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A new antioxidant stilbene and other constituents from the stem bark of Morus nigra L. a
a
a
Ghada M. Abbas , Fatma M. Abdel Bar , Hany N. Baraka , Ahmed a
a
A. Gohar & Mohammed-Farid Lahloub a
Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt Published online: 28 Mar 2014.
To cite this article: Ghada M. Abbas, Fatma M. Abdel Bar, Hany N. Baraka, Ahmed A. Gohar & Mohammed-Farid Lahloub (2014) A new antioxidant stilbene and other constituents from the stem bark of Morus nigra L., Natural Product Research: Formerly Natural Product Letters, 28:13, 952-959, DOI: 10.1080/14786419.2014.900770 To link to this article: http://dx.doi.org/10.1080/14786419.2014.900770
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Natural Product Research, 2014 Vol. 28, No. 13, 952–959, http://dx.doi.org/10.1080/14786419.2014.900770
A new antioxidant stilbene and other constituents from the stem bark of Morus nigra L. Ghada M. Abbas, Fatma M. Abdel Bar, Hany N. Baraka, Ahmed A. Gohar and Mohammed-Farid Lahloub* Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
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(Received 11 December 2013; final version received 2 March 2014) A new stilbene, 20 ,3,40 ,5,50 -pentahydroxy-cis-stilbene (1), along with 13 known compounds, resveratrol (2), oxyresveratrol (3), norartocarpetin (4), kuwanon C (5), morusin (6), cudraflavone A (7), kuwanon G (8), albafurane C (9), mulberrofuran G (10), 3-O-acetyl-a-amyrin (11), 3-O-acetyl-b-amyrin (12) ursolic acid-3-O-acetate (13) and uvaol (14), were isolated from the barks of Morus nigra. Compounds 2, 8, 10, 12 and 14 are reported for the first time from this plant. The isolated compounds were elucidated by means of 1D and 2D NMR, UV, IR and MS, as well as by comparison with the literature data. The isolated compounds and the different extracts were evaluated for their potential antioxidant activity using 2,20 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)þ radical-scavenging capacity assay and compared with ascorbic acid. The new stilbene (1) exhibited remarkable antioxidant capacity with IC50 of 4.69 mM. Keywords: Morus; Morus nigra; Moraceae; stilbenes; prenylated flavonoids; antioxidants
1. Introduction There is a great interest in determining the role of phytonutrients in promoting improved health and in reducing cancer, cardiovascular disease and the effects of ageing. It is widely believed that antioxidant phytonutrients can inhibit the propagation of free radical reactions that may ultimately lead to the development of diseases, especially those which are related to ageing. Many fruits and vegetables possess strong antioxidant capacities, and this capacity is due primarily to non-vitamin C phytochemicals (Wang et al. 1997; Prior et al. 1998). The fruits, roots and barks of Morus nigra have been used in folk medicine to treat diabetes, hypertension, anaemia and arthritis (Ozgen et al. 2009). It has been reported with a long history in Chinese herbal medicine as ‘Sang Bai-Pi’ (Nomura 1988). Morus contains a variety of phenolic compounds including isoprenylated flavonoids, stilbenes, 2-arylbenzofurans and a variety of Diels –Alder adducts (Nomura 1988; Nomura and Hano 1994). The bark of M. nigra was reputed for its use to expel tapeworm, and its extracts have been reported to possess antibacterial and fungicidal activities (Mazimba et al. 2011). In addition, it was used for toothache (Naderi et al. 2004) and as a purgative (Hanif and Singh 2012). The EtOAc extract of the stem bark of M. nigra has been reported to possess anti-inflammatory and antioxidative activities (Wang et al. 2010). In the course of our studies on antioxidant plants from Egypt, we investigated the methanol extract of the stem barks of M. nigra L. Moraceae. The extract exhibited a moderate antioxidant
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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activity against 2,20 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)þ (ABTS)þ radical (41.73%). The methanol extract was then partitioned with different solvent with increasing polarity to afford n-hexane, methylene chloride and ethyl acetate fractions. Consequently, the extracts were subjected to extensive chromatographic separation to explore their components as well as their antioxidant principles. In this study, a new antioxidant stilbene 1 was isolated together with 13 known compounds 2 –14, most of them exhibited remarkable antioxidant activities using ABTSþ radicalscavenging capacity in vitro assay relative to the standard ascorbic acid.
