134 Journal of Pharmacy and Nutrition Sciences, 2011, 1, 134-139 Flavonoids of Neotorularia aculeolata Plant Fatma A. Ahmed1, Inas M. Abd El–Wahab Khamis1 and Samar Y. Desoukey2,* 1 Medicinal and Aromatic Plant Dept., Desert Research Center, El–Matariya, Cairo, Egypt 2 Pharmacognosy Dept., Faculty of Pharmaceutical Sciences, Future University, New Cairo, Egypt Abstract: Neotorularia aculeolata belongs to the family Cruciferae that has several uses in the Egyptian folk medicine for many years. Nothing could be traced about the chemical composition of the plant. Extraction, isolation and purification of the air-dried plant material using different chromatographic techniques (PC, TLC & CC) provided seven 1 flavonoids. Identification of the isolated compounds using different chemical and physical techniques (UV, H-NMR and 13 C NMR spectroscopy) allowed to characterize these compounds as kaempferol, kaempferol-7-O-rhamnoglucoside {Kaempferol-7-neohesperidoside}, quercetin, rutin, quercetin-3-O- β-D-glucoside-7-O-α-L-rhamnoside-3`-methylether, quercetin-3,7-di-O-α-L-rhamnoside-3`-methylether and myricetin. Keywords: Neotorularia aculeolata, Cruciferae, kaempferol, quercetin, rutin, myricetin. 1. INTRODUCTION luteolin and apigenin and their derivatives were isolated [6-8]. Cruciferae (Brassicaceae) is one of the largest families in the plant kingdom that is rich in medicinal plants. It comprises approximately 380 genera and about 3350 species in 10 poorly defined tribes [1]. The family is represented in Egypt by 53 genera and 107 species mostly annual, biennial or perennial herbs. N. aculeolata (Boiss.) Hedge & J. Leonard is one of these annual plants in this family. It grows at Sinai proper; it always grows at the entire Sinai Peninsula including the coastal Mediterranean strip and El–Tih Desert east of Suez Canal, rock crevices and hillsides [2]. It is widely spread at Abo Egaila – El Qusayema road (North Sinai) from where it was collected for this study. Cruciferous plants have been used since ages and are grown as vegetables, sources of oils and as condiments. They are known for their stimulant, diuretic, thermogenic, depurative, rubefacient, galactogogue, emmenagogue, tonic, aphrodisiac, ophthalmic activities and are used for scurvy, peptic ulcers, hepatopathy, splenomegaly, dyspepsia, diarrhea, dysentery, lumbago, syphilis, leucorrhoea, seminal weakness, asthma, cough, hiccough, tenesmus, hemorrhoids as well as anticancer activity especially as androgen receptor antagonist in human prostate cancer [3, 4]. Cruciferous plants are inducers of microsomal cytochrome P450 enzyme [5]. It was reported that kaempferol, quercetin and isorhamnetin glycosides, in addition to myricetin, *Address corresponding to this author at the Pharmacognosy Dept., Faculty of Pharmaceutical Sciences, Future University, New Cairo, Egypt; Tel: 00202 22402046; E-mail: dr_samar_yehia@yahoo.com ISSN: 2223-3806 / E-ISSN: 1927-5951/11 The presence of phenolic acids as: caffeic, ferulic, P–coumaric and vanillic acids beside the presence of the previous flavonoids and rutin were also isolated from Brassica alba, B. oleraceae, B. campestris and other cruciferous species [9-14]. 2. RESULTS AND DISCUSIONS 2.1. Isolated Flavonoids Seven flavonoids were isolated, purified by CC, PC and TLC and identified through Rf-values, UV spectra in methanol with different shift reagents (Table 1) and 1 13 H & C-NMR. These compounds were coded as A1A7. 