Anthraquinone glycosides from Cassia roxburghii and evaluation of its free radical scavenging activity

Carbohydrate Research 360 (2012) 47–51

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Anthraquinone glycosides from Cassia roxburghii and evaluation of its free radical scavenging activity Sayed A. El-Toumy a,⇑, Sahar S. El Souda b, Tahia K. Mohamed b, Iñaki Brouard c, Jame Bermejo c a

Chemistry of Tannins Department, National Research Center, El-Bohouth St., Dokki, 12622 Cairo, Egypt Chemistry of Natural Compounds Department, National Research Center, El-Bohouth St., Dokki, 12622 Cairo, Egypt c Instituto de Productos Naturales y Agrobiología, Av. Astrofisico F. Sanchez 3, 38206 La Laguna, Tenerife, Spain b

a r t i c l e

i n f o

Article history: Received 21 June 2012 Received in revised form 28 July 2012 Accepted 30 July 2012 Available online 8 August 2012 Keywords: Cassia roxburghii Anthraquinones Free radicals scavenging activity

a b s t r a c t The methanolic extract of the leaves of Cassia roxburghii DC., was investigated for its anthraquinone glycosides and antioxidant activity. Two new anthraquinone glycosides named emodin 1-O-b-D-glucopyranosyl-(1?2)-glucopyranoside (1) and aloemodin 8-O-b-D-glucopyranosyl-(1?6)-glucopyranoside (2) along with aloemodin 8-O-b-D-glucopyranoside (3), emodin (4), aloemodin (5) and one flavonoid, quercetin-3-O-a-L-rhamnopyranoside, were isolated from the leaves of C. roxburghii. Structures of the isolated compounds were established by UV, HRESI-MS, and 1D/2D 1H/13C NMR spectroscopy. The total extract and some isolated compounds were determined against DPPH (2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazinyl radical, for their free radical scavenging activity, the total alcoholic extract showed strong antioxidant activity while the two new compounds showed weak antioxidant activity. Ó 2012 Elsevier Ltd. All rights reserved.

The use of natural products with therapeutic properties is an ancient as human civilization and for a long time. Recently, herbal medicines have increasingly been used to treat many human diseases.1 Natural antioxidants, especially phenolics and flavonoids are safe; they protect the human body from free radicals and retard the progress of many chronic diseases as well as lipid oxidative rancidity in foods.2 Numerous studies were carried out on plants with antioxidant properties.3,4 However, there is still great interest in finding new antioxidants from natural sources. The Cassia genus (Family: Fabaceae) represents one of the largest and most diverse group of flowering plants, including herbs to trees. Plants of the genus Cassia are widely distributed in most tropical and subtropical countries. Cassia species have biological and medical activities such as hepatoprotective, antibacterial, antioxidant, antitumor, antidiabetic, and antiparasitic.5–11 Species of the genus Cassia have been also used as laxative, purgative, antipyretic, antiviral, antibacterial, antifungal, as well as anti-inflammatory agents.12–14 Among those Cassia roxburghii is one of the medicinal plants used in ethnomedicine for the treatment of various liver ailments.15 Phytochemical analysis of certain Cassia species led to the isolation of flavonoids, anthraquinones, proanthocyanidins, and condensed tannins.16–18 Previous phytochemical investigation of C. roxburghii seeds have been isolated of chrysophanol, physcion, emodin, 1,3-dihydroxy-2-methyl anthraquinone 8-O-a-L-arabinopyranoside, physcion 8-O-a-L-xylopyranoside, and emodin O-a-Larabinopyranoside.19 ⇑ Corresponding author. E-mail address: [email protected] (S.A. El-Toumy). 0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2012.07.020

