Phytochemical Screening and Chemical Investigation of Lipoidal Matter of Arenga engleri Leaves

Objectives: In this study, a preliminary phytochemical screening and lipoidal matter of Arenga engleri Becc. leaves (family Arecaceae) were studied for the first time. Methods: Gas chromatography coupled with mass spectroscopy (GC/MS) was used for the identification of compounds of saponifiable and unsaponifiable content. Results: The preliminary phytochemical screening showed the presence of saponins, tannins, flavonoids, cardiac glycosides, carbohydrate and/or glycosides, unsaturated sterols and/or triterpenes and absence of anthraquinones, coumarins, volatiles and alkaloids or compound containing nitrogenous bases. GC/MS analysis revealed the higher percentage of unsaturated fatty acids (51.39%) than that of saturated ones (31.47%). The major unsaturated fatty acids present were linoleic acid (31.55%) and 7,10-hexadecadienoic acid (11.42%) while the major saturated one was palmitic acid (17.27%). The unsaponifiable matter was represented as hydrocarbons (41.19%), fatty alcohols (28.31%), terpenes (9.68%) and sterol (0.14%). 1-Octadecene (17.65%) and 1-hexadecene (12.41%) represented the major hydrocarbons while behenic alcohol (14.71%) was the major fatty alcohol, phytol (4.89%) was the major terpene and ethylisoallocholate (0.14%) was the only sterol identified.


INTRODUCTION
Arecaceae, previously called the Palmae family, comprises about 200 genera and 2600 species which are distributed throughout tropical and subtropical regions 1,2 .Palms are called the "Trees of Life" as they have a potential role in people's life supplying them with foods, fibers, shelter, fuels, oils, gums, waxes, poisons and medicines 2 .Genus Arenga contains 22 species, distributed in South China, the Ryukyu Islands and Taiwan in the North to Christmas Island in the South and from India in the West to Queensland, Australia in the East 3 .The genus is economically important as it is useful for sugar, starch, thatch production and potentially has an ornamental value 4 .Previous reports showed the identification of squalene, lutein, β-sitosterol and stigmasterol from Arenga tremula 5 and RP-HPLC analysis of Arenga wightii revealed the presence of caffeine and major phenolic compounds: gallic acid, ascorbic acid and chlorogenic acid 6 .Different species were reported to have hypocholesterolaemic 7 , antioxidant, antimicrobial 6 , anti-hypertensive, anti-inflammatory and analgesic activities 8 and treating skin allergies 9 , headache , malaria and tuberculosis 10 .
Arenga engleri Becc., commonly called the Formosa palm, Taiwan sugar palm or dwarf sugar palm, is an attractive medium-sized ornamental clustering  11 .The stems are covered with delicate black fibers which have been used for a long time to prepare coir raincoat, shoes, rope, fishing nets, brushes and pads 12 .The leaves are known to be useful in thatching and wickerwork 4 .The young ones and apical buds are edible, the stems pith used to produce starch while the sweet sap obtained from the inflorescence stem used to make sugar 13 and the flowers have a very pleasant fragrance.The mesocarp of the fruits are rich in irritant calcium oxalate raphides like other most species of this genus and this make them inedible and poisonous 4 .Since there are no reported studies about phytochemical constituents of Arenga engleri Becc.leaves and its lipoidal profile, it was found essential to carry out preliminary phytochemical screening and investigation of lipoidal matter of this palm.

Plant material
Fresh leaves of Arenga engleri Becc.were collected on October 2014 from Al Zohriya Garden, Cairo, Egypt and kindly identified by Dr. Terase Labib, Head of the Taxonomists at Orman Botanical Garden, Giza, Egypt.A Voucher specimen (03Aen/2019) was kept in the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Egypt.

Apparatus
TRACE™ 1310 Gas Chromatograph produced by Thermo Scientific™ provided by FID (Flame Ionization Detector), attached with ISQ LT single quadrupole Mass Spectrometer (Regional center for Mycology and Biotechnology, Al-Azhar University).

