J. Bio. & Env. Sci. 2014 Journal of Biodiversity and Environmental Sciences (JBES) ISSN: 2220-6663 (Print) 2222-3045 (Online) Vol. 5, No. 4, p. 264-278, 2014 http://www.innspub.net OPEN ACCESS RESEARCH PAPER Diversity of Varthemia candicans phytochemicals in response to growth habitat Mahmoud A. Elhaak1, Fatma A. Ahmed2, Mohamed E. Kashlana2 and Khalil M. SaadAllah1, * 1 Botany Department, Faculty of Science, Tanta University, Tanta, Egypt 2 Medical and Aromatic plants Department, Desert Research Center, El-Mataria, Egypt Article published on October 21, 2014 Key words: Growth habitat, carbohydrates, protein, amino acids, lipids, GC-MS, HPLC. Abstract Aerial parts of Varthemia candicans were collected seasonally for one year (winter, spring, summer and autumn) from Wadi Habbes (rocky habitat) and Sand Dunes habitat, West Marsa Matrouh, Egypt. The results of the plant photochemical analysis cleared out that the amount of soluble carbohydrates in the study habitats during the wet seasons (winter and autumn) was higher than that of the dry season, however the content of insoluble carbohydrates recorded a reverse trend. HPLC analysis of free sugars detected the occurrence of glucoronic acid, raffinose, glucose, galactose, fructose and fucose. The most abundant free sugar in WH was glucoronic acid but in SD was raffinose. Also, HPLC analysis of combined sugars detected the presence of glucose, mannose, fructose and maltose with the commonness of maltose in the two study habitats. The amount of soluble amino acids and soluble proteins were greater in SD habitat than WH habitat during all seasons, except autumn season. Free amino acids in WH habitat revealed the richness of the plant with asparagines, but in SD habitat with proline, but in case of protein amino acids proline was common in WH and aspartic acid was common in SD habitat. The total lipids content was greater in SD habitat than in WH. GC-MS analysis of fatty acids revealed that Hexadecanoic acid methyl ester was common in the plant aerial parts in WH habitat, however the fatty acid 6-Acetyl-8methoxy2,2-dimethyl-2H-chromen-5-ol was common in the plant aerial parts in SD habitat. *Corresponding Author: Khalil M. Saad-Allah  khalilmahfouz@yahoo.com 264 | Elhaak et al. J. Bio. & Env. Sci. 2014 Introduction 1993) together with several polymethoxylated The Western Mediterranean Coastal land of Egypt is flavonoids and some coumarins (Ahmed et al., 1994). relatively rich and diverse and it is one of the richest Plant sesquiterpenes are known to show diverse phyto-geographical regions in Egypt because of its biological and pharmacological actions, including relatively high rainfall, it contains about 50% of the anti-inflammatory activity (Recio et al., 2000). total flora of Egypt (Abbas et al., 2008). Sand dunes Recently, Ahmed et al. (2013) reported that the high along the Western Mediterranean coast of Egypt are content of terpenes, sesquiterpenes and flavonoids in formed of loose oval pseudo-oolitic grains of calcium the ethanolic extract of V. candicans could be carbonate. These dunes are close to the sea and as a responsible for the anti-cholinesterase activity, anti- result they are humid, exposed to northerly winds and inflammatory affected by sea spray. Plants growing in sand dunes neurotrophic effect as well as anti-amyloidogenic are highly adapted and have the ability to grow potential of these extracts. This suggests that V. vertically candicans and underground tolerate organs to the exposure of their action, ethanolic antioxidant extract capacity may and effectively sever water stress of ameliorate the inflammation and neurodegeneration calcareous sandy soil. The most important land-use in characterizing Alzheimer’s disease in the male rats this area is the grazing (Abbas et al., 2008). The plant (Ahmed et al., 2013). species in the Western Mediterranean desert area exhibit different growth and productivity characteristics (Elhaak, 1986). The aim of this study is to investigate