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Gas Chromatography-Mass Spectrometry Analysis of Agarwood Extracts from Mature and Juvenile Aquilaria malaccensis.

Byline: Phai Lee Jong Pascale Tsan and Rozi Mohamed

Abstract

Chemical composition of crude extracts from infected woods of Aquilaria malaccensis were compared to that of healthy wood and commercial agarwood. Infected woods were collected six months after drilling of wild mature trees or after fungal inoculation into the stem of 4-year-old trees. Agarwood substances were extracted in methanol and were subjected to GC-MS analyses. The major compounds were chromone derivative aromatic compounds sesquiterpenes monoterpenes sterols and fatty acid methyl ester. Aromatic compounds constituted of aldehyde phenol ether and ketone groups. In the agarwood extract of the juvenile fungal-elicited tree but not in the healthy wood some major compounds found were 2-(2-phenylethyl) chromone derivative 4-phenyl-2-butanone (1S4S7R)-14-dimethyl-7-(prop-1-en-2-yl)-12345678-octahydroazulene [guaiene] 1147-tetramethyl-2345677a7b-octahydro-1aH-cyclopropa[h]azulen-4a-ol [palustrol] and 4-(4- methoxyphenyl) butan-2-one [anisylacetone]. These were also found from agarwood of different grades and agarwood collected from the wild mature tree in addition to agarospirol alloaromadendre oxide (2) a-elemol -eudesmol and guaiol. This work demonstrated that in young A. malaccensis trees fungi may be associated to the formation of important agarwood compounds and can be detected as early as six months after inoculation. Copyright 2014 Friends Science Publishers

Keywords: Agarwood extracts; Aromatic compound; Chromone; Gaharu; GC-MS; Monoterpene; Sesquiterpene; Sterol

Introduction

Aquilaria malaccensis is the main producer of agarwood in Malaysia. The species can be found abundantly in the Peninsular but not in the eastern part of Malaysia. Agarwood is regarded as the most valuable resinous fragrant wood in the world because of its high price in the market which can reach up to US$100000 per kilogram for superior quality (Naef 2011). High demand for agarwood in the international market has seriously affected the natural resources of all Aquilaria species and as a result they were listed as endangered species in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) (CITES 2010].General understanding of agarwood formation normally associates the phenomenon with wounding and fungal infections. Studies have shown that a diverse group of fungi are commonly found in wounded wood of A. malaccensis (Tamuli et al. 2000; Mohamed et al. 2010). The resin is often regarded as a pathological product produced by fungal invasion on the host around affected areas as tree defense reaction (Oldfield et al. 1998; Van Beek and Phillips 1999). Agarwood contains aromatic terpenes with sesquiterpenes as the main active compounds

and chromones as another major contributor to the unique fragrance of agarwood (Naef 2011). In order to meet the demand for agarwood cultivation of Aquilaria trees in huge acreages are now being practiced in many countries. Artificial inoculation techniques using diverse methods such as deliberate wounding coupled with introduction of inducing-agents such as chemicals and microbes have been applied (Pojanagaroon and Kaewrak 2005; Zhang et al.2010).Different induction methods resulted in different agarwood qualities. Some reported that essential oils originated from agarwood induced by nail in setting and holing of Aquilaria stem contain high number of major sesquiterpenes and aromatic groups while those induced by trunk breaking contain high amount of fatty acids (Lin et al.2010). Chemicals such as sodium chloride has been shown to stimulate agarwood of similar quality to wild agarwood in A. sinensis (Chen et al. 2011). However artificial induction using microbes yielded less promising results. Chemical constituents of essential oils from A. agallocha infected with the fungi Chaetomium globosum and Fusarium oxysporum for 30 days had similar profiles to that of healthy trees and trees that were mechanically wounded with screws (Tamuli et al. 2005; Bhuiyan et al.

2009). Among the chemical constituents found fatty acids predominantly exist in both healthy and inoculated woods while 10-epi--eudesmol was identified in naturally infected wood (Tamuli et al. 2005). Aristolene a major compound in agarwood was detected in healthy (3.85%) and screw- wounded trees (5.36%) (Bhuiyan et al. 2009) while caryophyllene oxide an oxygenated sesquiterpene was identified in healthy (11.25%) and naturally infected A. sinensis (33%) (Lin et al. 2010). Here the chemical constituents in the extracts of fungal-elicited young wood and naturally infected mature wood from A. malaccensis were compared to the chemical profiles of commercial agarwood all of Malaysian origin. For extraction methanol was used and the crude extracts were subjected to GC-MS analyses.

