Antioxidant and Cytotoxic Flavonols from Calotropis Procera Mona A
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Antioxidant and Cytotoxic Flavonols from Calotropis procera Mona A. Mohameda,*, Manal M. Hameda, Wafaa S. Ahmedb, and Allia M. Abdoua a Medicinal Chemistry Department, Theodor Bilharz Research Institute (TBRI), P. O. B. 12411, Giza, Egypt. Fax: +20233388801. E-mail: [email protected] b Faculty of Science and Human Studies, Shaqra University, Kingdom of Saudi Arabia * Author for correspondence and reprint requests Z. Naturforsch. 66 c, 547 – 554 (2011); received May 24, 2010/April 15, 2011 Phytochemical investigations of Calotropis procera leaves have led to the isolation of two 4 new compounds: quercetagetin-6-methyl ether 3-O-β-D- C1-galacturonopyranoside (3) and 4 (E)-3-(4-methoxyphenyl-2-O-β-D- C1-glucopyranoside)-methyl propenoate (4), along with eleven known metabolites: nine fl avonol and two cinna mic acid derivatives. All metabolites were isolated for the fi rst time from the genus Calotropis, except for 1 isolated previously from Calotropis gigantea. The structures were determined by spectroscopic methods (UV, ESI-MS, 1H, 13C NMR, 1H-1H COSY, HSQC, and HMBC). The radical scavenging activity of the aqueous methanol extract and compounds 8 – 13 was measured by the 1,1-diphenyl- 2-picrylhydrazyl (DPPH) method. Cytotoxic screening of the same compounds was carried out on brine shrimps as well. Key words: Calotropis procera, Flavonols, Antioxidant Introduction 1999, 2001) activities. The milky white latex of this plant has been reported to exhibit potent Calotropis procera (Ait.) R. Br. is a xerophytic anti-infl ammatory and analgesic, and weak anti- shrub belonging to the Asclepiadaceae family and pyretic activities in various experimental models commonly known as Arka in India. It is widely (Kumar and Arya, 2006; Kumar and Basu, 1994; distributed in Asia, Africa, South America, and Dewan et al., 2000a, b). The latex also inhibits abundant in the northeast of Brazil. Asclepiada- the infl ammatory response elicited by various in- ceous plants are mainly either tropical or sub- fl ammation mediators (Arya and Kumar, 2005). tropical (South America and South Africa) with Besides, it has also been demonstrated to possess only a few species growing in temperate regions antioxidant, antihyperglycemic, and hepatopro- (Heywoo, 1978). C. procera is known to possess tective properties (Roy et al., 2005; Padhy et al., multifarious medicinal properties. It has been 2007). Recently, the aqueous extract of the latex used for the treatment of leprosy, ulcers, tumours, piles, and diseases of spleen, liver and abdomen has been shown to inhibit cellular infi ltration and (Kirtikar and Basu, 1935). It also has insecticidal afford protection against the development of neo- (Mours, 1997), larvicidal (Markouk et al., 2000), plastic changes in the transgenic mouse model of antibacterial, and antiparasitic activities (Larhsini hepatocellular carcinoma (Choedon et al., 2006), et al., 1999). The root of C. procera is used as a in addition to the application as an antidote for carminative in the treatment of dyspepsia (Kumar snake poisoning (Nandkarni, 1976). and Arya, 2006). Further, the root bark and leaves The plant has been investigated phytochemical- of C. procera are used by various tribes of central ly for cardenolides, anthocyanins, tannins, sterol, India as a curative agent for jaundice (Samvatsar hydrocarbons, a steroid, and triterpenoids (Mas- and Diwanji, 2000). The chloroform extract of the colo et al., 1988; Abdul Qasim and Abdul Malik, root also has been shown to provide protection 1989). The wide and diverse biological activit-ies against carbon tetrachloride-induced liver dam- of C. procera stimulated us to isolate and cha rac- age (Basu et al., 1992). An ethanolic extract of the terize polyphenols from the aqueous methanol fl owers of C. procera is reported to have antimi- extract (AME) of C. procera leaves. The antioxi- crobial, anti-infl ammatory, antipyretic, analgesic dant capacity of the extract and the major iso- (Mascolo et al., 1988), anticancerogenic (Smit et lated compounds 8 – 13 as well as their cytotoxic al., 1995), and antimalarial (Sharma and Sharma, activity towards brine shrimps have been studied. © 2011 Verlag der Zeitschrift für Naturforschung, Tübingen · http://znaturforsch.com 548 M. A. Mohamed et al. · Calotropis procera Material and Methods Antioxidant activity This activity was investigated in vitro for the Equipment aqueous methanol extract (AME) and com- The NMR spectra were recorded at 300, 400 pounds 8 – 13 using 1,1-diphenyl-2-picrylhydrazyl (1H) and 75, 100 (13C) MHz, respectively, on a (DPPH) free radicals, according to the method Varian Mercury 300 (Palo Alto, CA, USA) and of Peiwu et al. (1999). In a disposable cuvette