Pandanus amaryllifolius Roxb

Pandanus amaryllifolius Roxb. is an erect green plant with fan-formed splashes of long, restricted as such bladelike monocotyledon leaves. Its woody airborne roots are roughly 10 cm (4 inches) long (Laksanalamai and Ilangantileke, 1993). In the Asia including India, Thailand, Indonesia and Malaysia, pandan leaves are generally utilized as a source of natural flavouring such as in the preparations of the rice dishes (Ngadia and Yahya, 2014).

The essential oil of P. amaryllifolius yields terpenes and sesquiterpene hydrocarbons as well as a major aroma component, 2-acetyl-1-pyrolline (Yoshihashi, 2002). The mixtures are dominated by monoterpenes and sesquiterpene derivatives with insect-repellent properties. This plant is found to have therapeutic properties and is a best medium as cockroach repellent (Rusli, 2011).

Buttery et al. (1986), reported that only fresh pandan leaves have odour due to present of oxidative degradation product of yellow carotenoid pigment. The hydrodistillation of pandan leaves yielded essential oil, in which 2-acetyl-1-pyrolline as the aroma contributes that responsible for the aroma of pandan volatile oil and widely found in fragrant rice and basmati rice. This compound is widely use as flavouring agent for bakery (MacLeod and Pieris,1982).

Amount of chemical constituent in pandan leaves are differed by locality, amount of sunlight reached, type of soil and other external features. Pandanus had been reported to consist of various chemical constituent of terpenoids class of compound. Therefore, this study is proposed to identify the chemical constituents that present from essential oil and ethanol pandan leaves extract.
The chemical constituent in Pandan leaves is useful for further work on Pandan leaves for example application of Pandan leaves in nanoparticle application.

To extract and characterize the chemical constituent from ethanol Pandan leaves extract.
To distinguish the chemical constituent in essential oil and pandan leaves extract by using GC-MS analysis.

There are approximately 700 species of Pandanaceae distributed throughout the paleotropics in the order Pandanales that consist of trees, shrubs, epiphytes, and lianas from the arborescent or lianoid dioecious monocot family of Pandanaceae. Five genera have been recognized within the family which are Sararanga (2 spp.), Martellidendron (6 spp.), Benstonea (approximately 60 spp.), Freycinetia (approximately 250 spp.) and Pandanus Parkinson (approximately 450 spp.) (Callmander et al., 2013 and Rizki et al., 2015).

West Africa to the islands of the eastern Pacific is the regions where the Pandanaceace has been circulated throughout the Paleotropics (Figure 2.1). The genera have coincidental distributions centered on Malesia, includes Indonesia, Borneo, the Philippines, and New Guinea while for Martellidendron distributed in Madagascar and the Seychelles. In the Philippines, the Solomon Islands, northern mainland New Guinea, and on the nearby Yepen and Manus islands are narrowly circulated by Sararanga. In New Guinea and Indonesia, the distribution of Freycinetia reaches Hawai’i and Micronesia. The majority of species Benstonea found in Borneo.

There are two main Pandanus subclade which are “subclade 1” and “subclade 2”. “Subclade 1” has distribution of Benstonea while “subclade 2” has accomplished the most stretched out surviving dissemination. Africa, Madagascar, a lot of Southeast Asia, and almost the greater part of the islands of the tropical Indo-Pacific are the regions of the Pandanaceae ancestries (Gallaher et al., 2015).

The genus Pandanus (family Pandanaceae) consists of approximately 700 species circulated in tropical and sub-tropical districts (Tan et al., 2010). It has been divided into two large subgroups, which is fruit and and seeds. In Malaysia, Pandanus sp. leaves are widely used for making various type of handicraft while some of them are may split and unsplit into strip (approximately 2cm) which is dried for weaving baskets or mats and for roof thatching respectively. Usually, this kind of material from Pandanus is very durable for about two years (Othman, 1990). The content of Pandanus has been reported consist of phytochemicals like steroids, carbohydrates, phenols, isoflavones, coumestrol, lignans, alkaloids, glycosides, amino acids and vitamins (Gurmeet and Amrita, 2015).