Materials and Methods

Plant Material

Fungal-elicited wood (I) was harvested from 4-year-old trees grown in the nursery after 6 months of artificial inoculation. Naturally infected wood (W) was collected after 6 months of wounding from a wild growing mother tree while healthy wood (H) was collected from unwounded parts of the same tree. The wound was made 6 cm into the xylem using an electric drill with a diameter of16 mm. The commercial agarwood (Super A SA) was purchased from an international agarwood dealer in Kuala Lumpur and the grade was determined by the dealer. Fresh wood samples were air-dried and powdered followed by Soxhlet extraction for 6 hours using methanol as the solvent. After extraction the solvent was removed by means of a rotary evaporator under reduced pressure to yield the extracted compound.

Analysis of the Extracts

Extracts were analyzed on a Shimadzu GC-2010 equipped with a SGE BPX 5 fused silica capillary column 30 mA-0.25 mm 0.25 m film thickness. The injection port and detected temperatures were set at 230C and 250C respectively. Samples were injected by splitting and the split ratio was1:50. The carrier gas was He at a flow rate of 1.0 mL/min.Oven temperature was kept at 50C for 3 min increasing to320C at a rate of 5C/min and holding for 20 min then increasing to 340C at a rate of 15C/min and holding for 5 min. Injector temperature was 280C while the detector temperature was 340C. GC/MS analyses were carried out on a Shidmazu QP2010 Plus gas chromatography EI electron impact ion source 70 eV mass range 40-700 m/z with similar condition as described in GC programs. Identification of the chemical components was based on the comparison of the calculation of their retention indices and authentic mass spectra data with the existing National Institute of Standards and Technology (NIST) 2008 library

(U.S. Department of Commerce Gaithersburg MD) and literature.

Analysis of Chromone Derivative

Agarwood extracts from the SA and W samples were analyzed to determine the identity of the chromone derivative. Proton COSY (correlation spectroscopy) ROESY (rotating frame Overhauser effect spectroscopy)1H-13C HSQC (heteronuclear single quantum coherence) and HMBC (heteronuclear multi-bond correlation) NMR (Nuclear Magnetic Resonance) experiments were recorded in d4-methanol on a Bruker DRX 600 spectrometer.

Results

Table 1 shows the results of the identified compounds in all the wood samples. A total of 39 compounds were identified from the four samples with the major constituents being derivatives of 2-(2-phenylethyl) chromone aromatic compounds sesquiterpenes monoterpenes sterol compounds and fatty acid methyl ester. Besides chromone the dominant compounds were 4-phenyl-2-butanone (4.5%) palustrol (4.0%) benzaldehyde (1.9%) and benzenepropanoic acid methyl ester (0.9%). Compounds detected included major aromatic compounds (group of aldehyde phenol ether ketone) in addition to oxygenated sesquiterpenes monoterpene hydrocarbons oxygenated monoterpenes sesquiterpene hydrocarbons fatty acid methyl esters and sterols.This investigation revealed that chemical composition of the fungal-elicited young wood were very similar to that of high quality commercial agarwood and naturally infected agarwood from wild A. malaccensis. They were rich in important fragrant compounds such as benzaldehyde 4- phenyl-2-butanone benzenepropanoic acid methyl ester guaiene palustrol anisylacetone 8-napthol1-(benzyloxy)- and 2-(2-phenylethyl) chromone derivative. The chromone derivative was found at high percentages (between 17.6% to18.8%) in all agarwood samples except in the healthy wood (Table 1). This indicates that the chromone derivative is a major constituent of Malaysian agarwood. We further analyzed the compound to verify its identity using NMR (Fig. 1). The strongest signals observed in the NMR spectra of the SA and the W wood samples were characteristics of a2-(2-phenylethyl) chromone derivative (Shimada et al.1982).Indeed two coupled multiplets at dH 2.98 ppm (dC32.9 ppm) and dH 2.89 ppm (dC 35.5 ppm) were assignedrespectively to the H-7' and the H-8' of the CH2CH2fragment. On the one hand H-7' notably gives HMBCcorrelation with the phenyl C-1' quaternary carbon at dC140.7 ppm and NOE (nuclear Overhauser effect)correlations with the H-2'/H-6' phenyl protons at dH 7.18 ppm (dC 128.6) and with the H-3'/H-5' phenyl protons at dH7.21 ppm (dC 128.7 ppm). The latters appear to be coupled to the phenyl H-4' proton at dH 7.13 ppm (dC 126.8 ppm).

