Australian Journal of Basic and Applied Sciences, 4(10): 5038-5050, 2010 ISSN 1991-8178 © 2010, INSInet Publication

Glycosides of Verbascum letourneuxii, Asch. and its Antioxidant Activity
Shalabia Shahat Emam Professor Assistant of Phytochemistry Medicinal and Aromatic Plants Department, Desert Research Center El-Mataria, Cairo, Egypt.
Abstract: Five iridoid glycosides, 6-O-(α-L-rhamnopyranopranosyl)-catalpol, 6-O-(3``-O-acetyl-2``-O-ρmethoxy cinnamoyl-α-L-rhamnopyranosyl) catalpol, 6-O-(3``-O-ρ-methoxycinnamoyl-α-Lrhamnopyranopyranosyl) aucubin, 6-O-syringoyl-ajugol and harpagoside have been isolated from the methanolic extract of Verbascum letourneuxii, beside isolation of two phenylethanoide glucoside, eukovoside, martynoside, two phenylpropanoid glucoside, syringin and coniferin, from the ethyl acetate extract. Meanwhile two neolignan glucosides, dehydrodiconferyl alcohol-9-O-β-D-glucopyranoside and 4-O-methyl-dehydrodiconiferylalcohol-9`-O-β-D-glucopyranoside have been isolated from the butanol extract. These compounds were isolated, purified by column chromatography and PTLC then identified through UV, I.R., Mass, 13C and 1H-NMR spectral data. The antioxidant activity of the plant extract using diphenylpicryl hydrazyl (DPPH) radical scavenging method showed that the butanol, ethyl acetate and methanol (80%) extracts posses antioxidant activity. Key words: Verbascum species, Iridoid glycosides, Phenylethanoide glucoside, Phenylpropanoid glucoside, Neolignan glucosides and antioxidant activity. INTRODUCTION Verbascum L. is the largest genus of the family Scrophulariaceae, with about 2500 species worldwide. Verbascum species contain biologically, active compounds such as flavonoids, saponins, iridoid and alkaloids (Tatli and Akdemir, 2006). Several Verbascum species have been reported as antiseptic, astringent, demulcent, emollient, expectorant, sedative, diuretic, antimalarial and used in treatment of tumors, inflammations, migraine and spasmodic coughs (Tatli and Akdemir, 2004). Also Verbascum species are used in popular medicine for treating wounds, chilblains, respiratory ailments, acene and arthritic disturbances (Speranza et al., 2009). Guarino (2002) tested the antibacterial activity of extracts of Verbascum macrurum leaves and demonstrated that the ethanol (70%) extract was the most active one. The lyophilized infusion from flowers of Verbascum thapsiforme Schrad. (FVI) showed antiviral activity in vitro studies against Fowl plague virus, several influenza A strains, influenza B strain as well as Herpes simplex virus. Influenza viruses titer decreased by 1-3 log units, while of H. simplex virus by 2.3 log. FVI has shown virucidal activity on H. simplex virus at 300 micrograms'ml, but did not inactivate influenza viruses. Phytochemical investigations of FVI have shown the presence of flavonoids, irioids, phenolic acids, saponins, amino acids and free sugars (Zgórnik-Nowosielska et al., 1991). Also Khafagi and Dewedar (2000) have screened the hexane, ethyl acetate and ethanol extracts of Verbascum sinaiticum for antibacterial and antifungal activities and found promising antibacterial activity. Tadeg et al., (2005) reported that the hydroalcoholic extracts of Verbascum sinaiticum (Scrophulariaceae) traditionally used in the treatment of various skin disorders because it showed antimicrobial activity against different strains of bacteria and fungi which are known to cause different types of skin infections. The anti-inflammatory and antinociceptive properties of four major compounds from the flowers of Verbascum pterocalycinum var. mutense were investigated. Two saponin glycosides called ilwensisaponin A and C and iridoid glycosides known as ajugol and picroside IV were isolated from the methanolic extract (Akkol et al., 2007). A wide range of biological activities have been determined from Verbascum extracts, including antimicrobial, antioxidant, antitumor, immunomodulatory, antiulcerogenic activities, antinociceptive and
Corresponding Author: Shalabia Shahat Emam, Professor Assistant of Phytochemistry Medicinal and Aromatic Plants Department, Desert Research Center El-Mataria, Cairo, Egypt.