the effect of two different habitats of the plant on the chemical composition giving the plant its medicinal importance The genus Jasonia (=Varthemia) is a member of represented in soluble and insoluble carbohydrates, Asteraceae, is a small genus with about five species free and combined sugars, soluble amino acids and mainly distributed in the Mediterranean region proteins, type of amino and fatty acids, lipid profile, (Bremer et al., 1994). In Egypt, the genus Varthemia phenolics and alkakoids. comprises three species (Täckholm, 1974) namely V. candicans, V. montana and V. iphionoides. El-Kady Materials and methods (1993) reported that V. candicans is an aromatic Collection of Plant Material perennial plant and recorded it in rocky places, semi- The fresh aerial parts (shoot system) of Varthemia dry land and sand dunes in Egypt. Ali (2012) reported candicans were collected at winter, spring, summer that V. candicans is an unpalatable sub-shrub with a and autumn seasons (2011-2012 season) from the defense strategy depending on the odour generated by first habitat, Wadi Habbes (master up stream rocky the essential oils of the plant. In Egypt, V. candicans portion of Wadi Habbes habitats) situated about 18 is mainly distributed in Mareotic Sector, Isthmic km West of Marsa Matrouh. The second habitat was Desert, Galala Desert, Libyan Desert, Nubian Desert the Consolidated Oolitic Sand Dunes, situated about and Gebel Oweinat (Hepper and Friis, 1994 and 180 km West of Marsa Matrouh (the coastal of Abo Boulos, 2009). Zereba or west of Barany). The plant samples were washed, weighted as fresh, then dried in an oven at The plant is used in the earlier times in the folk 60°C to constant weight, then finally ground to fine medicine to treat some diseases. Earlier work on the powder and stored in paper bags for further analysis. chemistry of V. candicans revealed the presence of several sesquiterpenes and sesquiterpene-lactone Total soluble and insoluble carbohydrates content derivatives (De Pascuai et al., 1980) eudesmanoic The total carbohydrates in aerial parts of Varthemia acids and eudesmanolides (Ahmed et al., 1994) candicans were extracted and hydrolyzed by 2M HCl guaianolides and pseudoguaianolides (Ahmed et al., for 2-5 hrs at 100ºC. After neutralization, extracted 265 | Elhaak et al. J. Bio. & Env. Sci. 2014 sugars were estimated using the general phenol- Free amino acids and protein amino acids sulfuric acid assay (DuBois et al., 1956) and expressed The investigation of free amino acids and protein- as mg/g d.wt of the plant. amino acids after the hydrolysis of V. candicans were accomplished according to Pellet and Young (1980) Determination of Free Sugars by using Amino Acid Analyzer technique (Sykam Free sugars were extracted with 80% ethyl alcohol System 7130 Amino Acid Reagent organizer). from 10g of the plant dried material and filtered. The alcoholic extract was clarified on amberlite IR 120 Total lipids content and Saponifiable matter resin column and then evaporated. The residue was Total lipids of the plant aerial parts were extracted redissolved in 3ml of 10% aqueous isopropanol for with petroleum ether (40-60): ether (1:1) using chromatographic investigation according to Chaplin Soxhlet and Kennedy (1994). according to Christie (1982). After the removal of the apparatus. The lipids were quantified unsaponifiable fraction with ether, the soapy solution Determination of Combined Sugars was converted into the corresponding free fatty acids Combined sugars were extracted by refluxing of the by means of 2.5% sulphuric acid. When the fatty acids plant dry powder for 3 hours with 6N HCl. The acid were completely liberated they were removed by was evaporated under vacuum at 45ºC and the extracting with ether. Then ether extract was dried residue was dissolved in 10% isopropanol for over anhydrous Na2SO4. The extracted fatty acids chromatographic were converted to the corresponding methyl esters investigation as previously using ethereal solution of diazomethane (Farag et al., mentioned. 