a JEOL GX-400 NMR spectrometer (Akishima, methanolic solution of DPPH (2.95 ml, 100 μM) Japan), and δ values are reported in ppm relative was added to a 50-μl sample of AME or the re- to TMS in the convenient solvent. ESI-MS analy- spective compounds (in MeOH) in different con- ses were measured on a Finnigan LCQ deca LC/ centrations (2 – 100 mg ml–1 for quercetin, and MS and double focusing sector fi eld MAT 90 MS 20 – 100 mg ml–1 for AME and compounds 9 – 13). spectrometer (Finnigan, Bremen, Germany). UV The absorbance was measured at 517 nm at regu- spectra of pure samples were recorded, separate- lar intervals of 15 s for 5 min as described by Go- ly, in MeOH using different diagnostic UV shift vindarajan et al. (2003). Ascorbic acid was used reagents using a Shimadzu UV 240 spectropho- as standard (0.1 M). The percentage inhibition was tometer (Kyoto, Japan). For column chromatog- calculated according to: raphy (CC), Sephadex LH-20 (Pharmacia, Upp- % inhibition (reactive reaction rate) = sala, Sweden), microcrystalline cellulose (Merck, Darmstadt, Germany), polyamide 6S (Riedel de Abs.(DPPHsolution) · Abs.(sample) · 100 . Haën AG, Seelze, Germany), and silica gel pow- Abs.(DPPHsolution) der (G 60 E; Merck) were used. For paper chro- matography Whatman No. 1 sheets (Maidstone, Extraction and isolation England) were used. The air-dried powdered leaves of C. procera (750 g) were exhaustively extracted with 85% Plant material MeOH (5 x 1.5 l and 2 x 2.5 l, respectively), under Leaves of Calotropis procera were collected refl ux (70 °C). The AME was concentrated under in spring from fi elds near Cairo (Desert), Egypt. reduced pressure and defatted with light petrole- The plant was authenticated by Wafaa M. Amer, um ether (60 – 80 °C, 5 x 1 l) to give a dark brown Professor of Taxonomy, Department of Botany, viscous extract which was dissolved in water, and Faculty of Science, Cairo University, Giza, Egypt. the water-insoluble residue was removed by fi l- Voucher specimens (Reg. No. C–II) are kept in tration. The water-soluble portion was desalted the herbarium, Medicinal Chemistry Department, by precipitation with excess MeOH to give a dry Theodor Bilharz Research Institute, Giza, Egypt. brown residue (60 g) that was suspended in H2O and fractionated on a polyamide column (110 cm x 6 cm, 300 g) using a stepwise gradient from Brine shrimp lethality bioassay H2O, H2O/MeOH mixtures up to pure MeOH for Eggs of Artemia salina were allowed to hatch elution. Based on comparative paper chromatog- into their larvae (Fatope et al., 1993). Compounds raphy (Co-PC) with the use of UV light, 1% FeCl3, 8 – 13 were separately dissolved in distilled water or Naturstoff spray reagent for detection, the in- to give four assay concentrations (1000, 500, 100, dividual 75 fractions (each 1 l) were pooled into 5 –1 and 10 μg ml ); solubility was aided by Tween 80. collective fractions (A – E). Fraction A (H2O, 14 g) Each dose was examined in triplicate. Potassium was found to be a dark brown material with no dichromate was used as a reference drug and dis- phenolic character. Fraction B (10 – 40% MeOH, solved in sea water, to obtain concentrations of 11 g) was fractionated on cellulose C with 40% 1000, 100, and 10 μg ml–1. Assays were performed EtOH as an eluent, followed by a Sephadex LH- in test tubes with ten larvae each, and the fi nal 20 column using BIW (n-BuOH/2-propanol/H2O, volumes were adjusted to 5 ml sea water imme- 4:1:5 v/v/v, organic layer) to afford pure 1 (47 mg) diately after adding the shrimps. After 24 h, the and 2 (42 mg). Fraction C (40 – 60% MeOH, 10 g) number of surviving shrimps was recorded for was subjected to repeated CC on cellulose and Se- each dose. The LC50 values were calculated by the phadex LH-20 with 20 – 60% aqueous MeOH as use of the Instate computer program. an eluent, resulting in pure samples of 3 (32 mg) M. A. Mohamed et al. · Calotropis procera 549 and 4 (39 mg). Fraction D (70%, 11 g) was chro- 149.02 (C-4’), 146.17 (C-3’), 134.45 (C-3), 132.18 matographed on Sephadex with MeOH to give (C-6), 121.43 (C-1’), 121.06 (C-5’), 118.65 (C-6’), two major subfractions. With the use of UV light 115.97 (C-2’), 104.14 (C-10), 103.72 (C- 1”), 94.32 (365 nm) two major blue spots were detected in (C-8), 77.09 (C-5”), 74.08 (C-3”), 74.61 (C-2’’), the fi rst one which was applied to cellulose and 72.27 (C-2’’), 60.34 (CH3-6). – Negative ESI-MS: eluted with 30 – 50% aqueous MeOH followed m/z = 507.07 [M – H]–, 331.04 [M – H – galactu- by Sephadex LH-20 chromatography with EtOH ronic acid]–, 317.02 [M – H – galacturonic acid – – – to give pure 5 (10 mg) and 6 (15 mg). Repeated CH3] , 301.03 [M – H – glacturonic acid – OCH3] . CC of the second subfraction on Sephadex with 4 (E)-3-(4-Methoxyphenyl-2-O-β-D- C1-gluco- EtOH, afforded a pure sample of 7 (19 mg).