5038

The methanolic extract was subjected to successive chromatography on polyamide and silica gel columns. Some flavonoids. 2000). In addition to the known 6-O-{3-O-[(E)-p-coumaroyl]-α-L-rhamnopyranosyl}aucubin (2) three novel acyl iridoid diglycosides 3-5 have been isolated from the aerial parts of Verbascum nigrum. The sugar moiety was identified by comparison with authentic samples on TLC. MATERIALS AND METHODS 1. Polyamide column was eluted with water and mixtures of H2O-MeOH.) at 70°C for 3h. Vanillin ' H2SO4 Reagent: Two ml H2SO4 were added to 1 gm vanillin dissolved in 100 ml ethanol (Fried and Sherma. These structures were determined by spectral methods. anti-inflammatory. EtOAc 5039 .13C-NMR have been powerful tools for investigation the structures of natural products (Grycová et al. flavonoids and other phenolic compounds (Sarkhail et al.. 13C-NMR and MS spectra. 1996). 2001). The isolated pure compounds were subjected to mass spectrometric analysis. Sci. 1H-NMR.Aust. where the aqueous phase was evaporated under reduced pressure repeatedly by adding CH3OH-H2O (1:1) until pH 7 to afford sugar moiety. 6-O-{3-O-[(E)-isoferuloyl]-α-L-rhamnopyranosyl-}aucubin (4. Basic & Appl. Investigation of Iridoid Glycosides: Extraction and Isolation: Fresh plants of Verbascum letourneuxii were air-dried and powdered. mainly by 1D and 2D NMR spectroscopy (Magiatis et al. On further phytochemical investigation of the aerial parts of Verbascum undulatum. The aqueous solution of the methanolic extract was extracted with hexane to remove non-polar substances.5 ml) and then heated with 2M HCl (2ml. The obtained powder (1 kg) was maccrated at room temperature with 80% methanol. Ilwensisaponin A and C have antimicrobial activity. There are several isolated compounds from various Verbascum species. plants were collected from Wadi El-Arish. then 5 ml of H2O was added and the mixture was extracted with EtOAc (3x5ml).. 1H-NMR and 13C-NMR spectra were measured and reported in ppm using the residual solvent peak as an internal standaral. 2007): Pure compound (10 mg) was dissolved in MeOH (0. Phenylethanoid glycoside. nigroside V) (Vesper and Seifert.nigroside IV) and 6-O-{2-O-[(E)-isoferuloyl]-α-L-rhamnopyranosyl}aucubin (5. The received fractions were investigated by UV fluourescence and TLC analysis. Acid Hydrolysis (Yu et al. antitumor and immunostimulatory activities (Tatli and Akdemir. North Sinai during the growth season. where similar fractions were pooled together.4-dimethoxycinnamoyl)-α-L-rhamnopyranosyl] aucubin (1) and 6-O-[3-O(trans-p-methoxycinnamoyl)-α-L-rhamnopyranosyl]aucubin (2) were isolated. TLC Analysis: TLC analysis were carried out on precoated silica gel 60 F254 aluminum sheets using solvent system chloroform: methanol : water (61:32:7 v/v/v). two new acylated iridoid glycosides. samioside.1. showed scavenging properties towards the DPPH radical and antimicrobial activity against Gram-positive and negative bacteria (Kyriakopoulou et al. 2006). IR. Plant Materials: Verbascum letourneuxii. 4(10): 5038-5050. Silica gel column was used for further purification of the received fractions where elution was carried out by CHCl3-MeOH mixtures.. Biological effects of plants might be related to the presence of iriddoid glycosides.. Mass spectrometry. Asch. 2010 anticancer.. On the basis of their spectral data the structures of these iridoids have been elucidated as 6-O-{2-O-[(E)-p-coumaroyl]α-Lrhamnopyranosyl}aucubin (3. Identification: The structures of the isolated iridoid compounds were determined based on chemical investigation and spectrophotometric measurement. 2007). phenylethanoid and neolignan glycosides and saponins have antioxidative. J. aq. 2006). 2002). filtered and concentrated under reduced pressure. 1994).. Compounds were detected by using UV fluorescence and spraying with vanillin ' H2SO4 reagent followed by heating at 105°C for 1-2 min (Ersöz et al. such as saponins and iridoids which have aided in the healing of infections and were effective against ulcers in rats.. 6-O-[3-O-(trans-3. 1H.nigroside III).

1654 (c=c) cm-1. Their fractionation on polyamide column revealed the presence of five main fractions when subjected to TLC using solvent system chloroform : methanol : water (61:32:7 v/v/v). Identification of Compound (I1): TLC investigation of fraction (1) revealed the presence of one spot of iridoid glycoside (compound I1). The ethyl acetate extract was chromatographed over a sephadex LH-20 column using H2O/MeOH gradient and the obtained fractions were subjected to TLC investigation and purified on silica gel column using CHCl3/MeOH gradient. The percentage scavenging was calculated from the following equation:Ac-As % Scavenging = -----------Ac Ac: absorbance of control.. The main fractions were further purified on PTLC using (n-hexane ETOAc:acetone 90:8:2) system and the eluted bands were applied on the sephadex LH-20 column. Detection of Phenylethanoid and Phenyl Propanoid Glycosides: Phenylethanoid and phenylpropanoid glycosides were detected by UV fluorescence and spraying with AlCl3 5% (Sarkhail et al. ethyl acetate (5:1) and the obtained compound was identified by NMR data. All tests were run in duplicate and averaged. filtered.2) showed close relationship with those of catalpol.. UV spectral data. An a liquot of 0. then the solution of the sample was shaken.14. 2007). Detection of Neolignan Glucosides: Neolignan glucosides subjected to TLC. Investigation of Neolignan Glucosides: The air-dried powdered of Verbascum letourneuxii was extracted with methanol.2-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activities (Hamed et al.. Basic & Appl. 4(10): 5038-5050. As: absorbance of sample. (KBR).δppm: the 1H and 13CNMR spectrum of compound (I1) (Table 1. Mass spectrum showed a molecular ion peak at m'z 508 suggesting a molecular formula of C21 H32O14. so that the relative concentration of plant materials versus the stable radical in the cuvette was 0. IR spectrum. Each fraction was subjected to silica gel column chromatography using different ratios of chloroform ' methanol for further purification. RESULTS AND DISCUSSION Investigation of Iridoid Glycosides: The developed TLC of the iridoid glycosides of Verbascum letourneuxii revealed the presence of many spots of iridoid glycosides constituents. 2006). and methano (80%) extracts of Verbascum letourneuxii were determined using 2. Investigation of Phenylethanoid and Phenyl Propanoid Glycosides: The methanolic extract of Verbascum letourneuxii was partitioned with hexane and ethyl acetate..cm-1. 2002). evaporated under reduced pressure and than was extracted with n-butanol. showed absorption at λmax=206nm. γmax. Acid hydrolysis revealed the presence of rhamone and glucose as sugar moieties.. 1H and 13CNMR revealed the presence of C-4 non-substituted iridoid 5040 . examined under UV and spraying with 1% vanillin ' H2SO4 (Ersöz et al. showed peaks at γmax 3600 (OH). Methanol and DPPH served as control. absorbance was recorded at 517 nm. 1 H and 13CNMR spectral data. the developed chromatograms were air dried. The obtained fractions were subjected to TLC investigation. ethyl acetate. After incubation in the dark for 20 min.1 ml M methanol solution of DPPH was mixed with the methanolic solution of the sample. 2010 extract was applied on silica gel column eluted with petroleum ether. J.Aust. where similar fractions were pooled together. Sci. The n-butanol extract was fractionated on silica gel column eluting with hexane-ethyl acetate-MeOH mixtures. Antioxidant Activity: Antioxidant activity of butanol.