2.1.1. Compound A1 This compound was obtained as yellow crystals, soluble in methanol, Rf-values of 0.85 in BAW and 0.4 in acetic acid 15%. It showed two major absorption bands in MeOH; band I at 367nm and band II at 268nm, which indicated a flavonol nucleus with free hydroxyl group at the C-3 [15, 16]. Addition of sodium methoxide resulted in a bathochromic shift in band I (+49 nm), which proved the presence of a free OH\ group at C-4 . A bathochromic shift in band I (+53nm) with aluminum chloride, which was not affected by the addition of hydrochloric acid, indicating the presence of free hydroxyl group at C-3 and C-5. A bathochromic shift in band II (+7 nm) with sodium acetate indicated the presence of free hydroxyl group at C-7. Addition of H3BO3 gave no shift, which proved the absence of any catecholic hydroxyl group. From the UV analysis, compound A1 is probably kaempferol. The identity of © 2011 Lifescience Global Flavonoids of Neotorularia Aculeolata Plant Table 1: Journal of Pharmacy and Nutrition Sciences, 2011 Vol. 1, No. 2 135 UV Spectral Data of the Isolated Compounds UV Data AcONa/ H3BO 3 AcONa AlCl3/ HCl AlCl3 MeONa MeOH 274, 296 (sh), 320 (sh), 372 275, 302 (sh), 385 266, 305 (sh), 350, 420 266, 305 (sh), 350, 420 280, 318 (sh), 416 253 (sh), 268, 324 (sh), 367 A1 260, 325, (sh), 370 266, 323, 385, 418 (sh), 244 (sh), 258, 266, 300 (sh), 350, 422 259 (sh), 266, 299 (sh), 353, 424 245, 267, 335 (sh), 425 253, 266, 323, 354 A2 264, 292 (sh), 384 274, 320 (sh), 428 268, 300, (sh), 362 (sh), 428 272, 328, 445 262, 332, 440 267, 306 (sh), 370 A3 220, 298, 387 271, 325, 393 271, 300, 340 (sh), 402 275, 303, (sh), 433 272, 327, 410 259, 266 (sh), 299 (sh), 350 A4 262, 360 260, 360 270, 310, 350, 410 270, 310, 350, 410 270, 398 275, 355 A5 256, 310, 360, 400 256, 310, 360, 400 256, 360 256, 360 270, 410 256, 350 A6 258, 304 (sh), 382 269, 335 266, 275 (sh), 308 (sh), 366, 428 271, 316 (sh), 450 262 (sh), 285 (sh), 322, 423 254, 272, (sh),, 374 A7 R OH R3 O R1 R2 OH Comp. Name A1 Kaempferol O R R1 R2 R3 H H OH OH OH Oglrh A2 Kaempferol-7-O-neohesperidoside H H A3 Quercitin OH H OH OH A4 Rutin OH H Oglrh OH A5 Quercitin-3-O-β-D-gluc-7-α-L-rham-3`-methylether OMe H Ogl Orh A6 Quercitin-3,7di-O-α-L-rham-3`-methylether OMe H Orh Orh A7 Myrecitin OH OH OH OH Figure 1: Structures of compounds from Neotorularia aculeolata. compound A1 was further confirmed as kaempferol by 1 H-NMR spectrum in DMSO-d6, which showed signals at δ (ppm) 8.0 (2H, d, J= 8 Hz, H-2` and H-6`), 6.9 (2H, d, J= 8Hz, H-3` and H-5`), 6.4 (1H, d, J= 1.5 Hz, H-8), and 6.2 (1H, d, J= 1.5 Hz, H-6). Thus, from the above data and current literature this compound A1 is identified as kaempferol [17]. 2.1.2. Compound A2 This compound was obtained as dull yellow crystals, soluble in methanol, Rf-values 0.3 in BAW and 0.35 in acetic acid 15%. It showed two major absorption bands in MeOH; the absorption maximal in methanol, band I (354nm) indicated that it was a flavonol with a 3-OH free. The addition of NaOMe caused a bathochromic shift in both band I and II, a fact which proved the presence of a free OH at 4` position. After addition of AlCl3, a bathochromic shift proved the presence of a free OH at 5 positions. However, on addition of HCl, no change was observed indicating the absence of any catecholic hydroxyl groups. Meanwhile the addition of NaOAc caused no shift in band II thus suggesting the occupation of 7-position. Addition of H3BO3 caused no shift, this suggested the absence of any catecholic hydroxyl groups. 1 The H-NMR spectral data of compound A2 showed the signals characteristic for kaempferol with additional signal for the sugar moieties. Two signals for the two anomeric sugar protons at δ 5.4 (1H, d, J=2.5Hz, H-1`` 136 Journal of Pharmacy and Nutrition Sciences, 2011 Vol. 1, No. 2 glucose) and δ 5.2 (1H, d, J=2.5Hz, H-1``` rhamnose). The remaining sugar proton as m at 3.2-3.9, signal at 1.2 (3H, d, J=6Hz, CH3 rhamnose). 13 C-NMR spectrum data of compound A2 showed a ketonic carbon at 176.1 at C-4 and the most acidic carbon at C-7 at 162.4 followed by C-4` at 159.4 and C-3 at 135.9. Two anomeric sugar carbons at 98.4 and 100.5 for C-1`` and C-1```, respectively indicating the disaccharide nature of compound A2. One methyl carbon of rhamnose was shown at 20.9. In A2, C-2``` of rhamnose appeared at 70.5. Thus from the obtained 1 13 Rf-values, UV, H-NMR and C-NMR spectral data of compound A2, showed that it is identified as kaempferol-7-O-glucose (1→2)-rhamnose (Kaempferol-7-O-Neohesperidoside). 