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Detection of polyisoprenoids in the roots and stems of coastal grasses using a two-dimensional thin layer chromatography

Coastal plants are known to produce secondary metabolites including polyisoprenoid alcohols. Coastal plants have been shown to have phytomedicinal, biological, and pharmacological properties. The present study reports the detection of polyisoprenoids composition from roots and stems of selected coastal medicinal grasses, Cyperus rotundus, Distichlis spicata, and Spinifex littoreus. A two-dimensional thin layer chromatography (2D-TLC) method was used to analyse the content and distribution of polyisoprenoid alcohols (polyprenols/dehydrodolihols and dolichols) in coastal grasses. The presence of polyprenols and dolichols in the roots and stems were identified and grouped into two types. Type-I, showing a preponderance of dolichols over polyprenols, was detected in the roots of C. rotundus and D. spicata. Type-II, displaying the existence of both polyprenols and dolichols, was traced in S. littoreusroots and the stems of C. rotundus, D. spicata, and S. littoreus. The diversity of polyisoprenoids in the root and stem tissues even in the same species, suggesting the chemotaxonomic criterion of polyisoprenoids in coastal grasses.


Introduction
Plants that exist in the coastal environment and within mangroves as well are regarded as coastal plants or mangrove associates [1]. Coastal plants are widespread in the intertidal tropical and subtropical areas and have long been eminent a fountain ofsecondary metabolites including polyisoprenoid alcohols [2]. The selected coastal medicinal grasses, namely Cyperus rotundus, Distichlis spicata, and Spinifex littoreus are common species widespread in Tanjung Benoa and Taman Hutan Raya Ngurah Rai, Bali Province, Indonesia.
A number of studies have been reported for biological and medicinal activities of coastal plants. For example, C. rotundus has been shown to have traditional uses, phytochemical, biological, and  [3][4]. Anti-inflammatory and analgesic property of S. littoreus has also been reported [5]. Whereas, D. spicata has been used for food and cover by various species of snails and crabs [6]. Furthermore, polyisoprenoids have been reported to show some pharmacological activity such as anticancer [7], anti-dyslipidaemic [8], anti-influenza and antiviral agents [9]. In addition, dehydrodolichols from Ginkgo biloba leaves has been demonstrated to actively exhibit antibacterial and antifungal activity [10].
Supposed the essential biological and medicinal properties of C. rotundus, D. spicata, and S. littoreus as well as sources of plant polysioprenoids. However, the profile of polyisoprenoid in C. rotundus, D. spicata, and S. littoreus have not been studied yet. The occurrence and distribution of polyisoprenoid in mangrove and coastal plants have been reported [2,[11][12]. To obtain more understanding the function of polyisoprenoids, it is indispensable to acquire the data of allocation and profile of polyisoprenoid in selected coastal medicinal grasses. Therefore present study aimed to examine the polyisoprenoids (polyprenol and dolichol) profile and composition from selected coastal grasses namely C. rotundus, D. spicata, and S. littoreus to extend our previous works on the search of polyisoprenoids from coastal plants.

Chemicals
Dolichols (C 90 -C 95 ) and polyprenols (C 90 -C 100 ) standards as earlier reported [2] was used to detect the polyisoprenoids in this study. For at least three independent experiments were performed to determine the family compounds relating to polyprenols or dolichols. Silica gel 60 TLC glass plates and silica RP-18 HPTLC glass plates were obtained from Merck.

Plant materials
The roots and stems of coastal grasses, C. rotundus, D.spicata, and S. littoreus were collected from Taman Hutan Raya Ngurah Rai, Bali, Indonesia, in May 2017. These coastal florae are commonly growing exposer to sunlight. The mean temperature in the month of the sampling was 30-32ºC with an ordinary humidity of 75-77%.

Polyisoprenoid alcohols extraction
Extraction of polyisoprenoids' protocol was performed as previously described [11][12]. The roots and stems were dried at 65-70°C for 48 hours. The desiccated tissue (5 g each) was powdered and submerged in chloroform/methanol (2/1, v/v) for 2 days. The crude lipid extract of rootsand stems was saponified and removed physically with hexane, and the organic solvent was dried up and re-mixed in hexane as earlier reported [2]. The root (100-150 mg) and stem (150-200 mg) extracts were used to apply for each TLC plate.