Plants have played a major role in the introduction of new therapeutic agents. To the best of our knowledge, there is no scientific literature on the constituents and antioxidant activity of C. roxburghii leaves. The present study deals with the isolation and identification of anthraquinones glycosides from the leaves of C. roxburghii and evaluation of its free radical scavenging activity. The aqueous alcoholic extract MeOH–H2O (7:3) of C. roxburghii leaves was subjected to extensive repeated column chromatography on Polyamide 6 and Sephadex LH-20 resulted in isolation of two new anthraquinone glycosides 1 and 2 (Fig. 1), together with aloemodin 8-O-b-D-glucopyranoside, emodin, aloemodin, and one flavonoid, quercetin-3-O-a-L-rhamnopyranoside. Compound 1 was obtained as orange amorphous powder. It was positive to the Bornträger reaction, revealing that it was a hydroxyl anthraquinone compound.20 The UV spectrum gave the absorption maxima at 221, 271, and 415 nm. Its molecular formula was established as C27H30O15 from its HRESI-MS including a pseudomolecular ion at m/z 617.1487 [M+Na]+. The IR spectrum of the compound 1 showed characteristic absorption bands from OH (broad, 3442 cm1) and a,b-unsaturated ketone (1675 and 1625 cm1). On acid hydrolysis compound 1 afforded emodin and glucose, which identified by chromatography with authentic samples emodin and D-glucose. The 1H NMR spectrum showed two sets of two meta coupled aromatic hydrogens at d 7.16 (d, J = 1.8 Hz, H-2) and d 7.47 (d, J = 1.8 Hz, H-4); 6.91 (d, J = 2.1 Hz, H-5) and d 7.28 (d, J = 2.1 Hz, H-7) suggesting two sets a tetrasubstituted aromatic ring in addition to singlet for methyl group at d 2.4. Also, 1H NMR spectrum revealed a singlet at d 8.54, assigned to a hydroxyl group H-bonded to a carbonyl. The spectrum also showed two

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S. A. El-Toumy et al. / Carbohydrate Research 360 (2012) 47–51

HO OH OH

OH O

HO HO

O

1``

OH

13

12

7

HO

O

O

OH

O

1`

2

14

11 5

CH3

4

O

1 O 5

4 11

7

10

9

12

O

O

CH2OH

14

2

13

OH

1`

OH

O

OH

O HO HO

1``

O

OH HO OH

2 Figure 1. The structures of compounds 1 and 2.

distinct signals for anomeric protons, two hexose anomeric proton resonances at d 5.30 (1 H, d, J = 7.28 Hz) and a proton resonance at d 4.46 (1H, d, J = 7.28 Hz), indicating the presence of two sugars with b-configuration. The b-D-configuration was deduced from specific optical rotation [a]D +71.3 and confirmed by the acid hydrolysis with the comparison of D-glucose. The DEPT spectrum of compound 1 revealed the presence one methyl at d 21.74, four methane aromatic carbons. The downfield signals at (d 186.79 and d 182.55) were assigned to the two carbonyl carbons. Assignments of the aglycone carbons were aided by comparison with the reported values for emodin 1-O-b-glucoside.21 The application of HMQC and HMBC experiments (Fig. 2) led to full assignments of the 1H and 13C NMR chemical shifts of compound 1. From HMBC spectrum, the signals at d 7.47 and 6.91 simultaneously had correlation with the carbon at d 182.55 indicating that they were located at H-4 and H-5 respectively. In the HMBC spectrum (Fig. 2) the pat-

tern of 1H–13C correlation was observed between d 5.30 (H-10 ) with d 160.81 (C-1) and d 4.46 (H-100 ) in addition to d 83.07 (C-200 ) indicating a glucosyl moiety is attached to C-1 and the link is glucose (1?2). Based on the above data, compound 1 is deduced to be emodin 1-O-b-D-glucopyranosyl-(1?2)-glucopyranoside. To the best of the authors’ knowledge, this is the first report of isolation of this compound from any natural source. Compound 2 was obtained as yellowish-orange powder, which gave a positive Bornträger’s test for anthraquinone derivatives.20 The UV spectrum gave the absorption maxima at 219, 269, and 408 nm. Its molecular formula was established as C27H30O15 by positive HR-ESI-MS at m/z 617.1495 [M+Na]+. The IR spectrum of the compound 2 showed characteristic absorption bands from OH (broad, 3410 cm1) and a,b-unsaturated ketone (1645, 1638 cm1). On acid hydrolysis compound 2 afforded aloemodin and glucose, which identified by chromatography with authentic

