Analytical Methods Discrimination of young and mature leaves of Melicope ptelefolia using 1 H NMR and multivariate data analysis Nor Hassifi Shuib a,1 , Khozirah Shaari a,1 , Alfi Khatib a,b,⇑ , Maulidiani a , Ralf Kneer c , Seema Zareen a , Salahudin Mohd. Raof a , Nordin Hj. Lajis a , Victor Neto d a Laboratory of Natural Product, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia b Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia c Borneo Plant Technology Sdn Bhd, Kuching, Sarawak, Malaysia d Faculty of Forestry, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia article info Article history: Received 16 June 2010 Received in revised form 29 September 2010 Accepted 10 October 2010 Keywords: Metabolomics Multivariate data analysis Principle component analysis NMR Melicope ptelefolia abstract The ‘Ulam’, a traditional Malay dish, are plants that can be eaten raw, as a form of local salad. The shoots and young leaves of Melicope ptelefolia are among the popular species, believed to be high in nutritional and medicinal values. The metabolomic fingerprinting analysis of the ethanolic extracts of leaves of M. ptelefolia was carried out using 1 H Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate data analysis in order to differentiate young and mature leaves and to evaluate the variation of their chemical composition. Principle component analysis (PCA) of the 1 H NMR spectra showed a clear dis- crimination between the young and mature leaves extracts by PC3 and PC4. The compounds responsible for the differentiation were identified by comparison of 1 H NMR chemical shifts and qualitative HPLC. The young leaves were found to be richer in fatty acids and the levels of the three marker compounds, p-O- geranylcoumaric acid, 2,4,6-trihydroxy-3-geranylacetophenone and 2,4,6-trihydroxy-3-prenylacetophe- none, were clearly higher. The mature leaves contain higher levels of sugars and glycosidic components. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction A direct result of modern lifestyle, in developed and developing nations of the world, is the increase of diseases such as cancer, dia- betes and cardiovascular diseases. With an increased conscious- ness of the people of these health risks, the consumption of raw vegetables and fruits has increased significantly as individuals have become more health-conscious and aware of the importance of plant-based diets in combating the onset of such diseases. It is now a well known fact that plant-based diets offer nutritional ben- efits and therapeutic values which are connected with the presence of phytochemicals (flavonoids, isoflavones, lignans, cinnamic acids derivatives, steroids, carotenoids, terpenoids, etc.), vitamins, poly- saccharides, proteins and minerals contained in plants. Taking raw vegetables as a side dish at mealtimes is also a tradition that dates back to the ancient times of the Malay culture. The vegetable or ‘ulam’ which are normally taken together with a relish made up of pounded chillies and ‘belacan’ (shrimp paste), may consist of a variety of traditional vegetables deemed to be beneficial for health according to the Malay folklore. Some examples of these healthy vegetables include well known medicinal plants such as ‘petai’ (Parkia speciosa), ‘jering’ (Pithecellobium jiringga), ‘ulam raja’ (Cos- mos caudatus), ‘temu mangga’ (Curcuma mangga), ‘pudina’ (Mentha pudica), ‘selasih’ (Ocimum basilicum) and a myriad of other species (Mansor, 1988). Nutritionally, these vegetables have been shown to be rich sources of carbohydrates, proteins, minerals and vita- mins (Ismail, 2000). The fruit kernels, rhizomes, young leaves and shoots usually selected for consumption are mostly consumed in raw form but sometimes they are blanched or boiled (Norhanom, Ashril, & Mustafa, 1999). The shoots and young leaves of Melicope ptelefolia are also pop- ular as an ‘ulam’. The genus Melicope encompass around 233 spe- cies, occurring from the Malagasy and Indo-Himalaya regions east to the Hawaiian and Marquesas Islands and south to New Zealand (Hartley, 2001). The subject of our study is M. ptelefolia Champ Ex Benth, a common species of the genus with the vernacular names ‘tenggek burung’ and ‘pauh-pauh’. The latter name refers to the pleasant citrusy taste of the young leaves and shoots of the plant which are much favored as an ‘ulam’. In Vietnam, the leaves are medicinally used for treating itches and wounds, while the root and bark served as a digestive and an appetizer (Loi, 1977). A decoction of the roots is used to treat fever and rheumatism (Perry & Metzger, 1980). The leaf of the plant has been reported to show fungicidal (Kumar, Karunaratne, Sanath, 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.10.043 ⇑ Corresponding author at: Laboratory of Natural Product, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia. Tel.: +603 8946 8540; fax: +603 8942 3552. E-mail address: alfikhatib@hotmail.com (A. Khatib). 1 These authors equally contributed to the article. Food Chemistry 126 (2011) 640–645 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem