http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–9 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.948633
ORIGINAL ARTICLE
Two antigenotoxic chalcone glycosides from Mentha longifolia subsp. longifolia Zuhal Guvenalp1, Hilal Ozbek1, Mehmet Karadayi2, Medine Gulluce2, Ayse Kuruuzum-Uz3, Bekir Salih4, and Omur Demirezer3 Pharmaceutical Biology Downloaded from informahealthcare.com by University of Liverpool on 12/29/14 For personal use only.
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Department of Pharmacognosy, Faculty of Pharmacy, 2Department of Biology, Faculty of Science, Atatu¨rk University, Erzurum, Turkey, Department of Pharmacognosy, Faculty of Pharmacy, and 4Department of Chemistry, Faculty of Science, Hacettepe University, Ankara, Turkey
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Abstract
Keywords
Context: Mentha L. (Labiatae) species (mint) with their flavoring properties have been used in food industries for centuries. Besides they have a great importance in drug development and medicinal applications due to various bioactive compounds of several members of the genus. Objective: The aim of this study was to isolate bioactive compounds with antimutagenic potential by bio-guided fractionation and determine their structures by spectroscopic methods. Materials and methods: The structural elucidation of the isolated compounds was done based on spectroscopic methods, including MALDI-MS, UV, IR, and 2D NMR experiments, and the bioguided fractionation process was done by using the Ames/Salmonella test system. Henceforth, solely genotoxic and antigenotoxic potential of the new compounds were also confirmed up to 2 mM/plate by using the same test system. Results: Two new chalcone glycosides: (bR)-b,3,20 ,60 -tetrahydroxy-4-methoxy-40 -O-rutinosyldihydrochalcone and (bR)-b,4,20 ,60 -tetrahydroxy-40 -O-rutinosyldihydrochalcone, were isolated from Mentha longifolia (L.) Hudson subsp. longifolia, together with known six flavonoid glycosides and one phenolic acid: apigenin-7-O-glucoside, luteolin-7-O-glucoside, apigenin-7-O-rutinoside, luteolin-7-O-rutinoside, apigenin-7-O-glucuronide, luteolin-7-O-glucuronide, rosmarinic acid. According to the antimutagenicity results, both new test compounds significantly inhibited the mutagenic activity of 9-aminoacridine in a dose-dependent manner at the tested concentrations from 0.8 to 2 mM/plate. (bR)-b,4,20 ,60 -Tetrahydroxy-40 -O-rutinosyldihydrochalcone showed the maximum inhibition rate as 75.94% at 2 mM/plate concentration. Conclusions: This is the first report that two new chalcone glycosides were isolated from Mentha longifolia subsp. longifolia and their antimutagenic potentials by using mutant bacterial tester strains. In conclusion, the two new chalcone glycosides showed a significant antigenotoxic effect on 9-aminoacridine-induced mutagenesis at tested concentrations.
Ames/Salmonella test system, antimutagenicity, bioguided fractionation, chalcone glycoside derivatives, mint, natural bioactive compounds
Introduction Throughout the centuries, plants have been a unique resource for the basic needs of mankind such as the production of foodstuffs, shelters, clothes, flavors, and fragrance (GuribFakim, 2006). Furthermore, various plant species have been utilized as herbal medicines for thousands of years (Balunas & Kinghorn, 2005). In the beginning, crude application methods for specific plant species and their several parts were used to treat particular ailments. Then, the isolation of active compounds, beginning with the isolation of morphine from opium in the early nineteenth century, has dominated trends and researches associated with the use of plants as medicines
Correspondance: Mehmet Karadayi, Department of Biology, Faculty of Science, Atatu¨rk University, 25240 Erzurum, Turkey. Tel: +90 442 2314452. Fax: +90 442 2360948. E-mail: [email protected]
History Received 7 February 2014 Revised 21 May 2014 Accepted 21 July 2014 Published online 27 November 2014
in more recent history (Balunas & Kinghorn, 2005). Today, the concept of research studies on drug development from medicinal plants mainly includes isolation and characterization of pharmacologically active compounds (Balunas & Kinghorn, 2005; Gurib-Fakim, 2006). Mentha L. (Labiatae), represented by eight species and 15 taxa in the flora of Turkey, is one of the most important genera for herbal medicine. Besides the wide range usage of its species in many cuisines and food-manufacturing industries around the world, there has been a growing interest on new compound isolation studies due to many bioactive properties of several members of the genus (Baris et al., 2011; Duman, 2000; Harley, 1982). Some Mentha species are commonly used in Turkish folk medicine as carminative, aromatisan, and for the treatment of neural induced nausea (Baytop, 1984). They have also been traditionally used as an antiseptic for the treatment of cold, sinusitis, cholera, food poisoning, bronchitis, and tuberculosis, and also as antiflatulent, expectorant, diuretic,
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Z. Guvenalp et al.
antitussive, and menstruate (Barnes et al., 2007; Zargari, 1990). Besides, many bioactive properties of Mentha species, including antioxidant (Ko¸sar et al., 2004; She et al., 2010a), analgesic (Shalaby et al., 2000; Villasenor & Sanchez, 2009), antimutagenic (Baris et al., 2011), antihistaminic (Yamamura et al., 1998), and calcium channel blocking activities (Shah et al., 2010) have been also reported. It is known that bioactive properties are mainly associated with the chemical composition of medicinal plants (GuribFakim, 2006). In this context, the genus Mentha can be thought as one of the best candidates for new bioactive compound isolation studies because bicyclic lactones (Villasenor & Sanchez, 2009), triterpenes (Monte et al., 1997), flavonoids (Ko¸sar et al., 2004; Shalaby et al., 2000), phenolic acids (Ko¸sar et al., 2004; Shalaby et al., 2000), monoterpenes (She et al., 2010b), lignans (Zheng et al., 2007), and aliphatic glycosides (Yamamura et al., 1998) were reported from its various species in previous studies. Thus, the aim of the present study was determined as isolation and structure elucidation of bioactive compounds from Mentha longifolia (L.) Hudson subsp. longifolia, by using bio-guided fractionation process, and determination of their solely antigenotoxic potential.
Materials and methods General experimental procedures 1
H and 13C NMR spectra were recorded on Varian Mercury plus 400 MHz for proton and 100 MHz for carbon by using TMS as an internal standard. The solvents were DMSO-d6 and CD3OD. HR-(pos.) and MALDI-TOF-MS (reflectron pos., mass resolution 14 700) were performed on Applied Biosystems Voyager DETM (Applied Biosystems, Foster City, CA). Optical rotations were obtained on Rudolph-Research Analytical Autopol IV automatic polarimeter. UV spectra were measured with a Biotek mQuant MQX200 microplate spectrophotometer (Applied Biosystems, Foster City, CA). IR spectra were run on a Perkin Elmer FT-IR Spectrum Bx (Applied Biosystems, Foster City, CA). CD spectra were recorded on a Jasco Model J-815 Spectropolarimeter. Silica gel 60 (0.063–0.200 mm, Merck, Darmstadt, Germany) and Sephadex LH-20 (Fluka, Thessaloniki, Greece) were used for open column chromatographic separations. Lichroprep RP-18 (25–40 mm, Merck, Darmstadt, Germany) reversed phase material was used for vacuum liquid chromatography (VLC). TLC analyses were carried out on pre-coated Kieselgel 60 F254 aluminum sheets (Merck, Darmstadt, Germany). Compounds were detected by UV fluorescence and spraying 1% vanillin–H2SO4 reagent, followed by heating at 105 C for 1–2 min. In the Ames/Salmonella test system, direct acting mutagens sodium azide (NaN3) and 9-aminoacridine (9-AA) were obtained from Sigma-Aldrich (St. Louis, MO) and Merck (Darmstadt, Germany), respectively. Other solvents and pure chemicals including magnesium sulfate (MgSO4), sodium ammonium phosphate (Na2NH2PO4), D-glucose, D-biotin, sodium chloride (NaCl), L-histidine HCl, sodium phosphatedibasic (Na2HPO4), crystal violet, citric acid monohydrate, potassium phosphate-dibasic (K2HPO4), and sodium phosphate-monobasic (NaH2PO4) were also obtained from Sigma
Pharm Biol, Early Online: 1–9
(St. Louis, MO), Merck (Darmstadt, Germany), Difco (Oxoid, UK), and Fluka (Thessaloniki, Greece). Plant material The plant samples of M. longifolia subsp. longifolia were collected at the flowering stage from the Palando¨ken Mountain of Erzurum located in the eastern Anatolia in Turkey. The taxonomic identification of plant material was confirmed by Dr. Meryem Sengul Koseoglu, a senior plant taxonomist in Biology Department of Atatu¨rk University, Erzurum, Turkey. The voucher specimen has been deposited at the Herbarium of the Department of Biology (ATA Herbarium 9732), Atatu¨rk University. The collected plant materials were dried in the shade, then, the aerial parts of the plant samples were separated from the stem and ground in a grinder with a 2 mm in diameter mesh. Extraction, fractionation, and isolation Air-dried and powdered aerial parts of the plant (1000 g) were extracted four times with MeOH at 40 C (4 2 L). After evaporation of the combined extracts in vacuo, 140 g of MeOH extract was obtained. The crude extract was dissolved in water and subjected to liquid–liquid partitions successively with petroleum ether (25 0.5 L), CHCl3 (8 0.5 L), EtOAc (6 0.5 L), and n-butanol (15 0.5 L). Thereafter, the solvents were evaporated under reduced pressure in a rotary evaporator oven at 45 C. The residues obtained were 33.4, 14.7, 8.4, and 36.3 g. Fractionation of M. longifolia subsp. longifolia was done by using the Ames/Salmonella test system as a guide to obtain the mutagenic or antimutagenic extracts and compounds. According to the results, the n-butanol extract showed significant antimutagenic activity. Then, the n-butanol extract (36.3 g) was first subjected to a silica gel column and eluted with a solvent gradient of CHCl3:MeOH (100:0 ! 0:100) to afford five main fractions (Fr. A: 450 mg, Fr. B: 1.9 g, Fr. C: 11.8 g, Fr. D: 5.6 g, and Fr. E: 8.2 g). The mutagenic and antimutagenic potential was revaluated by using the same test system and three of them (A, C, and E fractions) were determined as antimutagenic. The fraction A (450 mg) was rechromatographed by Sephadex LH-20 CC; eluting with MeOH to give a subfraction (Fr. A1). Purification of subfraction A1 (50 mg) by Sephadex LH-20 CC using MeOH gave compound 3 (3.6 mg). The fraction C (11.8 g) was rechromatographed by Sephadex LH-20 CC; eluting with MeOH to give three subfractions (Fr. C1–3). Fraction C1 was further fractionated by vacuum liquid chromatography using reversed phase material with MeOH–H2O mixtures (0–50%) to give three subfractions (Fr. C1.1–1.3). Purification of subfractions C1.1 (253 mg) and C1.3 (35 mg) by Sephadex LH-20 CC using MeOH gave compound 1 (150 mg) and compound 5 (6.3 mg), respectively. Fraction C1.2 (207 mg) was also eluted with a gradient of CHCl3–MeOH (85:15 ! 80:20, v/v) by silica gel column chromatography to yield compound 6 (18.3 mg). On one hand, fraction C2 was subjected to vacuum liquid chromatography using reversed phase material and MeOH– H2O mixtures (0–50%) as a solvent to give Fr.C2.1 and Fr.C2.2. Purification of subfractions C2.1 (473 mg) and C2.2 (758 mg)
DOI: 10.3109/13880209.2014.948633
by Sephadex LH-20 CC using MeOH gave compound 9 (10 mg) and compound 2 (7.6 mg), respectively. On the other hand, fraction C3 directly gave pure compound 4 (7 mg). The fraction E (8.2 g) was rechromatographed by Sephadex LH-20 CC; eluting with MeOH to give two subfractions (Fr. E1–2). Repeated CC of the fraction E1 on Sephadex LH-20, silica gel, and reversed phase material gave compound 8 (8.8 mg). Finally, purification of subfraction E2 (20 mg) by Sephadex LH-20 CC using MeOH gave compound 7 (5.2 mg).
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Identification of compounds 1–9 Extraction, fractionation, and isolation steps of the present study led to the isolation of two new chalcone glycosides together with known six flavonoid glycosides and one phenolic acid (Figure 1). The structures of compounds were elucidated based on the analysis of their physical and spectroscopic data (1H NMR, 13C NMR, DEPT, 2D NMR, EIMS, and MALDI-MS). The known compounds isolated in the present study were identified by comparison of their physical and spectroscopic data with previous information in the literature. (bR)-,3,20 ,60 -Tetrahydroxy-4-methoxy-40 -O-rutinosyldihydrochalcone (1): Yellowish powder; ½30 D 139.39 (c 0.66, MeOH); IR max cm1: 3342, 1633; UV (MeOH) lmax (log ") 225 (4.03), 284 (3.92) nm; MALDI-MS: m/z 629.2068 [M + H]+ (theoretical 629.2076); CD (MeOH, c ¼ 3.18 104 M) []281 5325, []247 + 691.2; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data (Table 1). (R)-,4,20 ,60 -Tetrahydroxy-40 -O-rutinosyldihydrochalcone (2): Yellowish powder; ½30 D 40 (c 0.2, MeOH); IR max cm1: 3342, 1633; UV (MeOH) lmax (log ") 229 (4.57), 287 (4.65) nm; MALDI-MS: [M + H]+ at m/z 599.1966 (theoretical 599.1970); CD (MeOH, c ¼ 4.18 104 M) []281 6217, []250 + 516.1; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data (Table 1). Apigenin-7-O-glucoside (3): Yellow amorphous powder. EIMS m/z 269 [M Glc]+ (Calcd for C21H20O10). 1H NMR (DMSO-d6, 400 MHz): 7.93 (2H, d, J ¼ 8.8 Hz, H-20 , 60 ), 6.91 (2H, d, J ¼ 8.8 Hz, H-30 , 50 ), 6.84 (1H, s, H-3), 6.79 (1H, d, J ¼ 2.1 Hz, H-8), 6.42 (1H, d, J ¼ 2.1 Hz, H-6), 5.04 (1H, d, J ¼ 7.0 Hz, H-100 ), 3.68–3.86 (2H, m, Ha-600 , Hb-600 ), 3.14– 3.33 (4H, m, H-200 , 300 , 400 , 500 ). 13C NMR (DMSO-d6, 100 MHz): 182.7 (C-4), 164.9 (C-2), 163.6 (C-7), 162.1 (C5), 161.7 (C-40 ), 157.6 (C-9), 129.3 (C-20 , 60 ), 121.7 (C-10 ), 116.7 (C-30 , 50 ), 106.0 (C-10), 103.8 (C-3), 100.6 (C-100 ), 100.2 (C-6), 95.5 (C-8), 77.8 (C-300 ), 77.1 (C-500 ), 73.8 (C-200 ), 70.2 (C-400 ), 61.3 (C-600 ). 1H NMR and 13C NMR agree with data given in the literature for apigenin-7-O-glucoside (Baris et al., 2011; Gu¨venalp et al., 2006, 2009). Luteolin-7-O-glucoside (4): Yellow amorphous powder. EIMS m/z 285 [M Glc]+ (Calcd for C21H20O11). 1H NMR (DMSO-d6, 400 MHz): 7.42 (1H, dd, J ¼ 8.4/2.1 Hz, H-60 ), 7.39 (1H, d, J ¼ 1.8 Hz, H-20 ), 6.88 (1H, d, J ¼ 8.4 Hz, H-50 ), 6.77 (1H, d, J ¼ 1.8 Hz, H-8), 6.73 (1H, s, H-3), 6.42 (1H, d, J ¼ 1.8 Hz, H-6), 5.05 (1H, d, J ¼ 7.3 Hz, H-100 ), 3.69 (1H, dd, J ¼ 10.2/- Hz, H-600 b), 3.14–3.63 (5H, m, H-200 , 300 , 400 , 500 , 600 a). 13C NMR (DMSO-d6, 100 MHz): 182.6 (C-4), 165.1
Antigenotoxic chalcone glycosides of Mentha (L.)
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(C-2), 163.6 (C-7), 161.8 (C-5), 157.6 (C-9), 150.6 (C-40 ), 146.5 (C-30 ), 122.0 (C-10 ), 119.9 (C-60 ), 116.7 (C-50 ), 114.2 (C-20 ), 106.0 (C-10), 103.8 (C-3), 100.5 (C-100 ), 100.2 (C-6), 95.4 (C-8), 77.8 (C-500 ), 77.0 (C-300 ), 73.8 (C-200 ), 70.2 (C-400 ), 61.3 (C-600 ). 1H NMR and 13C NMR agree with data given in the literature for luteolin-7-O-glucoside (Kuruu¨zu¨m-Uz et al., 2008). Apigenin-7-O-rutinoside (5): Yellow amorphous powder. EIMS m/z 269 [M-(Glc + Rh)]+ (Calcd for. C27H30O14). 1H NMR (CD3OD, 400 MHz): 7.86 (2H, d, J ¼ 8.7 Hz, H-20 , 60 ), 6.93 (2H, d, J ¼ 8.7 Hz, H-30 , 50 ), 6.74 (1H, d, J ¼ 1.8 Hz, H-8), 6.63 (1H, s, H-3), 6.50 (1H, d, J ¼ 1.8 Hz, H-6), 5.03 (1H, d, J ¼ 7.3 Hz, H-100 ), 4.70 (1H, d, J ¼ 1.4 Hz, H-1000 ), 4.03 (1H, d, J ¼ 9.1 Hz, H-600 b), 3.89 (1H, dd, J ¼ 3.2/1.5 Hz, H-2000 ), 3.70 (1H, dd, J ¼ 9.5/3.6 Hz, H-3000 ), 3.29–3.66 (7H, m, H-200 , 300 , 400 , 500 , 600 a, 4000 , 5000 ), 1.17 (3H, d, J ¼ 6.2 Hz, H6000 ). 13C NMR (CD3OD, 100 MHz): 182.9 (C-4), 165.6 (C2), 163.5 (C-7), 161.8 (C-5), 161.7 (C-40 ), 157.7 (C-9), 128.5 (C-20 , C-60 ),121.9 (C-10 ),116.0 (C-30 , C-50 ),106.0 (C-10), 103.0 (C-3), 100.9 (C-100 ),100.3 (C-1000 ), 99.9 (C-6), 95.1 (C8), 76.6 (C-500 ), 76.0 (C-300 ), 73.5 (C-4000 ), 72.9 (C-200 ), 71.2 (C-2000 ), 70.9 (C-3000 ), 70.1 (C-400 ), 68.6 (C-5000 ), 66.2 (C-600 ), 16.7 (C-6000 ). 1H NMR and 13C NMR agree with data given in the literature for apigenin-7-O-rutinoside (Baris et al., 2011). Luteolin-7-O-rutinoside (6): Yellow amorphous powder. EIMS m/z 285 [M-(Glc + Rh)]+ (calc. for. C27H30O15). 