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D.M. Nazrul Hisham, J.M. Lip, J. Arif Zaidi and A. NormahJ. Trop. Agric. and Fd. Sc. 38(1)(2010): 97– 102

Main non-polar chemical constituent from Morinda citrifolia fruits(Pengekstrakan sebatian kimia utama tidak berkutub daripada buah Morinda citrifolia)

D.M. Nazrul Hisham*, J.M. Lip**, J. Arif Zaidi* and A. Normah*

Keywords: non-polar compound, Morinda citrifolia, coumarin, scopoletin

AbstractSeparation, isolation and purification of compounds from Morinda citrifolia fruits were done using solvent-solvent partitioning and chromatography techniques. The dichloromethane (DCM) soluble partition of the methanol (MeOH) extract had undergone a purification technique. One main non-polar constituent was successfully isolated and identified using modern spectroscopy method such as Nuclear Magnetic Resonance (NMR) and mass spectroscopy. This compound is a type of coumarin known as scopoletin and it can be a standard compound for non-polar constituent analysis of Morinda citrifolia fruits especially for products.

*Food Technology Research Centre, MARDI Headquarters, Serdang, P.O. Box 12301, 50774 Kuala Lumpur, Malaysia**Technical Services Centre, MARDI Headquarters, Serdang, P.O. Box 12301, 50774 Kuala Lumpur, MalaysiaAuthors’ full names: Mohd Nazrul Hisham Daud, Mohd Lip Jabit, Arif Zaidi Jusoh and Normah AhamadE-mail: nazrul@mardi.gov.my©Malaysian Agricultural Research and Development Institute 2010

IntroductionMorinda citrifolia L. (Rubiaceae), commonly called Noni or Indian mulberry, is a small evergreen tree or shrub of Polynesian origin (McClatchey 2002). Morinda citrifolia bears a lumpy, green to yellowish-white fruit, normally 5–10 cm long, with a surface covered in polygonal-shaped sections (Nelson 2001; McClatchey 2002). Morinda citrifolia has a long history of use as medicinal plant in part of Southeast Asia, Polynesia and Australia and is considered to be the second most important medicinal plant in the Hawaiian Island (Tabrah and Eveleth 1966; McClatchey 2002). The fruits have been used medicinally to treat a wide range of ailments. These include, but not limited to, diabetes, diarrhoea, hypertension, malaria, pain and infections (Morton 1992; McClatchey 2002). The fruits are also eaten as a food,

but primarily only in times of famine (Morton 1992). Products from M. citrifolia (fruits) has become widespread and commercially available in health food stores and chain grocery stores specializing in natural food. Usually products from M. citrifolia fruits are sold in the form of tablet, tea and juice. In the United States, the growth in popularity may in part be attributed to claim of Noni being a ‘cure-all’ or aid in relieving symptoms for a host of chronic conditions such as arthritis, cancer, diabetes and hypertension (McClatchey 2002). Past studies indicated that the major chemical constituents of Morinda being identified were anthraquinones (Perkin 1908), lipid glycosides (Wang et al. 1999) and flavonol glycosides, iridoid glucosides and triterpenoids (Sang et al. 2001, 2002). Purification procedure of scopoletin from M. citrifolia was not reported in

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previous studies. Latest study by Su et al. (2005) only reported the purification work on iridoid glucosides. GC trace analysis using column CP Wax 58 CB by Farine et al. (1995) on volatile components of M. citrifolia ripe fruits discovered 2% of scopoletin. The GC data interpretation was done by comparing with the library of Laboratoire de Recherches sur les Aromes (INFRA, Dijon, France). No further purification had been done and hence biological studies such as antioxidant, antimicrobial and other potential biological activities on this compound was not looked into. Identification work using other spectroscopy analysis to support this finding was also lacking. Although many major chemical constituents have been found, they are not easy to be isolated and identified even using modern spectroscopy technique. This study focused on the isolation technique to get a truly major component with a simple technique of isolation and identification especially in a mixture environment as in food products. Availability of these simple techniques would reduce the cost of analysis and standardization.