Pandanus amaryllifolius Roxb. (Pandanaceae) as shown in Figure 2.2, generally recognized as the fragrant screw pine, is circulated in parts of Southeast Asia. Ethnobotanical records of this Pandanus species show that it has a few restorative properties, for instance, antispasmodic, diuretic, and stimulant properties.

Screw pine is a small plant of around 50 cm expressed by very sweet odours leaves. It is spread broadly through tropical and subtropical districts and generally used as a food flavouring additives and folk medicine for curing the heart and as a diuretic (Busque et al., 2002).

Since the main features of Pandanus amaryllifolius is its fragrant leaves, it has been used as zest, nourishment shading and seasoning in culinary application. Its smell is unmistakable with a nutty to new feed emphasize, by one means or another like the scent found in specific assortments of fragrant rice developed in Southeast Asia for example, Thai jasmine rice (Linda et al., 2004).

P. amaryllifolius have a taro-like fragrance and is utilized as enhancing in Indochinese food. Customarily, this plant is also viewed as a therapeutic plant for the therapy of gout, hyperglycemia, hypertension, and stiffness. Futhermore, the antimicrobial, cancer prevention agent, antiviral, hypoglycemic, and tumor development inhibitive impacts of P. amaryllifolius were established through a progression of pharmacological examinations. Because of its different benefits, P. amaryllifolius has been widely refined in southern Taiwan since 1980s (Cheng et al., 2017).

The volatile compounds of the species belonging to the genus Pandanus has been rarely described (Table 2.1). The essential oil of P.latifolius consists of sesquiterpene hydrocarbons which is 6-42% and linalool is 6%. The main component of P. amaryllifolius is 2-acetyl-1-pyrolline (2-AP) (Lechat et al., 1996). A few novel alkaloids bearing c-butylidene-a-methyl ?,?-unsaturated c-lactone and pyrrolidinyl ?,?-unsaturated c-lactone themes were identified from the leaves of P. amaryllifolius as to scan for novel and naturally dynamic metabolites from the variety Pandanus (Tan et al., 2010). The isolation identified of two diastereoisomeric pyrrolidine alkaloids which are pandamarilactonine-A and – B. (Busque et al., 2002).

A total of 31 compounds were identified from the leaves of P. amaryllifolius that accounted for 85% of total volatiles by HS-SPME and 22 compounds by solvent extraction method. The major components are 2-AP (5.52%), hexanal (6.63%), 2-hexanal (21.87%) and nonanal (10.50%) (Wakte et al., 2010). The quantities of 2-AP (of the order of 1 ppm) is more than 10 times found in scented milled rice and 100 times found in common rice while in bread flower with a concentration of 0.53 ppm (Rusli, 2011). The composition of free amino acids and reducing sugars that could be precursors of 2-AP in pandan leaves consist of 2.38 mg/g fructose and 1.77 mg/g glucose for fresh leaves. Major free amino acid in pandan is glutamic acid (0.41 mg/g) and proline is 0.12 mg/g (Cheetangdee et al., 2006).

In the Pandanus extraction, the volatile compounds has been determined by liquid-liquid extraction using dichloromethane as solvent then subjected to GC-MS. There are 22 compounds that has been identified, which are 9 alcohols, 4 carboxylic acids, 3 ketones, 2 esters, 3 hydrocarbons and 1 furanone. The major component is over 70% of 3-Methyl-2-(5H)-furanone. The others component that have been found as major compounds are 3-hexanol, 4-methylpentanol, 3-hexanone and 2-hexanone (Jiang, 1999).

Pandanus has been reported to consist of the aroma principal 2-Acetyl-1-pyrroline (2-AP) (Figure 2.3) where this compound is responsible for pleasant aroma in basmati and other scented rice. 2-AP was analysed using continuous steam-distillation extraction of freeze-dried fresh leaves of Pandanus yields 12 ppm (based on dry weight of leaves) of steam-volatile oil. Gas chromatography-mass spectrometry (GC-MS) analysis showed 1 ppm of 2-AP in the volatile oil (Bhattacharjee et al., 2004). It is available in the rice endosperm at ten times more prominent in scented rice than in nonaromatic rice. Phytochemical examinations of this plant uncovered that it is rich in alkaloids, which were proposed to be biosynthesized from glutamate and leucine (Cheng et al., 2017).

