Journal of American Science 2015;11(9) http://www.jofamericanscience.org Comparative Investigation of Minerals, Amino acids, Lipoidal Contents and General Analysis of Chamaerops humilis L. Organs Growing in Egypt Shimaa A.Ahmed1, Hossam M. Hassan2, Sayed A. Ahmed1* and Ahmed H. Elghandour1. 1Department of Organic Chemistry, Faculty of Science, Beni-Suef University, Beni- Seuf, 62514, Egypt. 2Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, 62514, Egypt. [email protected], [email protected] Abstract: In this paper, a comparison of nutritional value of the pollen grains and the fruits of chamaerops humilis L. and comparison of lipoidal content of the pollen grains, the fruits and the leaves was carried out. The preliminary phytochemical screening of the petroleum ether extracts of different organs of chamaerops humilis (L) revealed that the petroleum ether of the leaf, the pollen grain, the fruit are rich in lipid content and sterols and/or triterpenes. Total protein, fats, ash, carbohydrates and moisture percentages were determined in pollen grains and fruits. Minerals analysis show that Potassium was the major composition of macro-elements in both organs followed by sodium. [Shimaa A. Ahmed, Hossam M. Hassan, Sayed A. Ahmed and Ahmed H. Elghandour. Comparative Investigation of Minerals, Amino acids, Lipoidal Contents and General Analysis of Chamaerops humilis L. Organs Growing in Egypt. J Am Sci 2015;11(9):94-100]. (ISSN: 1545-1003). http://www.jofamericanscience.org. 13 Keywords: Chamaerops humilis L., pollen grains, Phytochemical investigation, lipoidal, sterols. 1. Introduction 2. Material and Methods Arecaceae family are flowering plants. Plant material Arecaceae family (palm family) have been important Samples of chamaerops humilis L. used in this to human beings throughout history and about 200 study were collected during the years (2012-2013) genera with around 300 species are currently known. from Orman zoo in Giza, Cairo, Egypt. The plants [1,2] Arecaceae family(palm family) have been were kindly identified by Dr. Mohamed Gibali, Senior important to human throughout much of history. many Botanist and madam tries. common products and foods are derived from palms The Leaves, the pollen grains and the fruits were for example the pejibaye palm (Bactris gasipaes air-dried, powdered and stored for chemical and Kunth), also known as peach palm the fruit of this biological studies. palm can be used as a cereal for human consumption Preparation of the extract and has high food values for some nutrients[3 ] For the investigation of lipoidal matter content chamaerops is a genus of flowering plants in the the air-dried powdered leaves, pollen grains and fruits family Arecaceae (palm family), comprising asingle of chamaerops humilis L. (50 g for each) were species chamaerops humilis (European fan palm or exhaustively extracted with petroleum ether and the Mediterranean dwarf palm). solvent was evaporated under reduced pressure, then Chamaerops humilis L. is a shrub-like clumping saponification of petroleum ether extracts of the palm, with several stems growing from a single base. leaves, the pollen grains and the fruits to separate sap chamaerops humilis L. is a medicinal plant which and unsap matters. (1g) of defatted powder of the belong to the Arecaceae family. [4-6] The seed of C. pollen grains and the fruits for amino acid analysis. humilis showed high content of lipids, which implies (10g) of dried powder of the pollen grains and the an importance of the species for feeding wild life fruits for minerals analysis. (20g) of dried powder of during winter, and a high degree of unsaturation [7] but the pollen grains and the fruits for nutritional value no studies were done on fruits, pollen grains and analysis. leaves. Traditional medical practices survey carried Study of the Lipoidal Content out in Western Algeria (Tlemcen department) and The lipoidal content of the petroleum ether Morocco revealed that chamaerops humilis L. is taken extract of the leaves, the pollen grains and the fruits of as stipe or leaf extracts for the treatment of diabetes, chamaerops humilis L. was investigated by GLC after digestive disorders, spasm, toning and gastrointestinal saponification. disorders diseases. [8-10] Sterols, also known as steroid 1. Preparation of unsaponifiable (USM): [11] alcohols, are a subgroup of the steroids and an The petroleum ether extracts of the leaves, pollen important class of organic molecules. They occur grains and fruits of chamaerops humilis L. (1.5 g, 2.2 naturally in plants, animals, and fungi, with the most g a residues of pollen grains and leaves respectively familiar type of animal sterol being cholesterol. and 1.5 g oily residue of fruits) were separately saponified by heating under reflux with 100 ml 10 % 94 Journal of American Science 2015;11(9) http://www.jofamericanscience.org alcoholic KOH and 40 ml benzene for 24 hours to chromatographed. Identification of the fatty acids ensure complete hydrolysis. The saponified solutions (FA) was carried out by comparing the retention times were then concentrated under reduced pressure and of their methyl esters with those of the available suspended in 100 ml Distilled H2O followed by reference fatty acids similarity analyzed. extraction with diethyl ether (10×50 ml) till - Preparation of samples for General chemical exhaustion. the combined ethereal extracts in each analysis (total protein, crude fat, ash, crude fibre case, were washed several times with Distilled H2O and moisture: till free from alkalinity the dried over anhydrous Percentage of total protein, crude fat, ash, crude sodium sulphate, followed by evaporation to dryness. fibre and moisture were determined according to the The solvent-free residues obtained represent the methods outlined by AOAC (1980).