6. 1546. Sci. The presence of these sugar units were confirmed by 1HNMR spectrum (Table 1).dd. UV spectral data. The 13CNMR spectrum showed four aromatic carbon signals at δ 108. indicating that the benzoic acid derivative was attached to C-6 of ajugol. The location of α-Lrhamnopyranozyl was found to be at C-6 in the aucubin.8 (2C). UV spectral data.06 (1H. 13CNMR spectrum of compound (I4) (Table 2) indicated that signal due to C-6 of ajugol was shifted down fieled (δ 80. 1363 (aromatic ring) cm-1.6-O-(3``-O-acetyl-2``-O-P-methoxy cinnamoyl-α-L-rhamnopyranosylcatalpol).J=6Hz. J. shifted downfield. Compound (I4).6Hz). suggesting a molecular ion peak at m'z 528.m). 121. 1654(C=C). 2003). γmax.dd.3). Identification of Compound (I3): TLC investigation of fraction (3) revealed the presence of one spot of iridoid glycoside (compound I3). UV spectral data.S) and 2 methoxy proton signal at 3.. 142.J=14. Mass spectrum showed a molecular ion peak at m'z 528. 1708(C=O). 2010 skeleton and 2 sugar moieties.04 (H-6).J=14.2 (1H. 13CNMR indicated that rhamnose attached to C-6 hydroxyl group of the aglycone. 5041 . 292 (sh) nm. 277 nm. suggesting a molecular formula of C24H32O13.d. 2Hz) and two methylene protons at δ2. 6 signals for glucopyranose and 6 signals for di-substituted rhamnopyranose.2% sodium hydroxide gave syringic acid and ajugol which were identified with authentic samples using comparative TLC.J=2.2) showed signals similar to those of aucubin with additional signals of ρ-methoxy cinnamic acid and rhamnopyranozyl moieties.8 (brs).3. 1706 (C=O). Identification of Compound (I4): TLC investigation of fraction (4) revealed the presence of one spot of iridoid glycoside (compound I4). δ 1.5) in comparison to that reported for catalpol (δ75. IR.J=6.0. 1654 (C=C). showed absorption at λmax=216. 1 H and 13CNMR spectra data. a carbonyl carbon signal at δ 167. 1533. Thus the structure of (I3) was confirmed to be 6-O-(3``-O-ρ-methoxy cinnamoyl-α-L-rhamnopyranosyl aucubin). 2. Identification of Compound (I2): TLC investigation of fraction (2) revealed the presence of one spot of iridoid glycoside (compound I2). and 1363 (aromatic ring) cm-1.9. two cis olefinic protons at δ5. the attachment of the acetyl group is C-3`` and ρ-methoxy cinnamoyl group at C-2`` of rhamnose because1HNMR spectrum indicated that the signal of H-3`` was shifted down field and C-2`` signal shifted downfield in 13C-NMR specrtrum. Basic & Appl.9 (6H. Iridoid glycoside compounds were identified through UV. when hydrolyzed with 0. The site of esterification by ρ-methoxycinnamoyl was found to be C-3`` in the rhamnopyranozyl moiety. 13CNMR also showed signals for ρ-methoxy cinnamoyl ester and 2 signals for acetyl groups indicating that compound (I2) was iridoid derivative.Aust. 1 H and 13CNMR spectra data. 232 nm. δppm: 1HNMR spectral data of compound (I4) (Table 1) showed signals of two arbonyl protons at δ5. attributed to α-rhamnopyranosyl moiety.2Hz). Mass spectrum showed a molecular ion peak at m'z 652 suggesting a molecular formula of C31H40O15.5Hz).5 (1H. IR spectrum (KBR).5Hz).cm . Signal at 5.04 (1H. showed absorption bands at λmax=218. 1 H and 13CNMR spectral data of compound (I3) (Table 1.8) and the signals due to C`-5 and C`-3 shifted to upfield indicating that these positions are substituted. 1H and 13C spectroscopy (Saracoglu et al. the signals of the glucose were superimposable with those of catalpol.3. Mass spectrum showed a molecular ion peak at m'z 710 suggesting a molecular formula of C33H42O17. 1604. an anomeric proton signal at δ 4. showed absorption band at λmax=217.dd.. 148.28(1H. 4(10): 5038-5050. Comparison of spectral data of compound (I2) with published data. indicating its non-conjugated enol-ether function group. Mass.6. compound (I2) was identified as :. 5.35 (2H.J=6. Thus compound (I4) was identified as 6-O-syringoylajugol.1 : IR spectrum of compound (I2) revealed the presence of peaks at γmax 3600(OH).9 and methoxy group signal at δ 56. S). The site of the rhamnopyranosyl unit was found to be C-6 due to down field shift of C-6 resonance (δ 81. because1HNMR spectrum indicated that the signal of H-3`` was shifted downfield also the site of esterification was confirmed to be at C-3`` rhamnose from 13CNMR spectrum. δppm: 13CNMR spectrum of compound (I2) showed signals of catalpol (Table 2). showed peaks at γmax 3600 (OH). CH3 rhamnose). From the obtained results compound (I1) was identified as : 6-O-α-L-rhamnopyranosyl-catalpol. IR spectrum (KBr).14 (d.dd. These explained that benzoic acid derivative is syringic acid.0 (1H. 1 HNMR spectrum also showed two equivalent aromatic proton signals at δ 7.