Supercritical carbon dioxide (SC-CO2) and solvent extraction of components from pandan leaves gave 10 times lower extraction yield as compared to the hexane extracts. By decreasing particle size of pandan leaves, the total yield of extracts was increased up to 50%. The highest yield of extraction is coarsely ground freeze-dried by SC-CO2 obtained (0.88±0.06%) followed by oven dried (0.38±0.09%) and fresh leaves (0.34±0.01%) (Yahyaa et al., 2010).

There are two key methods in extraction of essential oil which is distillation (includes hydrodistillation) and expression. Besides, it also can be extracted via Solvent extraction, but it is rarely performed in the modern day (Schnaubelt, 2002).

There are various uses of essential oils. For examples, the mixtures consisting predominantly of mono- and sesquiterpene derivatives, accounts for the insect-repellent and attractant properties associated with some aromatic plants (Herout, 1970; Rice, 1983). Previous studies have established significant repellent activity of P. amaryllifolius against American cockroaches (Periplaneta americana L.) (Ahmad et al, 1995). The main chemical constituents of the essential oil were phytol (42.15%), squalene (16.81%), pentadecanal (6.17%), pentadecanoic acid (4.49%), 3, 7, 11, 15-tetramethyl-2-hexadecen-1-ol (3.83%), phytone (2.05%) and the other 74 chemical compositions which were firstly identified from the essential oil of Pandanus amaryllifolius leaves (Cai, 2014).

The first step in the analysis and isolation of natural products is extraction which is to categorize the compounds from the cellular matrix. Pre-purification step in extraction methods is also used as to eliminate interfering components and/or to isolate the active compounds. Extraction and recovery of a solute from a solid matrix may be regarded as a five-stage process: (i) desorption of the compound from the active sites of the matrix; (ii) diffusion into the matrix itself; (iii) solubilisation of the analyte in the extractant; (iv) diffusion of the compound in the extractant; (v) collection of the extracted solutes. Ideally, an extraction process should be exhaustive with respect to the constituents to be analysed or isolated, fast, uncomplicated, cheap, and – at least for routine analysis – amenable for automation (Sticher, 2007).

Solvent extraction is an efficient separation technique used in a variety of applications including analytical chemistry to industrial processes in pharmaceutical, food engineering, hydrometallurgy, and waste treatment. The advantage of solvent extraction is it takes the solubility difference of a solute in two immiscible liquid phases (usually one organic and one aqueous phase) in associate with each other to achieve separation (Li et al., 2016).

In this study, ethanol extract will be fractionated using vacuum liquid chromatography (VLC). For the fractionation of natural products prior to other separation steps like HPLC, VLC is mainly used. For purification process the sample will be subjected to solvents of varied polarity (Sticher, 2007).

There are two ways for purification process which are dry column vacuum chromatography and silica gel column. The advantages of dry column vacuum chromatography consist of great resolving power, simple applied to large scale chromatography (up to 100 g) and is faster. Furthermore, the method is economical and environmentally friendly due to significant reductions in solvent and the amount of silica used (Pedersen and Rosenbohm, 2001).

The chemical structure at secondary metabolites is analysed using spectroscopic technique. By analysing the data critically, it is possible to determine its molecular weight. The molecular interpretation helps to deduce the structure of the compound (Sasidharan et al., 2011).

Fresh pandan leaves (Pandanus amarylliofolius) about 1 to 2kg were collected from Sik, Kedah. Pandan leaves were cut into a small pieces and dried for one month. After that, pandan leaves were ground powder. Approximately 2kg of leaves mass were soaked for 1 week in 2L 95% ethanol. After that, the pandan leaves were filtered out from the solution and rotavap. The crude were obtained and used for perform TLC and some of crude were prepared for run in GC-MS

Silica gel (60 g, Merck) was in dry-packed 60 mm column using suction to make a 4–5 cm bed onto which the dried sample was added. The column was eluted with hexane in numbered 250 ml Erlenmeyer flasks. The fractions were collected from different solvent ratio from non-polar solvent to polar solvent system.