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2. Results and discussion Intensive repeated column chromatography of the n-hexane, CH2Cl2 and EtOAc fractions of the methanol extract of the barks of M. nigra, using silica gel or Sephadex LH20 columns and elution with different combinations of solvents, afforded two compounds 11, 12 from the n-hexane fraction, five compounds 5 – 7, 13, and 14 from the CH2Cl2 fraction and seven compounds 1 –4 and 8– 10 from the EtOAc fraction, respectively. Compound 1 was obtained as creamy white needles, Rƒ 0.47 [silica gel F254 plates, CH2Cl2/ MeOH (9:1)]. It yielded fluorescence under the UV light at l365 and a grey colour after heating with vanillin – sulphuric acid spray reagent. The molecular formula of compound 1 was established as C14H12O5 from its [M]þ ion at m/z 260.26 and [M 2 H2O]þ ion at m/z 242.05 (calcd for C14H12O5 260.2). The UV absorption maxima at 315 and 225 nm were characteristic of a stilbene skeleton (Gorham 1995). The IR spectrum displayed characteristic aromatic and olefinic absorptions at nmax 1510, 1579 and 1614 cm21, in addition to hydroxyl groups at nmax 3520 and 3431 cm21. The APT spectrum revealed the presence of 12 olefinic and/or aromatic carbon signals. The 1H NMR spectrum of 1 indicated two cis-olefinic proton signals at dH 6.74 (1H, d, J ¼ 8.4 Hz) and 7.37 (1H, d, J ¼ 8.4 Hz) assigned to H-a and H-b, respectively, and three aromatic proton resonances at dH 6.77 (2H, d, J ¼ 2.0 Hz, H-2/6) and 6.26 (1H, s, H-4) of a symmetric A-ring. The HMBC of 1 correlated the proton signal at dH 6.26 (H-4) with the quaternary carbon signal at dC 158.6 (H-3/5) and with the methine carbon signal at dC 102.5 (C-2/6). Furthermore, the proton signal H-2/6 is correlated in HMBC with the carbon signal C-3/5 and with the carbon signal C-2/6. All these findings confirmed the 3,5-dihydroxylation pattern of A-ring. Moreover, B-ring displayed two aromatic proton signals at dH 6.92 (1H, br s, H-30 ) and 6.93 (1H, br s, H-60 ) suggesting 20 ,40 ,50 -trihydroxylated phenyl moiety. The hydroxylation pattern of B-ring was confirmed through HMBC correlations of the proton signal at dH 6.92 (H-30 ) with the quaternary carbon signals at dC 155.8 (C-50 ) and 121.6 (C-10 ). In addition, the proton signal at dH 6.93 (H-60 ) revealed HMBC correlations with the carbon signals at dC 155.5 (C-40 ) and 111.8 (C-b). Both protons H-30 and H-60 indicated no correlations to the carbons of each other (97.1, C-30 ) or (100.8, C-60 ) which confirmed their para position to each other. Thus, 20 ,40 ,50 -trihydroxy substitution was confirmed for B-ring. From the previous data, compound 1 was confirmed to be 20 ,3,40 ,5,50 -pentahydroxy-cis-stilbene. This compound is reported for the first time from natural source. The structures of the remaining 13 compounds 2 –14 (Figure 1) were elucidated by comparing their 1H and 13C NMR spectral data with those reported in the literature as resveratrol (2) (Jayatilake et al. 1993; Sivakumar et al. 2013), oxyresveratrol (3) (Povichit et al. 2010), norartocarpetin (4) (Lin et al. 1995; Soekamto et al. 2009), kuwanon C (5) (Nomura et al. 1978; Agrawal et al. 1989), morusin (6) (Nomura et al. 1976, Agrawal et al. 1989; Tesing et al. 2010), cudraflavone A (7) (Fujimoto et al. 1984; Agrawal et al. 1989), kuwanon G (8) (Oshima et al. 1980; Takasugi et al. 1980; Nomura et al. 1981), albafuran C (9) (Takasugi et al. 1982; Hong et al. 2013), mulberrofuran G (10) (Fukai et al. 1984), 3-O-acetyl-a-amyrin (11) and 3-O-acetyl-
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H
β
α 2 HO
A
3
OH HO 2`
1`
1 6 6` 5
4
5`
6
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4´ 2
3 HO
2R=H 3 R = OH
OH
OH HO 1
3´
6´
4
4`
5
R
20
HO 8
O
3´
2´
16 O1
1´ 6´
5
10
OH
16
11
4
O
9
6
5
10
OH 14
12
O
8
7
2
4´
1´
5´
12
15
16´´ 17´´ HO 18´´ 19´´ 13´´ 12´´ HO
5 2´´ 3´´ 5 4
20´´ 4´´ 6 14´´ 8 9´´ O HO 9 10´´ OH 11´´
OH 3a
7a
8´´
2´ 3´
3
1´
HO
3a 2`
2
7a O
O
6´
5´ OH
10`` O
HO
3` H
1` 6`
4 5` OH 10
3``
H
2``
8
7
11 1 2
10
9
8
4
3 R1
O
23
5 24
6
2´
OH 11
3
O
14
12
13
9``O 16`` 17`` OH 8`` 18`` H 19`` 15`` 4`` 20`` 5``
15
6`` 1`` 7`` 30
29 19
20 12 11 25 1 2
O
15
7 4
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8
16 R2 27
2
10
5 OH
22 17 28
26 14
3´
9 6
21
18 13
12 25
19
4´ OH
O
30 29
5´
3´´ 6´
14``
3
6 7
4´
4´´
O HO
13``
11``
2 7
9´´
OH
1´´ 2´´
12``
4
1´´
6´´ 15´´5´´
11´´ 10´´
HO
7´´ OH
7
6´´
14´´ 5´´
12´´
15
OH
15´´
20´´ 13´´
HO
13
7
16´´
19´´
14
13
6
5 R = prenyl
17´´
11
O
4R=H
R
18´´
O
3
4
6`
3
HO
OH
2´ 6´
17
3
6
18 O
19
5´
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7
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OH
4 O
OH
4´ 9
10
OH 4` 5`
2
5´
2´
3`
2` 1`
O
9
6
3´ 17
18
8
7
OH
1´
β
HMBC correlation
19
HO
R
α
1
3`
B
11 R1 = acetyl R2 = CH3
10
C
O
4 23
22 17
28
16 27 15
7
5 24
18
14 8
3 H3C
21 13
26 9
20
6
12
13 R1 = acetyl R2 = COOH 14 R1 = H = CH2OH
Figure 1. Structures of compounds 1 – 14 isolated from M. nigra.
b-amyrin (12) (Mahato and Kundu 1994; Bandeira et al. 2007), ursolic acid-3-O-acetate (13) (Mahato and Kundu 1994) and uvaol (14) (Mahato and Kundu 1994; Zhang et al. 2012). Free radical-scavenging activities of the total MeOH extract and fractions were evaluated by the ABTSþ radical-scavenging assay (Figure 2). Among the investigated fractions, the EtOAc and the CH2Cl2 fractions exhibited remarkable free radical-scavenging activity (88.07% and 84.80% inhibition, respectively). Free hydroxyl groups in stilbenes 1 –3 increase the free radicalscavenging activity as indicated from their percentage inhibition of ABTSþ radical (88.46%, 86.17% and 87.69%, respectively). The new stilbene 1 isolated from the EtOAc fraction exhibited the highest antioxidant activity. A concentration – response curve of 1 (Figure 3) indicated that this compound has promising antioxidant capacity with IC50 of 4.69 mM. The free radical-scavenging profile of 1 indicated that it increases by time and becomes constant after 10 min of treatment (Figure 4). For the flavonoidal compounds, prenylation decreases the antioxidant activity since compound 4 which possesses free 3 and 8 positions exhibits higher free radical-scavenging activities (86.53%) compared with 5 with two prenyl groups at 3 and 8 positions (80.38%). Furthermore, cyclisation of the prenyl group decreases the antioxidant activity of the prenylated flavonoids, since 5 exhibited a higher antioxidant activity (80.38%) than 6 (61.34%), in which the prenyl group at position 8 is cyclised forming a pyran ring. This relationship was further established in the case of 7 where the two prenyl groups at 3 and 6
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Fractions and isolated compounds 100
88.07
90
84.8
88.46 86.17 87.69 86.53
87.5
87.11 85.96 85.19 80.38
80
% inhibition
70
61.34
60 50
41.73
40
34.42
30 20
15
10
5.76
9.42 5
Figure 2. Antioxidant evaluation of the different fractions and compounds 1 – 14 using ABTSþ radicalscavenging assay using ascorbic acid as a standard (at a concentration ¼ 50 mg, % inhibition ^ SD).
positions are cyclised; therefore, the antioxidant activity nearly disappeared (15%). Formation of Diels –Alder adducts between a flavonoid and a chalcone increases the antioxidant activity. This was obvious in the case of compound 8 which is formed through a Diels –Alder type reaction of a chalcone and dehydroprenylkuwanon C, 5 (Gunawan 2011) which results in higher antioxidant activity (87.11%) in 8 than in 5 (80.38%).
100
80
% Inhibition
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0
60
40
20
0 0
3.125
6.25
12.5
25
50
100
Concentration (µM)
Figure 3. Concentration – response curve of compound 1 for the inhibition of ABTSþ radical at l734 nm.
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Absorbance (734 nm)
0.25
50 µM 25 µM
0.2
12.5 µM 6.25 µM
0.15
3.125 µM
0.1
0.05
0 0
5
10
15
20
Time (min)
Figure 4. Time profile of the suppression of absorbance of ABTSþ radical (l734 nm) at different concentrations of compound 1.
3. Experimental 3.1. General methods Melting points were determined on Fisher-johns Scientific Co., U.S.A melting point apparatus (Waltham, MA, USA) and are uncorrected. UV spectra were recorded on a Shimadzu 1601 PC, model TCC240, spectrophotometer (Shimadzu, Kyoto, Japan). Mass spectrometry (EIMS) spectra were obtained using a Thermo Scientific DSQTM II instrument (Austin, TX, USA). FTIR spectra were obtained using a Thermo Scientific FT-IR spectrometer (Thermo Fisher Scientific Co., Madison, WI, USA). The 1D- and 2D-NMR spectra were performed on Bruker-500 and Bruker-400 AscendTM spectrometer (Bruker Daltonics, Bremen, Germany). Silica gel G60-230 (Merck, Darmstadt, Germany), Sephadex LH-20 Sigma-Aldrich (Missouri, USA) and RPC18 (BAKERBONDwOctadecyl, C18) 40 mm, Prep LC Packing (Phillipsburg, NJ, USA). Thin layer chromatography (TLC) was performed on silica gel F254 plates (Machery-Nagel, Germany) using vanillin-sulphuric acid, 5% AlCl3 and FeCl3 spray reagents. The solvents used were of reagent grade (El-Nasr Co., Cairo, Egypt). For the ABTSþ radical antioxidant assay: ABTS (2,20 -Azino-bis-(3-ethylbenzthiazoline-6sulfonic acid) diammonium salt (Fluka, Seelze, Germany) and MnO2 (Oxford Laboratory, Mumbai, India).
3.2. Plant material The plant material was collected in September 2009, from Mansoura University, Dakahlia, Egypt. The plant was identified by Prof. Ibrahim Mashaly, Systematic Botany Department, Faculty of Sciences, Mansoura University. The stem barks were separated from the plant, shade dried, powdered and kept for further investigations. A voucher specimen has been deposited in the herbarium of the College of Pharmacy, Mansoura University (009Mansoura-4).
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3.3. Extraction and isolation M. nigra stem barks (4.0 kg) were exhausted with methanol and the extract evaporated under reduced pressure to yield 570 g; the extract was partitioned successively with n-hexane, CH2Cl2 and EtOAc. The EtOAc fraction (90 g) was chromatographed over a silica gel column (54 cm £ 4.5 cm i.d., 470 g) using CH2Cl2 –EtOAc mixtures of increasing polarity starting with 100% CH2Cl2 and the effluents (250 mL each) were collected. Fractions 27–30 (220 mg) eluted with CH2Cl2 –EtOAc (8:2) were re-chromatographed over a silica gel column (40 cm £ 1.2 cm i. d., 125 g) packed in 100% CH2Cl2 and eluted with CH2Cl2 –MeOH mixtures of increasing polarity and 25 mL fractions were collected. Sub-fractions 25–92 (200 mg) eluted with CH2Cl2 –MeOH (9.8:0.2) were further purified by Sephadex LH-20 using MPLC column (20 cm £ 3 cm i.d., 5 g) and elution with CH2Cl2 –MeOH (8:2) and 15 mL fractions were collected. Fractions 40–43 afforded compound 2 (60 mg) and fractions 44–49 afforded compound 1 (80 mg); both compounds have the same Rƒ value (0.47) using CH2Cl2 –MeOH (9:1). However, spot of 1 on TLC revealed a blue fluorescence under UV365 light and a grey colour after heating with vanillin– H2SO4 spray reagent while, spot of 2 revealed a reddish brown colour with no fluorescence under UV365 light. (Isolation procedure for compounds 3–14 is supplied in the supplementary materials). 3.4. Antioxidant capacity assay The procedures employed were adapted to those described in Modak et al. (2007), Aliaga and Lissi (2000) and El-Gammal et al. (2012). For each of the investigated compounds, 2 mL of ABTS solution (60 mM) was added to 3 mL of MnO2 solution (25 mg/mL), all prepared in 5 mL aqueous phosphate buffer solution (pH 7, 0.1 M). The mixture was shaken, centrifuged and decanted. Formation of the radical is almost instantaneous and stable for several hours (Aliaga and Lissi 2000). The absorbance of the resulting blue/green solution (ABTSþ radical solution) at l734 nm was adjusted to approximately 0.5 (Acontrol). The absorbance (Atest) was measured upon the addition of 50 mL of 1 mg/mL solution of the test sample (isolated compounds and extracts) or aliquots of the new stilbene 1 at concentrations within the activity range of the assay (3.125, 6.25, 12.5, 25, 50 and 100 mM final concentrations) in spectroscopic grade MeOH – buffer (1:1 v/v) to the ABTS solution. Ascorbic acid, 50 mL of 1 mg/mL solution, was used as standard antioxidant. Duplicate of each sample was used in this assay. Blank sample was run without ABTS and using MeOH – phosphate buffer (1:1) instead of tested compounds. Negative control was run with ABTS and MeOH – phosphate buffer (1:1) only. The decrease in absorbance is expressed as % inhibition which is calculated from the equation: (Acontrol 2 Atest)/Acontrol £ 100. Compound 1: m.p. 250– 2528C; UV [MeOH] lmax 315 and 225 nm; IR nmax (KBr): 3520, 3431, 2957, 2922, 2852, 1659, 1579, 1510, 1436, 1142, 1121, 842, 818 and 800 cm21; 1H NMR (400 MHz, CD3OD): dH 6.26 [1H, s, H-4], 6.74 [1H, d, J ¼ 8.4 Hz, H-a], 6.77 [2H, d, J ¼ 2.0 Hz, H-2/6], 6.92 [1H, br s, H-30 ], 6.93 [1H, br s, H-60 ] and 7.37 [1H, d, J ¼ 8.4 Hz, b]; 13 C NMR (100 MHz, CD3OD): dC 97.1 (C-30 ), 100.8 (C-60 ), 102.1 (C-4), 102.5 (C-2/6), 111.8 (C-b), 120.6 (C-a), 121.6 (C-10 ), 132.4 (C-1), 154.7 (C-20 ), 155.5 (C-40 ), 155.8 (C-50 ) and 158.6 (C-3/5); EI-MS: m/z 260.2 [Mþ], 242.1 [M 2 H2O]þ (Calcd for C14H12O5: 260.2593). Spectroscopic data for compounds 2 –14 are supplied in the supplementary materials. 4. Conclusions In this study, we reported the isolation of a new stilbene (20 ,3,40 ,5,50 -pentahydroxy-cis-stilbene) from the stem bark of M. nigra L. Several chemically diverse stilbenes and prenylated flavonoids were also isolated. The isolated compounds and the different extracts from the stem bark of M. nigra were evaluated for their ABTSþ radical-scavenging capacity. The new stilbene 1 exhibited high antioxidant activity with IC50 4.69 mM.
