www.elsevier.com/locate/biochemsysecoBiochemical Systematics and Ecology 34 (2006) 509e513

Unusually high quantity of 4-hydroxybenzoic acid accumulationin cell wall of palm mesocarps

Moumita Chakraborty, Kingsuk Das, Gargi Dey, Adinpunya Mitra*

Natural Product Biotechnology Group, Agricultural & Food Engineering Department, Indian Institute of Technology Kharagpur,

Kharagpur 721 302, India

Received 10 June 2005; accepted 26 November 2005

Abstract

Presence of 4-hydroxybenzoic acid in the mesocarp walls of 22 genera of Arecaceae (Palmae) was investigated using a TLC/UVspectra analysis method and confirmed by HPLC and ESI-MS. The genera collected mainly belong to the Copryphoideae andArecoideae subfamilies. All the investigated genera possess an unusually high amount of 4-hydroxybenzoic acid, whichvaried from 5.6 mg/g dry wt cell wall material (CWM) (Areca catechu) to 1.0 mg/g dry wt CWM (Roystonea regia). Apartfrom 4-hydroxybenzoic acid, ferulic acid is also found in all the genera studied along with some traces of 4-coumarate. Thiswork presents an overview of the major wall-bound phenolics found in the mesocarp of different palms, and on the basis of thisoccurrence, a possible hypothesis for considering 4-hydroxybenzoic acid as a chemotaxonomic marker of this particular familycan be drawn.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Mesocarp; 4-Hydroxybenzoic acid; 4-Coumaric acid; Ferulic acid; Arecaceae

1. Introduction

4-Hydroxybenzoic acid is a natural component of plant-based foods and has been identified as an important dietaryanti-oxidant (Tomas-Barberan and Clifford, 2000). Apart from its role in plant defence responses against pathogens,4-hydroxybenzoic acid finds its application as preservatives for cosmetics, foods and drugs (McQualter et al., 2005).The biosynthesis of 4-hydroxybenzoic acid in plants remains unresolved (Abd El-Mawla and Beerhues, 2002). It ispossible that there are various pathways leading to the formation of individual hydroxybenzoate that depend on thetype of plant. Hence, in order to work out the biosynthetic pathway for 4-hydroxybenzoic acid, it is important to iden-tify plants that accumulate 4-hydroxybenzoic acid in their cytosol and cell walls. In this context, while exploringcheap alternatives for isolating phenolic acids of economic use, our group discovered 4-hydroxybenzoic acid asthe major phenolic compound accumulated in mesocarp wall of coconut husk (Dey et al., 2003). This observationhas raised the question as to whether this high amount of 4-hydroxybenzoic acid (0.2e0.5% dry wt of biomass) occursin the mesocarp of other species of palm (Dey et al., 2005). If so, it could then be considered a possible

* Corresponding author. Fax: þ91 3222 282 244/255 303.

E-mail address: adin@iitkgp.ac.in (A. Mitra).

0305-1978/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.bse.2005.11.011

510 M. Chakraborty et al. / Biochemical Systematics and Ecology 34 (2006) 509e513

chemotaxonomic marker of mesocarp wall of palms. The aim of this current study was to profile phenolic acids fromthe mesocarp of different species of palms available in the West Bengal region of India to see if they could be used astaxonomic markers. In this communication, we report on the high quantity of 4-hydroxybenzoic acid accumulationalong with ferulic acid and traces of 4-coumaric acid in the mesocarp of 22 different species of palm fruits. This ac-cumulation of 4-hydroxybenzoic acid in the mesocarp wall is discussed in the context of a possible chemotaxonomicmarker along with its ecological significance.

2. Materials and methods

2.1. Materials

Palm fruits were collected from the Indian Botanic Garden (Botanical Survey of India [BSI]), Howrah, West Ben-gal (India) and subsequently identified at the BSI. The voucher specimen numbers of all the plants analysed are shownin Table 1. The genera studied mainly belong to two subfamilies Arecoideae and Coryphoideae as shown in Table 1.After removal of the epicarp, mesocarp materials were separated out and subsequently used for phenolic extraction.

