al., 2001) and from the

retro Diels-Alder cleavage of the ring C in the flavonoids (Liu et al., 2005). Similar

ions were observed in the current work in negative ionisation mode for compounds 1

and 2.

1354 M. Arman

Dow

nloa

ded

by [

Muh

amm

ad A

rman

] at

04:

21 2

8 A

ugus

t 201

1

For compounds 3, 4 and 6, the fragment ions observed in positive and negativemodes were mainly due to the losses of OH and CO ions as well as the loss indicatingfrom the retro Diels-Alder cleavage. In compounds 5, 7 and 10 the major fragmentions observed in positive and negative modes were due to the losses of CO & CH3

ions and from the loss occurred due to the retro Diels–Alder cleavage.In compounds 8, 9, 11 and 12, the major fragment ions observed in the positive

and negative modes were due to the losses of CH2O2 and CH3 ions along with somedifferent fragmentation pathways. Representative fragmentation pattern of medi-carpin (Supplementary Figure S9 – online only) in negative mode showed sixfragments A to F in the MS/MS spectrum among which D and E are thecharacteristic fragments for identification of pterocarpans (Woodward, 1981, 1982).For compound 13, the product ions observed in positive and negative modes weremainly due to the losses of CH2O2 and CO ions as well as the losses indicated by theretro Diels–Alder cleavage. Although these ions exhibited low abundance at 25% inNormalised Collision Energy (NCE), they become predominant when the collisionenergy was increased to 35% (Table S3).

The metabolic grid of an enzyme sequence showing the conjugation reaction andthe metabolic turnover of isoflavonoids in chickpeas was established earlier andexplained the conjugation reaction of isoflavanone such as formononetin-7-Oglucoside and formononetin-7-O-glucoside-6-O-malonate. Formononetin is referredas an intermediate component for the biosynthesis of pterocarpan phytoalexinsmaackiain and medicarpin in this system. Barz et al. (1988) reported formononetinwith one sugar component. The present studies showed an early glycoconjugationreaction soon as the naringenin was formed in the primary biosynthetic pathway.

O

OH

OH O

OOH

OH HH

OH

OH

CH2OR

H

OOH

H HH

OHOH

H

H

CH3

O

OH

O

HO

O

O

HO

R4

R1

O

O

R1

R2

R 4O

1 R=H2 R=COCH

2COOH

3 R=H4 R=OH

5 R1=OCH

3, R

2=R

3=H, R

4=OH

6 R1=OH, R

2=R

3=R

4=H

7 R1=OCH

3, R

2=R

3=R

4=H

10 R1=R

3=OCH

3, R2=R

4=H

13 R1R

2=O-CH

2-O, R

3=R

4=H

R3

R2

R

R3

8 R1R

2=O-CH

2-O, R

3=R

4=H

9 R1=OCH

3, R

2=R

3=R

4=H

11 R1R

2=O-CH

2-O,R

4=CH

3, R

3=Nofunctional group,

6a 11a=Double bond

12 R1R

2=O-CH

2-O,R

3=OH, R

4=CH

3

1

4

6

6a

11a

9

Figure 1. Identified phytoalexins 1–13 from elicited tissues of chickpea and peas.

Natural Product Research 1355

Dow

nloa

ded

by [

Muh

amm

ad A

rman

] at

04:

21 2

8 A

ugus

t 201

1

LC-(MS)n data showed that isoflavanone naringenin conjugation reaction provided

two compounds tentatively identified as naringin malonate (naringin-7-O-digluco-side-6-O-malonate) of molecular mass [683] and naringin (naringin- 7- O-diglyco-

side) of molecular mass [580]. Mass calculation indicated the presence of two sugarmoieties, glucose and rhamnose in these two compounds which were detected and

confirmed by paper chromatography. Maackiain and medicarpin were obtainedfrom formononetin instead of following the intermediate compounds like calycosin,

pseudobaptigenin, 2-hydroxy pseudobaptigenin, 2-hydroxy dihydropseudobapti-

genin, 2-hydroxy formononetin and vestitone, may be due to the transit inductiontime that was possibly difficult to locate or produced in very small quantities that

could not be detected. These compounds are known (Zhang & Brodbelt, 2004) andfirst time identified during this interaction. These results support the investigation of

Sumner, Paiva, Dixon, and Geno (1998) that described the optimisation parametersin the identification of flavonoid glucosides in alfalfa and chickpea extracts using

HPLC/CF-LSI MS technique.Referring to Figure S4, the compounds corresponding peaks 1–5 were

characterised as afrormosin, anhydropisatin, pisatin, pseudobaptigenin and maack-

iain and considered as pea phytoalexins. Earlier studies showed that liquiritigenin,isoliquiritigenin and formononetin were present in the biosynthesis of pisatin

(Carlson & Dolphin, 1981) whereas these compounds were not found during thisinvestigation.