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Supplementary material Supplementary material relating to this article is available online, alongside Flowcharts S1 – S3 and Tables S1 –S6.
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Soekamto NH, Nafie NL, Mandey FW, Garson M. 2009. Norartocarpetin, flavone derivative from leaves of Artocarpus fretessi. Indo J Chem. 9:328–331. Takasugi M, Ishikawa S, Nagao S, Masamune T. 1982. Studies on phytoalexins of the Moraceae. Part 12. Albafuran-C, a natural Diels –Alder adduct of a dehydroprenyl-2-phenylbenzofuran with a chalcone from mulberry. Chem Lett. 8:1223–1224. Takasugi M, Ishikawa SI, Nagao S, Masamune T, Shirata A, Takahashi K. 1980. Albanins F and G, natural Diels–Alder adducts from mulberry. Chem Lett. 108:1577–1580. Tesing TH, Chuang SK, Hu CC, Chang CF, Huang YC, Lin CW, Lee YJ. 2010. The synthesis of morusin as a potent antitumor agent. Tetrahedron. 66:1335–1340. Wang H, Cao G, Prior RL. 1997. Oxygen radical absorbing capacity of anthocyanins. J Agric Food Chem. 45:304–309. Wang L, Yang Y, Liu C, Chen RY. 2010. Three new compounds from Morus nigra L. J Asian Nat Prod Res. 12:431–437. Zhang JY, Li N, Hu K, Tu PF. 2012. Chemical constituents from processed seeds of Strychnos nux-vomica. J Chin Pharm Sci. 21:187–191.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new antioxidant stilbene and other constituents from the stem bark of Morus nigra L. a
a
a
Ghada M. Abbas , Fatma M. Abdel Bar , Hany N. Baraka , Ahmed a
a
A. Gohar & Mohammed-Farid Lahloub a
Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt Published online: 28 Mar 2014.
To cite this article: Ghada M. Abbas, Fatma M. Abdel Bar, Hany N. Baraka, Ahmed A. Gohar & Mohammed-Farid Lahloub (2014) A new antioxidant stilbene and other constituents from the stem bark of Morus nigra L., Natural Product Research: Formerly Natural Product Letters, 28:13, 952-959, DOI: 10.1080/14786419.2014.900770 To link to this article: http://dx.doi.org/10.1080/14786419.2014.900770
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Natural Product Research, 2014 Vol. 28, No. 13, 952–959, http://dx.doi.org/10.1080/14786419.2014.900770
A new antioxidant stilbene and other constituents from the stem bark of Morus nigra L. Ghada M. Abbas, Fatma M. Abdel Bar, Hany N. Baraka, Ahmed A. Gohar and Mohammed-Farid Lahloub* Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
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(Received 11 December 2013; final version received 2 March 2014) A new stilbene, 20 ,3,40 ,5,50 -pentahydroxy-cis-stilbene (1), along with 13 known compounds, resveratrol (2), oxyresveratrol (3), norartocarpetin (4), kuwanon C (5), morusin (6), cudraflavone A (7), kuwanon G (8), albafurane C (9), mulberrofuran G (10), 3-O-acetyl-a-amyrin (11), 3-O-acetyl-b-amyrin (12) ursolic acid-3-O-acetate (13) and uvaol (14), were isolated from the barks of Morus nigra. Compounds 2, 8, 10, 12 and 14 are reported for the first time from this plant. The isolated compounds were elucidated by means of 1D and 2D NMR, UV, IR and MS, as well as by comparison with the literature data. The isolated compounds and the different extracts were evaluated for their potential antioxidant activity using 2,20 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)þ radical-scavenging capacity assay and compared with ascorbic acid. The new stilbene (1) exhibited remarkable antioxidant capacity with IC50 of 4.69 mM. Keywords: Morus; Morus nigra; Moraceae; stilbenes; prenylated flavonoids; antioxidants
1. Introduction There is a great interest in determining the role of phytonutrients in promoting improved health and in reducing cancer, cardiovascular disease and the effects of ageing. It is widely believed that antioxidant phytonutrients can inhibit the propagation of free radical reactions that may ultimately lead to the development of diseases, especially those which are related to ageing. Many fruits and vegetables possess strong antioxidant capacities, and this capacity is due primarily to non-vitamin C phytochemicals (Wang et al. 1997; Prior et al. 1998). The fruits, roots and barks of Morus nigra have been used in folk medicine to treat diabetes, hypertension, anaemia and arthritis (Ozgen et al. 2009). It has been reported with a long history in Chinese herbal medicine as ‘Sang Bai-Pi’ (Nomura 1988). Morus contains a variety of phenolic compounds including isoprenylated flavonoids, stilbenes, 2-arylbenzofurans and a variety of Diels –Alder adducts (Nomura 1988; Nomura and Hano 1994). The bark of M. nigra was reputed for its use to expel tapeworm, and its extracts have been reported to possess antibacterial and fungicidal activities (Mazimba et al. 2011). In addition, it was used for toothache (Naderi et al. 2004) and as a purgative (Hanif and Singh 2012). The EtOAc extract of the stem bark of M. nigra has been reported to possess anti-inflammatory and antioxidative activities (Wang et al. 2010). In the course of our studies on antioxidant plants from Egypt, we investigated the methanol extract of the stem barks of M. nigra L. Moraceae. The extract exhibited a moderate antioxidant
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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activity against 2,20 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)þ (ABTS)þ radical (41.73%). The methanol extract was then partitioned with different solvent with increasing polarity to afford n-hexane, methylene chloride and ethyl acetate fractions. Consequently, the extracts were subjected to extensive chromatographic separation to explore their components as well as their antioxidant principles. In this study, a new antioxidant stilbene 1 was isolated together with 13 known compounds 2 –14, most of them exhibited remarkable antioxidant activities using ABTSþ radicalscavenging capacity in vitro assay relative to the standard ascorbic acid.