1986). The methyl esters of the fatty acids were HPLC analysis of sugars analyzed with gas chromatographic apparatus. The Samples (free and combined sugars) were filtered fractionation through a 0.45 µm membrane. Analysis of the conducted using GC-MS analyzer HP 6890 Series Gas carbohydrate in the filtrate was performed using Chromatograph System with an HP 5973 Mass HPLC, Shimadzu Class-VPV 5.03 (Kyoto, Japan) Selective Detector. The column is TR-FAME (Thermo equipped with refractive index RID-10A Shimadzu 260 M142 P) (30 m, 0.25 mm ID, 0.25υm Film) (70% detector, LC-16ADVP binary pump, DCou-14 A Cyanopropyl –Polysilphphenylene siloxane) capillary degasser and Shodex PL Hi-Plex Pb column (Sc 1011 column with No. H706081), Guard column Sc-Lc Shodex, and temperature transfer line 250°C. The carrier gas was heater set at 80oC. Separation and quantitation were carried out on an amino-bonded column with a of fatty acid methyl esters was injector Temperature 200°C and He (1.5 ml/min) and the ionization energy was 70eV. The sample inject was 1μl (5 μl/1 ml solvent). mobile phase of CH3CN and H2O (80:20 v/v). Identification of the different constituents was Determination of total soluble proteins and free performed by comparison of the relative retention amino acids times and mass spectra with those of authentic The total soluble proteins content of the plant dried reference compound and probability merge search samples were estimated quantitatively using the libraries software of NIST11.L, wiley7n.l and Pest l method described by Bradford (1976) and the protein (Adams, 2004). content was calculated as mg/g d.wt. Total free amino acids were assayed by the method described by Lee Statistical Analysis and Takahashi (1966) and amino acids content was Statistical analysis of the obtained results was carried calculated as mg/g d.wt. out according to the technique adopted by Norusis (2006 and 2007) using SPSS statistical package. 266 | Elhaak et al. J. Bio. & Env. Sci. 2014 Results and discussion was in winter and the lowest content (38 mg/g d.wt) Total soluble and insoluble carbohydrates was in spring. Total insoluble carbohydrates in this The variation in carbohydrates content in the aerial habitat recorded the highest content (352 mg/g d.wt) parts of V. candicans in WH and SD habitats during in summer and the lowest one (130 mg/g d.wt) was in the dry and wet seasons indicated changes in soluble winter. So, in this habitat, the amount of soluble and insoluble carbohydrates with high significant carbohydrates in the wet seasons (winter and value (P<0.001) with the changes in the study autumn) was higher than that of the dry season, habitats, seasons and the interaction between the however the content of the insoluble carbohydrates habitats and seasons (Table 1). The highest amount of recorded a reverse trend. total soluble carbohydrates in WH (215 mg/g d.wt) Table 1. The content of total soluble and insoluble carbohydrates in V. candicans aerial parts in the study habitats during the period of investigation. Habitat Season Wadi Habbes Sand Dunes Soluble Insoluble Ratio Soluble Insoluble Ratio Wet seasons Winter 215 ±13 130±15 1.65 121 ±7 271 ±64 0.44 Spring 38±6 268±70 0.14 13±4 418±66 0.03 Dry seasons Summer 89±9 352±78 0.25 139±26 108±45 1.28 Autumn 173±16 311±33 0.55 152±6 449±17 0.34 Mean 128.75 265.25 0.65 106.25 311.50 0.52 The highest amount of total soluble carbohydrates in metabolic pathways leading to accumulation or SD (152 mg/g d.wt) was in autumn and the lowest depletion of metabolites, (b) alterations in activities of content (13 mg/g d.wt) was in spring. Total insoluble conveniently assayable enzymes, and (c) changes in carbohydrates in this habitat recorded the highest the pattern of synthesis of proteins of unknown content (449 mg/g d.wt) in autumn and the minimum function. These changes include the accumulation of one (108 mg/g d.wt) was in summer. The trend of