Ecological and Phytochemical Studies on Euphorbia retusa (Forssk.) from Egyptian Habitat

This study deals with the ecology, phytochemistry, and biological activity investigation of Euphorbia retusa, belonging to Euphorbiaceae family, obtained from Egypt. Ecologically, Euphorbia retusa secretes white sap inhibiting the growth of the other species, so Euphorbia retusa is forming complete patches. Phytochemical study of the plant was visualized intensively based on its extraction with a protic organic solvent, working up and purifying its entire bioactive compounds using a series of different chromatographic techniques. A broad range of diverse compounds were isolated, namely, 1-hexacosanol (1), 3β-hydroxy-24-methylene-9,19-cyclolanostane; 24-methylenecycloartanol (2), 3β-hydroxy-9,19-cyclolanostane; cyclolaudanol (3), 3β,24S-Ergost-5-en-ol (4), and methyllinoleate. Additionally, GC-MS analysis of the unpolar fractions detected the existence of n-dodecane, methyllaurate, 6,10,14-trimethyl-pentadecan-2-one (5), 6,10-dimethyl-undecan-2-one (6), 2-methyl-hexadecanal (7), methylpalmitate, methyl-9,12,15-octadecatrienoate (8), and n-heneicosane (9). A full assignment for compounds 2 and 3 using 1 and 2 DNMR was carried out herein for the first time. The antimicrobial activity of the strain extract and obtained compounds was studied using a panel of pathogenic bacterial strains. The in vitro cytotoxicity of the compounds as well as the crude extract was studied against the human cervix carcinoma cell line (KB-3-1).


Introduction
Euphorbiaceae (spurge family) is a large and polymorphic genus with about 8100 species, out of which 2000 Euphorbia species were found.
is family was reported recently to comprise about 300 genus and 10,000 species, which are used in folk medicine against the venomous bites and trichiasis and known as wart remover [1]. Species of Euphorbia are characterized by high ecological amplitudes in tropical, subtropical, and warm temperate regions, and they are widely spread around the world, and distributed mainly in North Africa and in the temperate parts of Asia, but mainly in the Mediterranean region [2][3][4][5].
can be recognized as coastal sand dunes dominated with Ammophila arenaria and Euphorbia paralias and inland areas dominated with Haloxylon scoparium and Asphodelus aestivus [9][10][11]. Phytochemically, Euphorbia retusa was reported as a rich source of diverse bioactive compounds. Di erent classes of compounds have been isolated from various parts of Euphorbia retusa, of which the main groups are terpenoids and avonoids [12]. Additionally, topical pharmaceutical compounds comprising a dry extract of Euphorbia species or a drug or a active agent with excipients useful for the treatment of anorectal diseases and colon diseases such as hemorrhoids, ssures, cracks, stulas, abscesses, and in ammatory bowel disease are provided [13].
In the present study, measuring of the density, frequency, and owering sequences of Euphorbia retusa and its associated species was investigated. Additionally, further studies to identify the secondary metabolites of Euphorbia retusa collected from Egyptian habitats and evaluate their antimicrobial activities was presented as well.
e analysis was carried out at programmed temperatures with initial temperature 40°C (kept for 1 min) which is then increased at a rate of 10°C/min to reach the nal temperature 280°C (kept for 10 min), injector temperature was 250°C, and detector (mode of ionization: EI) temperature was 250°C, with He as a carrier gas at a ow rate of 1 mL/min, a total run time of 27 min, and an injection volume of 0.2 µL. Characterization of individual unpolar compounds by GC-MS was performed in triplicate. R f values were determined on Polygram SIL G/UV 254 (Macherey & Nagel, Düren, Germany). Size exclusion chromatography was performed on Sephadex LH-20 (Lipophilic Sephadex; Amersham Biosciences, Ltd., purchased from Sigma-Aldrich Chemie, Steinheim, Germany).

