A New Acylated Flavonol from the Aerial Parts of Asteriscus Maritimus (L.) Less (Asteraceae)

Natural Product Research Formerly Natural Product Letters

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

Marwa I. Ezzat, Shahira M. Ezzat, Kadriya S. El Deeb, Ahlam M. El Fishawy & Sayed A. El-Toumy

To cite this article: Marwa I. Ezzat, Shahira M. Ezzat, Kadriya S. El Deeb, Ahlam M. El Fishawy & Sayed A. El-Toumy (2016): A new acylated flavonol from the aerial parts of Asteriscus maritimus (L.) Less (Asteraceae), Natural Product Research, DOI: 10.1080/14786419.2016.1138298

To link to this article: http://dx.doi.org/10.1080/14786419.2016.1138298

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Download by: [Ahlam Elfishawy] Date: 03 February 2016, At: 17:10 Natural Product Research, 2016 http://dx.doi.org/10.1080/14786419.2016.1138298

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

Marwa I. Ezzata, Shahira M. Ezzata, Kadriya S. El Deeba, Ahlam M. El Fishawya and Sayed A. El-Toumyb

aFaculty of Pharmacy, Pharmacognosy Department, Cairo University, Cairo, Egypt; bChemistry of Tannins Department, National Research Centre, Cairo, Egypt

ABSTRACT ARTICLE HISTORY Phytochemical investigation of the flowering aerial parts of Received 16 February 2015 Asteriscus maritimus (L.) Less (Asteraceae) led to the isolation of a Accepted 14 December 2015 new compound: patuletin 7-O-β-D-[(2″′S) 6″(3″′-hydroxy-2″′-methyl- KEYWORDS propanoyl)] glucopyranoside, together with five known metabolites; Asteriscus maritimus; β-sitosterol 2, chlorogenic acid 3, P-hydroxy -methylbenzoate 4, acylated flavonol; luteolin 5 and protocatechuic acid 6. The structures of the isolated antioxidant; anti- compounds were determined by comprehensive analyses of its 1D inflammatory 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). Downloaded by [Ahlam Elfishawy] at 17:10 03 February 2016

1. Introduction

Over the past 20 years, considerable attention has been given to Asteriscus genus in which mainly sesquiterpenes, as asteriscunolides and aquatolide were isolated (San Feliciano

al. 1984, 1989; El-Dahmy et al. 1985; Jakupovic et al. 1987; Rauter et al. 2001). 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), antimicrobial (Zaki et al. 1984; Sarg 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.

2. 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, 1H and 13C 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 AlCl3/HCl and + 18 nm by NaOAc/H3BO3. 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 C26H27O15 from its 1 HRESI-MS at m/z 579.1346. The H NMR spectrum of 1 (DMSO-d6) 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, 1H NMR showed one signal anomeric proten, a hexose anomeric proton resonance at δ 5.17 Downloaded by [Ahlam Elfishawy] at 17:10 03 February 2016 (d, J = 8 Hz, H-1″) indicating the presence of sugar with β-configuration. The β-D-configuration 20 13 was deduced from specific optical rotation[ ]D + 69.4º. C DEPT experiments showed 2 methyl groups, 2 methylene group, 10 methine and 12 quaternary carbons together with molecular 1 formula C26H27O15. Accordingly, the H NMR spectrum of 1 displayed signals for one second- ary 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 13C NMR spectrum showed two singlets at δ 176.68 and 174.70 assigned to the carbonyls carbons. A comparison of 13C 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 13C NMR spectrum that pointed to the presence of four carbon atoms and a 1H 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 13C NMR Natural Product Research 3

4'''

3''' 2''' OH OH O 1''' 3' OH O 2' 4'

6'' O 1 H H O 8 1' O 5' 1'' 2 5'' 4'' 7 9 6' 2'' OH H H 10 3 HO 3'' HO 6 4 H CO H 3 5 OH

OH O

HO OH

OH O

HO HO 3 2 OH OH O O OH OCH3 OH

HO O

OH O OH OH 4 5 6

Downloaded by [Ahlam Elfishawy] at 17:10 03 February 2016 Figure 1. Chemical structure of the compounds isolated from A. maritimus.