31) of glucose indicating that the substitution at these carbons. IR spectrum showed absorption at γmax cm-1 3391 (OH). 1604. 4(10): 5038-5050.5 in the 13CNMR spectrum (Table 3). 2. 1703 (ester).Aust. 2007). 2004). also the absence of H-8 indicated the presence of methyl group attached to C-8. Basic & Appl.8 ppm (3H. 5042 . 1H and13CNMR revealed the presence of an isoferuloyl-3'. Identification of Compound (P2): TLC investigation of the fraction (2) indicated the presence of one spot of phenolic nature (compound P2).62) and C-4" (70. Fraction 3 was found to be phenylpropanoid glycoside.88 ppm (S) indicating the presence of 2 methoxy groups and their corresponding carbons resonating at δ56.78 (2H.S.32 (2H) and 7.5Hz.d. 7. The 1H and 13CNMR data (Table 3) were found to be similar to those of Eukovoside (β-(3'. 1363 (aromatic ring) cm-1. Mass spectrum gave a molecular ion peak at m'z 494 suggesting a molecular formula of C24H30O11. Mass spectrum showed a molecular ion peak at m/z 652 corresponding to the formula C31H40O15. 3. CH3 rhamnose). rhamnose).J=6Hz. J.8Hz. 4.H-7). 1982).35 (1H.4'dihydroxyphenyl) ethyl-O-α-L-rhamnopyranosyl (1-3)-β-D-(4-O-isoferuloyl) glucopyranoside (Sticher and Lahloub. J=16 Hz) and 5 aromatic protons at δ 7. harpagoside and phenylethanoide glycoside were isolated from the bioactive methanolic extract of Verbascum pycnostachyum flowers (Tatli et al.d. thus compound (P2) was identified as: β-(3'-hydroxy-4'-methoxy-phenyl)ethylO-α-L-rhamnosyl (13)-β-D-(4-O-isoferuloyl)-glucopyranoside. From the obtained data compound (I5) was identified as harpagoside (Akdemir et al.. revealed the presence of peaks at γmax 3600 (OH). aucubin. Sci.. where it was purified by PTLC using CHCl3:MeOH (5:1) system.31 (1H). showed absorption band at λmax=228 nm indicating the presence of non conjugated system. IR spectrum showed absorption bands at γmax cm-1 3415 (OH). 1592 and 1518 (aromatic). The chemical shift values of (CH3) at position 10 indicated the presence of methyl group attached to C8.. 1. 1515 (aromatic ring) cm-1. 1705 (C=O). Iridoid glycosides.d. where they were purified on silica gel column using CHCl3'MeOH system.J=15.87 and 3. TLC investigation of these fractions indicated that fractions 1&2 were phenylethanoid glycosides. The iridoid and phenylethanoid glycosides are widely distributed in the genus Verbascum. 13 CNMR spectrum (Table 3) showed signal at δ56.47 and 7. UV spectral data. glucose. 1H and 13CNMR indicating that the attachment of rhamnose at C-3 ''' and hence of the isoferuloyl moiety at C-4" of glucose. 1 H and 13CNMR spectral data: 1HNMR spectrum (Table 3) of compound (P1) showed signals at δ3. IR spectrum (KBr).H-7 and H-8.t. ajugol. Mass spectrum showed a molecular ion peak at m/z 639 coresponding to the formula C30H39O15.4 indicating the presence of methoxy group. 1637 (C=C).6 and 56. ajugoside. 2010 Identification of Compound (I5): TLC investigation of fraction (5) revealed the presence of one major spot of iridoid glycoside (compound I5). 1HNMR spectrum revealed the presence of two olefinic protons at δ 6.2-3.56 (1H.8Hz. Identification of Compound (P1): TLC investigation of the fraction (1) indicated the presence of one spot of phenolic nature (compound P1).53 (d. J=7.α-CH2. 1632 (C=C) and 1605. 3. 1706 (conjugated ester).S.18 (1H. 1 H and 13CNMR spectral data: 1HNMR spectrum (Table 3) of compound (P2) showed signals at δ3.d. The downfield chemical shift of C-3" (81.6 (m).6-39 (m) indicating the presence of βCH2.62 (2H) indicating the presence of cinammoyl moiety. 6. Investigation of Phenylethanoid and Phenylpropanoid Glycosides: Fractionation of the ethyl acetate extract of Verbascum letourneuxii on Sephadex LH-20 column using H2O ' MeOH system yielded 3 fractions. H7´ ) indicating the presence of α-CH2 at C-7`.J=15. 7. δ5. indicating the presence of two olefinic protons. 1625 (C=C).H-8). Iridoids showed anti-inflammatory activity while phenylethanoid glycosides possess antioxidant activity.4'disubstituted phenylethanol.1 (3H. 1 HNMR spectral data of compound (I5) (Table 1) showed signals characteristic for Harpagoside.OCH3). The downfield chemical shift of C-1" of glucose (δ104) indicating that phenylethanol moiety was attached to C-1" of glucose. glucose and rhamnose moieties. The obtained spectral data were found to be indicatal to those of martynoside.24(1H.