3. Experimental

3.1. Plant materials

Plant collection, extraction and dry weight of High Molecular Weight Crude ElicitorPolysaccharides (HMWCEP) from hot water extract of H.musciformis, red algae of

Karachi Coast was described earlier (Bi & Iqbal, 1999). The voucher specimen wasdeposited at the Herbarium of the Botanical Garden of PCSIR Laboratories

Complex Karachi, Pakistan (M.A. R1130.E026). These crude polysaccharides were

purified by anion exchange chromatography on DEAE cellulose column and twopurified fractions PF-A and PF-B were collected. HMW PF-A was further examined

for its elicitor activity.

3.2. Elicitor activity and ethanolic extraction for induced secondarymetabolites (ISMs)

A general method of elicitor application was employed in all experiments

(Whitehead, Dey, & Dixon, 1982). Elicitor preparations of 5, 25, 50, 75 and100 mg glu. eqmL�1 of PF-A were used for dose response elicitor activity. Treated

and control samples were prepared by application of 20 mL of these elicitorpreparations and sterile water (control) on the cut surfaces of cotyledons and

incubated for 6, 12, 24, 48 and 72 h with control samples. Treated and control

samples were extracted with alcohol, defated with hexane and residual alcoholicextract were stored at �20�C in the dark.

1356 M. Arman

Dow

nloa

ded

by [

Muh

amm

ad A

rman

] at

04:

21 2

8 A

ugus

t 201

1

3.3. HPLC separation of phytoalexins

HPLC separation of phytoalexins of chickpea and peas were performed on FinniganSurveyor Liquid Chromatography equipped with PDA plus detector and auto-sampler plus (Thermo Electron Corporation, San Jose, CA USA). A (TeknokromaKromasil 100) C18 5 mm 25�0.46 cm analytical column was used for both chickpeaand peas analysis. A guard column of pellicular C18 hydrocarbon chemically bondedto glass beads was placed before the analytical column. Flow rate was 1mLmin�1.For the analysis of chickpea tissues, the gradient elution employed was acetonitrileand water; both solvents contained 1% acetic acid. Initially a solvent system consistof 80 : 20 Water:Acetonitrile was run for 5min, then a gradient of 40 : 60 reached into15min and further goes to 100% acetonitrile in 5min, stays there for another 5minand returned to initial solvent system, while for pea tissues the gradient elutionemployed was acetonitrile and 0.1% phosphoric acid in aqueous phase. Initially asolvent system consist of 40 : 60 Acetonitrile: 0.1% phosphoric acid in aqueous phasewas run for 3min, then a gradient of 95 : 05 reached into 17min and further goes to100 % acetonitrile in 5min, stays there for another 5min and returned to initialsolvent system. The whole chromatograms were monitored at 254 nm and 365 nm forchickpea and peas, respectively.

3.4. Sample preparations

Dry alcoholic extracts of elicited and control tissues of chickpea and peas weredissolved into 2mL of 40:60 following solvent system. (a) for chickpea, water:ace-tonitrile, both phases contain 1% acetic acid; (b) for peas, acetonitrile:0.1%phosphoric acid in aqueous phase. 100 mL of each solution was further diluted with1mL of their respective solvents, centrifuged and filtered through 0.2mm filters.20 mL of clear filtrates were applied to the column.

3.5. Identification of phytoalexins by LC-ESI MSn technique

Chromatograms run under the optimided HPLC conditions were reproduced onLC-MS system of Finnigan LCQ Advantage Max mass spectrometer (ThermoElectron Corporation, San Jose CA USA) and monitored by total ion current (TIC)in positive and negative modes in the mass range of 50–1500 u by using ElectrosprayIonisation source. Sheath and auxiliary gases were set at 80 and 20 units, needlevoltage of 4.5 kV and the capillary temperature was set at 250�C. (MS)n spectrawere recorded by selecting the ions obtained by applying 35% of normalised collisionenergy.

4. Conclusion

It is concluded from the present study that polysaccharides fromHypnea musciformis(red algae) are active elicitor of plant defense responses and induced biologicallyactive secondary metabolites ‘phytoalexins’. HPLC method employed gave a goodseparation and quantification of induced polyphenolic compounds in elicitedchickpea tissues while pea tissues were not quantified due to low profile of elicitoractivity. LC-ESI-(MS)n technique is helpful in determining the molecular ions and to

Natural Product Research 1357

Dow

nloa

ded

by [

Muh

amm

ad A

rman

] at

04:

21 2

8 A

ugus

t 201

1

identify the structures of aglycones (flavonoids) and different types of glycosidessuch as Naringin, Naringin malonate, Liquiritigenin, Naringenin, Biochanin A,Daidzein, Formononetin, Maackiain and Medicarpin from elicited chickpea tissueswhile Afrormosin, Anhydropisatin, Pisatin, Pseudobaptigenin and Maackiain fromelicited pea tissues. Methods and techniques involved have an advantage of reducedtime of analysis which make these techniques one of the promising tools available forlooking at initial changes in a particular plant pathogen or plant elicitor interactionsand also facilitates the routine processing of large number of elicited samples forquantitation and identification.