carbohydrates, organic and amino acids, quaternary soluble and insoluble carbohydrates in this habitat is ammonium compounds and ABA. The accumulation like that of the former habitats. So our results support of osmolytes may ensure the maintenance of the the hypothesis that the change in growth habitat and structural integrity of membranes (Conroy et al., growth season has a major effect on the metabolic 1988). There are some evidences that plants are more pathways of the plant tissues. tolerant to water shortage when water is withheld under conditions that favour osmotic adjustment Prado et al. (2000) and Gill et al. (2001) reported (Moinuddin and Chopra, 2004). that the accumulation of sugars in plants is enhanced in response to a variety of environmental stresses. Free sugars Siddique et al. (2000) found that the adaptation of Data represented in Table (2) and high performance plants to water stress has been attributed to the liquid chromatography (HPLC) photogram (1) show stress-induced the increase in carbohydrate levels. difference in free and combined sugars Hanson and Hitz (1982) concluded that plant cells composition in the aerial parts of V. candicans and tissues show several metabolic responses to water growing in WH and SD habitats. The detected free stress, some of which may have adaptive implication. sugars in the two habitats were glucoronic, raffinose, These responses are usually described at one or glucose, galactose, fructose and fucose. another of three levels: (a) perturbation of whole 267 | Elhaak et al. J. Bio. & Env. Sci. 2014 Table 2. Free and combined sugars (%) of the aerial parts of V. candicans in the study habitats using HPLC technique. No R.T Sugar 1 2 3 4 5 6 7 8 9 10 11 2.700 4.417 4.567 5.750 6.717 7.133 7.450 8.383 9.717 11.650 13.300 Unknown Glucornic Raffinose Glucose Galactose Mannose Fructose Fucose Unknown Maltose Unknown Habitat Wadi Habbes Sand Dunes Free Combined Free Combined 0.969 39.227 57.241 18.768 2.415 12.153 14.160 12.124 9.479 0.354 12.727 0.281 9.037 14.763 12.090 0.405 96.545 85.840 1.421 - Photogram 1. Free sugars (%) of the aerial parts of V. candicans in the study habitats using HPLC technique. The plant growing in WH habitat was characterized The accumulation of free soluble sugars seem to play by the presence of five free monosaccharides; an important role in osmotic regulation of cells glucoronic acid, glucose, galactose, fructose and (Bolarin et al., 1995) and regulate the expression of fucose. The most abundant free sugar was glucornic some genes (Yu et al., 1996). Also, these sugars acid (39.3%), while the most scant free sugar was appear to be central to the development of desiccation galactose (12.1%). Also, the plant growing in SD tolerance (Hoekstra et al., 2001). On the other hand, habitat was characterized by the presence of five Herbinger et al. (2002) concluded that the decrease sugars, four free monosaccharides; glucose, galactose, in soil moisture content resulted in the decrease of fructose and fucose and one trisaccharide; raffinose. photosynthesis, which The most abundant free sugar was the trisaccharide increase in respiration rate and led to the reduction in raffinose (57.2%) and the most scant one was the carbohydrates concentration in plant and hence the monosaccharide fructose (9.0%). The obtained results breakdown of starch into free soluble sugars needed showed that the plant aerial parts in the two habitats for osmoregulation (Elhaak and El Sayed, 1990). was associated with an have four common monosaccharides. In WH habitat the free monosaccharide glucoronic acid is found and Combined sugars replaced in SD habitat by the trisaccharide raffinose. The combined sugars (glycosides) of the aerial parts Also, in this habitat, two unknown sugars were of V. candicans were determined after hydrolysis detected but with small percentages. using HPLC (Table 2 and Photogram 2). The detected sugars were the monosaccharides glucose, mannose and fructose and the disaccharide maltose. 