1H NMR (CD3OD, 400 MHz): 7.34 (1H, d, J ¼ 2.2 Hz, H-20 ), 7.32 (1H, dd, J ¼ 8.7/2.2 Hz, H-60 ), 6.88 (1H, d, J ¼ 8.7 Hz, H-50 ), 6.65 (1H, d, J ¼ 2.1 Hz, H-8), 6.52 (1H, s, H-3), 6.45 (1H, d, J ¼ 2.1 Hz, H-6), 5.00 (1H, d, J ¼ 7.3 Hz, H-100 ), 4.72 (1H, d, J ¼ 1.5 Hz, H-1000 ), 4.05 (1H, d, J ¼ 9.5 Hz, H-600 b), 3.91 (1H, dd, J ¼ 3.5/1.6 Hz, H-2000 ), 3.73 (1H, dd, J ¼ 9.5/ 3.3 Hz, H-3000 ), 3.29–3.66 (7H, m, H-200 , 300 , 400 , 500 , 600 a, 4000 , 5000 ), 1.18 (3H, d, J ¼ 6.2 Hz, H-6000 ). 13C NMR (CD3OD, 100 MHz): 182.8 (C-4), 165.6 (C-2), 163.5 (C-7), 161.7 (C5), 157.6 (C-9), 149.9 (C-40 ), 145.7 (C-30 ), 122.3 (C-10 ), 119.5 (C-60 ), 115.7 (C-50 ), 113.2 (C-20 ), 105.9 (C-10), 103.1 (C-3), 100.9 (C-100 ), 100.4 (C-1000 ), 100.0 (C-6), 95.0 (C-8), 76.6 (C500 ), 76.0 (C-300 ), 73.6 (C-4000 ), 72.9 (C-200 ), 71.3 (C-2000 ), 70.9 (C-3000 ), 70.2 (C-400 ), 68.6 (C-5000 ), 66.4 (C-600 ), 16.7 (C-6000 ). 1 H NMR and 13C NMR agree with data given in the literature for luteolin-7-O-rutinoside (Wang et al., 2003). Apigenin-7-O-glucuronide (7): Yellow amorphous powder. EIMS m/z 269 [M-Glu]+ (Calcd for. C21H18O11). 1H NMR (DMSO-d6, 400 MHz): 7.87 (2H, d, J ¼ 8.8 Hz, H-20 , 60 ), 6.88 (2H, d, J ¼ 8.8 Hz, H-30 , 50 ), 6.80 (1H, s, H-3), 6.77 (1H, d, J ¼ 1.8 Hz, H-8), 6.42 (1H, d, J ¼ 1.8 Hz, H-6), 5.05 (1H, d, J ¼ 7.3 Hz, H-100 ), 3.61 (1H, d, J ¼ 10.3 Hz, H-500 ), 3.13–3.40 (3H, m, H-200 , 300 , 400 ). 13C NMR (DMSO-d6, 100 MHz): 182.6 (C-4), 172.6 (C-600 ), 164.9 (C-2), 163.7 (C-7), 162.4 (C5), 161.7 (C-40 ), 157.6 (C-9), 129.4 (C-20 , 60 ), 121.3 (C-10 ), 116.7 (C-30 , 50 ), 105.9 (C-10), 103.5 (C-3), 100.3 (C-100 ), 100.2 (C-6), 95.3 (C-8), 77.2 (C-300 ), 74.4 (C-500 ), 73.6 (C-200 ), 72.6 (C-400 ). 1H NMR and 13C NMR agree with data given in the literature for apigenin-7-O-glucuronide (Baris et al., 2011; Guvenalp et al., 2009). Luteolin-7-O-glucuronide (8): Yellow amorphous powder. EIMS m/z 285 [M-GlcA]+ (Calcd for. C21H18O12). 1H NMR (DMSO-d6, 400 MHz): 7.40 (1H, d, J ¼ 2.2 Hz, H-20 ), 7.37 (1H, dd, J ¼ 8.4/2.2 Hz, H-60 ), 6.84 (1H, d, J ¼ 8.4 Hz, H-50 ),
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Figure 1. Chemical structures of compounds 1–9.
Pharm Biol, Early Online: 1–9
HO
HO
HO
4''' 6'''
H3C
2'''
3'''
O
1'''
R1
5'''
O 4''
HO
O
6'' 5''
HO
3'
O
OH
4'
1''
2''
α
1'
5'
5
1
6'
6
β O
OH
R1 OH H
1 2
4
2
2'
OH
3''
3
OH
R2 OCH3 OH
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R2 3'
OH
2'
O
R1 HO
4''
5''
HO
3''
8
O 7
1''
2''
OH
6
4
O
R1 CH2OH CH2OH COOH COOH
R2 H OH H OH R
HO
HO
3'
2'''
3'''
6'''
H3C
6'
3
10 5
3 4 7 8
HO
5'
2
OH
4'''
1'
O
9
4'
O
2'
1'''
5'''
O
HO
O
6''
4''
5''
HO
3''
OH
O
9
7
1''
2''
8
O
6
6'
3
10 4
5
O
R H OH
5 6
5' 9'
COOH
O 2 3
1
4
HO
7'
9
7 8
6 5
9
O
6'
OH 4'
3'
1'
8'
5'
1' 2
OH
HO
OH 4'
2'
OH
R2
Antigenotoxic chalcone glycosides of Mentha (L.)
DOI: 10.3109/13880209.2014.948633
Table 1. 1H and
13
C NMR spectroscopic data for compounds 1 and 2. Compound 1
No.
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1 2 3 4 5 6 A B C¼O OCH3 10 20 30 40 50 60 100 200 300 400 500 600 1000 2000 3000 4000 5000 6000
5
H (J in Hz) 6.78 d (2.1) 6.70 d (8.1) 6.94 dd (8.1, 2.1) 2.75 dd (17.0, 2.9) 3.11 dd (17.0, 12.4) 5.31 dd (11.4, 2.9) 3.85 s 6.18 d (2.2) 6.16 d (2.2) 4.93 d (7.0) 3.41–3.48 m 3.45–3.60 m 3.60–3.70 m 3.40–3.50 m 3.58–3.65 m 3.98 dd (9.5, 2.5) 4.68 d (1.83) 3.88 dd (3.48, 1.83) 3.60–3.65 m 3.30–3.40 m 3.55–3.65 m 1.18 d (6.2)
C 130.4 113.7 145.2 145.7 115.2 118.2 42.9 79.5 197.4 55.3 103.8 163.3 95.9 165.6 96.8 163.7 101.0 73.5 75.9 71.2 76.7 66.3
Compound 2
3,4,1, b 3, 1, 6 3,4,1, b C¼O C¼O, 1, b 2,6,1, a, C¼O
7.32 d (8.4) 6.81 d (8.4) 6.81 d (8.4) 7.32 d (8.4) 2.74 dd (17.1, 2.9) 3.15 dd (16.9, 12.4) 5.39 dd (12.4, 2.9)
C 129.7 128.0 115.2 157.9 115.2 128.0 42.9 79.4 197.3
HMBC 4, 1, b 4, 1, 5, b 4, 1, 3, b 4, 1, b C¼O C¼O, b C¼O,1, 2, 6
4 10 ,50 ,20 ,40 , C¼O
6.18 d (2.2)
10 ,30 ,40 ,60 , C¼O
6.16 d (2.2)
99.9 70.2 70.8 72.9 68.6 16.8