2.1.3. Compound A3 This compound was obtained as yellow crystals, soluble in methanol, Rf-values 0.73 in BAW and 0.29 in acetic acid. Compound A3 showed two major absorption bands in MeOH; band I at 370nm and band II at 267nm, which indicated a flavonol nucleus with free hydroxyl group at the 3 position [15, 16]. Addition of sodium methoxide resulted in a bathochromic shift in band I (+70nm), which proved the presence of a free \ OH-group at 4 -position. A bathochromic shift in band I (+75nm) with aluminum chloride, indicated the presence of free hydroxyl group at C-3 and C-5. The hypthochromic shift of AlCl3 spectrum in band I (-17 nm) after the addition of HCl indicated the presence of orthodihydroxy group in B-ring (3`, 4` position). A bathochromic shift in band I (+14 nm) with sodium acetate indicating the presence of free hydroxyl group at C-7, which was detected by H3BO3 addition, indicates the presence of orthodihydroxy group (3`, 4` position). Thus, from the UV analysis and Rf-values, compound A3 may be identified as quercetin. The compound A3 was further confirmed as quercetin by 1 H-NMR spectrum in DMSO-d6, which showed signals at δ (ppm) 7.7 (1H, d, J = 8.5 Hz, H-2`), δ 7.5 (1H, dd, J= 2.5, H-6`) and δ 6.8 (1H, d, J = 8.5, H= 5`), indicated the presence of aromatic ring with two substitutions, in m, p-substitution {δ 6.5 (1H, d, J = 1.5 H-6) and δ 6.2 (1H, d, J= 1.5, H-8)}. Thus, from the above mentioned data, compound A3 is Quercetin [17]. 2.1.4. Compound A4 This compound was obtained as yellow crystals, Rfvalues of 0.49 in BAW and 0.54 in acetic acid respectively. The absorption maxima in methanol, band Fatma et al. I at 350 nm, indicates that it is a flavonol with 3-OH substitution. The remaining UV spectral data were found to be similar to that of quercetin type compound. 1 H-NMR spectrum of the compound A4 in DMSOd6, showed signals at δ (ppm) 7.6 (1H, d, J = 2.5 Hz, H-2`), δ 7.5 (1H, dd, J = 8.5, 2.5 H-6`), δ 6.8 (1H, d, J = 8.5, H= 5`), δ 6.4 (1H, d, = 1.5 H-8), δ 6.2 (1H, d, J = 1.5, H-6) and for sugar moiety δ (ppm): 5.3 (1H, d, J= 8Hz, H-1`` glucose), 4.5 (1H, d, J= 2.5Hz, H-1``` rhamnose), 3.4 (m, remaining sugar protons) and 0.8 13 (3H, d, J= 6Hz, CH3 rhamnose). C-NMR of A4 gave the following peaks in DMSO-d6: δ (ppm): 146.9 (C-2), 135.5 (C-3), 175.8 (C-4), 160.7 (C-5), 98.2 (C-6), 163.9 (C-7), 93.3 (C-8), 156.2 (C-9), 103.1 (C-10), 122.1 (C1′), 115.3 (C-2′), 145.0 (C-3′), 147.6 (C-4′), 115.6 (C5′), 120.0 (C-6′), and for sugar moiety, 101.5 (C-1′′), 74.3 (C-2′′),75.9 (C-3′′), 70.2 (C-4′′), 76.2 (C-5′′), 67.4 (C-6′′), 101.2 (C-1′′′), 70.8 (C-2′′′), 71.0 (C-3′′′), 72.2 (C-4′′′), 69.1 (C-5′′′) and 18.1 (C-6′′′). Complete acid hydrolysis yielded glucose and rhamnose in the aqueous phase and quercetin in the organic phase in (a), (e) and (f) using specific spray reagents. From the above data and by comparison with published data, compound A4 is identified as Rutin (quercetin-3-O-α-Lrhamnoside (1→6)-β-D-glucoside) [17]. 2.1.5. Compound A5 This compound was obtained as yellow crystals, Rfvalues of 0.51 in BAW and 0.7 in acetic acid respectively. The absorption maxima in methanol, band I at 355 nm, indicates that it is a flavonol with 3-OH substitution. The remaining UV spectral data were found to be similar to that of quercetin type compound. 