Plant Material.
e plant samples were collected during the spring of 2014 from 5 homogeneous sites in their oristic composition, but they are di erent in the habitats (Table 1) 10]) visualized by UV and then spraying with anisaldehyde/sulphuric acid and heating, ve fractions were obtained: FI (3.6 g), FII (5.23 g), FIII (7.2 g), FIV (7.7 g), and FV (8.2 g). Fractions III-V showed similarity in their containing of a major UV nonabsorbing band, which was detected as intensive pink-violet on spraying with anisaldehyde/ sulphuric acid. Puri cation of this band using a silica gel column eluted with cyclohexane a orded two colorless solids of 1-hexacosanol (1, 200 mg) and 24-methylenecycloartanol (2, 2.11 g). An increase in the polarity of the eluent system by gradual addition of CH 2 Cl 2 to cyclohexane a orded 3β-hydroxy-9,19-cyclolanostane (3, 6 mg) and 3β,24S-Ergost-5-en-ol (4, 20 mg) as colorless solids, in addition to a colorless oil of methyllinoleate (25 mg). An application of the unpolar fractions to I-II to GC-MS analysis detected the existence of further nine compounds ( Table 2).

Biological Activities
2.5.1. Antimicrobial Assay. Antimicrobial assays using the agar di usion test were performed as described previously [14]. Origin of test strains: M. miehei Tü 284 and Streptomyces viridochromogenes Tü 57 were obtained from the  [15] has been performed. 20 mL of the medium seeded with test organisms was poured into 9 cm sterile Petri dishes. After solidi cation, the paper disks (6 mm diameter) were placed on inoculated agar plates and allowed to di use the loaded substances into refrigerator at 4°C for 2 h. e plates were incubated for 24 h at 35°C. Bacteria were grown on the nutrient agar medium: 3 gL −1 beef extract, 10 gL −1 peptone, and 20 gL −1 agar. e pH was adjusted to 7.2. After incubation, the diameters of inhibition zones were measured.

Cytotoxicity Assay.
e KB-3-1 cells were cultivated as a monolayer in DMEM (Dulbecco's modi ed Eagle medium) with glucose (4.5 gL −1 ), L-glutamine, sodium pyruvate, and phenol red, supplemented with 10% (KB-3-1) foetal bovine serum (FBS). e cells were maintained at 37°C and 5.3% CO 2 -humidi ed air. On the day before the test, the cells (70% con uence) were detached with trypsinethylenediamine tetraacetic acid solution (0.05%; 0.02% in DPBS) and placed in sterile 96-well plates in a density of 10,000 cells in 100 μL medium per well. e dilution series of the compounds were prepared from stock solutions in DMSO of concentrations of 100 mm, 50 mm, or 25 mm. e stock solutions were diluted with culture medium (10% FBS) down to the picomolar (pM) range. e dilution prepared from the stock solution was added to the wells. Each concentration was tested in six replicates. Dilution series were prepared by pipetting the liquid from well to well. e control contained the same concentration of DMSO as the rst dilution. After incubation for 72 h at 37°C and 5.3% CO 2 -humidi ed air, 30 μL of an aqueous resazurin solution (175 μM) was added to each well. e cells were incubated under the same conditions for 5 h. Subsequently, the uorescence was measured. e excitation was measured at a wavelength of 530 nm, whereas the emission was recorded at a wavelength of 588 nm. e IC 50 values were calculated as a sigmoidal dose-response curve using GRAPHPADPRISM 4.03.

1. Ecological Study.
Euphorbia retusa is a glabrous glaucous perennial species, with high range (20-60 cm), sometimes owering in the rst year, and stems erect, many from a woody base. Leaves are sessile, and the cauline is 1-5 × 0.3-0.6 cm oblong-linear, alternate, and base-rounded. Apex is acute to retuse. Margin is acutely serrate and umbrella-like, and oral leaves are opposite. Cyanthia are 2.5-3 mm long in forked umbels. e data shown in Table 1 represent 5 sites which were selected for eld studies. ese sites are located about 30 km west of Sidi Barrani city, comprising 4 plant communities and three habitats, namely, plateau, slopes, and desert plains. Selection of the sites was depending on habitat heterogeneity and the difference of oristic composition along transect from the north to south for about 25 km. e study area has been extensively used for barely cultivation and grazing of livestock. Apart from that, Euphorbia retusa is neither grazed nor eaten by humans, and its patches remain abundant inside the pasture land.