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2. Results and discussion Intensive repeated column chromatography of the n-hexane, CH2Cl2 and EtOAc fractions of the methanol extract of the barks of M. nigra, using silica gel or Sephadex LH20 columns and elution with different combinations of solvents, afforded two compounds 11, 12 from the n-hexane fraction, five compounds 5 – 7, 13, and 14 from the CH2Cl2 fraction and seven compounds 1 –4 and 8– 10 from the EtOAc fraction, respectively. Compound 1 was obtained as creamy white needles, Rƒ 0.47 [silica gel F254 plates, CH2Cl2/ MeOH (9:1)]. It yielded fluorescence under the UV light at l365 and a grey colour after heating with vanillin – sulphuric acid spray reagent. The molecular formula of compound 1 was established as C14H12O5 from its [M]þ ion at m/z 260.26 and [M 2 H2O]þ ion at m/z 242.05 (calcd for C14H12O5 260.2). The UV absorption maxima at 315 and 225 nm were characteristic of a stilbene skeleton (Gorham 1995). The IR spectrum displayed characteristic aromatic and olefinic absorptions at nmax 1510, 1579 and 1614 cm21, in addition to hydroxyl groups at nmax 3520 and 3431 cm21. The APT spectrum revealed the presence of 12 olefinic and/or aromatic carbon signals. The 1H NMR spectrum of 1 indicated two cis-olefinic proton signals at dH 6.74 (1H, d, J ¼ 8.4 Hz) and 7.37 (1H, d, J ¼ 8.4 Hz) assigned to H-a and H-b, respectively, and three aromatic proton resonances at dH 6.77 (2H, d, J ¼ 2.0 Hz, H-2/6) and 6.26 (1H, s, H-4) of a symmetric A-ring. The HMBC of 1 correlated the proton signal at dH 6.26 (H-4) with the quaternary carbon signal at dC 158.6 (H-3/5) and with the methine carbon signal at dC 102.5 (C-2/6). Furthermore, the proton signal H-2/6 is correlated in HMBC with the carbon signal C-3/5 and with the carbon signal C-2/6. All these findings confirmed the 3,5-dihydroxylation pattern of A-ring. Moreover, B-ring displayed two aromatic proton signals at dH 6.92 (1H, br s, H-30 ) and 6.93 (1H, br s, H-60 ) suggesting 20 ,40 ,50 -trihydroxylated phenyl moiety. The hydroxylation pattern of B-ring was confirmed through HMBC correlations of the proton signal at dH 6.92 (H-30 ) with the quaternary carbon signals at dC 155.8 (C-50 ) and 121.6 (C-10 ). In addition, the proton signal at dH 6.93 (H-60 ) revealed HMBC correlations with the carbon signals at dC 155.5 (C-40 ) and 111.8 (C-b). Both protons H-30 and H-60 indicated no correlations to the carbons of each other (97.1, C-30 ) or (100.8, C-60 ) which confirmed their para position to each other. Thus, 20 ,40 ,50 -trihydroxy substitution was confirmed for B-ring. From the previous data, compound 1 was confirmed to be 20 ,3,40 ,5,50 -pentahydroxy-cis-stilbene. This compound is reported for the first time from natural source. The structures of the remaining 13 compounds 2 –14 (Figure 1) were elucidated by comparing their 1H and 13C NMR spectral data with those reported in the literature as resveratrol (2) (Jayatilake et al. 1993; Sivakumar et al. 2013), oxyresveratrol (3) (Povichit et al. 2010), norartocarpetin (4) (Lin et al. 1995; Soekamto et al. 2009), kuwanon C (5) (Nomura et al. 1978; Agrawal et al. 1989), morusin (6) (Nomura et al. 1976, Agrawal et al. 1989; Tesing et al. 2010), cudraflavone A (7) (Fujimoto et al. 1984; Agrawal et al. 1989), kuwanon G (8) (Oshima et al. 1980; Takasugi et al. 1980; Nomura et al. 1981), albafuran C (9) (Takasugi et al. 1982; Hong et al. 2013), mulberrofuran G (10) (Fukai et al. 1984), 3-O-acetyl-a-amyrin (11) and 3-O-acetyl-
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26 9
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13 R1 = acetyl R2 = COOH 14 R1 = H = CH2OH
Figure 1. Structures of compounds 1 – 14 isolated from M. nigra.