6.75 (1H, d, J ¼ 1.8 Hz, H-8), 6.68 (1H, s, H-3), 6.39 (1H, d, J ¼ 1.8 Hz, H-6), 5.06 (1H, d, J ¼ 7.3 Hz, H-100 ), 3,61 (1H, d, J ¼ 9.8 Hz, H-500 ), 3.14–3.31 (3H, m, H-200 , H-300 , H-400 ); 13C NMR (DMSO-d6, 100 MHz): 182.5 (C-4), 172.8 (C-600 ), 165.2 (C-2), 163.7 (C-7), 161.7 (C-5), 157.6 (C-9), 151.1 (C40 ), 146.6 (C-30 ), 121.6 (C-10 ), 119.7 (C-60 ), 116.7 (C-50 ), 114.1 (C-20 ), 105.9 (C-10), 103.5 (C-3), 100.4 (C-100 ), 100.3 (C-6), 95.3 (C-8), 77.1 (C-300 ), 74.5 (C-500 ), 73.6 (C-200 ), 72.6 (C-400 ). 1H NMR and 13C NMR agree with data given in the literature for luteolin 7-O-glucuronide (Gulluce et al., 2012). Rosmarinic acid (9): White-yellow amorphous solid; EIMS m/z 360 [M] (Calcd for. C18H16O8). 1H NMR (CD3OD, 400 MHz): 7.50 (1H, d, J ¼ 15.7 Hz, H-7), 7.02 (1H, d, J ¼ 1.8 Hz, H-2), 6.90 (1H, dd, J ¼ 8.0/1.8 Hz, H-6), 6.76 (1H, d, J ¼ 8.0 Hz, H-5), 6.76 (1H, d, J ¼ 1.8 Hz, H-20 ), 6.67 (1H, d, J ¼ 8.0 Hz, H-50 ), 6.62 (1H, dd, J ¼ 8.0/1.8 Hz, H-60 ), 6.26 (1H, d, J ¼ 15.7 Hz, H-8), 5.06 (1H, dd, J ¼ 9.7/ 3.4 Hz, H-80 ), 3.09 (1H, d, J ¼ 14.1/3.1 Hz, H-70 b), 2.93 (1H, d, J ¼ 14.1/9.7 Hz, H-70 a), 13C NMR (CD3OD, 100 MHz): 176.5 (C-90 ), 167.9 (C-9), 148.2 (C-4), 145.5 (C-7), 145.5 (C-30 ), 144.7 (C-40 ), 143.6 (C-3), 130.0 (C-10 ), 126.7 (C-1), 121.7 (C-60 ), 120.6 (C-6), 116.3 (C-20 ), 115.3 (C-50 ), 115.0 (C-5), 114.4 (C-2), 114.0 (C-8), 76.6 (C-80 ), 37.6 (C-70 ). 1H NMR and 13C NMR agree with data given in the literature for rosmarinic acid (Ly et al., 2006). Bacterial strains Õ
H (J in Hz)
HMBC
Salmonella typhimurium TA1535 (ATCC number: 29629) Õ and S. typhimurium TA1537 (ATCC number: 29630) strains
40
600
4.93 d (7.3) 3.41–3.48 m 3.45–3.60 m 3.60–3.70 m 3.40–3.50 m 3.58–3.65 m 3.98 dd (9.5, 2.5) 4.68 bs 3.88 dd (3.48, 1.83) 3.60–3.65 m 3.30–3.40 m 3.55–3.65 m 1.18 d (6.2)
103.8 163.2 95.9 165.7 96.8 163.3 101.0 73.5 75.9 71.2 76.7 66.2
10 , 50 , 20 , 40 10 , 30 , 40 , 60
99.9 70.1 70.9 72.9 68.6 16.7
40
600
were provided by The American Type Culture Collection – Bacteria Department of Georgetown University, Washington, DC. Both strains were stored at 80 C. Working cultures were prepared by inoculating nutrient broth with the frozen cultures, followed by an overnight incubation at 37 C with gentle agitation (Oh et al., 2008). Viability assays and determination of test concentrations Toxic levels of the test materials toward S. typhimurium TA1535 and 1537 strains were determined as described in detail elsewhere (Yu et al., 2001). These tests confirmed that there was normal growth of the background lawn, spontaneous colony numbers within the regular range, and no significant reduction in cell survival. Thus, for the concentrations and conditions reported here, no toxicity or other adverse effects were observed. The Ames/Salmonella test The bacterial mutagenicity and antimutagenicity assays were performed according to described procedures (Mortelmans & Zeiger, 2000). The known mutagens NaN3 (in distilled water – 1 mg/plate) for S. typhimurium TA1535 and 9-AA (in methanol – 10 mg/plate) for S. typhimurium TA1537 were used as positive controls and 10% DMSO was used as a negative control. The plate incorporation method was used to assess the results of mutagenicity and antimutagenicity assays (Mortelmans & Zeiger, 2000).
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For the mutagenicity assays, the mutagenic index was calculated for each concentration, which is the average number of revertants per plate divided by the average number of revertants per plate with the negative (solvent) control. A sample was considered as mutagenic when a dose– response relationship and a two-fold increase in the number of revertants with at least one concentration were observed (Gulluce et al., 2010). For the antimutagenicity assays, the inhibition rate of mutagenicity was calculated by using the following equation (M: the number of revertants/plate induced by mutagen alone, S0: the number of spontaneous revertants, S1: the number of revertants/plate induced by the extract plus the mutagen): inhibition% ¼ {1 [(M S1)/(M S0)]} 100 About 25–40% inhibition was defined as moderate antimutagenicity; 40% or more inhibition as strong antimutagenicity; and less than 25% inhibition as no antimutagenicity (Gulluce et al., 2010). Statistical analysis The results are presented as the average and standard error of three experiments with duplicate plates/dose experiment. The data were further analyzed for statistical significance using analysis of variance (ANOVA), and the difference among means was compared by high-range statistical domain using Tukey’s test. A level of probability was taken as p50.05 indicating statistical significance (Gulluce et al., 2010).