Vegetation.
A total of 30 species (22 perennials and 8 annuals) were recorded in the study area (Table 3). e richest sites are 2, 3, and 4 with 15 species per site, whereas site 1 is the poorest site in vegetation. e mean of species per site is 14.4 ± 0.89.
Besides Euphorbia retusa species, there are three species presented in all sites, namely, ymelaea hirsuta and Haloxylon scoparium as perennials and Medicago intertexta as annuals. ey followed with another medicinal plant (Asphodelus aestivus) with frequency (80%). Site (1) occupies the inland plateau and is dominated by Haloxylon scoparium followed by Asphodelus aestivus, Deverra tortuosa, and Euphorbia retusa. Sites (2,3,4) are similar in oristic composition with 15 species in each. Site 5 represents desert plain habitat and supports 14 species with di erent frequencies. e most common species located in the last site are Asphodelus aestivus (density � 33) and Euphorbia retusa (density � 24) (Table 3).

Phenological Aspects.
Phenological sequences of Euphorbia retusa are summarized in Figure 1. e most common growth stage around the year is the vegetative stage which started in the beginning of February to April, and the plant becomes vegetative and dormant from August to November. e seedling stage started at the beginning of December to February, whereas owering and fruiting stages started from April to August, respectively. e general trends and ranking of vegetation are similar to the prevailing type in the western Mediterranean coast of Egypt where there are ve main aliens predominate, namely, Haloxylon scoparium, Asphodelus aestivum, Atriplex halimus, ymelaea hirsuta, and Salsola tetrandra [18]. Vegetation is mostly represented with shrubs and undershrubs or perennial herbs. Phanerophytes are less represented except some individuals especially in catchment areas in wadi beds.
e phenological aspects of our species are mostly represented with the vegetative stage whatever, before or after raping. is sequence is playing a very important role in chemical investigation studies.  e importance of Euphorbia retusa as an interest source of diverse bioactive compounds served in treatment of diverse infectious diseases [12,13] encouraged the authors herein to study the entire constituents of its organic extract. In accordance, an air-dried sample of the plant was applied to intensive extraction with methanol followed by concentration in vacuo, and the obtained dark green crude extract was then applied to a series of chromatographic puri cations monitored by TLC and visualizing agents delivering ve diverse compounds, namely, 1-hexacosanol (1), 3β-hydroxy-24-methylene-9,19-cyclolanostane (2), 3β-hydroxy-9,19-cyclolanostane (3), 3β,24S-ergost-5-en-ol (4), and methyllinoleate ( Figure 2). Alternatively, analysis of the individual di erent unpolar fractions by GC-MS revealed the existence of further nine compounds as listed in Table 2 utilizing alkane internal standards (C8-C22) during the analyses for the determination of linear retention indices (LRIs) and assay performance.

1-Hexacosanol.
As colorless needles, compound 1 was obtained as an extremely unpolar major substance from the fast fractions during a silica gel column eluted with cyclohexane. It showed no UV activity on TLC; however, it has been detected as violet and turned later as blue on spraying with anisaldehyde/ sulphuric acid. e molecular weight of 1 was deduced as 382 Dalton according to EI-MS, displaying a further ion peak at m/z 364 due to the expulsion of a water molecule. e HREI-MS of 1 proved the corresponding molecular formula as C 26 H 54 O, displaying no degree of unsaturations, and hence, an aliphatic open chain of sp 3 hybridization nature for 1 was concluded. e 1 H NMR spectrum exhibited a triplet oxymethylene signal at δ 3.62, and its corresponding carbon signal was con rmed at δ C 63.1. Additional quartet methylene signal was visible at δ H 1.54 and its corresponding carbon value at δ C 32.9. is was in addition to a broad multiplet signal with integration of 36 protons being for 18 methylene groups, and their corresponding carbons were visible in the range of δ 32.0-22.8, terminated by a triplet methyl group at δ 0.85 (δ C 14.2). Based on the revealed chromatographic and spectroscopic features, compound 1 was con rmed as 1-hexacosanol.
Biologically, n-hexacosanol, as long-chain fatty alcohol, was reported to promote the maturation of central neurons, and hence, it is known as neurotrophic factor [19], reducing the neuronal damage induced by the neurotoxin, kainic acid [20]. Alternatively, hexacosanol showed a stimulation of insulin secretion in vivo and in vitro, inducing a reduction of the insulin response to an intravenous glucose tolerance test with a consequent increase in hyperglycaemia, and hence, it has antidiabetic e ects [21,22].