The slightly purified compound were subjected to glass column (10x2cm) packed with 60-120 mesh silica gel washed with hexane. Care should be taken to keep away air bubbles and prevent the crack into the column. The silica gel was mixed with the slightly purified sample (w/v) and filled on the top of the packed column. The adsorbed compound was eluted with hexane and chloroform in the ratio of 10:0 under the flow rate of 3ml/min. The targeted fractions were collected in pre-weighed container, concentrated. Fractions were pooled together and monitored using TLC and visualized under UV light at 250 nm and 400 nm.

The fractions collected from column chromatography were subjected to thin layer chromatography for observation and identification. Sample must be carefully spot on the plates and promptly dried with hair drier. Different solvent systems were used. The desired solvent system was used to obtained good separation. The developed TLC was observed under UV light and/or iodine vapour for the detection of any other spot.

Based on Figure 3.1, dried pandan leaves have been soaked with ethanol for 24 hours for four times was resulted as ethanol crude extract. Approximately 10mg of the crude was subjected in the Vacuum Liquid Chromatography (VLC) and washed with hexane, ethyl acetate and methanol. The fraction was collected for every ratio of the solvent system and was labelled as PE1 until PE73. TLC was performed for every fraction using different solvent system to obtain good separation and fraction PE5-7 was selected. This fraction was subjected to silica gel column or column chromatography (CC) and wash up with hexane, chloroform and methanol. The CC has been resulted fraction PE (CC) 1 until PE (CC) 23 and these fractions also was performed TLC. The fraction of PE (CC) 11 and PE (CC) 12 was selected and combined with weight of 11.9 mg for further isolated by using TLC. Then the mixture was isolated by preparative thin layer chromatography (PTLC) and characterized using NMR.

NMR spectral data was obtained by Bruker ultrashield 500 MHz NMR spectrometer. The sample was dissolved in about 1.5 ml of CDCl3 solvent and transferred to a NMR standard tube for further assessment.

The extracts and essential oil obtained were subjected to GC-MS. The spectrums were analyzed using GC-MS. The model was used is Agilent 19091S-433 series: 70eV with capillary column (30m x 250 µm x 0.25 nm) at intermediate polarity (HP5MS). The carrier gas was used is helium: the pressure is 8.71 psi, the flow rate is 1.0 m3/min and the average velocity is 37cm/s. The temperature of the column was initially set at 60?C (2 min hold) followed by 60-120?C (2 min hold) with temperature programming of 3?C/min. Then further by 120-270? (2 min hold) with temperature programming of 2?C/min. The injection temperature was set at 250?C. The major compounds in extracts were identified by comparing their mass spectra with the library in the GC-MS database which is observing the molecular ion peak and fragmented pattern

Hydrodistillation was used to extract the essential oil from fresh Pandan leaves. After the Pandan leaves were weighed, and then leaves were cut into smaller pieces and put into round bottom flask. Then the distilled water were added and heated until the temperature reached the boiling point. Hexane (10ml) was added into the mixture of water and oil produced. Hexane were retained the oil because it is non-polar. Then the mixture undergoes separation by using separatory funnel to remove water and only pure oil was obtained. Anhydrous sodium sulphate was added into the hexane layer to remove excess water in the oil. Hexane was removed from essential oil by using rotary evaporator. The pure oil of Pandan leaves was weighed and recorded. This oil was dilute with 3ml of hexane and transferred into vials and was subjected to the GC-MS.