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S. A. El-Toumy et al. / Carbohydrate Research 360 (2012) 47–51

HO OH OH

OH O

HO

O

HO

O

OH OH

O

O

HO

CH3 O

1 O CH2OH

O OH

O

O

OH

OH

O HO O

HO OH

HO OH

2 Figure 2. HMBC correlations H–C for 1, 2.

samples aloemodin and D-glucose. The 1H NMR spectrum showed two meta coupled aromatic hydrogens at d 7.24 (d, J = 1.2 Hz, H2) and d 7.60 (d, J = 1.2 Hz, H-4), suggesting a 1,3,5,6-tetrasubstituted aromatic ring. The presence of a 1,2,3-trisubstituted aromatic ring was evident from the signals at d 7.83 (dd, J = 8.2, 1.2 Hz, H-7), d 7.86 (t, J = 8.2, H-6) and d 7.87 (dd, J = 8.2, 1.2 Hz, H-5). The 1H NMR spectra of compound 2 indicated the presence of a methylene bearing a hydroxyl group at d 4.60 (2 H, s). The 1H NMR spectrum also showed two distinct signals for anomeric protons, two hexose anomeric proton resonance at d 5.10 (1 H, d, J = 7.28 Hz) and a proton resonance at d 4.22 (1 H, d, J = 7.28 Hz), indicating the presence of two sugars with b-configuration. The b-D-configuration was deduced from specific optical rotation [a]D +48.4 and confirmed by the acid hydrolysis with the comparison of D-glucose. The 13C NMR spectrum of compound 2 (Table 2) showed 15 carbon signals of the anthraquinone, two carbonyl carbons (d 187.87

and d 182.74), five quaternary sp3 carbons, five methine aromatic carbons, and one sp3 methylene group. The application of HMQC and HMBC experiments (Fig. 2) led to full assignments of the 1H and 13C NMR chemical shifts of compound 2. Both hydrogens at d 7.60 (H-4) and d 7.87 (H-5) showed long range correlations with the carbon at d 182.74 establishing this ketone at C-10. The cross peak in the HMQC between the hydrogens at d 4.60 (s, 2H) and the carbon at d 64.4 suggested a hydroxymethyl group at C-3. The HMBC and HMQC data confirmed the location of the hydroxymethyl group at C-3 through the observed correlations among H2 and C-1, C-3, and C-4. In the HMBC spectrum (Fig. 2) the pattern of 1H–13C correlation was observed between d 5.10 (H-10 ) with d 159.59 (C-8) and d 4.22 (H-100 ) with d 69.1 (C-600 ) indicating a glucosyl moiety is attached to C-8 and the link is glucose (1?6). Based on the above data, compound 2 is deduced to be aloemodin 8-O-b-Dglucopyranosyl-(1?6)-glucopyranoside. To the best of the authors0

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knowledge, this is the first report of isolation of this compound from any natural source. The other three known compounds (3–5) were identified as aloemodin 8-O-b-glucoside 3, emodin 4, aloemodin 5 and one flavonoid, quercetin-3-O-a-L-rhamnopyranoside by comparison of their 1D and 2D NMR spectral data with the reported data in the literature.21–23 The total alcoholic extract and pure compounds were tested for their free radical scavenging activity on DPPH (2,2-di(phenyl)(2,4,6-trinitrophenyl)iminoazanium). The alcoholic extract and quercetin-3-O-a-L-rhamnopyranoside exhibited potent antioxidant scavenging activity toward DPPH, with IC50 values of 37.96 ± 0.42 and 29.49 ± 0.98 lg/mL, respectively, while the two new compounds exhibited very weak antioxidant scavenging activity against DPPH, with IC50 values of 138.01 ± 2.82 and 132.01 ± 2.82 lg/mL and the remaining compounds showed moderate activity (Table 3). Ascorbic acid was used as a positive control. 1. Experimental 1.1. General methods 1