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 1H and 13C 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 1H–13C 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 observ- able 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. 4 M. I. Ezzat et al.

Table 1. Acute anti-inflammatory activity of the ethanol extract of the flowering aerial parts of A. maritimus (L.) Less.

% Oedema Group (n = 6) Dose mg/kg b.wt. Mean ± S.E. % of Change Potencya Control 1 mL saline 59.4 ± 1.6 – – The ethanolic extract of A. maritimus 100 36.8 ± 1.3* 38 0.61 Indomethacin 20 22.6 ± 0.4* 62 1 Note: % of change calculated as regard the control group. aPotency calculated as compared to the standard anti-inflammatory drug Indomethacin. *p < 0.01 vs. control group.

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 acti- vation 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.

3. Experimental

Downloaded by [Ahlam Elfishawy] at 17:10 03 February 2016 3.1. General Silica gel H (Merck, Darmstadt, Germany) for vacuum liquid chromatography (VLC), silica gel 60 (70–230 mesh ASTM; Fluka, Steinheim, Germany), Diaion HP-20 AG for column

Table 2. Antioxidant activity of the ethanol extract of the flowering aerial parts of A. maritimus (L.) Less and vitamin E drug in male albino rats (n = 6).

Group Blood glutathione (mg %) % Change from control Control (1 mL saline) 36.3 ± 1.4 – Daibetic 21.4 ± 0.5a 41 Diabetic + vitamin E (7.5 mg/kg.b.wt.) 35.9 ± 1.2 1.1 Diabetic + the ethanolic extract 31.2 ± 1.4 14 of Asteriscus maritimus (100 mg/ kg.b.wt.) aStatistically significant different from control group at p < 0.01. Natural Product Research 5

chromatography (CC) (75–150 μm, Mitsubishi Chemical Industries Co. Ltd), Lichrprep RP-18 column, size A, 40–63 μm, 240 × 10 mm (Merck, Darmstadt, Germany) and Sephadex LH-20 (Pharmacia, Stockholm, Sweden) were used for CC. Thin-layer chromatography (TLC) was

performed on silica gel GF254 precoated plates (Fluka) and precoated plates, RP silica gel using the following solvent systems: S1, n-hexane-ethyl acetate (90:10 v/v); S2, n-hexane-ethyl acetate (80:20 v/v); S3, chloroform/methanol (95:5 v/v); S4, chloroform/methanol (90:10 v/v); S5 chloroform/methanol (80:20 v/v) and S6 methanol/water (55:45 v/v). The chromatograms were visualised under UV light (at 254 and 366 nm) before and after exposure to ammonia vapour and spraying with natural products/polyethylene glycol reagent (diphenylboryloxyethyl-amin, NP/PEG), as well as after spraying with anisaldehyde/sulfuric acid spray reagent. UV spectra were recorded using a Shimadzu UV 240 (P/N 204-58000) spectrophotometer (Kyoto, Japan). BRUKE NMR was used for 1H NMR (400 MHz) and 13C NMR (125 MHz) measurements. The NMR

spectra were recorded in CDCl3 and DMSO and chemical shifts are given in δ (ppm) relative to TMS as internal standard. Acquity UPLC system (Waters Corp., Milford, MA, USA) equipped with MicroTOF-Q hybrid quadrupole time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) and fitted with an Apollo II electrospray ion source in negative modes was used. CD spectra were obtained with a JASCO J-720 spectropolarimeter (JASCO, Tokyo, Japan). Optical rotation values were measured by the use of an ATAGO POLAX-D polarimeter. Gallic acid and Folin/Ciocalteau were purchased from Sigma-Aldrich. Indomethacin was purchased from Epico, Egyptian Int. Pharmaceutical Industries Co., glutathione kit from Wak-Chemie Medical, Germany, vitamin E from Pharco Pharmaceutical Co., Egypt, and carrageenan and alloxan were purchased from Sigma Co., USA.