b-amyrin (12) (Mahato and Kundu 1994; Bandeira et al. 2007), ursolic acid-3-O-acetate (13) (Mahato and Kundu 1994) and uvaol (14) (Mahato and Kundu 1994; Zhang et al. 2012). Free radical-scavenging activities of the total MeOH extract and fractions were evaluated by the ABTSþ radical-scavenging assay (Figure 2). Among the investigated fractions, the EtOAc and the CH2Cl2 fractions exhibited remarkable free radical-scavenging activity (88.07% and 84.80% inhibition, respectively). Free hydroxyl groups in stilbenes 1 –3 increase the free radicalscavenging activity as indicated from their percentage inhibition of ABTSþ radical (88.46%, 86.17% and 87.69%, respectively). The new stilbene 1 isolated from the EtOAc fraction exhibited the highest antioxidant activity. A concentration – response curve of 1 (Figure 3) indicated that this compound has promising antioxidant capacity with IC50 of 4.69 mM. The free radical-scavenging profile of 1 indicated that it increases by time and becomes constant after 10 min of treatment (Figure 4). For the flavonoidal compounds, prenylation decreases the antioxidant activity since compound 4 which possesses free 3 and 8 positions exhibits higher free radical-scavenging activities (86.53%) compared with 5 with two prenyl groups at 3 and 8 positions (80.38%). Furthermore, cyclisation of the prenyl group decreases the antioxidant activity of the prenylated flavonoids, since 5 exhibited a higher antioxidant activity (80.38%) than 6 (61.34%), in which the prenyl group at position 8 is cyclised forming a pyran ring. This relationship was further established in the case of 7 where the two prenyl groups at 3 and 6
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Fractions and isolated compounds 100
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60 50
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Figure 2. Antioxidant evaluation of the different fractions and compounds 1 – 14 using ABTSþ radicalscavenging assay using ascorbic acid as a standard (at a concentration ¼ 50 mg, % inhibition ^ SD).
positions are cyclised; therefore, the antioxidant activity nearly disappeared (15%). Formation of Diels –Alder adducts between a flavonoid and a chalcone increases the antioxidant activity. This was obvious in the case of compound 8 which is formed through a Diels –Alder type reaction of a chalcone and dehydroprenylkuwanon C, 5 (Gunawan 2011) which results in higher antioxidant activity (87.11%) in 8 than in 5 (80.38%).
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Figure 3. Concentration – response curve of compound 1 for the inhibition of ABTSþ radical at l734 nm.
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Figure 4. Time profile of the suppression of absorbance of ABTSþ radical (l734 nm) at different concentrations of compound 1.
3. Experimental 3.1. General methods Melting points were determined on Fisher-johns Scientific Co., U.S.A melting point apparatus (Waltham, MA, USA) and are uncorrected. UV spectra were recorded on a Shimadzu 1601 PC, model TCC240, spectrophotometer (Shimadzu, Kyoto, Japan). Mass spectrometry (EIMS) spectra were obtained using a Thermo Scientific DSQTM II instrument (Austin, TX, USA). FTIR spectra were obtained using a Thermo Scientific FT-IR spectrometer (Thermo Fisher Scientific Co., Madison, WI, USA). The 1D- and 2D-NMR spectra were performed on Bruker-500 and Bruker-400 AscendTM spectrometer (Bruker Daltonics, Bremen, Germany). Silica gel G60-230 (Merck, Darmstadt, Germany), Sephadex LH-20 Sigma-Aldrich (Missouri, USA) and RPC18 (BAKERBONDwOctadecyl, C18) 40 mm, Prep LC Packing (Phillipsburg, NJ, USA). Thin layer chromatography (TLC) was performed on silica gel F254 plates (Machery-Nagel, Germany) using vanillin-sulphuric acid, 5% AlCl3 and FeCl3 spray reagents. The solvents used were of reagent grade (El-Nasr Co., Cairo, Egypt). For the ABTSþ radical antioxidant assay: ABTS (2,20 -Azino-bis-(3-ethylbenzthiazoline-6sulfonic acid) diammonium salt (Fluka, Seelze, Germany) and MnO2 (Oxford Laboratory, Mumbai, India).
3.2. Plant material The plant material was collected in September 2009, from Mansoura University, Dakahlia, Egypt. The plant was identified by Prof. Ibrahim Mashaly, Systematic Botany Department, Faculty of Sciences, Mansoura University. The stem barks were separated from the plant, shade dried, powdered and kept for further investigations. A voucher specimen has been deposited in the herbarium of the College of Pharmacy, Mansoura University (009Mansoura-4).