H NMR and 13C NMR spectra were obtained on Bruker AMX400, Avance 400, and Avance 300 spectrometers with standard pulse sequences operating at 400, 300 MHz in 1H NMR and 100, 75 MHz in 13C NMR. Chemical shifts are given in d values (ppm) using tetramethylsilane as the internal standard. IR spectra were recorded with Nexus 670 FT-IR FT-Ramen spectrometer as potassium bromide disks. UV spectra were recorded with Schimadzu UV-1601. HRESI-MS was taken on a Micromass Autospec (70 eV) spectrometer. Optical rotation values were measured by the use of an ATAGO POLAX-D polarimeter. Column chromatography (CC) was carried out on Polyamid 6S and Sephadex LH20. 1.2. Plant materials Leaves of C. roxburghii were collected in April 2007 from the Orman Garden. Identification of the plants was confirmed by the Department of Flora Agricultural Museum, Ministry of Agriculture and Herbarium of the Department of Botany, Faculty of Science, Cairo University. Voucher Specimens were kept in herbarium, National Research Centre, El-Tahrir Str., Dokki, Cairo, Egypt. 1.3. Extraction and isolation The leaves of C. roxburghii (1.25 g) were crushed and extracted with the aqueous alcoholic (MeOH–H2O, 7:3) by soaking at room temperature and the methanol extract was evaporated under reduced (diminished) pressure then defatted using successive extraction by pet ether then chloroform; the residue was extracted with n-BuOH, affording a dry extract (40 g), which was purified by chromatography on Polyamide 6 CC. The column was eluted with H2O and with H2O–EtOH step gradient and 4 fractions (1 L, each) were collected. Separation of fraction 2 on Sephadex LH-20 CC using n-BuOH saturated with H2O gave three subfractions I, II, and III, further purification of II on Sephadex CC using MeOH– H2O (3:7) afforded the new compound 1 (18 mg), while subfractions I and III have been separated on paper chromatography then purified on Sephadex CC using MeOH–H2O (1:1) as eluent, gave aloemodin 8-O-b-glucoside 3. Consecutive CC on Sephadex (nBuOH satd. with H2O for elution) of fraction 3 gave two subfractions, then one major of them has been applied on TLC Silica Gel plates using EtOAc–methanol–H2O (100:13.5:10) to afford the new compound 2 (20 mg). Separation of fraction 1 on preparative paper chromatography using n-BuOH–HOAc–H2O (4:1:5) gave fla-

Table 1 1 H NMR (400 MHz) assignments [d (ppm), J in Hz] of compounds 1and 2 Position

1

2

2 4 5 6 7 3-CH3 3-CH2OH 10 100 The rest sugar protons

7.16 d (1.8) 7.47 d (1.8) 6.91 d (2.1)

7.24 7.60 7.87 7.86 7.83

7.28 d (2.1) 2.4 s 5.30 d (7.2) 4.46 d (7.2) 3.72–3.26 m

d (1.2) d (1.2) dd, (8.2, 1.2) t (8.2) dd, (8.2, 1.2)

4.60 s 5.11 d (7.28) 4.23 d (7.28) 3.70–3.19 m

Table 2 13 C NMR data of compounds 1 and 2 in DMSO-d6 at 100 MHz Atom

1

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 3-CH3 Glucosyl 10 20 30 40 50 60 Glucosyl 100 200 300 400 500 600

160.81 124.61 147.18 119.56 108.35 162.21 107.39 164.65 186.79 182.55 136.86 113.08 114.76 132.36 21.75

160.06 12159 152.53 116.11 121.01 136.66 123.56 158.59 187.87 182.74 132.85 116.11 121.18 135.20 62.47 (3-CH2OH)