38(1H.H3`.6 70.3 9 CH 41.5 99.d.d.8 Hz) 3-3.d.d.J=6. 2010 Table 1: 1HNMR Position Aglycone 1 3 4 5 6 7 7` 9 10a 10b β-D-Glucose 1` 2` 3` 4` 5` 6`a 6`b α-L-Rhamnose 1`` 2`` 3`` 4`` 5`` 6`` Ester moiety 2``` 3``` 4``` 5``` 6``` α β OCH3 OCOCH3 spectral data (δ ppm) of compounds (I1.7Hz) 6.H6`a.2(m)( H2``.59(1H.dd.7 43. Basic & Appl.3Hz) I3 5.7 3`` CH 70. I4).d.73(1H.J=6.43m 3.3 60.36(1H.5 98.8 121.7Hz) 3.15(1H.80s 1.04(1H.6Hz) 2.6Hz) 3.2 62.60(m) 7.4 145.73(1H.68(1H.8Hz) 3-3. H6`b) 3.7Hz) 3.56(1H.J=16Hz) 3.01(m) 6.J=4.d.J=8Hz) 3.06(1H.3 115.d.J=8.d.92(1H.32(d.3-3.J=7.5Hz) 6.96(1H.t.6 55.77(d. H6`a.1 104.d.J=8.19(1H.8 - 5043 .12(1H.5(m)( H3`.22(1H.78(m) 6.94s 7. H5`.31m 3.2 114.2 115.98(1H.2 51.6 I3 97 141.62(d.5Hz) 2.63(1H. H5`.7Hz) 6.J=6Hz) 5.6(m) 1.68d 4.J=8Hz) 3.dd..59(1H.91(1H.3 4`` CH 71.d.13. J=6Hz) 4.47(d.2 127.J=12Hz) 7.60brs 2.4 Hz) 1.J=16Hz) Table 2: 13CNMR spectral data (δppm) of compounds (I1.J=1. H4`.J=7.dd. 4(10): 5038-5050.J=6.31(t) 7.5Hz) 6.7Hz) 1.J=7. H6`a) 3.8Hz) 3.14(1H.d.2Hz) I4 5.d.9 2` CH 73.25m 3.7 72. I2 I1 4.9 58.J=9. H3``.2 Hz) 3.9 17.3 162.J=6. H5``) 1.d.2Hz) 4.J=7.6Hz) 4.69(1H.41(3H.89brs 2.94(1H.1 69.9 2`` CH 70.d.83brs 3.dd.1Hz) 5brs 3. 2.43(1H.8 47.d.6 108.d.d.H3`.1 74 77.J=14.J=5.7 100.J=1.84s 7.16(d.J=11.3Hz) 2.d.6 130.s) 7.d.82m 4.5Hz) 1.5 8 C 65.4.4 70.7 26.d.88(1H.7 79.2Hz) 5(1H.47brs 5.56(S) 6.d.91(m) 1.6.2 115.45(1H.J=6.7 74.8 21.J=8.65(1H.3 70.24(1H.J=16Hz) 7.4 167.d.d.7.3 131.6Hz) 6.J=8.1Hz) 3.8 47.6 162.J=15.68brs 2.2Hz) 5.2Hz) 4.69(d.7Hz) 3. J.7Hz) 6.4 4` CH 70. H4`.d.8 61.5 39.s) 4. H3``.dd.dd.d.7 78 71.d.2Hz) 3(1H.1-3.J=8.J=7.5 7 CH 57.J=9.J=8.J=6.70Hz) 3.66(m) 3.9(m)( H2``.1 - I4 93.J=7.J=15.14Hz) 1.J=7.1.3 88.5Hz) 2.J=6.8 59.62(1H.2 99. Sci.d. I2.38(1H.3 115.3 42.04(1H.62(d. Position CATOM I1 Aglycone 1 CH 93.J=7.32(d.5 80.d.J=9.dd.d.3 131.5 3` CH 7.2 96.H6`b) 3.46(d.2 70. I4 and I5 ).7Hz) 3.7 6 CH 81.6Hz) 3.2-3.J=16Hz) 7.7-5. H5``) 4.96Hz) 7.4 114.4 130.2 3 CH 140. H4``.7Hz) 6.60(1H.J=6. H6`a) 3.3 83 58.37(1H.8.J=8.J=16Hz) 4. I2.53(d.6 18.9Hz) 3.90(1H.70(1H.95(1H.4 146.J=8.4 6` CH2 α-L-Rhamnose 1`` CH 98. I3.35(2H.9 142 167.9 56.67(1H.5 127.7Hz) 5.7Hz) 7.J=7.89brs 3.J=8.J=16Hz) 3.2 66.7Hz) 7.5(1H.J=5.J=8.Aust.J=14.8 74.8(d.89(dd.9(d.J=8Hz) 3.2 170.d.3 71.3 62.28(1H.97(1H.J=6.t.3.06(1H.9 5`` CH 68.3 17.J=3.m) 5.90(1H.24(1H. H4`.J=9.s) 7.3 148. H4``.J=2.2 104.19(1H.9Hz) 2.4 61.J=5.5(m)(H2`.7Hz) 6.60(1H.41(1H.J=16Hz) I5 5.1 78.7 78.m) 2.7 77.d.30m 3.d.d.J=8. 3.d.6 69.9Hz) 6.9 6`` CH3 Ester moiety 1``` C 2``` CH 3``` CH 4``` C 5``` CH 6``` CH α CH β CH C=O C CH3 OCH3 C OCOCH3 CH3 OCOCH3 I2 94 142 103 36.7Hz) 4.d.dd.3 126.J=6.2 148.J=9.95(1H.97(1H.d.7(m)(H2`.96Hz) 7.J=8Hz) 3.9 4 CH 102.4 74.d.39(1H.5Hz) 4.J=14.4 141.4 71.3 77.6Hz) 4.6 5` CH 76.27(m) 3.J=8.t.7 56.9(m)(H5`.5 5 CH 35.4(S) 4. H5`.14(1H.9 10 CH2 β-D-Glucose 1` CH 97.23(1H.8Hz) 3. I3.3 162.98(1H.14.d.dd.1Hz) 2.dd.89(6H.