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3.3. Extraction and isolation M. nigra stem barks (4.0 kg) were exhausted with methanol and the extract evaporated under reduced pressure to yield 570 g; the extract was partitioned successively with n-hexane, CH2Cl2 and EtOAc. The EtOAc fraction (90 g) was chromatographed over a silica gel column (54 cm £ 4.5 cm i.d., 470 g) using CH2Cl2 –EtOAc mixtures of increasing polarity starting with 100% CH2Cl2 and the effluents (250 mL each) were collected. Fractions 27–30 (220 mg) eluted with CH2Cl2 –EtOAc (8:2) were re-chromatographed over a silica gel column (40 cm £ 1.2 cm i. d., 125 g) packed in 100% CH2Cl2 and eluted with CH2Cl2 –MeOH mixtures of increasing polarity and 25 mL fractions were collected. Sub-fractions 25–92 (200 mg) eluted with CH2Cl2 –MeOH (9.8:0.2) were further purified by Sephadex LH-20 using MPLC column (20 cm £ 3 cm i.d., 5 g) and elution with CH2Cl2 –MeOH (8:2) and 15 mL fractions were collected. Fractions 40–43 afforded compound 2 (60 mg) and fractions 44–49 afforded compound 1 (80 mg); both compounds have the same Rƒ value (0.47) using CH2Cl2 –MeOH (9:1). However, spot of 1 on TLC revealed a blue fluorescence under UV365 light and a grey colour after heating with vanillin– H2SO4 spray reagent while, spot of 2 revealed a reddish brown colour with no fluorescence under UV365 light. (Isolation procedure for compounds 3–14 is supplied in the supplementary materials). 3.4. Antioxidant capacity assay The procedures employed were adapted to those described in Modak et al. (2007), Aliaga and Lissi (2000) and El-Gammal et al. (2012). For each of the investigated compounds, 2 mL of ABTS solution (60 mM) was added to 3 mL of MnO2 solution (25 mg/mL), all prepared in 5 mL aqueous phosphate buffer solution (pH 7, 0.1 M). The mixture was shaken, centrifuged and decanted. Formation of the radical is almost instantaneous and stable for several hours (Aliaga and Lissi 2000). The absorbance of the resulting blue/green solution (ABTSþ radical solution) at l734 nm was adjusted to approximately 0.5 (Acontrol). The absorbance (Atest) was measured upon the addition of 50 mL of 1 mg/mL solution of the test sample (isolated compounds and extracts) or aliquots of the new stilbene 1 at concentrations within the activity range of the assay (3.125, 6.25, 12.5, 25, 50 and 100 mM final concentrations) in spectroscopic grade MeOH – buffer (1:1 v/v) to the ABTS solution. Ascorbic acid, 50 mL of 1 mg/mL solution, was used as standard antioxidant. Duplicate of each sample was used in this assay. Blank sample was run without ABTS and using MeOH – phosphate buffer (1:1) instead of tested compounds. Negative control was run with ABTS and MeOH – phosphate buffer (1:1) only. The decrease in absorbance is expressed as % inhibition which is calculated from the equation: (Acontrol 2 Atest)/Acontrol £ 100. Compound 1: m.p. 250– 2528C; UV [MeOH] lmax 315 and 225 nm; IR nmax (KBr): 3520, 3431, 2957, 2922, 2852, 1659, 1579, 1510, 1436, 1142, 1121, 842, 818 and 800 cm21; 1H NMR (400 MHz, CD3OD): dH 6.26 [1H, s, H-4], 6.74 [1H, d, J ¼ 8.4 Hz, H-a], 6.77 [2H, d, J ¼ 2.0 Hz, H-2/6], 6.92 [1H, br s, H-30 ], 6.93 [1H, br s, H-60 ] and 7.37 [1H, d, J ¼ 8.4 Hz, b]; 13 C NMR (100 MHz, CD3OD): dC 97.1 (C-30 ), 100.8 (C-60 ), 102.1 (C-4), 102.5 (C-2/6), 111.8 (C-b), 120.6 (C-a), 121.6 (C-10 ), 132.4 (C-1), 154.7 (C-20 ), 155.5 (C-40 ), 155.8 (C-50 ) and 158.6 (C-3/5); EI-MS: m/z 260.2 [Mþ], 242.1 [M 2 H2O]þ (Calcd for C14H12O5: 260.2593). Spectroscopic data for compounds 2 –14 are supplied in the supplementary materials. 4. Conclusions In this study, we reported the isolation of a new stilbene (20 ,3,40 ,5,50 -pentahydroxy-cis-stilbene) from the stem bark of M. nigra L. Several chemically diverse stilbenes and prenylated flavonoids were also isolated. The isolated compounds and the different extracts from the stem bark of M. nigra were evaluated for their ABTSþ radical-scavenging capacity. The new stilbene 1 exhibited high antioxidant activity with IC50 4.69 mM.
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Supplementary material Supplementary material relating to this article is available online, alongside Flowcharts S1 – S3 and Tables S1 –S6.
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