[13] unsaponifiable matters (USM). Total protein was determined by the Micro- 2. Isolation of the fatty acids from the saponifiable Kjeldahl method, which involves digestion or fractions: oxidation of the sample with conc. H2SO4 followed The aqueous alkaline solution left after by steam distillation. Ammonia formed at high pH separation of the unsaponifiable matters was, in each was received in 4% boric acid solution then titrated case, acidified with dilute HCL (10%) to librate the with 0.1N HCl. The percent of protein was obtained free fatty acids (FA). These were extracted with by multiplying N% by the factor of 6.25. diethyl ether (5×50ml). the combined ethereal extracts Determination of total crude fat depends on the were separately washed with Distilled H2O till free difference in weight between the original sample and from acidity then dried over anhydrous sodium the hexane-extracted sample in Soxhlet apparatus. sulphate and evaporated to dryness. Total ash obtained by heating a crucible 3. Preparation of the fatty acid methyl esters containing the sample in a muffle furnace at 550 oC (FAME): [12] overnight (till constant weight. Aliquots of the fatty acid mixtures (0.5 g) were, Total crude fibre (edible fibre) was determined separately, dissolved in 50 ml methanol, 2.5 ml of by boiling the sample (after extraction of the fats, as sulphuric acid were added and the mixture refluxed they form gelatinous layer with the alkali used in the for one hour on a boiling water bath. The cooled test) for 30 minutes with dilute acids followed by mixture was diluted with 20ml of H2O and then boiling for another 30 minutes with dilute alkali to get extracted with ether (5×50 ml). the combined ethereal rid of proteins, starches and carbohydrates, leaving extracts, in each case, were washed with water till only some carbohydrates which resist acid and alkali neutral to litmus paper, dried over anhydrous sodium and some ash minerals. Ashing will then cause the sulphate and the solvent was evaporated under fibre only to volatilize, while the ash will not. reduced pressure. The dried residue was, in each case, Total carbohydrates content (as % of dry weight) saved in a dessicator for GLC analysis. was obtained by difference. 4. GLC analysis of the USM and FAME: Total moisture was estimated (for the fresh USM analysis was carried out on Hewlett- samples) by heating at 105 oC overnight (till constant Packard HP-6890 N network GC system equipped weight). with a FID detector. Analysis was performed on an -Amino acids determination: HP-5 column; using N2 as a carrier gas, injection Determination of all amino acids other than temperature 250oC, detector temperature 300oC. tryptophan was carried out according to the method Aliquots, 2 μL each, of 2% chloroformic solutions of described by Winder and Eggum (1966). [14] the analyzed USM and reference samples were co- - Preparation of the protein for amino acid chromotographed. Identification of the components analysis: [15] was based on comparison of the retention times of One g of the defatted powdered of each of the their peaks with those of the available authentic pollen grains and the fruits were stirred in10% sodium samples. The relative amount of each component was chloride solution for one hour then filtered. An equal calculated via peak area measurement by means of volume of 10% trichloroacetic acid (TCA) solution computing integrator. was added to the filtrate of each sample. The protein FAME were analysed on a pye Unicam 304 was precipitated as white flocculent amorphous series gas chromatograph equipped with a FID precipitate, collected by centrifugation, washed with detector. Analysis was performed on 10% PEGA (on 5% TCA solution, then with ether and absolute chromosorb W-AW, 100-120 mesh) column; using N2 ethanol and dried in a vacuum desiccator. The crude as carrier gas, injection temperature 250oC, detector dried protein was dialyzed by parchment membrane. temperature 280oC. Aliquots, 2 μL each, of 2% The non-dialyzed fraction was collected and chloroformic solutions of the analyzed FAME and lyophilized by freeze drying. reference fatty acid methyl esters were co- - Amino acid analysis: 95 Journal of American Science 2015;11(9) http://www.jofamericanscience.org The protein (1mg) of each sample was are non essential amino acids.