2.2. Extraction methods

Dry mesocarp materials (ca. 500 mg) of each sample were washed with commercial detergent Surf� (1.5e2% w/v)and rinsed thoroughly with distilled water. The mesocarp material was then subjected to sequential alkaline hydrolysisunder progressively more vigorous conditions to release the wall-bound ether and ester-linked phenolics (Parr et al.,1996) as follows. The first extraction was with ca. 50 ml of 0.1 M NaOH for 12 h at room temperature in the dark torelease the ester-linked phenolics. The suspension was filtered and the residue retained for further extraction with ca.50 ml of 2 M NaOH under the same conditions to release ether-linked phenolic acids. Each of the extracts was

Table 1

Detection of 4-hydroxybenzoic acid and ferulic acid in mesocarp walls of different Arecaceae members studied here

Subfamily Species Voucher Phenolic acid contents (mg/g dry

wt of cell wall material)

4-Hydroxybenzoic acid Ferulic acid

Arecoideae Areca catechu L. P-03/01, NPBG 5.6 1.0

Areca triandra L. P-04/01, NPBG 2.7 0.1

Arenga sp. P-03/04, NPBG 2.3 1.7

Bentinckia nicobarica (Kurz) Becc. P-04/02, NPBG 2.9 1.1

Carpentaria sp. P-03/05, NPBG 2.3 1.6

Caryota mitis L. P-03/08, NPBG 2.7 2.9

Caryota urens L. P-03/07, NPBG 3.6 2.1

Cocos nucifera L. P-03/02, NPBG 3.4 1.2

Dictyospermum sp. P-03/03, NPBG 3.1 1.3

Elaeis guineensis L. P-04/03, NPBG 2.1 1.4

Roystonea regia (HBK) O.F. Cook P-03/06, NPBG 1.0 0.8

Veitchia sp. P-03/10, NPBG 1.9 1.3

Coryphoideae Borassus flabellifer L. P-03/08, NPBG 3.1 2.3

Kentia belmoreana F. Muell. P-03/09, NPBG 2.4 2.0

Kerriodoxa sp. P-04/14, NPBG 1.9 1.0

Licuala grandis Roxb. P-03/11, NPBG 1.8 1.0

Livistona chinensis R. Br. ex Mart. P-03/16, NPBG 1.5 1.0

Livistona jenkinsiana Griff. P-03/16, NPBG 1.9 1.0

Phoenix paludosa Roxb. P-04/05, NPBG 1.8 1.4

Phoenix sylvestris (L.) Roxb. P-03/15, NPBG 1.8 1.4

Rhapis sp. P-03/13, NPBG 2.8 1.9

Sabal sp. P-03/12, NPBG 1.8 1.4

The data shown consist of means of triplicate analyses from three different samples; the standard deviations of these were within 10% and are not

shown.

511M. Chakraborty et al. / Biochemical Systematics and Ecology 34 (2006) 509e513

collected separately after filtering the solution. The alkaline solution was acidified (pH 2.0) and the acidified solutionswere extracted with two volumes of ethyl acetate (ca. 100 ml). The organic phase containing the phenolic compoundwas evaporated under vacuum at 30 �C in a rotary evaporator. The dried sample was re-suspended in 1 ml of 50% (v/v)aqueous methanol, prior to TLC analysis.

2.3. Analytical methods

2.3.1. TLC and UV e spectral analysesThe TLC analysis was performed with microcrystalline cellulose plate (French et al., 1976; Dey et al., 2003). The

plates were developed in 2% aqueous formic acid. The phenolic acids were viewed under a dual wavelength (254/310 nm) UV-lamp (UVItec, Cambridge, UK). The bands corresponding to authentic standards were detected onthe plate and the scrapped bands were further processed to obtain a spectral scan (Dey et al., 2003). The identificationof the samples was done by comparison of the absorption spectra of the sample with those of the authentic standards(Waldron et al., 1996). The absorption at 254 nm and 310 nm was recorded for quantifying 4-hydroxybenzoic acid andfeulic acid, respectively, using standard plots for the individual phenolic acids.

2.3.2. HPLC and ESI-MS analysesAnalyses of the saponified extract were done to verify the identity of the phenolic acids extracted from the meso-

carp wall. HPLC separation was performed on a Phenomenex� (Torrance, CA, USA) C18 column (RP-Hydro 4 mm,250� 4.6 mm) using a BREEZE� HPLC system (Waters, Milford, USA) as described earlier (Sachan et al., 2004).ESI-MS was carried out to confirm further the chemical identities of these wall-bound phenolic acids. Mass spectrom-etry of the HPLC-purified samples was performed in a Micromass LCT� Mass Spectrometer (Micromass, Manches-ter, UK) as essentially described earlier (Mitra et al., 2002).

