A new acylated flavonol from the aerial parts of Asteriscus maritimus (L.) Less (Asteraceae)

Abstract Phytochemical investigation of the flowering aerial parts of Asteriscus maritimus (L.) Less (Asteraceae) led to the isolation of a new compound: patuletin 7-O-β-D-[(2″′S) 6″(3″′-hydroxy-2″′-methyl-propanoyl)] glucopyranoside, together with five known metabolites; β-sitosterol 2, chlorogenic acid 3, P-hydroxy -methylbenzoate 4, luteolin 5 and protocatechuic acid 6. The structures of the isolated compounds were determined by comprehensive analyses of its 1D and 2D NMR, HRMS and compared with previously known analogues. The ethanolic extract of the flowering aerial parts of A. maritimus was found to be safe (LD50 = 4.6 mg/kg) and possess significant antioxidant and anti-inflammatory activities and this was in accordance with its high phenolic content (107.36 ± 0.051 mg GAE/g extract). Graphical abstract Patuletin 7-O- β-D-[(2ˋˋˋS) 6ˋˋ(3ˋˋˋ-hydroxy-2ˋˋˋ-methyl-propanoyl)]glucopyranosideAsterisc

. Previous phytochemical analysis of certain Asteriscus species led to the isolation of flavonoids (Ahmed et al. 1991;Youssef et al. 1995), bisabolone hydroperoxides (Sarg et al. 1994), farnesol and thymol derivatives (Ahmed 1992). Sulphur-containing metabolites, asterisulphoxide, and asterisulphone were isolated from the root of A. maritimus (Medimagh-Saidana et al. 2014). Few studies focused their attention on the composition of the oil prepared from other species (Aboutabl & Hammerschmid 1989;znini et al. 2011), while the essential oil of A. maritimus was investigated (Fraternale et al. 2001). It was found that other Asteriscus species possess a wide spectrum of biological activities, such as, molluscicidal cercaricidal (Shabana et al. 1988(Shabana et al. ), antimicrobial (zaki et al. 1984Sarg et al. 1994) and insecticidal (Pascual-Villalobos and Robledo 1999;Fraternale et al. 2001) activities. The present study deals with the isolation and identification of a new acylated flavonol in addition to five known compounds that were isolated for the first time from the aerial part. Also, the acute toxicity, antioxidant and anti-inflammatory effects of the ethanolic extract were investigated.