5 5 6.72 bs 117 147.4 2' 6.3 7.J= 7Hz.8) 115 9 168.5 3.8) 104 2'' 75.3 6. 4. 4` are occupied. 7.81 d (7.8(S.17) 117.1 2.3) 121. 1589 and 1510 (aromatic C=C) and 1630 cm-1 (C=C). The 1HNMR spectrum (Table 4) of compound (P3) revealed the presence of signals at δ 6.3H.4.4 Rhamnose moiety 5'' 75.9 6.80 s (P2) -----------------------------------------------------------δH (ppm) δC (ppm) 127.2) 36.1.6. 1587 and 1507 (aromatic C=C). dd.J=2Hz. I.4 56.78 t (7. 5044 .J=5.6) 121.5.04 dd (16. H-4` indicated that positions 3`. The 1HNMR spectrum of compound (P4) (Table4) revealed the presence of signals at δ7.1 76. anomeric proton of glucose).ddd.4.0) 18.5.35 d (7. Mass spectrum of compound (P4) showed a molecular ion peak at m/z 342 suggesting a molecular formula of C16H22O8.8) 111.dd.7 OMe 5''' 70.08 (1H. 6.J=1. 281 nm..1. H2`.H-3). Basic & Appl.H-6`).5 70.3 56.5 2 7.2 7 7.4 Glucose moiety 8' 3. dd.9 72.3 3' 144.0) 18.40 d (8.06 d (6.9 (S.J=7Hz anomeric proton of glucose). J=1.5. eluted with acetone'MeOH (1:1).H-2).62d(15.63 m 62.5 3.36 d (7.R.H5`).6. 6.1) 116.5Hz.Aust.9 (m).OCH3).8Hz.5.34d(15.93 d (8.18 s 102.8) 149.6`).7 6'' 3.1 168.2 Hz..5 7.79 d(8.65(2H. J=6. spectrum of compound (P3) showed peaks at γmax 3391 cm-1 (OH).3 81. Each band was scraped off.9 6.1 1'' 4.0) 124.8) 115.5.3 6''' 1. which showed a single spot of phenyl praponoid glycoside in each band (compound P3 and P4). J. 4.5 (1H. 3395 (OH). OCH3).16Hz. The absence of H-3`.9 72 1. 3-3.1 7' 2. H-3). 15.1) 124.9 (m).H-2`).2 1''' 5.ddd.R.8) 147.9 8 6.6 75.5Hz.9 3.80 t (7.J=2. 6. 3.60 m 62.9 3'' 81.19 s 102. 3. Mass spectrum of compound (P3) showed a molecular ion peak at m ' z 372 suggesting a molecular formula of C17H24O9. filtered and the filtrate was concentrated under reduced pressure and subjected to TLC investigation.J=8Hz.5 147.6 7.58 (2H. I. P2) C/H (P1) ------------------------------------------δC (ppm) δH (ppm) Isoferulic acid moiety 1 127.19 bs 112. UV spectral data of compound (P3) in methanol showed absorption bands at λ max = 236.54dd(8.06 d (7. 16.88s Identification of Compound (P3): PTLC of fraction 3 gave 2 bands.67 d(7. S. 2010 Table 3: 1HNMR and13CNMR spectral data (δppm) of compounds (P1. 1.9 5' 6.8Hz.4(1H.2 (1H.J=1.9 147. dt.2 132. Identification of Compound (P4): UV spectral data of compound (P4) in methanol showed absorption bands at λmax 228.5. H1`).55(2H.4 4' 145. H-1).4.d. H-2). spectrum of compound (P4) showed peaks at. Sci.1 3''' 72. 7.78 m 72. 6.16Hz. 3-3.d.4 4.8 150.87s 56.1) 111..3 5.75 d (8.07 d (6.3 Aglycone moiety 1' 131.8 6 6. H-5` and H-4` indicated these position were occupied.63d (15.6) 116.9) 104. The absence of H-3`.35d(15.8) 72 4.65 d (8. 2000).8 3 149.4 3.2 76.1 4''' 73. thus compound (P3) was identified as syringin (Munkombwe et al.3 6.5 4'' 70.3 75.0 (1H.6 (1H.d. Thus from these data compound (P4) was identified as coniferin (Gűner et al. 6.5. 2003). dt (ddd) J=1. 1631 cm-1 (C=C).8 2''' 72.9(1H. 273 nm.1 6' 7.8 (1H.5.8(d. The obtained spectral data were similar to those reported for syringin.18 bs 115.2 4 150.6H. 4(10): 5038-5050.1) 36.7 6.

5) 116.0) 62.7.4 5` C 134. 276.3) 9` CH2 4.66 (s) 1` 6.86 br s 7 CH 88. Mass spectrum of compound (N1) indicated the presence of a molecular ion peak at m'z 521 corresponding to a molecular formula of C26H33O11.63dd (11.5.10 br dd Ome at C-3 and C-5(6H) 3.5.96 d (1.80 d (7.80 br s 3` C 145. N2) (N2) C'H (N1) --------------------------------------------------------------------------------------------------------δcppm δH ppm.31) 63.1 4. Sci.J(Hz) 1 C 134.0) 88.55 6.2 6` CH 116.4) 5`` CH 77.4 3. n-hexan:EtOAc:acetone (90:8:2) system and the eluted bands were applied on the sephadex LH-20 column.5) 8 CH 52.0 3.16Hz) 4.5.12 d (8Hz) 7.82(overlapped) 6`` CH2 3.2d (7.5 132.22 6.70 d (7. 5045 .5Hz) 3.22 dd (10.42 Investigation of Neolignan Glucosides: Fractionation of the n-butanol extract of Verbascum letourneuxii on silica gel column using hexane-ethyl acetate-methanol mixtures yielded 3 main fractions.93 3. 1.3 5.0) 6 CH 119.Aust.9) 70.74 (s) 6``a 3. 2.22 d (5.10 -3.3 136.5.3-3.4 4 C 147.53 br dd (6.0) 9a CH2 9b 3.25 ddd(dt) (5.70 dd(5.5 6.0) 134.224.5) 3. 4(10): 5038-5050.9 2 CH 109.00 d (1.9.6 (2H) 6.45 2` CH 111.9 3.56 dd (7.42 (overlapped) 71.25t (9.90 dd (12.70 (m) 55.8 7.21 66. IR spectrum of compound (N1) showed peaks at γmax 3403 (OH).30 (m) 78.4 6.52 d (7.7 5. 8.32 ddd (dt) (5.6 3.7 3.0) 75.1 3. UV spectral data of compound (N1) in methanol gave absorption bands at λmax.94 4.5.8 130. 2010 Table 4: 1HNMR spectral data (δppm) of the compounds (P3.1 150.J(Hz) δcppm δH ppm.87 dd (2.3.5) 3`` CH 77.0) 78.93 6.3 6. 6.6 (m) Table 5: The 13C and 1HNMR spectral data (δppm) of the compounds (N1.0 3.47 6.11Hz) 2``-5`` 3. J.5.16 Hz) 1`` 4. 6.7 6.5 4.20 dd (7..12Hz) 3.0) 71.2) 119.6 6.7.4 m Proton 5` 2` 6` 1` 2` 1`` 3` (2H) Ome at C-3 6``a 6``b 2``-5`` (P4) 7.0) Table 6: Antioxidant activity of different plant extracts Extract Ethyl acetate Butanol Methanol (80%) % Antioxidant activity 72.2 3.5 d (6.2) 110. P4) Proton (P3) 2.33 t (9.84 (s) 56.3) 2`` CH 81.8 3. for further purification each fraction was subjected to PTLC using.2 3.8Hz) 6.16Hz) 2` 6. 9.93 d (7Hz) 4.0 150.57 t (9.23 dt (16.8 6.48 t (7. 343 nm.65 d (11) 8` CH 127.2) 112.5.8 3.21 dd (11.5.80(overlapped) CH3 55.42ddd(dt)(1.1.32t (8.5 3.3) CH3 56.5) 103.12Hz) 3. Basic & Appl.55 4` C 149.18 4.1 150.11Hz) 6``b 3.11 d (2Hz) 6.80 dd (6.13 81.65 br dd (2.0 3.31 (m) 62.72 d (8.66 d (7Hz) 3`(2H) 4.0) 3 C 149. Identification of Compound (N1): Fraction (1) after purification on PTLC and sephadex LH-20 column gave one spot on TLC investigation (compound N1).26 t (7.5) 65.91 (s) 56.3 7.54 3.0.91 dd (7.0. 1560.2 6.5.91 d (2.65 5 CH 115.5. 16Hz) 6.22 dd (1.1.7 3.55 d (16. 1459 (aromatic ring).5.88 (s) 3.60 ddd (dt) (1.92 d (8. 1628 (olefinic c=c) cm-1.04 d (1.5) 4`` CH 70.88 dd (10.81 (s) 3-OCH3 1`` CH 102.6 145.9) 124.2) 119.5.0 4.73 (s) 4-OCH3 1` C 132.66 br s 7` CH 131.76 (s) 3-OCH3 CH3 56. 1609.31 6.3 3.5 3.

