Pandanaceae Research in Support for Industrial Plantation 2C Mohsin et...Pandanus tectorius...

2nd International Plantation Industry Conference and Exhibition (IPiCEX 2008), Blue Wave Hotel Shah Alam, from 18th till 21st November 2008. Theme IPiCEX 2008: "Reinventing the Plantation Sector: Globalisation, Diversification & Environmental Issues".

Pandanaceae Research in Support for Industrial Plantation

Mohsin, H. F., Abdul Wahab, I., Naz, H., Ali Shah, S. A. and Weber, J. F. F. Faculty of Pharmacy, Universiti Teknologi MARA, 40450 Shah Alam, Selangor Darul Ehsan.

e-mail: [email protected]

Abstract

The Pandanaceae is a predominantly tropical plant family. It contains three genera (Freycinetia, Pandanus and Sararanga) with approximately 700 species. From the literature review, various studies were performed on this plant family, however, only involving one percent of the known species. This includes conservation proposals, phytochemical work, chemosynthetic, bioinformatic, biogenetic and taxonomic researches, mycological, zoological and entomology observation plus ethnobotany and social studies. Polypeptides, phenolics, sterols, alkaloids and terpenoids are among the chemical constituents. Recently, we have reported the procedure of both neutral and acid-base extractions of the leaves sample from a Pandanus species. Here, the chromatographic profile of the root extract is presented (Agilent Technologies' Zorbax® C-18; 4.6 x 250 mm, 5 μm, CH3CN:H2O, 10 to 90%; 38 min, flow rate: 1 ml/min, λ=210, 280, 360 nm). Significant peaks which corresponded to chemical compounds were observed. Biological studies of the root extracts will also be carried out. It is anticipated that Pandanaceae will gain more attention in pharmaceutical investigations, food research and urban landscape improvement. From such continuous and supportive scientific evidences, it is hoped that future planning for a Pandanaceae plantation could encourage research and development in industrial, environmental and eco-tourism sectors. Keyword: Pandanaceae

Introduction The Pandanaceae is a predominantly tropical plant family, which is specific from Asia Pacific region. It contains three genera (Freycinetia, Pandanus and Sararanga) with approximately 700 species. Pandanus is a large shrub or small tree of immense cultural, health, and economic importance in the Pacific, second only to coconut. A highly variable species complex, it grows wild mainly in semi-natural vegetation, where it can withstand drought, strong winds and salt spray. It propagates readily from seed, but it is also widely propagated from branch cuttings for farms and home gardens. It grows fairly quickly, and all parts are used, from the nutritious fruits of edible varieties, to the poles and branches in construction, to the leaves for weaving and garlands (Thomson, L. A. J. et al. 2000). However, only one percent of the known species were chemically investigated. The Pandanaceae is commonly focused on their traditional uses e.g. in cooking and for some medicinal purposes. Several species were reported to act as a remedy for toothache and rheumatism (Takayama et al. 2000). In Malaysia, the antioxidant property of Pandanus extracts (Mohd. Nor et al., 2008), encapsulation process of pandan powder (Loh et al., 2005) and alpha-tocopherol content in Pandanus odorus (Soon Ching et al., 2001) were studied. Yaacob, M. et al. (2005) has described the plantation techniques for P. odorus. The root extract of this species was also subjected to several studies (Peungvicha et al., 1990; 1996; 1997; 1998).


Literature review From the literature review, wide areas of studies were performed on this plant family. This includes conservation proposal (St. John, 1963), phytochemical work, chemosynthetic, bioinformatic, biogenetic (Callmander 2001, 2003) and taxonomic researches (Stone 1983), mycological (McKenzie, E. et al. 2001), zoological (Hunt 2004; Clayton 2007) and entomology observation (Samarawickrema, W. A. et al. 2008), plus ethnobotany and social studies (Novellino et al. 2005). Polypeptides, phenolics, sterols, alkaloids (Figure 1) and terpenoids are among the chemical constituents (Ooi, L. S. M. et al. 2004; Inada, A. et al. 2005; Takayama et al. 2000; Macleod, A, J. et al. 1982).

NO

O CH3

OO

CH3

N

OOCH3

OH3C

O

H

Pandamarilactone-1C18H25NO4

PandanamineC18H23NO4

NO

O

CH3

Pandamarilactonine-AC18H25NO4

O

HH

OH3C

O

OH3C

O

O

CH3

An compound which is not a "natural product"C18H23NO4

16

16

N

NH

O

HH

OH3C

norpandamarilactonine-A norpandamarilactonine-B

NH

O

HH

OH3C

Figure 1: Examples of alkaloids from Pandanus species. The unique feature in our research plan involved one Pandanus species, Pandanus sanderi (P. sanderi). Unlike the green Pandanus amaryllifolius (pandan wangi), P. sanderi is variegated or having streaks of a different color or colors. It is characterized by longitudinal yellow and green stripes of the leaves. The yellow stripe is at the middle of two green stripes (Figure 2). Living samples of P. sanderi can be observed at IOI Mall, Puchong, Selangor Darul Ehsan, Malaysia.

Figure 2: Pandanus sanderi is also cultivated as an ornamental plant (left). The leave is about 6 centimeter in optimal diameter. The ratio of green to yellow stripe is 2:1

(middle). The opposite arrangement of the yellow stripes in Pandanus tectorius cultivar (right, NParks FloraWeb).