Results and discussion Chemical results Compound 1 was obtained as a yellowish powder. The molecular formula was established as C28H36O16, by positiveion and reflectron MALDI-MS, which showed molecular ion [M + H]+ at m/z 629.2068 (theoretical 629.2076). The IR spectrum displayed intense absorbtion bands for hydroxyl (3342 cm1), and carbonyl (1633 cm1) functionalities. Analysis of the 13C NMR and DEPT spectra indicated the presence of 28 carbon signals of which 15 carbon atoms were assigned to be aglycon, 12 carbons to the sugar moiety, and one carbon to the methoxy group. 1H NMR spectrum indicated the presence of an ABX-type aromatic proton system appearing at 6.70 (1H, d, J ¼ 8.1 Hz), 6.94 (1H, dd, J ¼ 8.1/2.1 Hz), and 6.78 (1H, d, J ¼ 2.1 Hz) due to a ring protons as well as a methoxyl proton signal at 3.85, a pair of meta-coupled aromatic protons at 6.18 and 6.16 (1H each, both d, J ¼ 2.2 Hz) and a set of three peculiar aliphatic protons 2.75 (1H, dd, J ¼ 17.0/2.9 Hz), 3.11 (1H, dd, J ¼ 17.0/ 12.4 Hz), and 5.31 (1H, dd, J ¼ 11.4/2.9 Hz) together with one glucopyranosyl part and one rhamnopyranosyl part. The large coupling constant (7.0 Hz) of the anomeric proton 4.93 indicated the presence of the b-glucose moiety. The small coupling constant (1.83 Hz) of the anomeric proton 4.68 indicated the presence of the a-rhamnose moiety. The 1H and 13 C NMR signals of compound 1 were completely assigned by a combination of HMQC, HMBC, and 1H–1H COSY experiments. The 1H–1H COSY correlation between H2-a (H 2.75, 3.11) and H-b (H 5.31) and the HMBC correlations of H2-a to C¼O (C 197.4), C-b (C 79.5), and C-1 (C 130.4);
Pharm Biol, Early Online: 1–9
H-b to C¼O (C 197.4), C-a (C 42.9), C-1, C-2 (C 113.7), and C-6 (C 118.2); H-100 (H 4.93) to C-40 (C 165.6); H-1000 (H 4.68) to C-600 (C 66.3), and H-OMe to C-4 (C 145.7) were determined (Figure 2). The absolute configuration at the b position was assigned as R from the CD spectrum that showed a positive Cotton effect at 247 nm and a negative Cotton effect at 281 nm (Nel et al., 1999). Thus, the structure of compound 1 was characterized as (bR)-b,3,20 ,60 -tetrahydroxy-4-methoxy-40 -O-rutinosyldihydrochalcone. Compound 2 was also isolated as a yellowish powder. The molecular formula was established as C27H34O15, by positiveion and reflectron MALDI-MS, which showed molecular ion [M + H]+ at m/z 599.1966 (theoretical 599.1970). IR spectra of 2 showed similar bands compared to those of 1. The 13C NMR and DEPT spectra indicated the presence of 27 carbon signals of which 15 carbon atoms were assigned to be aglycon, and 12 carbons to the sugar moiety. The 1H NMR spectrum showed a pair of A2B2 type signals at 7.32 and 6.81 (2H each, both d, J ¼ 8.4 Hz) attributed to a parasubstituted phenyl group, a pair of meta-coupled aromatic protons at 6.18 and 6.16 (1H each, both d, J ¼ 2.2 Hz) and a set of three peculiar aliphatic protons 2.74 (1H, dd, J ¼ 17.1/ 2.9 Hz), 3.15 (1H, dd, J ¼ 16.9/12.4 Hz), and 5.39 (1H, dd, J ¼ 12.4/2.9 Hz) together with one glucopyranosyl part and one rhamnopyranosyl part. The b-configuration of the glucopyranosyl and a-configuration of the rhamnopyranosyl moieties were deduced from the coupling constants of the anomeric protons and the 13C NMR data of the sugar unit. The 1H–1H COSY spectrum of compound 2 showed correlations between H2- (H 2.74, 3.15) and H-b (H 5.39) and the HMBC spectrum of compound 2 indicated correlations of H2-a to C¼O (C 197.3), and C-b (C 79.4); H-b to C¼O (C 197.3), C-1 (C 129.7), and C-2,6 (C 128.0); H-100 (H 4.93) to C-40 (C 165.7), and H-1000 (H 4.68) to C-600 (C 66.2) (Figure 2). The CD spectrum showed a positive Cotton effect at 250 nm and a negative one at 281 nm which suggested R-configuration at the b carbon (Nel et al., 1999). Thus, the structure of compound 2 was established as (bR)-b,4,20 ,60 tetrahydroxy-40 -O-rutinosyldihydrochalcone. Genotoxic and antigenotoxic activity evaluation Chalcone derivatives have a great importance for medicinal studies due to their various bioactive properties (Chen et al., 2012; Kaur et al., 2009). Antigenotoxic activity is a good example for the most important biological effects of these molecules, which is mainly related to their antioxidant capabilities (Kaur et al., 2009). Recent studies have focused on isolation of new natural compounds with antigenotoxic potential from plant components owing to increasing concerns about mutagenesis and related disorders such as cancer (Baris et al., 2011; Gulluce et al., 2012). In the present study, bacterial tester strains for determination of mutagenic and antimutagenic potentials of bioactive chalcone derivatives were S. typhimurium TA1535 and TA1537. Salmonella typhimurium TA1535 is known as a tester strain capable for detection of mutagenic chemicals cause base-substitution type of mutagenesis. In contrast, S. typhimurium TA1537 is known as a tester strain capable for
Antigenotoxic chalcone glycosides of Mentha (L.)
DOI: 10.3109/13880209.2014.948633
HO
4'''
HO
HO 2'''
3'''
6'''
H3C
7
O
1'''
OH
5'''
O 4''
HO
6'' 5''
HO
O 2''
3'
O 4'
1''
α
1'
5' 6'
5
1 6
β O
OH
OCH3
4
2
2'
OH
3''
3
OH
OH
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1
HO
4'''
HO
6'''
H3C
HO 2'''
3'''
O
1'''
5'''
O
HO
6''
4''
5''
HO
3''
O 2''
3'
O 1''
3
OH
4'
2'
OH
α
1'
5' 6'
4
2
5
1 6
β
OH
O
OH
OH
2 Figure 2. HMBC correlations of compounds 1 and 2.
detection of mutagenic chemicals cause frame-shift mutagenesis. Furthermore, these strains also can be used for detection of antimutagenic chemicals which inhibit hazardous activities of mutagenic substances cause base-substitution and frameshift mutagenesis (Mortelmans & Zeiger, 2000). According to the results of viability assays, the concentrations up to 2 mM were determined as applicable for both chalcone glycosides without any adverse effect or toxicity on bacterial tester strains. Thus, 0.4, 0.8, 1.2, 1.6, and 2 mM concentrations were chosen to determine mutagenic and antimutagenic potential of the test compounds. In the mutagenicity assays performed with S. typhimurium TA1535 and TA1537 strains, both test compounds did not give any mutagenic activity on the tester strains (Table 2). In the antimutagenicity assays performed with the same strains, both compounds did not show an antimutagenic effect against sodium azide (NaN3) induced mutagenesis in S. typhimurium TA1535. NaN3 is known as a powerful mutagen, which affects several organisms including bacteria, plants, and animals, and generally causes base-substitutions. Previous studies showed that the mutagenicity of NaN3 is related to the production of L-azidoalanine (Sadiq & Owais, 2000). One reason for observing no antimutagenicity against NaN3 may be that both the tested chalcones do not have sufficient capability to inhibit formation of L-azidoalanine. However, these compounds showed significant antimutagenic activity against 9-aminoacridine (9-AA)-induced mutagenesis in S. typhimurium TA1537. Inhibition rates of mutagenesis in TA1537 ranged from 49.14% to 65.41% for
Table 2. The mutagenicity assay results of the novel chalcone derivatives for S. typhimurium TA1535 and TA1537 bacterial tester strains. Number of revertants
Test items
Concentration (mM/plate)
S. typhimurium S. typhimurium TA1535 TA1537 Mean ± S.E. Mean ± S.E.
Compound 1 0.4 0.8 1.2 1.6 2.0
25.00 ± 01.29 25.83 ± 01.17 25.00 ± 01.53 24.00 ± 00.58 25.67 ± 01.23
25.50 ± 01.28 24.50 ± 01.26 23.50 ± 01.54 23.50 ± 01.98 27.33 ± 00.92
0.4 0.8 1.2 1.6 2.0
24.50 ± 00.92 25.67 ± 01.20 24.33 ± 00.67 26.17 ± 01.35 23.67 ± 01.02
23.83 ± 00.95 25.33 ± 01.67 24.33 ± 01.54 23.17 ± 01.35 25.50 ± 01.31
364.17 ± 08.66 26.17 ± 01.17
444.67 ± 10.37 23.33 ± 01.28
Compound 2
Controls NaN3a 9-AAa DMSOa (ml/plate) a
NaN3 (1 mg/plate) and 9-AA (40 mg/plate) were used as positive controls for S. typhimurium TA1535 and TA1537 strains, respectively. DMSO (dimethylsulfoxide; 100 ml/plate) was used as a negative control.