Table 1: Chemical constituents of various agarwood extracts from Malaysian agarwood and from infected mature and

juvenile Aquilaria malaccensis

Compounds###RIa###Relative Peak Area (%)

###SAb Wc###Id###He

2-Propenoic acid2-methyl-ethyl ester###752###-###-###-###03

2(3H)-Furanonedihydro-###825###-###-###-###03

Benzaldehyde###982###06###06###03###04

3(2H)-Furanone4-hydroxy-25-dimethyl-###1022###-###-###-###07

2-Methoxyphenol [Guaiacol]###1090###-###-###-###13

4-Phenyl-2-butanone [Benzylacetone]###1228###10###22###13###-

Benzenepropanoic acid methyl ester###1259###02###05###02###-

13-Dimethoxy-2-hydroxybenzene [Syringol]###1279###-###-###03###20

2-Methoxyhydroquione###1311###-###-###-###03

Benzenepropanoic acid###1349###-###-###06###-

4-Hydroxy-3-methoxybenzaldehyde [Vanillin]###1360###-###-###-###04

2H-1-Benzopyran-2-one34-dihydro-###1392###-###-###03###09

Phenol2-methoxy-4-propyl-###1402###-###-###-###08

1H-Cyclopropa[a]naphthalene1a245677a7b-octahydro-1177a-tetramethyl- (1aR7R7aR7bS)-[(-)-Aristolene]###1403###20###27###-###-

4-(4-methoxyphenyl) butan-2-one [Anisylacetone]###1417###03###-###03###-

Alloaromadendrene oxide-(2)###1462###03###05###-###-

Caryophyllene oxide###1507###03###-###-###-

-Elemol###1522###16###11###-###-

(1S4S7R)-14-Dimethyl-7-(prop-1-en-2-yl)-12345678-octahydroazulene [Guaiene]###1523###03###-###07###-

1147-tetramethyl-2345677a7b-octahydro-1aH-cyclopropa[h]azulen-4a-ol [Palustrol]###1530###14###15###11###-

2-propanone1-(4-hydroxy-3-methoxyphenyl)-###1538###-###-###-###11

gamma.Gurjunenepoxide-(2)###1558###10###-###-###-

2669-Tetramethyl-14-8-cycloundecatriene [Humulene]###1579###03###-###-###-

Agarospirol###1598###03###07###-###-

Guaiol###1614###09###14###-###-

Benzopheone###1603###-###-###05###30

-Eudesmol###1626###31###38###-###-

2-Butanone4-(4-hydroxyl-3-methoxyphenyl)-###1638###-###-###-###03

Phenol 4-(3-hydroxy-1-propenyl)-2-methoxy-###1653###-###-###-###66

6-Isopropenyl-48a-dimethyl-123456788a-octahydro-napthalen-2-ol###1690###-###13###-###-

37-Cyclodecadiene-1-methanolalpha.alpha. 48-tetramethyl-[s-(Z-Z)]###1694###-###18###-###-

Benzenepropanoic acid25-dimethoxy###1727###-###-###33###63

Hexadecanoic acidmethyl ester###1878###-###-###02###-

9-Octadecenoic acid methyl ester(E)-###2085###-###-###04###-

8-Napthol1-(benzyloxy)-###2314###02###-###06###-

2-(2-phenylethyl) chromone derivative###2389###176 188 185 -

Stigmastanol###2720###-###-###03###04

Stigmast-5-en-3-ol(3.beta.)###2731###-###-###-###18

Stigmasterol###2739###-###-###09###05

On the other hand H-8' gives a NOE correlation with the distinctive singlet of the H-3 chromone moiety at dH 6.08 ppm as well as HMBC correlations with C-3 (dC 113.3 ppm) and the C-2 quaternary carbon at dC 140.7 ppm. Then H-3 gives an HMBC correlation with C-10 at 120.9 ppm. Unfortunately the quality of the NMR spectra did not allow us to characterize further the second cycle (C-5 to C-8) of the chromone moiety. A precise and unambiguous identification of this 2-(2-phenylethyl) chromone derivatives would require the purification of the compounds but we did not pursue this matter as it is not within the scope of this present study. Besides agarwood extracts are known to contain many derivatives of 2-(2-phenylethyl) chromone with a total of thirty-nine differentones have been identified in various agarwood qualities(Naef 2011). At least four 2-(2-phenylethyl)-4H-chromen-

4-one derivatives have been reported from A. malacensis and an additional new derivative has been described recently from methanol extracts (Wu et al.2012).