J=7.H-6) suggesting the presence of a trisubstituted aromatic moiety. Other uses include the treatment of diarrhea.55 (d. Free radical scavenging and cell aggregation inhibitory activities of 36 secondary metabolites isolated from the methanolic extracts of Verbascum species were investigated. IR spectrum of compound (N2) showed peaks characteristic for OH. chills and flu.66 (brs) was indicative of the presence of a tetrasubstituted aromatic moiety. neolignans. gastrointestinal bleeding.d.76. These compounds demonstrated scavenging properties toward d the DPPH radical in TLC autographic assays. The proton resonances at δ 6. compound (N1) was identified as dehydrodiconiferyl alcohol-9-O-B-Dglucopyranoside. 1 HNMR spectral data. bruises and inflamed mucosa.5Hz) and 3.H-4 and H-3` indicated that these positions are occupied. Antioxidant activity of butanol. 1 5046 . The absence of the H-3.0 (d. phenylethanoid. herpes simplex viruses.J=7.J=2Hz. Mullein has in vitro antiviral activity against influenza. Five phenylethanoid glycosides (verbascoside. 1HNMR spectrum of (N2) showed 3 methoxy singlets at δ 3.86 (br S. 2004). The glycosidic linkage was determined to be at C-9` due to the downfield shift of the C-9`. Fragoso et al.73 and an aromatic proton signal at δ 4.3Hz) indicating the presence of a glucose unit. Thus from the previous data. signals due to glucose unit and due to 3 methoxy groups.91 (d. δppm: The 1HNMR spectrum (Table 5) revealed the presence of 5 aromatic proton signals. The presence of 2 proton signals δ 6. Identification of Compound (N2): Fraction (2) after purification on PTLC and sephadex LH-20 column gave one spot on TLC investigation (compound N2).J=11.J=16Hz) and 6.1).5. burns.79 (d. 2010 H and 13CNMR spectral data.92 (d. (2008) reported that flowers and leaves of Verbascum densiflorum are used for a wide range of ailments. Mass spectrum of compound (N2) revealed the presence of a molecular ion peak at m ' z 534 corresponding to a molecular formula of C27H34O11. J.J=7.J=16. Antioxidant Activity: The radical scavenging effect of the tested extracts on DPPH free radical (Table 6) showed that the butanol extract of Verbascum letourneuxii had the highest antioxidant activity followed by ethyl acetate extract. Basic & Appl. 13 CNMR spectrum of compound (N2) (Table 5) showed 18 carbon atoms consistent with the neolignan structure. Moreover. colds.H-5) and 6. 7.J=11Hz) and 6. The proton resonances at δ 7.9 and 3. asthma. migraines. hemorrhoids. and martynoside) as well as two neolignan glycosides (dehydrodiconiferyl alcohol-9`-O-β-D-glucopyranoside and dehydrodiconiferyl alcohol-9-O-β-D-glucopyranoside) were isolated from the aerial parts of Verbascum salviifolium). 2 methoxy signals at δ3. They were found to have significant antioxidant properties.5.55 (d. tuberculosis..2Hz. 3. phenyl propanoid glycoside and iridoid glycoside.8. colic. 6... The glycosidic linkage was determined to be at C-9 due to the downfield shift of the C-9 (71.J=7. 4(10): 5038-5050. 2007).9Hz). sleep disorders. kidney dosirdes and chronic inflammation. signals due to glucose unit and due to 2 methoxy groups. Thus from the previous data.H-2). The 13CNMR spectrum of compound (N1) showed 18 carbon atoms consistent with the neolignan structure. which indicated their ability to efficiency scavenge free radicals (Akdemir et al.2 (d.H-5) suggesting the presence of a trisubstituted aromatic moiety. 3. angoroside A.1-3.Aust. wounds. tonsillitis and tracheitis). The 2H resonances at δ 6. forsythoside B.82 ppm and anomeric proton signal at δ4.H-2). based on the experiments with DPPH.21 (1H. ethyl acetate and methanol (80%) extracts of Verbascum letourneuxii due to the presence of phenolic constituents. such as inflammatory ailments in respiratory tract (cough.23 (dt.3Hz) indicated the presence of olefinic bond.5Hz. 6.9 (m) indicating that the sugar moiety was glucose. gout. Sci.δppm : The 1HNMR spectrum of compound (N2) (Table 5) revealed the presence of 5 aromatic proton signals. Also 1HNMR spectrum showed 2 trans olefinic proton signals at δ 6. bronchitis. The isolated compounds exhibited a dose dependent inhibition of bioautographic and spectrophotometric DPPH activities (Tatli et al. 1. b-hydroxyacteoside.H-6) and 6.J=1. compound (N2) was identified as: 4-o-methyl-dehydrodiconiferylacohol-9`-o-βD-glucopyranoside.65 (1H. aromatic ring and olefinic c=c.dd.9 (dd.J=8Hz. pneumonia. herpes simplex virus type 1 and also an anti-bacterial activity..2Hz. while methanol extract had lower antioxidant activity.

J. Basic & Appl.. 2010 Table 7: Isolated iridoid glycosides 5047 .Aust. Sci. 4(10): 5038-5050.

2010 Table 8: Isolated phenylethanoid and phenyl propanoid glycosides 5048 . Sci. Basic & Appl. J.Aust.. 4(10): 5038-5050.