Supplementary material

Figures S1–S9 and Tables S1–S3 relating to this article are available online.

Acknowledgement

The author is thankful to Mr Muhammad Sadiq Ali, Technical Officer for his assistance in thelaboratory work.

References

Arnold, A., & Merlin, L. (1990). Lipophilicity-antifungal activity relationship for some

isoflavonoid phytoalexin. Journal of Agricultural and Food Chemistry, 38, 834–838.Barz, W., Daniel, S., Hinderer, W., Jaques, U., Kessmann, H., Koster, J., . . . , Tiemann, K.

(1988). Elicitation and metabolism of phytoalexins in plant cell cultures. Applications of

plant cell and tissue culture (pp. 178–198), Wiley: Chichester (Ciba FoundationSymposium 137).

Bednarek, P., Franski, R., Kerhoas, L., Einhorn, J., Wojtaszek, P., & Stobiecki, M. (2001).Profiling changes in metabolism of isoflavonoids and their conjugates in Lupinus albus

treated with biotic elicitor. Phytochemistry, 56, 77–85.Bi, F., & Iqbal, S. (1999). Chemical investigation and elicitor activity of polysaccharides of red

algaeHypnea musciformis and Botryocladia leptopoda. Pakistan Journal of Scientific and

Industrial Research, 42, 223–226.Bi, F., Iqbal, S., Ali, A., Arman, M., & Hassan, M.U. (2008). Induction of secondary

metabolites in Chickpea, Carrot and Potato tissues in response to elicitor ofH. musciformis. Indian Journal of Plant Physiology, 13, 101–106.

Careri, M., Corradini, C., Elviri, L., & Mangia, A. (2007). Optimization of a rapid microwaveassisted extraction method for the liquid chromatography-electrospray-tandem mass

spectrometry determination of isoflavonoid aglycones in soybeans. Journal of

Chromatography, A. 1152, 274–279.Carlsen, S.C.K., & Understrup, A. (2008). Flavonoids in roots of white clover: interaction of

arbuscular mycorrhizal fungi and a pathogenic fungus. Plant Soil, 302, 33–43.Carlson, R.E., & Dolphin, D.H. (1981). Chromatographic analysis of isoflavonoid accumu-

lation in stressed Pisum sativum. Phytochemistry, 20, 2281–2284.Castoria, A.R., Maria, F.M., Anna, T.A., & Marisa, C.F. (1995). Inter-relationship between

browning and phytoalexins accumulation elicited by arachidonic acid. Journal of PlantPhysiology, 145, 209–214.

Cooper, L.D., Doss, R.P., Price, R., Peterson, K., & Oliver, J.E. (2005). Application of

Bruchin B to Pea Pods results in the up regulation of CYP93C18, a putative isoflavone

1358 M. Arman

Dow

nloa

ded

by [

Muh

amm

ad A

rman

] at

04:

21 2

8 A

ugus

t 201

1

synthase gene and an increase in the level of pisatin, an isoflavone phytoalexin. Journal

of Experimental Botany, 56, 1229–1237.Fang, T., Wang, Y., Ma, Y., Su, W., Bai, Y., & Zhao, P. (2006). A rapid LC/MS/MS

quantitation assay for naringin and its two metabolites in rats plasma. Journal of

Pharmaceutical and Biomedical Analysis, 40, 454–459.Gong, T., Wang, D.X., Chen, R.Y., Liu, P., & Yu, D.Q. (2009). Novel benzyl and isoflavone

derivative from Millettia dielsina. Planta Medica, 75, e102–e104.Grayer, R.J., & Kokubun, T. (2001). Plant – fungal interactions: the search for phytoalexins

and other antifungal compounds from higher plants. Phytochemistry, 56, 253 – 263.

Ho, C.Y., Lin, T.Y., Labbe, R.G. & Shetty, K. (2003). Session 29F, food microbiol-

ogy:control of food born microorganisms by antimicrobials, IFT Annual Meeting-

Chicago.

Kim, H.K., Park, S.Y., Lim, H.K., Park, A.Y., Kim, J.S., Kang, S.S., . . . , Han, S.B. (2007).