3. Results and discussion

The ferulic acid and 4-hydroxybenzoic acid contents of the investigated genera are listed in Table 1, based on TLCanalyses. Identities were validated by subsequent HPLC analysis (data not shown). The chemical structure of thesecompounds was further confirmed by ESI-MS {4-hydroxybenzoic acid: [M�H]� m/z 137 (M.W. 138); ferulic acid:[M�H]� m/z 193 (M.W. 194)}. Areca catechu was found to accumulate a maximum amount of 4-hydroxybenzoicacid (5.6 mg/g dry wt), whereas Roystonea regia accumulates a minimum amount (1.0 mg/g dry wt). Genera underArecoideae showing a relatively high amount of 4-hydroxybenzoic acid were Cocos nucifera (3.4 mg/g drywt), Borassus flabellifer (3.1 mg/g dry wt) and Caryota urens (3.6 mg/g dry wt). The average content of 4-hydrox-ybenzoic acid for Arecoideae was 2.8 mg/g and for Coryphoideae was 2.1 mg/g. Ferulic acid was detected in allthe genera studied with an average quantity of 1.6 mg/g dry wt. In almost all the cases, 4-coumaric acid wasfound in very low amounts and was detected only by HPLC analysis. However, in case of Dictyospermum sp.,Veitchia sp., Bentinckia nicobarica and Carpentaria sp., the content of p-coumaric acid was relatively higherand was detected on the TLC plates as well as in the HPLC chromatograms (data not shown).

Occurrence of 4-hydroxybenzoic acid has also been reported in many plant species, apart from Arecaceae. An ex-ample for this is Pandanus odorus (Pandanaceae), where 4-hydroxybenzoic acid was found to accumulate in the roottissues (Peungvicha et al., 1998). Presence of 4-hydroxybenzoic acid was also reported from the pericarp of Virolamultinervia (Myristiaceae) (Nunomura and Yoshida, 2002). In these studies, 4-hydroxybenzoic acid was extractedwith organic solvents from dried powdered pericarps, which appears to be in cytosol, not wall-bound. However, inan earlier study, extraction with different organic solvents did not release any phenolic acids from the mesocarp tissueof C. nucifera (Dey et al., 2003). In fact, our observation in mesocarps of different palm species revealed that 4-hydroxybenzoic acid predominantly exists as cell wall-bound forms and not as soluble conjugated forms.

The predominance of 4-hydroxybenzoic acid in the mesocarp wall of all the genera studied here is interesting. Thisis because the monocot cell walls are usually characterised by an abundance of hydroxycinnamates, such as, ferulicacid (Harris and Hartley, 1980). It is well known that environmental factors play a vital role in determining the phe-nolic composition of fruits and vegetables (Parr and Bolwell, 2000). Water availability and soil composition like min-eral and organic nutrients have a marked effect on the phenolic contents of the plants. It has been shown that anincreased calcium uptake leads to an excess production of 4-hydroxybenzoic acid in carrot cell cultures

512 M. Chakraborty et al. / Biochemical Systematics and Ecology 34 (2006) 509e513

(Bach et al., 1993). Thus in natural habitat there is every possibility that mineral content of the soil will play somerole in the accumulation of the 4-hydroxybenzoic acid. In carrot cell culture system, it was also proven that 4-hydroxybenzoic acid productions could be induced by elicitation with pathogenic fungus Pythium aphanidermatum(Schnitzler et al., 1992). Inoculation of the date palm roots with Fusarium oxysporum f. sp. albedinis induced several-fold more 4-hydroxybenzoic acid production in the resistant cultivar than those in the susceptible cultivars (Modafaret al., 2000). Hence, the accumulation of 4-hydroxybenzoic acid in the mesocarp wall of palm fruits may be consid-ered as a possible response to the ecological factors as well as to combat pathogenic attack.

Highly expressed metabolites, often referred to as biomarkers, can provide an exclusive basis for chemotaxonomicidentity for the taxa (Sumner et al., 2003). There are many instances where phenolic compounds were used as taxo-nomic markers (Bate-Smith, 1962). For example, in cultivated Amazonian coca, flavonoid compounds are used aschemotaxonomic markers (Johnson et al., 2002). Recently, the presence of 60-O-coumaroylaloesin in six species ofAloe was considered a unique chemotaxonomic marker of these species (van Heerden et al., 2000). While analysingthe accumulation of 4-hydroxybenzoic acid in the cell wall within a genus, we observed the same accumulationpattern at the family level. It is evident that all members of the Arecaceae family studied here share the same 4-hydroxybenzoic acid profile which would favour its used as a taxonomic marker (Wink and Waterman, 1999).However, detailed investigations in all six subfamilies are required to confirm the hypothesis.

Acknowledgement

This work was funded by the Council of Scientific and Industrial Research (CSIR), India, in the form of an extra-mural research grant [no. 38 (1065)/03/EMR-II to A Mitra]. Saibal Basu of Indian Botanic Garden (BSI) has suppliedall the palm fruits used in this study, we are indeed grateful to him. Gargi Dey thanks CSIR for the award of anindividual research associateship. M. Chakraborty and K. Das contributed equally to this work.

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