1 H-NMR spectrum of the compound A5 in DMSO-d6, showed signals at δ (ppm) 7.95 (1H, d, J= 8.5 Hz, H2′), 7.65 (1H, dd, J= 8.5 Hz, H-6′), 6.94 (1H, d, J= 8.5Hz, H-5′), 6.75 (1H, d, J= 2.5Hz, H-8), 6.45(1H, d, J=2.5 Hz, H-6), 5.6 (1H, d, J= 2.5Hz, H-1′′ rhamnose), 5.4 (1H, d, J=2.5 Hz, H -1′′′ glucose), 3.92 (s, OCH3) and 1.2 (3H, d, J=6Hz, OCH3 rhamnose). The isolated compound A5 when subjected to partial acid hydrolysis afforded quercetine-3′-methoxide and the sugars were glucose and rhamnose. On the other hand, a known amount of the compound A5 was subjected to complete acid hydrolysis using 2N HCl. It was observed that compound A5 resisted acid hydrolysis, which coincided with C-glycoside flavonoid. From the data above and by comparison with published data, compound A5 is identified as Quercetin-3-O-β-D-glucoside -7-O-α-Lrhamnoside-3`-methyether [17]. Flavonoids of Neotorularia Aculeolata Plant Journal of Pharmacy and Nutrition Sciences, 2011 Vol. 1, No. 2 137 2.1.6. Compound A6 3. SUMMERY AND CONCLUSION This compound was obtained as yellow crystals, Rfvalues 0.52 in BAW and 0.69 in acetic acid. The absorption maxima in methanol, band I at 350 nm, indicated that it was a flavonol with 3-OH substitution. The remaining UV spectral data was found to be similar 1 to that of quercetin type compound. H-NMR spectrum of the compound A6 in DMSO-d6, showed signals at δ (ppm) 7.6 (1H, d, J= 8.5 Hz, H-2′), 7.4 (1H, dd, J= 8.5 Hz, H-6′), 6.9 (1H, d, J= 8.5Hz, H-5′), 6.7 (1H, d, J= 2.5Hz, H-8), 6.4 (1H, d, J=2.5 Hz, H-6), 5.6 (1H, d, J= 2.5Hz, H-1′′ rhamnose), 5.5 (1H, d, J=2.5 Hz, H -1′′′ rhamnose), 3.9 (s, OCH3), 1.2 (3H, d, J=6Hz, OCH 3 rhamnose) and 0.8 (3H, d, J=6Hz, CH3 rhamnose). The isolated compound A6 when subjected to partial acid hydrolysis afforded quercetine-3′-methylether and the sugar was rhamnose. On other hand a known weight of the compound A6 was subjected to complete acid hydrolysis using 2N HCl, which gave the aglycone quercetine-3′-methoxide and the sugar was rhamnose. From above data and by comparison with the published data, compound A6 is identified as Quercetin-3,7-di-Oα-L-rhamnoside-3`-methylether [17]. In the present study, the defatted desalted methanolic extract of the air-dried whole plant of Neotorularia aculeolata was purified by CC, PC and TLC ,to afford seven flavonoids {A1 to A7} these compounds are identified as:- kaempferol, kaempferol7-O-rhamnoglucoside {Kaempferol-7-Neohesperidoside}, Quercetin, Rutin, Quercetin-3-O- β-D-glucoside7-O-α-L-rhamnoside-3`-methylether , Quercetin-3,7-diO-α-L-rhamnoside-3`-methylether and Myricetin. 2.1.7. Compound A7 This compound was obtained as yellow crystals, Rfvalues of 0.31 in BAW and 0.16 in acetic acid respectively. It showed two major absorption bands in MeOH; band I at 374 nm and band II at 254 nm, which indicated a flavonol nucleus with free hydroxyl group at the 3 position [15, 16]. Addition of sodium methoxide resulted in a bathochromic shift in band I (+49nm), which proved the presence of a free OH-group at C4`position. A bathochromic shift in band I (+76nm) with aluminum chloride, indicated the presence of a free hydroxyl group at C-3 and C-5. The hypthochromic shift of AlCl3 spectrum in band I (-22 nm), after the addition of HCl, indicated the presence of orthodihydroxy group in B-ring (3`and 4` position). A bathochromic shift in band II (+15 nm) with sodium acetate indicated the presence of a free hydroxyl group at C-7, which was detected by H3BO3 addition, that indicated the presence of orthodihydroxy group (3`and 4` position). Thus, from the UV analysis and Rf-values compound 1 A7 may be identified as myricetin. H-NMR spectrum of the compound A7 in DMSO-d6, showed signals at δ (ppm) 6.25 (1H, d, J= 2.5 Hz, H-6), 6.33 (1H, d, J= 2.5 Hz, H-8), 7.32 (s, H-2′ and H-6). From the above data and by comparison with published data, Compound A7 is identified as Myricetin [17]. To our knowledge this represents the first report for the isolation of these compounds from N. aculeolata. 