compound 1 and from 30.06 to 75.94% for compound 2 (Table 3). 9-AA, a member of acridine family, is known as a model frame-shift mutagen (Ferguson & Denny, 1991). In the frame-shift mutagenesis mechanism, it binds to DNA non-
8
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Table 3. The antimutagenicity assay results of the novel chalcone derivatives for S. typhimurium TA1535 and TA1537 bacterial tester strains. Number of revertants
Test items
Concentration (mM/plate)
S. typhimurium TA1535 Mean ± S.E.
S. typhimurium TA1537 Mean ± S.E.
Compound 1 0.4 0.8 1.2 1.6 2.0
367.50 ± 10.51 359.00 ± 12.35 355.67 ± 12.28 349.00 ± 08.24 372.00 ± 07.86
458.83 ± 12.61 226.17 ± 08.86y 199.50 ± 08.55y 177.00 ± 07.43y 153.83 ± 07.70y
0.4 0.8 1.2 1.6 2.0
369.00 ± 12.84 348.83 ± 08.99 368.00 ± 07.21 367.83 ± 08.08 357.83 ± 12.77
447.33 ± 07.39 311.00 ± 09.96y 266.00 ± 06.06y 171.67 ± 04.42y 107.00 ± 05.11y
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Compound 2
Controls NaN3a 9-AAa DMSOa (ml/plate)
364.17 ± 08.66 444.67 ± 10.37 26.17 ± 01.17 23.33 ± 01.28
a
NaN3 (1 mg/plate) and 9-AA (40 mg/plate) were used as positive controls for S. typhimurium TA1535 and TA1537 strains, respectively. DMSO (dimethylsulfoxide; 100 ml/plate) was used as a negative control. yp50.05 to positive control.
covalently by intercalation. Through this way, 9-AA induces frameshift mutations at hot spots where a single base, especially guanine, is repeated (Hoffmann et al., 2003). They performed experiments to determine antimutagenic properties of the test materials by using S. typhimurium TA1537 strain and 9-AA depend on inhibition of the intercalation mechanism. The results showed that both chalcone derivatives used in this study have antimutagenic activity against 9-AA. This activity may be due to their inhibition capabilities by blocking 9-AA binding to DNA. In contrast, it is known that antimutagenic activity of phytophenols is mainly dependent on their antioxidant activity (Nagy et al., 2009). The antimutagenic potencies of these molecules mainly depend on the number and the position of the polar hydroxyl groups (OH) within the molecule (Edenharder & Gru¨nhage, 2003). Chalcone derivatives are also known as antioxidative molecules with polar hydroxyl groups. Both chalcone derivatives have the same numbers of OH groups in their structures. Probable reasons for the difference between antimutagenic potentials of the test compounds may be explained by the different positions of OH between R1 and R2 groups, and sterical effect of OMe group in the structure of compound 1.
Conclusions Both chalcone derivatives isolated from M. longifolia subsp. longifolia can be considered as genotoxically safe at tested concentrations owing to the fact that they did not show any mutagenic activity on the mutant tester strains. Both of them also provided significant antimutagenic activity against known mutagen 9-AA in the same test system. These activities are valuable for further investigations focusing on protective strategies development against 9-AA induced
genotoxicity. The data obtained from the present study can also be supported by performing studies with more complex test systems resulting in more reliable results for human health applications.
Declaration of interest This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK: 107T203).
References Baris O, Karadayi M, Yanmis D, et al. (2011). Isolation of 3 flavonoids from Mentha longifolia (L.) Hudson subsp. longifolia and determination of their genotoxic potentials by using the E. coli WP2 test system. J Food Sci 76:T212–7. Balunas MJ, Kinghorn AD. (2005). Drug discovery from medicinal plants. Life Sci 78:431–41. Barnes J, Anderson LA, Phillipson JD. (2007). Herbal Medicines. London, UK: Pharmaceutical Press. Baytop T. (1984). Therapy with Medicinal Plants in Turkey (Past and _ _ Present). Istanbul, Turkey: Publication of Istanbul University. Chen J, Li Y, Yang LQ, et al. (2012). Biological activities of flavonoids from pathogenic-infected Astragalus adsurgens. Food Chem 131: 546–51. ¨ zhatay N, Ekim T, Duman H. (2000). The genus Mentha. In: Gu¨ner A, O Ba¸ser KHC, ed. Flora of Turkey and the East Aegean Islands. Vol. 11. Edinburgh: University Press, 209. Edenharder R, Gru¨nhage D. (2003). Free radical scavenging abilities of flavonoids as mechanism of protection against mutagenicity induced by tert-butyl hydroperoxide or cumene hydroperoxide in Salmonella typhimurium TA102. Mutat Res 540:1–8. Ferguson LR, Denny WA. (1991). The genetic toxicology of acridines. Mutat Res 258:123–60. Gulluce M, Agar G, Baris O, et al. (2010). Mutagenic and antimutagenic effects of hexane extract of some Astragalus species grown in the Eastern Anatolia region of Turkey. Phytother Res 24:1014–8. Gulluce M, Karadayi M, Guvenalp Z, et al. (2012). Isolation of some active compounds from Origanum vulgare L. ssp. vulgare and determination of their genotoxic potentials. Food Chem 130:248–53. Gurib-Fakim A. (2006). Medicinal plants: Traditions of yesterday and drugs of tomorrow. Mol Aspects Med 27:1–93. ¨ zbek H, U ¨ nsalar T, et al. (2006). Iridoid, flavonoid and Gu¨venalp Z, O phenylethanoid glycosides from Wiedemannia orientalis. Turk J Chem 30:391–400. ¨ zbek H, Kuruu¨zu¨m-Uz A, et al. (2009). Secondary Gu¨venalp Z, O metabolites from Nepeta heliotropifolia. Turk J Chem 33:667–75. Harley RM. (1982). Mentha L. In: Davis PH, ed. Flora of Turkey and the East Aegean Islands. Vol. 7. Edinburgh: University Press, 384–94. Hoffmann GR, Calciano MA, Lawless BM, Mahoney KM. (2003). Frameshift mutations induced by three classes of acridines lacZ reversion assay in Escherichia coli: Potency of responses and relationship to slipped mispairing models. Environ Mol Mutagen 42: 111–21. Kaur P, Kaur S, Kumar N, et al. (2009). Evaluation of antigenotoxic activity of isoliquiritin apioside from Glycyrrhiza glabra L. Toxicol In Vitro 23:680–6. Ko¸sar M, Dorman HJD, Ba¸ser KHC, Hiltunen R. (2004). Screening of free radical scavenging compounds in water extracts of Mentha samples using a post column derivatization method. J Agric Food Chem 52:5004–10. Kuruu¨zu¨m-Uz A, Gu¨venalp Z, Stroch K, et al. (2008). Antioxidant potency of flavonoids from Vitex agnus-castus L. growing in Turkey. FABAD 33:11–6. Ly TN, Shimoyamada M, Yamauchi R. (2006). Isolation and characterization of rosmarinic acid oligomers in Celastrus hindsii Benth leaves and their antioxidative activities. J Agric Food Chem 54: 3786–93. Monte EJQ, Kintzinger JR, Trendel JM, Poinsot J. (1997). Mixture of closely related isomeric triterpenoid derivatives: Separation and purification by reversed-phase high-performance liquid chromatography. Chromatographia 46:251–5.