268 | Elhaak et al. J. Bio. & Env. Sci. 2014 Photogram 2. Combined sugars (%) of the aerial parts of V. candicans in the study habitats using HPLC technique. The plant growing in WH habitat was characterized tolerance strategies in plants is the overproduction of by the presence of three combined monosugars different types of compatible organic solutes (Serraj glucose and the disaccharide maltose in the plant in and Sinclair, 2002). Singh (2004) proved that a the two greater accumulation of sugar lowers the osmotic monosaccharides mannose and fructose in the plant potential of cells and reduces loss of turgidity in of WH habitat. The most abundant combined sugar in plants. The other possible role of sugar may be as a the plant aerial parts in this habitat was the readily available energy source. two habitats, disaccharide maltose in addition (96.5%), to while the the most inadequate sugar was the monosaccharide fructose Soluble amino acids and soluble protein contents (0.3%). However, the plant growing in SD habitat was Data in Table (3) represent the variation in soluble characterized by lower number of combined sugars, amino acids and soluble protein contents in V. one combined monosaccharide; glucose (14.2%) and candicans aerial parts during the study seasons in the one disaccharide, maltose (85.8%). The data revealed studied habitats (WH and SD). The variation in the that the two monosaccharides mannose and fructose contents of soluble amino acids and soluble proteins have been vanished in SD habitat, so the plant varied statistically with highly significant value compensated the deficiency of these two sugars by the (P<0.001) by each habitats, seasons and the abundance of the monosaccharide glucose. interaction between them. The content of soluble amino acids of WH plant varied slightly between a Li et al. (2013) found a slight increase in soluble sugar maximum value (6.4 mg/g d.wt) in autumn and a concentration in Eremosparton songoricum with the minimum value (6.2 mg/g d.wt) in winter. On the increase in the osmotic potential. They reported that opposite, the soluble amino acids content in SD one of the osmotic stress defense mechanisms is the habitat plants recorded the highest content (6.7 mg/g accumulation of organic osmolytes (such as proline, d.wt) in spring and the lowest one (6.2 mg/g d.wt) in soluble sugars, glycine betaine, and organic acids) in autumn. However, the content of soluble protein in the cytoplasm to maintain the plant water potential the WH habitat recorded the maximum content (32.8 during drought stress. The severity of drought is mg/g d.wt) in summer and the minimum one (9.5 unpredictable as it depends on many factors such as mg/g d.wt) in winter through the period of occurrence and distribution of rainfall, evaporative investigation. Also, the amount of soluble proteins in demands and moisture storing capacity of soils (Wery SD reached to its highest content (48.9 mg/g d.wt) in et al., 1994). One of the most common stress spring and the lowest one (12.2 mg/g d.wt) in autumn 269 | Elhaak et al. J. Bio. & Env. Sci. 2014 throughout the period of investigation. In general, SD habitat than in the WH habitat during all seasons, both soluble amino acids and proteins were greater in except autumn. Table 3. Free amino acids and free protein contents (mg/g d.wt) in the aerial parts of V. candicans in the study habitats during the period of investigation. Habitat Season Wet seasons Dry seasons Winter Spring Summer Autumn Mean Wadi Habbes Amino acids Protein 6.2 ±0.02 9.5 ±0.96 6.3 ±0.08 25.9 ±2.21 6.2 ±0.04 32.8 ±2.18 6.4 ±0.03 17.3 ±4.54 6.30 21.38 Ratio 0.65 0.25 0.19 0.37 0.36 Sand Dunes Amino acids Protein 6.5 ±0.00 17.8 ±0.76 6.7 ±0.07 48.9 ±3.09 6.3 ±0.05 35.7 ±4.63 6.2 ±0.02 12.2 ±2.31 6.43 28.65 Ratio 0.37 0.14 0.18 0.51 0.30 Vyas et al. (1996) observed that water stress causes during drought stress (Ahire et al., 2005 and both reductions in the rate of protein synthesis as well Kottapalli