Preliminary phytochemical screening
Air dried powdered leaves of A. engleri were screened for its constituents using standard protocols in the mentioned references 14,15,16 .

Preparation of lipoidal matter
The air-dried powder of A. engleri leaves (100 g) were extracted by n-hexane.The solvent was evaporated at 40 o C under reduced pressure to give 7 g residue of lipoidal matter 17 .

Fractionation of lipoidal matter
Two gm of lipoidal matter were saponified by refluxing with 50 ml of 10% alcoholic potassium hydroxide solution for 2 hr followed by evaporating the alcohol, diluting with distilled water and extracting with ether exhaustively.The collected ethereal extract was washed with distilled water till being free from alkalinity, dried over anhydrous sodium sulphate, and then evaporated to give 1.16 g (58%) unsaponifiable matter (USM) residue.
The remaining saponifiable aqueous layer left after extraction with ether was acidified with 10 2 N hydrochloric acid and the liberated fatty acids were extracted exhaustively with ether.The collected ethereal extract was washed with distilled water until neutralization, dried over anhydrous sodium sulfate, and then evaporated to give 0.54 g (27%) total fatty acids (TFA) residue 18 .

Preparation of fatty acid methyl esters
The preparation of methyl esters of free fatty acids (0.54 g) was carried out by refluxing with 100 ml of absolute methanol and 5 ml sulphuric acid for 2 hr, extracting with ether and drying the ethereal layer over anhydrous sodium sulfate followed by evaporation of ether to give residue of the fatty acid methyl esters (FAME), kept for GC-MS analysis 19 .

GC-MS analysis of the unsaponifiable matter
The analysis was carried out under the following conditions: DB-17 P/N 122-1751 (30 m, 0.

Identification of USM and TFA
After GC-MS analysis of USM and TFA, the compounds were identified by comparing their retention times and mass fragmentation patterns with those of the reference standard data of WILEY and NIST libraries 20 .Quantitative determination was based on peak area integration 21 .

Preliminary phytochemical screening
Preliminary phytochemical screening of A. engleri leaves showed the presence of saponins, tannins, flavonoids, cardiac glycosides, carbohydrate and/or glycosides, unsaturated sterols and/or triterpenes while anthraquinones, coumarins, volatiles and alkaloids or compound containing nitrogenous bases were absent.

DISCUSSION
The results of preliminary phytochemical screening of Arenga engleri leaves showed the presence of sterols and triterpene that was confirmed by the GC-MS analysis of its lipoidal matter.As revealed in GC-MS analysis, the percentage of unsaponifiable matter was higher than saponifiable one and unsaturated fatty acids were more than saturated ones.Linoleic acid and palmitic acid represented the major identified unsaturated and saturated fatty acids, respectively.For unsaponifiable matter, hydrocarbons represented the major component then fatty alcohols and then terpenes while the only sterol identified was ethylisoallocholate.The most abundant compounds identified in hydrocarbons content were 1-octadecene and 1hexadecene.Behenic alcohol was the major fatty alcohols and total terpenes identified were phytol and squalene.
Many biological activities have been reported for linoleic acid as antioxidant, anti-inflammatory by cyclooxygenase-I (COX-I) and cyclooxygenase-II (COX-II) inhibition 22 and hypocholesterolemic activities 23 .Studies revealed that elevated levels of linoleic acid in the plasma prevented and controlled hypertension 24,25 .It helped in glycemic control and reduced the risk of Type 2 diabetes 26 and also had anticancer activity 27 Injector temperature: 200 o C; temperature transfer line: 250 o C; initial column temperature 80 o C, programmed by 3 o C/ min up to the final temperature 230 o C within 50 min and the ionization energy was 70 ev.

Table 2 . GC-MS analysis of total fatty acids of A. engleri leaves
RRT*: Relative retention time of linoleic acid with RT = 22.85 min