Polyisoprenoids were analyzed by two-dimensional thin layer chromatography (2D-TLC)
2D-TLC method contained two steps: a silica gel glass plate (20 × 3 cm) was used for firstdimensional TLC (1D-TLC) for about 45 min with toluene-ethyl acetate (9:1) solvent as earlier reported [11]. The second phase, acetone as the solvent was used to 2D-TLC analysis with for about 40 min. Theoblique ladder of polyprenols and dolichols along with the standard are separated and then recognized and visualised with iodine vapour. The enhanced chromatographic was imaged and scanned with a Canon Pixma G2000 series printer. The dehydrodolichols and dolichols family was analyzed by the corresponding to movability on chromatograms with those of certain standards of polyisoprenoids that were practised in the two-plate run. The polyprenols and dolichols contents were assessed using ImageJ version 1.46r [13], with dolichol and dehydrodolichol standards as criterions.

Distribution and profile of polyisoprenoids in selected coastal grasses
The selected coastal grasses, namely C. rotundus, D. spicata, and S. littoreus for long-chain polyisoprenoids was analysed by 2D-TLC [2]. Polyisoprenoids of different chain-lengths were divided into polyprenols and dolichols. Tables 1-2 compile the analytical results of the distribution of the dehydrodolichols and dolichols with the carbon (C)-chain lengths addicted for every species.
TL content of coastal grasses roots and stems ranged 562 to 657 and 569 to 715 mg/g dw, respectively ( Table 1). The quantity of PI was highest in D. spicata roots (157 mg/g dw) and S. littoreus stems (63 mg/g dw). The lowest content of PI was in the roots of C. rotundus (13 mg/g dw) and the stems of D. spicata (7 mg/g dw) ( Table 1).
The comparable results on TL and PI contents of this study were also have been previously supported for North Sumatran coastal leaves [1] and Nephellium lappaceum various tissues [14]. The TL and PI concentrations in the coastal and mangrove associates plants presented in this study were relatively higher than those shown from major and minor components of mangrove plants [11][12] and generative and vegetative tissues of oil palm [15]

Polyisoprenoid analyzed by 2D-TLC
The spreading of polyprenols and dolichols in the roots were determined and classified into two types.
Type-I, which shows a numerousness of dolichols over dehydrodolichols (100%), was found in the roots of C. rotundus and D. spicata (Figure 1 A-B, Table 2). Type-II, which has the existence of both dehydrodolichols and dolichols, was identified in S. littoreus roots (Figure 1 C, Table 2). However, in the stems of C. rotundus, D. spicata, and S. littoreus, the dolichols were not detected as a case in the roots but contained both polyprenols and dolichols as Typed-II ( Figure 1 D-F, Table 2). The abundance of dolichols in the roots of both species of C. rotundus and D. spicata well agreed with previous results on mangrove plant roots [11][12], coastal plant roots [2], N. lappaceum roots [14], rubber roots [16], and soybean roots [17]. It is noteworthy that the existence of both polyprenols and dolichols in the stems of selected coastal grasses indicated the similar pattern of some coastal plants leaves such as Acanthus ilicifolius [11], Casuarina equisetifolia [2], Melastoma candidum [2], Morinda citrifolia [2], Sesuvium portulacastrum [2]. The existence of both polyprenols and dolichols also have been reported in a few mangrove leaves [11][12], N. lappaceum rinds, fruits, and seeds [14], young and old leaves of Hevea brasiliensis [16], Ginkgo seeds, embryo, young and old leaves [16], and pine (Pinus sylvestris) [16]. The presence both polyprenols and dolichols are typical of oil palm pisifera and tenera-type fruit [15].  Table 2. Chain-lengths distribution of polyprenol and dolichol of coastal grasses roots and stems Furthermore, the physiological function of polyisoprenoids remains obscure. It has been reported recently that salinity changes the polyisoprenoid contents in four salt-secretor and non-salt-secretor mangroves [18]. The alteration of polyisoprenoids including in polyprenols, dolichols, and bombiprenone as well. The content of polyisoprenoid also altered upon abiotic stress as well as with senescence has been shown [11][12]19]. The similar function has been demonstrated in triterpenoids as 5 a defence mechanism to external stress such as salinity [20]. In this context, polyisoprenoids are known to have some pharmacological and biological activities [7][8][9].

Conclusions
This study confirmed the presence of polyisoprenoids profile in the roots and stems of selected coastal grasses: dolichols dominated in the roots and the incidence of both polyprenols and dolichols in the stems. The significant findings showing the diversity of polyisoprenoids in the root and stem tissues even in the same species, suggesting the chemotaxonomic marker of polyisoprenoids in coastal grasses for the potential use of phytomedicinal, biological, and pharmacological activities.