Materials and methodsCollection and preparation of sampleA total of 10 kg of M. citrifolia fruits were collected from MARDI Bukit Ridan station. All the fresh fruits were cut into small pieces and finely ground with an electric blender, prior to extraction with methanol.

Extraction and isolationThe ground sample was soaked in 100% methanol (MeOH) for 4 days at room temperature after which the extract was decanted. It was then replenished with fresh MeOH and the same extraction procedure was repeated twice. The extracts collected from each soaking were pooled and evaporated to dryness. The crude methanolic extract was then resuspended in distilled water and solvent partitioned with dichloromethane (DCM).

The DCM partition or fraction was then subjected to chromatographic technique for the isolation and purification of compounds from the plant extract. It was performed using open column chromatography and monitored by thin layer chromatography (TLC) respectively. Open column chromatography was carried out using regular glass chromatography columns, slurry-packed with normal phase silica gel and eluted using organic solvent starting with 100% hexane, followed by mixtures of hexane and chloroform of increasing polarity. A compound in the form of white crystalline needles was obtained. This compound was further analysed with modern spectroscopy such as gas chromatography mass spectrometer (GC-MS) using direct injection and nuclear magnetic resonance (NMR) for structure identification.

Results and discussionIdentification of scopoletinIsolation and purification of non-polar fraction (DCM fraction) of M. citrifolia fruits led to discovery of scopoletin using three simple techniques. Starting with sample extraction followed by fractionation with organic solvent and finally, the fraction underwent one time separation with column chromatography using a mixture of hexane and chloroform gradiently from non-polar to polar (Figure 1).

Sample extractionMorinda citrifolia (fruits)

MeOH

Fractionation of extract

Dichloromethane

Dichloromethane fraction undergo column chromatographic technique using mixtures of hexane

and chloroform gradiently

Figure 1. Isolation and purification technique of scopoletin

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Plate 1. Scopoletin in the form of white crystalline needles

From isolation and purification work on M. citrifolia (fruits), 0.02% of scopoletin was obtained as fine white crystalline needles as shown in Plate 1. Study by Aisha (2002) indicated that the melting point for scopoletin was 204 –206 °C with blue fluorescent colour under UV light. The UV lmax (CHCl3); 244, 260, 296, 345 nm while the infrared spectrum, IR nmax ( KBr disc) cm-1; 3448 (hydroxyl, OH), 1728 (carbonyl, C=O), 2939, 2850 (CH), 1490 and 1589 (aromatic). GC-MS analysis found that the EI-MS (Figure 2) exhibited a molecular ion peak at m/z 192.0 [M+], 177 [M+-CH3], 164 [M+-CO], 149 [M+- CH3-2CO], 121, 107, 69 and 51 corresponding to the molecular formula C10H8O4. The structure of scopoletin comprising 10 carbons with four oxygenated aromatic quaternary carbon, five non-oxygenated aromatic carbon and one methyl carbon (Figure 3). The general functional groups that could be observed on this compound include carbonyl group (C=O), a hydroxyl (OH), an aromatic and a methoxy (OMe/ OCH3) group. Cheng et al. (2007) reported that the presence of methoxy and hydroxyl groups could contribute to antioxidant properties.