4. EXPERIMENTAL 4.1. Material, Methods and Techniques 4.1.1. Plant Materials Neotorularia aculeolata (Boiss.) plant was collected from North Sinai (Abo Egaila–El Qusayema road) in June 2006 and identified by Prof. Dr. Nahed El-Hadidi, Botany Department, Faculty of Science, Cairo University and by comparison with herbarium specimens at the Desert Research Center {DRC}. A voucher specimen was deposited in the Pharmacognosy lab, Future University, Egypt. The plant material was ground to fine powder and kept in an amber light proof container. 4.1.2. Extraction and Purification of Flavonoids: The whole plant {2 kg} was defatted with petroleum ether (B.p. 40 -60°C): ether (1:1 v/v). The defatted powder was percolated with methyl alcohol (80%) till exhaustion to obtain a brownish alcoholic extract. This extract was concentrated by evaporation under reduced pressure at not more than 45°C to yield a sticky brownish residue (90g) which was suspended in hot distilled water, filtered and desalted. 4.1.3. Chromatographic Investigation: 4.1.3.1. PC The concentrated aqueous extract was applied on Whatmman No.1 paper chromatography using descending technique with suitable solvent systems [15]. 4.1.3.2. TLC Precoated TLC plates silica gel 60SF254 (E- Merck) 20 x 20 cm were used for investigation of the 138 Journal of Pharmacy and Nutrition Sciences, 2011 Vol. 1, No. 2 flavonoids using suitable solvent systems precoated preparative TLC silica gel F254 and • Solvent systems used for PC and TLC: a) n-butanol-acetic acid- water ( BAW) (4:1:5) organic phase. b) Acetic acid 15%. c) Ethyl acetate-methanol-water (30:5:4)and (30:2.5:2)one phase, for TLC only. d) Phenol-water(80:20) • Spray Reagent for PC and TLC Aluminum chloride reagent: 1 % aluminum chloride reagent solution in ethanol [18]. 4.1.3.3. Isolation and Purification of The Flavonoidal Compounds The desalted alcoholic extract was submitted to column chromatography and gradient elution was performed, using firstly chloroform with increasing concentration of ethyl acetate and methanol, respectively. Fractions of 50 ml were collected, then subjected to TLC using system (c) and similar fractions were pooled together. The pooled fractions were subjected to preparative TLC using system (a), the bands corresponding to the flavonoidal compounds were visualized under UV, eluted with methanol and water {1:1}. The eluted bands were purified on a Sephadex LH-20 column using methanol and water as eluting system where seven compounds could be isolated. 4.1.3.4. Identification of Flavonoids: 4.1.3.4.1. Spectral data Ultraviolet (UV) with shift reagents and nuclear I 13 magnetic resonance ( H-NMR and C-NMR). 4.1.3.4.2. Acid Hydrolysis • Partial and Complete Acid Hydrolysis 5 mg of each compound were dissolved in 5 ml methanol and an equal volume of 0.1N and 2N HCl aqueous solution {each separately} was added for mild and complete acid hydrolysis, respectively. The mixture was refluxed on a boiling water bath for 3 hours. The methanol was evaporated and the aglycone was extracted with ether. The aqueous layer containing the Fatma et al. sugar part was evaporated to dryness and the residue was dissolved in 10% isopropanol solution. • Identification of the Aglycone: The ether extract of each compound containing the aglycone moiety was applied on paper chromatogram along side with authentic flavonoid aglycones using the solvent systems (a) and (b), visualized under UV, exposed to ammonia vapor and sprayed with AlCl3. • Identification of the Sugars: The isopropanol solution of each compound containing the sugar moiety was applied on PC along side with authentic sugars using the solvent systems (a) and (d). After development, they were visualized by spraying with aniline hydrogen phthalate reagent and heating at 110°C for 10 min.. 5. AUTHENTIC REFERENCE 5.1. Flavonoid Kaempferol ,Quercetin & myricetin were available at the medicinal and aromatic plants department, Phytochemistry unit, Desert Research Center Egypt. 5.2. Sugars Glucose and rhamnose, (Sigma). 5.3. 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