Based on gas chromatography-mass spectroscopy (GC-MS) analysis, there were twenty nine chemical compounds that have been identified in the essential oil of fresh pandan leaves. The chemical composition was classified into four types of hydrocarbons such as diterpenes, oxygenated compound, alkaloid and miscellaneous compound. Based on Table 4.1, there are two major compounds from diterpenes which are neophytadiene (17.70%) and phytol (17.40%). The chemical structure for neophytadiene and phytol were shown in the Figure 4.2. The oxygenated compound that has been identified was pentadecanal (2.30%), sandaracopimarinal (1.88%) and tetradecanal (0.35%). Besides, some alkaloid compound also has been identified such as acetonanil (2.53%), 7-ethyl-2,4-dimethyl-10H-benzob1,8naphthyridin-5-one (0.73%), mesitylene (0.61%) and N,N-Dimethyl-5,6,7,8-Tetrahydronaphthalen-1-Amine (0.36%). The highest percentage for the miscellaneous compound was octadecane (5.05%) followed by tetracosane (2.27%), eicosane (2.09%) and nonadecane (1.18%). The total percentage for the four types of hydrocarbons where diterpenes (35.1%), oxygenated compound (4.53%), alkaloid (4.23%) and miscellaneous compounds (11.69%) (Figure 4.1).
From Figure 4.1 has shown that the essential oil of pandan leaves was dominated by diterpenes hydrocarbon group. Therefore, essential oil of pandan leaves was highly consisting of compounds with C20. Previous study have shown that there were main chemical constituent of essential oil which is phytol (42.1%) (Chen and Ge, 2014) and eight major volatile compounds that has been contributed to pleasant aroma in Pandanus amaryllifolius such as hexanal, 2-hexanal, nonanal, 2-acetyl-1-pyrroline, benzaldehyde, linalool, 3-methylpyridine and 2-penten-1-ol (Jimtaisong and Krisdaphong, 2013).

Based on gas chromatography-mass spectroscopy (GC-MS) analysis, there were ten chemical compounds that have been identified in the crude of pandan leaves. The chemical composition was classified into two types of hydrocarbons such as oxygenated compound and miscellaneous compound. Based on Table 4.2, there are two major compounds which are 3-nitro-1-phenylpropan-1-one (45.73%) and cyclomethicone 4 (12.05%). The chemical structure for 3-nitro-1-phenylpropan-1-one and cyclomethicone 4 were shown in the Figure 4.4. Besides, the oxygenated compound that has been identified was ionol 2 (6.13%), ethyl palmitate (2.68%) and p-methoxybenzil (1.33%). For the miscellaneous compound that has been identified was cyclomethicone 5 (11.51%), heptacosane (9.42%), cyclomethicone 6 (5.36%) and tris(trimethylsilyl) arsorite (1.57%). The total percentage for the two types of hydrocarbons where oxygenated compound was 55.87% and miscellaneous compound was 39.91% (Figure 4.3).

From Figure 4.3 has shown that the ethanol pandan leaves extract was dominated by oxygenated compound. Therefore, ethanol pandan leaves extract was highly consisting of compounds with oxygen. Previous study have shown that there were two major volatile compounds that has been contributed to pleasant aroma in Pandanus amaryllifolius such as oxygenated compound for example 3-methyl-2(5H)-furanone, 3-hexanone and 2-hexanone and miscellaneous compound (Jiang,1999).

Phytochemical investigation on Pandanus amaryllifolius was resulted one chemical compound. This compound were analysed using 1H NMR and resulted as a mixture only. The compound has been assumed to be a terpene group.

As conclusion, chemical compositions identified in essential oil and ethanol pandan leaves extract has showed a different of chemical constituent. The major composition of essential oil of pandan leaves extract were neophytadiene (17.70%) and phytol (17.40%) while for the ethanol pandan leaves extract were dominated by 3-nitro-1-phenylpropan-1-one (45.73%) and Cyclomethicone 4 (12.05%). These chemical constituents are responsible as aromatic compound that give the fragrant to the pandan leaves.

Further studies are required to explore and investigate more about the extraction of essential oil and pandan leaves extract to get more chemical constituent. It is recommended that pandan leaves extract can be performed by using other methods such as soxhlet extraction method and can extract using different solvent system such as methanol in order to get more accurate results. The compositions of chemical constituent in essential oil of pandan leaves can be improved by using latest extraction method likes Microwave Assisted Hydrodistillation. Besides, it is recommended to identify the marker aromatic compound in pandan leaves extract such as 2-acetyl-1-pyrolline by using supercritical carbon dioxide (SC-CO2) extraction.