3.2. Plant material The flowering aerial parts ofA. 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).

3.3. Extraction, fractionation and isolation Downloaded by [Ahlam Elfishawy] at 17:10 03 February 2016 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 (S1–S2). 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, Rf = 0.57 in S2). 6 M. I. Ezzat et al.

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

(S3–S5). 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, Rf = 0.36 in S4,). 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, Rf = 0.52 in S5). 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, Rf = 0.77 in S5) and 6 (12 mg, white needles, Rf = 0.53 in S5). The second subfraction was purified on a RP column using methanol-water (4:6) as eluent to

give compound 1 (35 mg, yellow powder, Rf = 0.42 in S6). Patuletin 7-O-β-D-[(2″′S) 6″(3″′-hydroxy-2″′-methyl-propanoyl)] glucopyranoside 1: UV

λmax nm (MeOH): 371 and 258 nm, (NaOMe): 458, 285 and 241 nm, (AlCl3): 459 and 275 nm, (AlCl3/HCl): 429 and 264 nm, (NaOAC): 394 and 266 nm, (NaOAC/H3BO3): 389 and 264 nm. 20 – 1 [ ]D + 69.4º (c 0.1, MeOH). HRMS [M–H] at m/z: 579.1346. H NMR: δ 7.73, (d, J = 2.1 Hz, H-2′), 7.57, (dd, J = 2.1 and 8.4 Hz, H-6′), 6.97 (d J = 8.4 Hz, H-5′), 6.88 (s, H-8), 5.17 (d, J = 8 Hz, 1″),

4.44 (d, J = 13.1 Hz, H-6″ a), 3.96 (m, H-6″ b), 3.8 (m, H-5″), 3.77 (s, OCH3), 3.41, (d, J = 6.3 Hz, H-3″′), 3.4 (m, H-2″), 3.4 (m, H-3″), 3.2 (m, H-4″), 2.48 (m, H-2″′), 0.94 (d, J = 6.3 Hz, H-4″′). 13C NMR: δ 148.4 (C-2), 136.1 (C-3), 176.7 (C-4), 151.5 (C-5), 132.2 (C-6), 156.6 (C-7), 94.2 (C-8), 151.9 (C-9), 105.5 (C-10), 122.3 (C-1′), 115.9 (C-2′), 145.5 (C-3′), 148.3 (C-4′), 115.2 (C-5′), 120.3

(C-6′), 60.8 (OCH3), 100.2 (C-1″), 73.5 (C-2″), 76.8 (C-3″), 70.4 (C-4″), 74.6 (C-5″), 63.9 (C-6″), 174.7 (C-1″′), 42.5 (C-2″′), 63.3 (C-3″′), 13.7 (C-4″′).

3.4. 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 Na2CO3 (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 Downloaded by [Ahlam Elfishawy] at 17:10 03 February 2016 were carried out in triplicate. The total phenolic concentration was expressed as gallic acid equivalents.

3.5. Biological activity 3.5.1 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 pro- cedures 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). Natural Product Research 7

3.5.2 Determination of LD50 LD50 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 (LD100) 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.

3.5.3 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.

3.5.4. 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).

Acknowledgements The authors would like to thank Associate Professor Dr Mohamed Farag, Pharmacognosy Department, Faculty of Pharmacy, Cairo University, for performing HRES-MS. The authors also are indebted and Downloaded by [Ahlam Elfishawy] at 17:10 03 February 2016 grateful to Professor Dr. Amani A. Sleem, Pharmacology Department, National Research Center for carrying out the pharmacological and toxicological testing of the plant extracts.

Disclosure statement No potential conflict of interest was reported by the authors.

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