Discussion

The compounds reported here specifically benzaldehyde benzenepropanoic acid and anisylacetone were also reported by Lin et al. (2010). In addition they also found a chromone 8-methoxy-2-(2-phenylethyl)-4H-1-benzopyran-4-one in the ether extract of infected wood from A. sinensis after six months and one year of inoculation with the fungus Melanotus flavolivens. These findings show promising results for microbe induction method unlike in previous experiments (Tamuli et al. 2005; Bhuiyan et al. 2009). Chromones are reported to be responsible for the warm balsamic sweet and long-lasting odour when agarwood is burnt (Naef 2011).Interestingly the fungal-elicited wood shared some similarities with healthy wood in the aromatic and sterol compounds. Among them are benzopheone benzenepropanoic acid 25-dimethoxy 2H-1-benzopyran-2-one34-dihydro- 1-3 dimethoxy [syringol] stigmasterol and stigmastanol. Several terpene types such as aristoleneagarospiral alloaromadendrene oxide-(2) and guaiol werenot identified from the young infected wood (Table 1) indicating that these major compounds contribute to the unique odor formed in high quality agarwood (SA and W) while gamma.gurjunenepoxide-(2) was found exclusively in the SA agarwood. Both stigmasterol and stigmastanol are important pharmaceutical intermediates since they are known phytosterols which have potent anti-inflammatory effects and anti-catabolic properties (Gabay et al. 2010).When looking at available data from Malaysian agarwood 4-phenyl-2-butanone and agarospirol were identified consistently in A. malaccensis agarwood samples (Nor Azah et al. 2008; Saiful and Mashitah 2010; and this present study). Two compounds Jinkohol II and Kusunol were not detected in our samples which had originated from A. malaccensis populations in the states of Malacca and Terengganu. They are also absent in agarwood samples from Kelantan Pahang and Terengganu states but are present from Selangor population (Nor Azah et al. 2008). This indicates that environment and genetic factors may have roles in causing the varying aromatic compounds. Some major terpenes that are present in Malaysian agarwood are jinkoh-eremol caryophellene oxide eudesmol and a-guaiene (Saiful and Mashitah 2010).Chromone although a major compound in agarwood has not been reported from solvent-extracted A. malaccensis agarwood from Malaysia (Nor Azah et al. 2008; Saiful and Mashitah 2010) but has been reported from A. malaccensis of Laos origin (Wu et al. 2012). Here we report the presence of 2-(2-phenylethyl) chromone derivatives in all Malaysian agarwood tested (including grades A B C and D; data not shown) and at high percentages (between 17% to 19%). This compound was not detected in non-infected wood. Other important compounds such as AY-agarofuran a-agarofuran and 10-epi--eudesmol were not detected in this study perhaps because these were methanol extracts and not hydrodistilled (Tamuli et al. 2005; Nor Azah et al.2008; Saiful and Mashitah 2010; Chen et al. 2011; Pripdeevech et al. 2011). Different extraction methods employed could result in different compounds.In conclusion the analysis of the extracts obtained from the six-month fungal-elicited wood has high similarity to that of naturally infected and commercial agarwood. Thissuggests that agarwood could be produced artificially byfungi inoculation method. Indeed the role of fungi in forming agarwood compounds need to be studied further.

Acknowledgments

This project was supported by the Universiti Putra Malaysia Research University Grant Scheme (Project No. 03-03-11-1438RU). Access to the Bruker DRX 600 (NMR facilities of the SCBIM and IFR 111 BioingACopyrightnierie UniversitACopyright de Lorraine) was deeply appreciated.