H O

M e O

O O

Figure 3. Chemical structure of scopoletin with molecular formula C10H8O4

107

69

51

25 50 75 100 125 150 175 200

121

149

164

177

192

Figure 2. EI-MS spectra of scopoletin

Through 1H NMR spectrum, the presence of one methoxy (–OCH3) group at d 1.6 was observed (Figure 4). Abundant peaks that represent hydrogen on the non-oxygenated aromatic ring could be seen between d 6.0 and 7.6. The 13C NMR spectrum as showed in Figure 5 indicates the presence of a carbonyl (C=O) group at dc 161.5 and methoxy carbon at dc 56.4. A number of non-oxygenated aromatic carbon could be

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Figure 4. 1H NMR spectrum of scopoletin

Figure 5. 13C NMR spectrum of scopoletin

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observed within the range dc 103.2 to 113.4 of the carbon spectrum. Other than the carbonyl group that is present within the dc range of oxygenated aromatic carbon, the rest of the oxygenated aromatic carbon appears in the range dc 143.3–150.2. Sibanda et al. (1988) also discovered scopoletin from Xeromphis obovata root bark (arboreal plant of central and southern Africa utilized in traditional medicine). Together with scopoletin, one new diglucocoumarin, named xeroboside and a known compound, scopolin were also isolated from this species. Comparing this literature (Sibanda et al. 1988) with this study, it was confirmed that the compound isolated in this study was scopoletin. The comparison of the 13C NMR value for compound isolated from M. citrifolia and the compound which was isolated by Sibanda et al. (1988) is shown in Table 1. All the 13C values for compound isolated from M. citrifolia and X. obovata match with slight differences based on the different type of instrument used.

ConclusionPhytochemical analysis consisting of isolation and purification on Morinda citrifolia fruits led to the discovery of a main non-polar chemical constituent that belongs to coumarin group called scopoletin. Although screening of this compound in the same species has been done using GC-MS, no purification and identification

using detailed spectroscopy techniques had been done. Present discovery also suggests that scopoletin could be a good indicator or standard for M. citrifolia fruits main non-polar chemical constituents. A simple extraction, isolation and purification technique had been developed. As a result, chemical analysis procedure for isolation and purification of coumarin will be more efficient in terms of time, equipment and cost, in order to fulfil analytical needs.

AcknowledgementThe authors thank MARDI for sponsoring this research.

ReferencesAisha, H.S.A.Z. (2002). Stress metabolites from

Corchorus olitorius L. leaves in response to certain stress agents. Food Chemistry 76(2): 187–195

Cheng, J.C, Fang Dai, Bo Zhou, Li Yang, Zhong-Li Liu (2007). Antioxidant activity of hydroxycinnamic acid derivatives in human low density lipoprotein: Mechanism and structure–activity relationship. Food Chemistry 104(1): 132

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Table 1. Comparison of 13C NMR value13C value (dc) 13C value (dc)[this study] [Sibanda et al. 1988]161.4 160.2113.4 112.5144.0 142.3107.5 107.0143.3 143.2149.7 149.2103.1 102.5111.5 110.5150.2 149.8 56.4 55.2

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Su, B.N., Alison, D.P., Hyun-Ah Jung, Keller, W.J., McLaughlin, J.L. and Kinghorn, A.D. (2005). Chemical constituents of the fruits of Morinda citrifolia (Noni) and their antioxidant activity. J. Nat. Prod. 68(4): 592–595

Tabrah, F.L. and Eveleth, B.M. (1966). Evaluation of the effectiveness of ancient Hawaiian medicine. Hawaii Medical Journal 25: 223–230

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Accepted for publication on 4 January 2010

AbstrakPengasingan dan penulenan sebatian kimia daripada buah Morinda citrifolia telah dilakukan dengan menggunakan kaedah pemisahan pelarut-pelarut dan teknik kromatografi. Bahagian larut di dalam pelarut diklorometana (DCM) daripada ekstrak metanol (MeOH) telah melalui teknik penulenan. Satu sebatian tak berkutub yang utama telah berjaya dipencilkan dan dikenal pasti melalui teknik analisis spektroskopi moden iaitu Nuklear Magnetik Resonan (NMR) dan spectroskopi jisim. Sebatian kimia ini ialah jenis kaumerin dikenali sebagai skopoletin dan ia boleh dijadikan sebatian kimia rujukan bagi analisis sebatian kimia tak berkutub untuk kajian buah Morinda citrifolia terutama dalam penghasilan produk.