et al., 2009). as changes in the type of proteins produced. He also reported that increasing water stress progressively Free and protein amino acids Vigna The separation of free amino acids and protein amino aconitifolia leaves. Garg et al. (2001) also found that acids of the aerial parts of Varthemia candicans were increasing water stress progressively decreased plant achieved using amino acid analyzer and each water potential, leaf area, net photosynthetic rate, component was detected quantitatively (Table 4). The starch and soluble protein contents and nitrate data illustrated that the aerial parts of the plant in the reductase activity in Vigna aconitifolia. Al-Jebory WH habitat exhibited twenty seven free amino acids (2012) reported that the content of protein in Pisum and twenty five in SD habitat. The most abundant free sativum decreased with increasing of drought stress, amino acid in WH habitat was asparagine (63.576 whereas proline content increased with increasing of µg/g d.wt), however the most abundant one in SD drought stress. The alternation in protein synthesis or habitat was proline (136.649 µg/g d.wt). The lowest degradation is one of the fundamental metabolic concentrations of free amino acids (0.065 and 0.064 processes that may influence water stress tolerance µg/g d.wt) given by hydroxy-proline in WH and SD (Jiang and Huang, 2002). Both quantitative and habitat, respectively. decreased soluble protein content in qualitative changes of proteins have been detected Table 4. Free and protein amino acids content of V. candicans aerial parts in the study habitats (WH and SD) using amino acid analyzer. No 1 2 3 4 5 6 7 8 9 10 11 12 13 Amino acid Phospho-serine Aspartic acid Hydroxy-Proline Threonine Serine Asparagine Glutamine α-Aminoadepic acid Proline Glycine Alanine Citrulline α-Aminobutyric acid Free amino acids (µg) Habitat RT WH SD 4.075 0.605 0.491 17.563 2.012 7.102 23.275 0.065 0. 064 25.317 1.591 3.520 27.752 4.880 3.944 32.683 63.576 95.287 36.704 0.183 1.533 41.579 0.723 1.121 46.048 24.913 136.649 49.011 2.703 0.795 51.259 10.255 18.170 55.501 0.163 55.736 0.144 270 | Elhaak et al. Protein amino acids (mg) Habitat RT WH SD 9.053 54.015 108.577 10.347 5.615 12.225 11.192 17.335 32.401 15.048 102.189 42.238 19.576 24.853 51.471 21.016 22.819 38.665 - J. Bio. & Env. Sci. 2014 No Amino acid 14 Valine 15 Cystine 16 Methionine 17 Isoleucine 18 Leucine 19 Tyrosine 20 Phenylalanine 21 β-Alanine 22 β-Aminobutyric acid 23 γ-Aminobutyric acid 24 Lysine 25 3-Methylhistidine 26 Histidine 27 Argenine 28 Glutamic acid Total number of amino acids Free amino acids (µg) Habitat RT WH SD 58.987 4.573 16.391 60.704 9.500 8.189 62.888 0.291 0.378 66.704 1.811 7.022 68.352 1.087 4.305 71.675 0.921 3.393 75.616 0.159 5.681 77.019 2.166 1.377 79.853 1.729 0.129 85.333 0.181 14.362 97.165 63.037 21.580 99.389 6.499 19.693 102.365 53.555 24.187 123.928 9.524 18.070 25 27 Protein amino acids (mg) Habitat RT WH SD 23.611 8.446 13.929 22.613 2.250 25.683 5.509 9.982 27.717 5.186 9.990 28.667 17.013 32.267 31.016 6.960 14.038 32.227 7.007 14.906 38.549 12.437 19.301 35.203 37.810 55.706 42.749 16.417 23.491 12.816 39.481 61.783 17 16 Also, all of the detected amino acids were found in the habitat was aspartic acid (108.577 mg) and the less aerial parts of the plant growing in SD habitat, but the abundant protein-amino acids were methionine and two amino acids citrulline and α-aminobutyric acid isoleucine (9.982 and 9.990 mg). The data also were vanished in WH habitat. It is also important to revealed that the amino acid cystine was unique and note that the content of most detected amino acids in characteristic to the plant growing in WH habitat. SD habitat was greater than the total content of free amino acids in WH habitat. Table 5. Total lipids content of the aerial parts of Varthemia candicans in two different habitats (WH The investigation of hydrolyzed protein-amino acids in and SD) during the period of investigation (2011- the