Results and discussion
Phytochemical investigation of the flowering aerial parts of A. maritimus afforded a new flavonoid 1 in addition to five known metabolites ( Figure 1). The structures of the known compounds were established by comparing their UV, 1 H and 13 C NMR spectroscopic data with those in the literature review and confirmed through co-chromatography with authentic samples. They were identified as β-sitosterol 2 (Goad & Akihisa 1997), chlorogenic acid 3 (Sook et al. 2010), P-hydroxy-methylbenzoate 4 (Pouchert 1993;Pouchert & Behnke 1993), luteolin 5 (Mabry et al. 1970) and protocatechuic acid 6 (Lee et al. 2010).
Compound 1 was obtained as a yellow microcrystalline powder showing chromatographic properties (yellow spot under UV light) . The UV spectrum in MeOH exhibited characteristic absorbance bands of flavonol at 258 and 371 nm. The band at 371 nm was shifted + 58 nm by AlCl 3 /HCl and + 18 nm by NaOAc/H 3 BO 3 . These results suggested that compound 1 was a flavonol with a hydroxyl group at C-5 and an ortho-dihydroxyl group in the B ring (Mabry et al. 1970;Harborne et al. 1975). Its molecular formula was established as C 26 H 27 O 15 from its HRESI-MS at m/z 579.1346. The 1 H NMR spectrum of 1 (DMSO-d 6 ) showed aromatic signals at δ 7.73 (d, J = 2.1, H-2′), 7.53 (dd, J = 8.4, 2.1 Hz, H-6′), 6.9 (d, J = 8.4 Hz, H-5′) and 6.89 (s, H-8). Also, 1 H NMR showed one signal anomeric proten, a hexose anomeric proton resonance at δ 5.17 (d, J = 8 Hz, H-1″) indicating the presence of sugar with β-configuration. The β-D-configuration was deduced from specific optical rotation [ ] 20 D + 69.4º. 13 C DEPT experiments showed 2 methyl groups, 2 methylene group, 10 methine and 12 quaternary carbons together with molecular formula C 26 H 27 O 15 . Accordingly, the 1 H NMR spectrum of 1 displayed signals for one secondary methyl at δ 0.94 (d, J = 6 Hz, H-4″′), one methine at δ 2.48 (m, H-2″′) and one oxygenated methylene at δ 3.41 (d, J = 6.3 Hz, H-3″′a,b). These data were in accordance with published values for 3-hydroxy-2-methylpropanoyl esters (Materska et al. 2003). The 13 C NMR spectrum showed two singlets at δ 176.68 and 174.70 assigned to the carbonyls carbons. A comparison of 13 C NMR spectra with the literature review data (Roitman & James 1985) points to its great similarity to patuletin 7-O-β-glucopyranoside. However, there were some differences in compound 1 additional resonance was found in 13 C NMR spectrum that pointed to the presence of four carbon atoms and a 1 H NMR analysis confirmed of a 2-methyl-3-hydroxyl propanoyl substituent. A downfield shift at C-6″ (δ 63.90) of the glucopyranosyl residue in the 13 C NMR spectrum proved that this substituent was located at C-6 ( Charia et al. 1977). The application of HMQC and HMBC experiments led to full assignments of the 1 H and 13 C NMR chemical shifts of compound 1. HSQC experiments which correlated all proton resonances with those of each corresponding carbon. In the HMBC spectrum, the pattern of 1 H-13 C correlation was observed between δ H 5.17 (H-1″) with δ C 156.6 (C-7) indicating a glucosyl moiety is attached to C-7. The HMBC and HSQC data confirmed the location of the propinyl unit at C-6″ through the observed correlations among H-6″ (δ 3.96, 4.44) and C-1″′ (δ 174.7). The substitution at C-2″′ by methyl and methine groups was confirmed by the long-range correlations between the protons of these groups and carbonyl group (δ 174.7). The positive cotton effect observable in the circular dichroism (CD) spectrum indicated that the absolute configuration at the 2″′-position of compound 1 was S. Based on the above data, compound 1 is deduced to be patuletin 7-O-β-D-[(2″′S) 6″(3″′-hydroxy-2″′-methyl-propanoyl)] glucopyranoside. To the best of the authors' knowledge, this is the first report of isolation of this compound from any natural source. The phenolic content of the ethanolic extracts of the flowering aerial parts of A. maritimus was 107.36 ± 0.051 mg GAE/g extract.
The ethanolic extract of the flowering aerial parts of A. maritimus was safe up to 4.6 mg/kg b. wt. The ethanolic extract exhibited acute anti-inflammatory activity at the tested doses represented by a significant decrease in the weight of the oedema comparing its activity to that of indomethacin (Table 1). This could be attributed to the presence of sterol compound (β-sitosterol). These data are in agreement with those reported of β-sitosterol (Loizou et al. 2010).
It is well known that there is a strong relationship between total phenol content and the antioxidant activity, as phenols possess strong scavenging ability for free radicals due to their hydroxyl groups. Therefore, the phenolic content of plant may directly contribute to their antioxidant action (Bendini et al. 2006;Dlugosz et al. 2006;Wojdylo et al. 2007). The ethanolic extracts exhibited pronounced antioxidant activity at a concentration of 100 mg/kg b.wt. (Table 2). Phenolic compounds are also believed to have chemopreventive and suppressive activities against cancer cells by the inhibition of the metabolic enzymes involved in the activation of potential carcinogens or arresting the cell cycle (Newman et al. 2002). Nevertheless, a compound with strong antioxidant potential can also contribute to DNA protection and prevent apoptosis (Rajkumar et al. 2011). Further studies are therefore required to detect potential anticancer activities of the extract reported here.

Plant material
The flowering aerial parts of A. maritimus were collected during the spring 2010 from the Experimental Station of Medicinal Plants, Faculty of Pharmacy, Cairo University, Giza, Egypt. The plant was authenticated by Dr M. Gibali (Senior Botanist). A voucher specimen was deposited at the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt (30-5-2013-1).