References

Bhuiyan M.N.I. J. Begum and M.N.H. Bhuiyan 2009. Analysis of essential oil of eaglewood tree (Aquilaria agallocha Roxb.) by gas chromatography mass spectrometry. Bangl. J. Pharm. 4: 2428Chen H.Q. Y. Yang X. Jian J.H. Wei Z. Zhang and H.J. Chen 2011.Comparison of compositions and antimicrobial activities of essential oils from chemically stimulated agarwood wild agarwood and healthy Aquilaria sinensis (Lour.) Gilg trees. Molecules 16: 48844896CITES 2010. Appendix II of Conventional on Internal Trade in Endangered Species of Wild Fauna and Flora. http://www.cites.org/eng/app/appendices.phpGabay O. C. Sanchez C. Salvat F. Chevy M. Breton G. Nourissat C.Wolf C. Jacques and F. Berenbaum 2010. Stigmasterol: a phytosterol with potential anti-osteoarthritic properties. Osteo. Cart.18: 106116Lin F. W.L. Mei J. Wu and H.F. Dai 2010. GC-MS analysis of volatile constituents from Chinese eaglewood produced by artificial methods. J. Chin. Med. Mater. 33: 222225Mohamed R. P.L. Jong and M.S. Zali 2010. Fungal diversity in woundedstems of Aquilaria malaccensis. Fungal Div. 43: 6774Naef R. 2011. The volatile and semi-volatile constituents of agarwood the infected heartwood of Aquilaria species: a review. Flav. Frag. J. 26:7387Nor Azah M.A. Y.S. Chang J. Mailina A. Abu Said J. Abd Majid H.S.Saidatul H. Hasnida and Y. Nik Yasmin 2008. Comparison of chemical profiles of selected gaharu oils from Peninsular Malaysia. Malay. J. Anal. Sci. 12: 338340Oldfield S. C. Lusty and A. MacKinven 1998. The Word List ofThreatened Trees. Heart of the matter: Agarwood use and trade and CITES implementation for Aquilaria malaccensis. TRAFFIC InternationalPojanagaroon S. and C. Kaewrak 2005. Mechanical methods to stimulate aloes wood formation in Aquilaria crassna Pierre Ex H. LEC. (Kristsana) trees. In: III WOCMAP Congress on Medicinal and Aromatic Plants Vol. 2: Conservation Cultivation and Sustainable Use of Medicinal and Aromatic Plants. Jatisatienr A. T. Paratasilpin S. Elliott V. Anusarnsunthorn D. Wedge L.E. Craker and Z.E. Gardner (eds.). ISHS Acta Horticulturae Chiang Mai ThailandPripdeevech P. W. Khummueng and S.K. Park 2011. Identification of odor-active components of agarwood essential oils from Thailand by solid phase microextraction-GC/MS and GC-O. J. Essent. Oil Res.23: 4653Saiful N.T. and M.Y. Mashitah 2010. Chemical composition of volatile oils of Aquilaria malaccensis (Thymelaeaceae) from Malaysia. Nat. Prod. Comm. 5: 19651968Shimada Y. T. Tominaga T. Konishi and S. Kiyosawa 1982. Studies on the agarwood (Jinko) I Structures of 2-(2-phenylethyl) chromone derivatives. Chem. Pharm. Bull. 30: 37913795Tamuli P. P. Boruah S.C. Nath and P. Leclercq 2005. Essential oil of eaglewood tree: a product of pathogenesis. J. Essent. Oil Res. 17:601604Tamuli P. P. Boruah S.C. Nath and R. Samanta 2000. Fungi from diseased agarwood tree (Aquilaria agallocha Roxb.). In: Advances in Forestry Research p: 22. Parkash R. (ed.). New Delhi IndiaVan Beek H. and D. Phillips 1999. Agarwood. Trade and CITES Implementation in Southeast Asia. Report TRAFFIC Southeast Asia MalaysiaWu B. J.G. Lee C.J. Lim S.D. S.W. Jia Kwon G.S. Hwang and J.H.Park 2012. Sesquiterpenoids and 2-(2- phenylethyl)-4H-chromen-4- one (=2-(2-phenylethyl)-4H-1-benxopyran-4-one) derivatives from Aquilaria malaccensis agarwood. Helv. Chim. Act. 95: 636642Zhang Z. Y. Yang H. Meng C. Sui J.H. Wei and H.Q. Chen 2010.Advances in studies on mechanism of agarwood formation in Aquilaria sinensis and its hypothesis of agarwood formation induced by defense response. Chin. Trad. Herbal Drugs. 41: 156160
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Author:Jong, Phai Lee; Tsan, Pascale; Mohamed, Rozi
Publication:International Journal of Agriculture and Biology
Article Type:Report
Date:Jun 30, 2014
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