L. 2004. I. Shams and F. Tatli. E. A. Khan. E. Fragoso. Basic & Appl. Torres.A. Chem. J.A. 2007. Taşdemir. 227(1): 125-135. Ireland and I. Z. and J.A. Practical Thin Layer Chromatography. 2000.I.. Neolignan glucosides from Phlomis chimera Boiss. Harvala.. Quaternary protoberberine alkaloids. S.. N.L. Ruiz and E. Mor. 2007. pp: 279... N. 11: 73. Khafagi.I. Mitaku. N. Naturforsch C. D. Phytochemistry. University Press. J. H. eCAM. 62(11-12): 813-20. Dönmez. Hamed. 57(c): 221-225. 1996. Nat. I. Abdel-Azim. Ethnopharmacol. Dewedar.A. Z. 71(3): 365-76. Tatli and Z. E. 2000. Z. Turk. Samioside. 64(8): 1095-7. K. Toxicol Appl Pharmacol.Aust... Tatli. Guarino. Antinociceptive and anti-inflammatory effects of saponin and iridoid glycosides from Verbascum pterocalycinum var. Khan. Prod. J. Risks and benefits of commonly used herbal medicines in Mexico. Z. Chem. P.. Magiatis. Tűbingen. 2002. a new phenylethanoid glycoside with free-radical scavenging and antimicrobial activities from Phlomis samia. E. Naturforsch. Fried. 4(10): 5038-5050. C. Antimicrobial activity of Verbascum macrurum Ten.. Ekim and K. Neolignan and phenylethanoid glycosides from Verbascum salviifolium. Dostál and R.. Melliou. A. I.H. L. 2007. Turk. C.. I. A. Tsitsa. Bedir and I. 5049 . mutense Hub.M. Esparza. Kirmizibekmez. I. J. T. Hammouda. 28: 227. 2001.. C. Buchie. Marek. 2002. 28: 621-628. Sherma.I. Verlag der Zeitschrift fűr Naturforshung. Sci. 4(S1): 25-28. N. Bedir and I. Başer. Kyriakopoulou. B. Edinburgh. J. Boll Chim Farm. Caliş. Magiatis. I. Gűner. 68: 150175..S. and A.R.. Shafeek..M.H. Grycová.. Chemical investigation of some Capparis species growing in Egypt and their antioxidant activity.S. 2010 Table 9: Isolated neolignan glucosides REFERENCES Akdemir. T. Skaltsounis. 141(3): 238-42. (Scrophulariaceae). E. P. Flora of Turkey and the East Aegean Islands..S.R. Akkol. Iridoid and phenylethanoid glycosides from Verbascum lasianthum. 2008.R. Akdemir.K. J.C.S.K. The efficiency of random versus ethno-directed research in the evaluation of Sinai medicinal plants for bioactive compounds. J. Akdemir. Two new acylated iridoid glycosides from Verbascum undulatum. 55(c): 667-6699. Saracoğlu. Aligiannis and C. I. Őzhaty. D. Ersöz. 2000. 2004. Charvala and S.

5050 . M. Sticher. Saracoglu. Neiolignan. and Z. 2005.. phenylethanoid and iridoid glycosides from Phlomis integrifolia. Screening for free radical scavenging and cell aggregation inhibitory activities by secondary metabolities from Turkish Verbascum species. 55(12): 1744-1747. Takamatsu. Shafiee. Vinciguerra. Surmaghi and A. 1991. M.I. 31: 85-96. Phenylpropanoid glycosides of Gnidia polycephala. G.. 100(1-2): 16875. Tatli. N.S. 14(3): 115-119. 1982. 27: 739-747. G. FABADI J. E. Li. 2006. L. Gebre-Mariam..I. and M.. J. Sarkhail. Antiviral activity of Flos Verbasci infusion against influenza and Herpes simplex viruses. P. Flavonoid. Chem. and Z. 2010 Munkombwe. Akdemir. Varel. Pharm. Sci. 27(1): 23-32.. 2003. K. Tatli. Planta Med. Felaco and A.G. Bull. I. Pesce. Pharm. W. C. Seifert. 2007. 64: 1401-1404.H. J. 2009. Tatli. 2006.R. Ethnopharmacol. H. J... Fabadi J. K. Sci. Esfehani.. and K. Asres and T. 62(9-10): 673-8..F. Grzybek. Calis. Fng and G.&Helder flowers. L. Galebotswe.M. Liebigs Annalen der Chemie.S. I. Zgórnik-Nowosielska. S. Abietane lactones and iridoids from Goldfussia yunnanensis.M. Naturforsch C.S. Secondary metabolities from bioactive methanolic extract of Verbascum pycnostachyum Boiss. B.. Phytochem. J.. MA. I. Anti-inflammatory properties of the plant Verbascum mallophorum.Y.W.. I. species.Ş. Hacettepe University Journal of the Faculty of Pharmacy. Morebodi. Khan and Z. Modibesane and N. H. H. 29: 93-107. M. Tadeg. Franceschelli. P. S. 23(3): 189-95.Ş.M. Tatli.S.. O.. I. Basic & Appl. Phenolic glycosides of Paulownia tomentosa. Pharm. Yu. Felaco.L. Traditional uses and biological activities of Verbascum species. Biol Regul Homeost Agents. Zhang. N. Speranza. Mohammed.. Schuhly. Li. Akdemir. Z. I.I. Grilli.. Akdemir. 2003. M.I. Arch Immunol Ther Exp (Warsz). Daru. Iridoids from Verbascum nigrum. Vesper. Sci. D. Phytochemical study of Phlomis olivieri Benth and Phlomois persica Boiss. 2004. I.. 39(1-2): 103-8. Z. Antimicrobial activities of some selected traditional Ethiopian medicinal plants used in the treatment of skin disorders. Menghini. 2007. 4(10): 5038-5050. Amin. Serkedjieva and B. 7: 751753. Patruno. Zawilińska. Chem. Li. Chemical constituents of Verbascum L. I. Lahloub. P.Aust. Turk J. Akdemic. De Lutiis. A. 1994. Manolova. I.. T. 46: 145148. 2007.

Sign up to vote on this title
UsefulNot useful