Quantitative evaluation of Radix Astragali through the simultaneous determination of

bioactive isoflavonoids and saponins by HPLC/UV and LC-ESI-MS/MS. Bulletin of

Korean Chemical Society, 28, 1187–1194.Kite, G.C., Porter, E.A., Denison, F.C., Grayer, R.J., Veitch, N.C., Buttler, I., . . . , Simmonds,

M.S.J. (2006). Data directed scan sequence for the general assignment of C-

glycosylflavon-O-glycosides in plant extracts by liquid chromatography ion trap mass

spectrometry. Journal of Chromatography, 1104, 123–131.Li, X., Xiao, H., Liang, X., Shi, D, & Liu, J. (2004). LC-MS/MS determination of naringin,

hesperidin and neohesperidin in rat serum after orally administrating the decoction of

Bulpleurum falcatum L. and Fractus auranti.i. Journal of Pharmaceutical and Biomedical

Analysis, 34, 159–166.Li, Y., Liu, L., & Cheng, Y. (2007). Identification of components of traditional Chinese

medicine preparation ‘‘Jing-Zhi-Guan—Xin’’ Troche by medium pressure LC and

HPLC-DAD-MS. Chromatographia, 65, 749–755.Liu, C.J., Deavours, B.E., Richard, S.B., Ferrer, J.L., Blount, J.W., Huhman, D., . . . , Noel,

J.P. (2006). Structural basis for dual functionality of isoflavonoid O-methyl transferases

in the evolution of plant defense responses. Plant Cell, 18, 3656–3669.Liu, R., Ye, M., Guo, H., Bi, K., & Guo, D. (2005). Liquid chromatography/electrospray

ionization mass spectrometry for the characterization of twenty-three flavonoids in the

extract of Dalbergia odorifera. Rapid Communication in Mass Spectrometry, 19,

1557–1565.Melotto, E., & Labavitch, J.M. (1994). Biologically active cell wall materials. Revista

Brasileira de Fisiologia Vegetal, 6, 75–82.Mercier, L., Lafitte, C., Borderies, G., Briand, X., Tugaye, M.T.E., & Fournier, J. (2001). The

algal polysaccharide carrageenans can act as an elicitor of plant defence. New

Phytologist, 149, 43–51.Mizuno, M., Tanaka, T., Katsuragawa, M., Saito, H., & Iinuma, M. (1990). A new

pterocarpan from the heartwood of Cladrastis Platycarpa. Journal of Natural Products,

53, 498–499.Rao, R.S., Sarada, R., & Ravishankar, G.A. (1996). Phycocyanin, a new elicitor for capsaicin

and anthocyanin accumulation in plant cell cultures. Applied Microbiology and

Biotechnology, 46, 619–621.Smith, D.A., & Banks, S.W. (1986). Biosynthesis, elicitation and biological activity of

isoflavonoid phytoalexins. Phytochemistry, 25, 979–995.

Sumner, L.W., Paiva, N.L., Dixon, R.A., & Geno, P.W. (1998). High performance liquid

chromatography/continuous-flow liquid secondary ion mass spectrometry of flavonoid

glycosides in leguminous plant extracts. Journal of Mass Spectrometry, 31, 472–485.

Tsiri, D., Chinou, I., Halabalaki, M., Haralampidis, K., & Ganis-Spyropoulos, C. (2009). The

origin of copper-induced medicarpin accumulation and its secretion from roots of

Natural Product Research 1359

Dow

nloa

ded

by [

Muh

amm

ad A

rman

] at

04:

21 2

8 A

ugus

t 201

1

young fenugreek seedlings are regulated by copper concentration. Plant Science, 176,367–374.

Whitehead, T.M., Dey, P.M., & Dixon, R.A. (1982). Differential patterns of phytoalexinaccumulation and enzyme induction in wounded and elicitor treated tissues of Phaseolus

vulgaris. Planta, 154, 156–164.Woodward, MD. (1981). Differentiation of dihydroxy mono methoxy isoflavones by gas

chromatography mass spectroscopy. Phytochemistry, 20, 532–534.

Woodward, M.D. (1982). Gas chromatography/Mass spectroscopy of isoflavones and relatedcompounds. Phytochemistry, 21, 1403–1407.

Wu, Q., Wang, M., & Simon, J.E. (2003). Determination of isoflavones in red clover and

related species by high-performance liquid chromatography combined with ultravioletand mass spectrometric detection. Journal of Chromatography A, 1016, 195–209.

Zhang, J., & Brodbelt, J.S. (2004). Screening flavonoid metabolites of naringin and narirutin

in urine after human consumption of grapefruit juice by LC-MS and LC MS/MS.Analyst, 129, 1227–1233.

1360 M. Arman

Dow

nloa

ded

by [

Muh

amm

ad A

rman

] at

04:

21 2

8 A

ugus

t 201

1