98.58 83.07 75.62 69.38 79.53 60.69

101.07 74.10 76.96 70.60 77.14 69.10

104.70 75.48 76.85 69.24 77.50 60.03

103.96 73.79 76.74 70.30 77.32 61.52

vonoid a quercetin-3-O-a-L-rhamnopyranoside. Using the same procedures fractions 4 and 5 gave chromatographically pure samples emodin 4 and aloemodin 5. 1.4. Acid hydrolysis of compounds 1 and 2 A solution of 5 mg of 1 in 10 mL MeOH–H2O (1:1) mixed with 5 mL 2 N HCl was heated at reflux at 60 °C for 6 h. After evaporation of the organic solvent under vacuum and threefold extraction of the aqueous phase with ethyl acetate, the aglycone was identified as emodin by NMR and by comparison with an authentic sample. The neutralized aqueous portion resulted in TLC detection of glucose by comparison with standard sugar (TLC: n-BuOH– EtOAc-i-PrOH–HOAc–H2O, 7:20:12:7:6 aniline phthalate spray reagent). Using the same procedure compound 2 gave aloemodin and glucose, which identified by comparison with authentic sample. 1.5. Identification 1.5.1. Emodin 1-O-b-D-glucopyranosyl-(1?2)-glucopyranoside (1) Orange amorphous powder; Rf = 0.66 and 0.70 (cellulose paper) in n-BuOH–HOAc–H2O (upper layer, 4:1:5) and 15% HOAc, respec-

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51

Table 3 DPPH radical scavenging activity of the total extract and compounds Compound

DPPH free radical scavenging activity IC50a (lg/mL)

Alcoholic extract Emodin 1-O-b-D-glucoside (2?1)glucopyranoside Aloemodin 8-O-b-D-glucoside (6?1)glucopyranoside Aloemodin 8-O-b-D-glucopyranodide Emodin Aloemodin Quercetin-3-O-a-L-rhamnopyranoside Ascorbic acid

37.96 ± 0.42 138.01 ± 2.82 132.01 ± 2.82 96.32 ± 0.32 71.65 ± 0.42 86.42 ± 0.92 29.49 ± 0.98 7.90 ± 0.21

a Values of IC50 were calculated as mean of triplicate determination ± standard deviation, concentrations in lg/mL required scavenging the DPPH radical (100 lg/mL) by 50%.

tively; [a]D +71.3 (c, 0.1, MeOH); UV kmax nm: 221, 271, 282, 415, IR, (KBr) cm1 3442, 1675, 1625, 1590, 1480, 1040; HRESI-MS m/z 617.1487 [M+Na]+; For 1H NMR (DMSO-d6) data see Table 1; For 13 C NMR data see Table 2. 1.5.2. Aloemodin 8-O-b-D-glucopyranosyl-(1?6)glucopyranoside (2) Yellowish-orange powder; Rf = 0.45 and 0.73 (cellulose paper) in n-BuOH–HOAc–H2O (upper layer, 4:1:5) and 15% HOAc, respectively; [a]D +48.4 (c, 0.1, MeOH); UV kmax nm: 219, 254, 269, 408; IR, (KBr) cm1 3410, 1645, 1638, 1602, 1458, 1070; HRESI-MS m/z 617.1495 [M+Na]+; For 1H NMR (DMSO-d6) data see Table 1; For 13 C NMR data see Table 2. 1.6. Scavenging activity towards DPPH radicals The DPPH assay was performed as described by Shirwaikar et al.24 This method depends on the reduction of the purple DPPH radicals to a yellow colored diphenyl picrylhydrazine and the remaining DPPH radicals, which showed maximum absorption at 517 nm were measured; 2 mL of various concentrations of each compound were added to 2 mL solution 0.1 mM DPPH. An equal amount of methanol and DPPH served as control. After 20 min of incubation at 37 °C in the dark, the absorbance was recorded at 517 nm. The experiment was performed in triplicates. The DPPH radical scavenging activity was calculated according to the following equation: % DPPH radical scavenging activity = 1[Asample/Acontrol]  100 where Asample and Acontrol are absorbance of sample and control. The SC50 (concentration of sample required to scavenge 50% of DPPH radicals) values were determined. Decreasing of the DPPH solution absorbance indicated as increase of the DPPH radicals scavenging activity.

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