However, this occurrence is in contrast with other Pandanus species, e.g. P. tectorius cultivar. The green stripe of P. tectorius cultivar leaves is in between the two yellow stripes (Chih Min, B. et al., 2003). Tan, M. A. et al. (2008) recently published antitubercular triterpenes and phytosterols from P. tectorius (mengkuang). Previously, Beentje, H. (1993) reported that the taxonomy of the Pandanus species is difficult due to the lacking of correlation between male and female plants and extensive widely distributed of the species. This resulted with the variability of Pandanus tectorius. Vahirua-Lechat, I. et al. (1996) documented isoprene related esters as the significant components of Pandanus tectorius Parkinson. In contrast, P. sanderi (synonym Pandanus tectorius “Sanderi) is still unexplored, though more lately; it is noticeable in urban area as a landscaper. The extraction procedure of the leaves from this species will be reported elsewhere (Mohsin, H. F. et al. 2008). In this paper, the chromatographic profile of the root extract is presented. Materials and method (a) Plant sample The sample of P. sanderi (Figure 3) was obtained from a nursery garden at Seri Kembangan, Selangor Darul Ehsan. The root was cut, dried and weighed (89.6 g). Later, it was ground and added with non-aqueous solvents (hexane, chloroform and methanol), consecutively. All extracts were subjected to Thin Layer Chromatography (TLC). The methanolic extract was not introduced to any primary fractionation. On the other hand, High Performance Liquid Chromatography (HPLC) technique was performed, as described in the following page.

(i) (ii)

Figure 3: Pandanus sanderi (i) roots with leaves and (ii) roots with stems.


(b) Chromatography apparatus (i) HPLC instrumentation All analytical separations were performed with a quartenary gradient HPLC pumps (Agilent Technologies G1311A), connected with a penta wavelength UV-Visible detector (Agilent Technologies MWD multiple wavelength detector G1365B), allowing three simultaneous detections at λ=210, 280 and 360 nm (Figure 4).

Figure 4: The HPLC set-up. A manual injection unit (Agilent G1328B) was used, the injection loop volume being 20 μl. The peak area was computed by the software, Agilent ChemStation 2001-2007 on a Pentium® D PC. The samples were injected by a 50 μl syringe. They were filtered through Agilent 0.45 μm membrane filters prior to injections (regenerated cellulose filters for water and nylon filters for acetonitrile). (ii) HPLC columns Two HPLC columns were employed. Both Agilent Technologies' Zorbax® Stable Bond (SB)-C18 column (4.6 x 150 mm and 4.6 x 250 mm, with a particle size of 5 μm) were used to analyze the fractions’ profile. It was provided by Agilent Technologies Inc., Santa Clara, California, U.S.A. (iii) Solvents HPLC-grade acetonitrile (CH3CN) from Fischer (UV cut off: 190 nm) and water were used. The mobile phase was a gradient mixture of acetonitrile and water (CH3CN:H2O, 10 to 90%, pressure = 70 bar). The solvents were eluted at a flow rate of 1.0 ml/min in the analytical scale for 38 minutes. Results The weights from the root extraction procedure are found below (Table 1). The most significant weight was retrieved from methanol extract.

Table 1: The root extracts of P. sanderi. No. Root Extracts Weight (g) Percentage (%) 1 Hexane 0.271 0.3 2 Chloroform 0.533 0.6 3 Methanol 4.265 4.8

Several spots appeared from every extracts, when they were introduced to the TLC plates (hexane:acetone) (Figure 5).


(i) (ii)

Figure 5: The visualization of the TLC profiles of hexane, chloroform and methanol extracts, respectively, under ultraviolet rays;

(i) short wave (hexane:acetone = 80:20) (ii) long wave (hexane:acetone = 50:50). Our focused was turned to the methanolic extract. Preliminary injections of the methanolic extract by using a shorter column (Agilent Technologies' Zorbax® Stable Bond (SB)-C18 column (4.6 x 150 mm) produced chromatograms with low resolution [Figure 6(i)]. Relatively, more separated peaks were observed, when a longer column was used. The three chromatograms with significantly good resolutions are presented below [Figure 6(ii) – (iv)].

(i) λ=210, 280 and 360 nm. (ii) λ=210 nm

(iii) λ=280 nm (iv) λ=360 nm

Figure 6: The chromatograms that were produced by using different lengths of C18 columns. (i) 4.6 x 150 mm; (ii) – (iv) 4.6 x 250 mm.


Figure 7 showed two overlapping chromatograms of the methanolic root extract, with and without the utilization of the analytical guard column [Agilent Technologies' Zorbax® Stable Bond (SB)-C18 analytical guard column (4.6 x 12.5 mm, with a particle size of 5 μm)]. From both chromatograms, it was observed that all targeted eight peaks were eluted. However, the injection with the presence of the guard column resulted with an earlier retention time (tR = 18 – 36 minutes, ± 4 minutes).

Figure 7: The chromatograms that were produced, with and without the

utilization of the analytical guard column (4.6 x 12.5 mm).

Discussion From Figure 3 - 5, important signals which corresponded to a number of phytochemical compounds, were observed. The HPLC peaks within the retention time, tR = 18 – 36 minutes could be isolated and subjected for fractionation. The collection methods (at λ=210 nm) are currently in progress, with the employment of the analytical guard column. Biological studies of the root extracts will also be carried out. Conclusion It is anticipated that Pandanaceae will gain more attention in pharmaceutical investigations, food research and urban landscape improvement. From such continuous and supportive scientific evidences, it is hoped that future planning for a Pandanaceae plantation could encourage research and development in industrial, environmental and eco-tourism sectors. Acknowledgments We would like to thank Mr. Mohamad Soliheen Bin Mohd. Zaki and Mr. Mohd Rizal Bin Salleh for their technical assistance, under the university’s Student Employment Scheme. We would also like to acknowledge Ms. Suriana Hanim bt. Ariffin @ Sukri for the literature search.

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Pandanaceae Research in Support for Industrial Plantation 2C Mohsin et...Pandanus tectorius...

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