aerial parts of V. candicans growing in the two 2012). study habitats (WH and SD) was achieved (Table 5 and Photogram 3a and b). The data reflected that there were seventeen amino acids with different concentrations in the two habitats. The most abundant protein-amino acid in WH habitat was proline (102.189 mg) and the scarcer one was cystine (2.250 mg). Season Winter Spring Summer Autumn Mean Total lipids (mg/g d.wt) Wadi Habbes Sand Dunes 10.870 ±0.91 52.855 ±0.78 71.945 ±0.84 31.555 ±1.00 41.806±26.41 21.110 ±0.69 87.330 ±1.00 82.135 ±0.85 52.420 ±0.76 60.749±30.57 However, the most abundant protein-amino acid in SD Photogram 3a. Free amino acids of V. candicans aerial parts in the study habitats (WH and SD) using amino acid analyzer. 271 | Elhaak et al. J. Bio. & Env. Sci. 2014 Photogram 3b. Protein amino acids of V. candicans aerial parts in the study habitats (WH and SD) using amino acid analyzer. Proline is one of the osmolytes, which increase faster and isoleucine (Eid et al., 2011). do Amarante et al. than other amino acids in plants under water deficit (2006) proved that legumes use asparagine rather stress and help the plants to maintain cell turgor than ureides as nitrogen transport compounds in the pressure (Valentovic et al., 2006). Thus, proline xylem. accumulation can be used as a criterion for drought conditions of stress as proline in many plant species resistance assessment (Gunes et al., 2008). Najaphy as soybean (King and Purcell, 2005), alfalfa (Fougere Asparagine also accumulates under et al. (2010) explained that the increased proline et al., 1991), pearl millet (Kusaka et al., 2005) and content in chickpea leaves exposed to water deficit wheat (Carillo et al., 2005). This may be a direct was due to protein breakdown. Increasing proline biological response to the stress conditions, for content of leaves with decreasing available water example, by contributing to the maintenance of means that an efficient mechanism for osmotic osmotic pressure or indirect result of the restriction of regulation, stabilizing sub-cellular structures and protein synthesis under stress conditions (Lea and cellular adaptation to water stress was provided Miflin, 2003). (Valentovic et al., 2006 and Gunes et al., 2008). Total lipids content Asparagine and glutamine connect the two important The total lipids content of V. candicans aerial parts in metabolic cycles of the plant, the carbon and nitrogen the study habitats (WH and SD) was recorded in cycles, and they have an influence both on sugars and Table (5). The recorded data were statistically highly proteins. In plants, aspartate is the precursor to significant (P<0.001) and revealed that the total several amino acids, including methionine, threonine lipids content was greater in SD habitat than in WH 272 | Elhaak et al. J. Bio. & Env. Sci. 2014 one during all seasons. The highest lipid content in (Monteiro et al., 1990) and a stimulation of lipolytic SD habitat (87.330 mg/g d.wt) and in WH habitat and peroxidative activities (Matos et al., 2001). (71.945 mg/g d.wt) was recorded during spring and summer, respectively. However, the lowest lipid Fatty acids constituents content in the two habitats (10.870 and 21.110 mg/g The fatty acids content of the aerial parts d.wt, respectively) was recorded during winter candicans in the study habitats, WH and SD, season. estimated using GC-MS technique was calculated and of V. tabulated (Table 6 and Photogram 4a and b). The Membranes are main targets of degradative processes obtained results revealed that V. candicans growing induced by drought and it has been shown that, under in the two habitats contained twenty eight fatty acids; water stress, a decrease in membrane lipid content is twenty four fatty acids were identified and four fatty correlated to an inhibition of lipid biosynthesis acids remained nameless. Table 6. GC-MS analysis of fatty acids composition of the aerial parts of V. candicans in the study habitats. Habitat No RT (min) IUPAC