24-Methylenecycloartanol.
As further colorless needles, compound 2 was obtained as the most predominant substance in the plant's extract with additional low polarity. e compound displayed no UV activity on TLC as well, while it was detected as pink on spraying with anisaldehyde/sulphuric acid and turned later as violet, referring most likely to a terpenoidal moiety [23]. According to the EI-MS, compound 2 exhibited a molecular ion peak at m/z 440 followed by expulsion of the methyl group to a ord a fragment ion peak at m/z 425. e molecular weight of 2 was further established as 440 Dalton by ESI-MS showing a quasi-ion peak at m/z 463 [M + Na] + and the corresponding molecular formula as C 31 H 52 O, bearing six DBE. e 1 H NMR spectrum displayed two resonating signals with a tiny coupling constant (J∼1.7) being for an exomethylene group, whereas its carbon was deduced at δ 105.9 according to HMQC experiment and the complementary quaternary carbon at δ 156.6. Additionally, a multiplet signal for an oxymethine was at δ 3.25 and its carbon at δ 78.7. Furthermore, two highly up eld shifted doublet signals (each of J∼4.2) were observed at δ 0.52 and 0.30, corresponding to the highly strained methylene group (δ C : 29.9) existence in cyclopentane moiety. An additional NMR study ( 1 H, 13 C, HMQC, Table 4 and Figure 3) of compound 2 con rmed the existence of further seven methyls (classi ed into four singlet and three doublet signals), twelve methylenes (among them those of exomethylene and cyclopentane one), six methines (including the 3-hydroxy-methine one), and six quaternary carbons among them the ole nic one (156.6) as matched with the revealed molecular formula. An intensive study of compound 2 on the bases of H,H COSY, and HMBC experiments ( Figure 3) con rmed its structure de nitely as 3β-hydroxy-24methylene-9,19-cyclolanostane.

3β-Hydroxy-9,19-cyclolanostane; Cyclolaudanol.
As closely related to compound 2, with identical chromatographic properties, compound 3 was obtained exhibiting a tiny higher polarity than compound 2, indicating a further triterpene system. According to the ESI-MS, the molecular weight of 3 was deduced as 442 Dalton due to the existence of two quasi-ion peaks at m/z 465 [M + Na] + and 442 [M + H] + , with higher 2 amu than compound 2. e corresponding molecular formula was subsequently established as C 31 H 54 O bearing ve DBE (i.e., less double bonds than in compound 2). Based on that, it clearly appeared that compound 3 is a hydrogenated analogue of compound 2, and this was de nitely con rmed from the 1 HNMR spectrum, at where the ole nic protons of the exomethylene group in compound 2 disappeared, while a new doublet methyl visible at 0.93 was created. Detailed NMR study of compound 3 using 1 ( 1 H, 13 C) and 2D (H,H COSY, HMQC, and HMBC) ( Table 4 and Figure 4) con rmed its structure as 3β-hydroxy-9,19-cyclolanostane [32].
Pharmaceuticals which contain cycloartanol, 24methylcycloartanol, and/or 24-methylenecycloartanol in addition to highly unsaturated oils containing more than 3 double bonds have the capability to control the lipid metabolism in serum, and hence, they showed anticholesteremic e ects [33].

3β,24S-Ergost-5-en-ol.
As an alternative colorless solid, exhibiting no UV activity during TLC, compound 4 was a orded; nevertheless, it exhibited a violet coloration on spraying with anisaldehyde/sulphuric acid and turned later as blue, indicating a fatty acid or steroidal compound's nature. According to intensive spectroscopic study and comparison with corresponding literature, the structure of compound 4 was deduced as 3β,24S-Ergost-5-en-ol (Table  5). Compound 4 was reported previously as a constituent of rapeseed oil (Brassica napa), soybean oil (Glycine max), and  wheat-germ oil (Triticum spp.) and found in virtually all plant oils [34][35][36].

Complementing Chromatographic and Spectroscopic
Results for Compounds 1-        (Table 6). Based on that, the plant extract exhibited weak activity against Staphylococcus warneri (8 mm) and Escherichia coli (7 mm), meanwhile compounds 1-4 displayed no antibacterial activity. Alternatively, the plant extract and the corresponding compounds were tested for in vitro cytotoxicity against the human cervix carcinoma cell line (KB-3-1) in comparison with griseofulvin. e results revealed that the plant extract and the a orded compounds have no cytotoxicity (Table 7).