Extraction, fractionation and isolation
The air-dried flowering aerial parts of A. maritimus (1.5 kg) were powdered, then extracted with ethanol 95% by cold maceration till exhaustion. The ethanol extract was evaporated under reduced pressure to yield 172 g dried extract. The dried residue was suspended in water (250 mL) and partitioned successively between petroleum ether (6 × 500 mL), chloroform (6 × 500 mL), ethyl acetate (6 × 500 mL) and n-butanol saturated with water (4 × 500 mL). The petroleum ether, chloroform, ethyl acetate and n-butanol fractions were evaporated to yield 20.8, 6.3, 6.2, and 16 g, respectively.
The petroleum ether (20 g) was chromatographed over a VLC column (6 × 20 cm, silica gel H, 250 g). Gradient elution was carried out using n-hexane/chloroform mixtures and chloroform/ethyl acetate mixtures. Fractions of 200 mL each were collected and monitored by TLC using the solvent systems (S 1 -S 2 ). Fraction (20% ethyl acetate/chloroform) was rechromatographed over a silica gel 60 column, using n-hexane-ethyl acetate (9.8: 0.2 v/v) as eluent, to give compound 2 (50 mg, white needle crystals, R f = 0.57 in S 2 ). Ethyl acetate fraction (6 g) was loaded to a diaion HP-20 AG (250 g, 5 × 120 cm) packed in water. Elution was carried out with water, followed by methanol/water (1:1) and methanol (100%) to give three fractions (A-C), which were monitored by TLC, using the solvent systems (S 3 -S 5 ). The chromatograms were examined under UV light at 365 nm and 254 nm before and after exposure to ammonia vapor. Fraction A was purified on Sephadex LH-20 column and eluted with methanol/water (1:1 v/v) to give compound 3 (45 mg, white amorphous powder, R f = 0.36 in S 4 ,) . Fraction B was purified on silica column and eluted with chloroform/ methanol (9.9:0.1 v/v) to give compound 4 (16 mg, white needle crystals, R f = 0.52 in S 5 ). Fraction C was rechromatographed on silica column using chloroform/methanol (9.8:0.2 v/v) as an eluant to give two subfractions. The first subfraction was further purified on a Sephadex LH-20 column using 50% methanol in water as eluent to give two compounds 5 (20 mg, yellow powder, R f = 0.77 in S 5 ) and 6 (12 mg, white needles, R f = 0.53 in S 5 ). The second subfraction was purified on a RP column using methanol-water (4:6) as eluent to give compound 1 (35 mg, yellow powder, R f = 0.42 in S 6 ).

Total phenolics content
The concentration of total phenolics of the plant extract and fractions was determined according to the method described by (Kumar et al. 2008). Gallic acid was used as standard. Briefly, a mixture of 100 μL of plant extract (100 μg mL −1 ), 500 μL of Folin/Ciocalteu reagent and 1.5 mL of Na 2 CO 3 (20%) was shaken and diluted up to 10 mL with water. After 2 h, the absorbance was measured at 765 nm (using a spectrophotometer. All determinations were carried out in triplicate. The total phenolic concentration was expressed as gallic acid equivalents.

Animals
Animals were obtained from the animal house colony, supplied by central services of the Laboratory National Research Center, Giza, Egypt. They were kept on standard laboratory diet under hygienic conditions. This study was conducted in accordance with ethical procedures and policies approved by Animal Care and Use Committee of Faculty of Pharmacy, Cairo University which follows the World Medical Association Declaration of Helsinki (WMA General Assembly 1964).

Determination of LD 50
LD 50 of the ethanol extract of the flowering aerial parts of A. maritimus was performed by oral treatment of male albino mice (25-30 g) adopting Karber's procedure (1931). Preliminary experiments were carried out to determine the minimal dose that kills all animals (LD 100 ) and the maximal dose that fails to kill any animal. Several doses at equal logarithmic intervals were selected in between these two doses; each dose was injected in a group of six animals by subcutaneous injection. The mice were observed for 24 h and symptoms of toxicity and mortality rates in each group were recorded and calculated.

In vivo anti-inflammatory activity
The anti-inflammatory activity of the extract was determined, in vivo, by adopting the carrageenan-induced oedema in the hind paws of rats as described by (Winter et al. 1962). Eighteen male albino rats, weighing 130-150 g, were divided into three groups (each of six), and orally treated one hour before induction of oedema. Group 1 receiving saline and served as negative control. Group 2 was administered the total ethanol extract, at a dose of 100 mg/kg b.wt, Group 3 received indomethacin, as standard anti-inflammatory drug (20 mg/kg b.wt). Induction of oedema was performed by subplanter injection of 0.1 mL of 1% Carrageenan (Penn & Ashford 1963), in saline into the pad of experimental animal right hind paw and 0.1 mL saline in its left hind paw. Four hours after drugs administration, the rats were sacrificed. Both hind paws were excised and weighed separately; the difference in weight between both represents the weight of the oedema.

In vivo antioxidant activity
Antioxidant activity was assessed by measuring the ability of the extract to restore glutathione levels in the blood of alloxan-induced diabetic rats after the oral administration of 100 mg/kg body weight (Beutler et al. 1963). Induction of diabetes mellitus was carried out according to the method described by Eliasson and Samet (1969). Vitamin E was used as a standard (7.5 mg/kg b.wt., positive control).