Name WH (%) SD (%) 1 18.62 Methyl tetradecanoate 0.77 0.77 2 19.57 Unknown 14.14 7.71 3 20.96 Trimethylurea 9.08 5.23 4 21.22 Pentadecanoic acid, methyl ester 0.33 - 5 21.80 Unknown - 0.82 6 22.46 Unknown - 3.32 7 22.81 Unknown 12.01 - 8 23.81 Hexadecanoic acid, methyl ester 12.01 8.62 9 24.04 2,5,10-Trimethyl-6,7,8,9-tetradehydrocyclopentadecenone 7.17 - 10 24.14 6-Acetyl-8methoxy-2,2-dimethyl-2H-chromen-5-ol - 23.92 11 24.88 3-Oxo-isocostic acid 6.69 7.61 12 25.89 8,9-Dihydrocyclohepta[a]phenalene-6 (10H) - one - 2.53 13 26.15 Hexadecanoic acid, 15-methyl-, methyl ester 1.11 1.67 14 26.42 Ethanone 3.77 - 15 26.45 2,2,4-Trimethyl-4,5-dihydro-1,3,8H-azulene-6,7dicarboxylic anhydride - 5.77 16 26.63 Methyl ester of Encecalol - 2.71 17 28.50 Stearic acid, methyl ester 2.05 1.43 18 29.11 6-Octadecadienoic acid, methyl ester (Z)- (CAS) 8.33 4.76 19 30.41 9,12-Octadecadienoic acid, methyl ester 5.28 3.71 20 31.75 Benzene, hexaethyl- 6.00 7.02 21 31.98 9,12,15-Octadecatrienoic acid, methyl ester 1.48 2.20 22 32.88 Eicosanoic acid, methyl ester 1.94 1.35 23 33.44 Cis-11-Eicosenoic acid, methyl ester 0.33 - 24 34.94 Heneicosanoic acid, methyl ester 0.32 - 25 36.98 Docosenoic acid, methyl ester 6.51 4.37 26 37.50 13-Docosenoic acid, methyl ester, (Z)- 1.20 - 27 38.86 Tricosanoic acid, methyl ester 0.68 - 28 40.76 Tetracosanoic acid, methyl ester 5.74 4.51 273 | Elhaak et al. J. Bio. & Env. Sci. 2014 Photogram 4a. GC-MS analysis of fatty acids composition of the aerial parts of V. candicans in WH habitat. Photogram 4b. GC-MS analysis of fatty acids composition of the aerial parts of V. candicans in SD habitat. 274 | Elhaak et al. J. Bio. & Env. Sci. 2014 The fatty acid Hexadecanoic acid methyl ester carbohydrates in the body are converted to palmitic presented the higher percentage (12.01%) in the aerial acid and it is a major body component of animals, it parts of the plant growing in WH habitat, however the comprise 21–30% of human depot fat (Kingsbury et fatty al., 1961) and it is a major, but highly variable, lipid acid 6-Acetyl-8methoxy-2,2-dimethyl-2Hpercentage component of human breast milk (Jensen et al., (23.92%) in the aerial parts of the plant growing in 1978). Palmitate negatively feeds back on acetyl-CoA SD habitat. On the other hand, the fatty acid carboxylase (ACC), Heneicosanoic acid methyl ester represented the converting acetyl-CoA to malonyl-CoA, which in turn lowest percentage (0.32%) in the aerial parts in WH is used to add to the growing acyl chain, thus habitat; however the fatty acid Methyl tetradecanoate preventing further palmitate generation. In biology, represented the lowest percentage (0.77%) in SD some proteins are modified by the addition of a habitat. palmitoyl group in a process known as palmitoylation chromen-5-ol presented the highest which is responsible for which is important for membrane localization of The results cleared out that the aerial parts of the many proteins. Wei et al. (2011) reported that plant in WH habitat exhibited unique characteristic Octadecatrienoic fatty acids like Pentadecanoic acid Heneicosanoic acid, Oleic acid, β-sitosterol and methyl ester, 2,5,10-Trimethyl-6,7,8,9- acid, Pentadecanoic acid, Phytol may be responsible for the antioxidant activity tetradehydrocyclopentadecenone, Cis-11-Eicosenoic of Adrographis paiculata leaf extract. acid methyl ester, Heneicosanoic acid methyl ester, 13-Docosenoic acid, methyl ester, (Z) and Tricosanoic References acid methyl ester. Also, the plant growing in SD Abbas MS, El-Morsy MH, Shahba MA, Moursy habitat exhibited some unique fatty acids like 6- FI. 2008. Ecological studies in coastal sand dune Acetyl-8methoxy-2,2-dimethyl-2H-chromen-5-ol, rangelands in the north west of Egypt Options 8,9-Dihydrocyclohepta[a]phenalene-6 (10H) – one, Méditerranéennes Series A 79, 389-393. Methyl ester of Encecalol and 2,2,4-Trimethyl-4,5dihydro-1,3,8H-azulene-6,7-dicarboxylic anhydride. Adams RP. 2004. 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