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She GM, Xu C, Liu B, Shi RB. (2010a). Polyphenolic acids from mint (the aerial of Mentha haplocalyx Briq.) with DPPH radical scavenging activity. J Food Sci 75:359–62. She G, Xu C, Liu B, Shi R. (2010b). Two new monoterpenes from Mentha haplocalyx Briq. Helv Chim Acta 93:2495–8. Villasenor IM, Sanchez AC. (2009). Menthalactone, a new analgesic from Mentha cordifolia Opiz. leaves. Z Naturforsch C 64:809–12. Wang M, Simon JE, Aviles IF, et al. (2003). Analysis of antioxidative phenolic compounds in Artichoke (Cynara scolymus L.). J Agric Food Chem 51:601–8. Yamamura S, Ozawa K, Ohtani K, et al. (1998). Antihistaminic flavones and aliphatic glycosides from Mentha spicata. Phytochemistry 48: 131–6. Yu Z, Xu M, Santana-Rios G, et al. (2001). A comparison of whole wheat, refined wheat and wheat bran as inhibitors of heterocyclic amines in the Salmonella mutagenicity assay and in the rat colonic abberant crypt focus assay. Food Chem Toxicol 39:655–65. Zargari A. (1990). Herbal Medicines. Tehran: Publication of Tehran University. Zheng J, Chen GT, Gao HY, et al. (2007). Two new lignans from Mentha spicata L. J Asian Nat Prod Res 9:445–9.
ORIGINAL ARTICLE
Two antigenotoxic chalcone glycosides from Mentha longifolia subsp. longifolia Zuhal Guvenalp1, Hilal Ozbek1, Mehmet Karadayi2, Medine Gulluce2, Ayse Kuruuzum-Uz3, Bekir Salih4, and Omur Demirezer3 Pharmaceutical Biology Downloaded from informahealthcare.com by University of Liverpool on 12/29/14 For personal use only.
1
Department of Pharmacognosy, Faculty of Pharmacy, 2Department of Biology, Faculty of Science, Atatu¨rk University, Erzurum, Turkey, Department of Pharmacognosy, Faculty of Pharmacy, and 4Department of Chemistry, Faculty of Science, Hacettepe University, Ankara, Turkey
3
Abstract
Keywords
Context: Mentha L. (Labiatae) species (mint) with their flavoring properties have been used in food industries for centuries. Besides they have a great importance in drug development and medicinal applications due to various bioactive compounds of several members of the genus. Objective: The aim of this study was to isolate bioactive compounds with antimutagenic potential by bio-guided fractionation and determine their structures by spectroscopic methods. Materials and methods: The structural elucidation of the isolated compounds was done based on spectroscopic methods, including MALDI-MS, UV, IR, and 2D NMR experiments, and the bioguided fractionation process was done by using the Ames/Salmonella test system. Henceforth, solely genotoxic and antigenotoxic potential of the new compounds were also confirmed up to 2 mM/plate by using the same test system. Results: Two new chalcone glycosides: (bR)-b,3,20 ,60 -tetrahydroxy-4-methoxy-40 -O-rutinosyldihydrochalcone and (bR)-b,4,20 ,60 -tetrahydroxy-40 -O-rutinosyldihydrochalcone, were isolated from Mentha longifolia (L.) Hudson subsp. longifolia, together with known six flavonoid glycosides and one phenolic acid: apigenin-7-O-glucoside, luteolin-7-O-glucoside, apigenin-7-O-rutinoside, luteolin-7-O-rutinoside, apigenin-7-O-glucuronide, luteolin-7-O-glucuronide, rosmarinic acid. According to the antimutagenicity results, both new test compounds significantly inhibited the mutagenic activity of 9-aminoacridine in a dose-dependent manner at the tested concentrations from 0.8 to 2 mM/plate. (bR)-b,4,20 ,60 -Tetrahydroxy-40 -O-rutinosyldihydrochalcone showed the maximum inhibition rate as 75.94% at 2 mM/plate concentration. Conclusions: This is the first report that two new chalcone glycosides were isolated from Mentha longifolia subsp. longifolia and their antimutagenic potentials by using mutant bacterial tester strains. In conclusion, the two new chalcone glycosides showed a significant antigenotoxic effect on 9-aminoacridine-induced mutagenesis at tested concentrations.
Ames/Salmonella test system, antimutagenicity, bioguided fractionation, chalcone glycoside derivatives, mint, natural bioactive compounds
Introduction Throughout the centuries, plants have been a unique resource for the basic needs of mankind such as the production of foodstuffs, shelters, clothes, flavors, and fragrance (GuribFakim, 2006). Furthermore, various plant species have been utilized as herbal medicines for thousands of years (Balunas & Kinghorn, 2005). In the beginning, crude application methods for specific plant species and their several parts were used to treat particular ailments. Then, the isolation of active compounds, beginning with the isolation of morphine from opium in the early nineteenth century, has dominated trends and researches associated with the use of plants as medicines
Correspondance: Mehmet Karadayi, Department of Biology, Faculty of Science, Atatu¨rk University, 25240 Erzurum, Turkey. Tel: +90 442 2314452. Fax: +90 442 2360948. E-mail: [email protected]
History Received 7 February 2014 Revised 21 May 2014 Accepted 21 July 2014 Published online 27 November 2014
in more recent history (Balunas & Kinghorn, 2005). Today, the concept of research studies on drug development from medicinal plants mainly includes isolation and characterization of pharmacologically active compounds (Balunas & Kinghorn, 2005; Gurib-Fakim, 2006). Mentha L. (Labiatae), represented by eight species and 15 taxa in the flora of Turkey, is one of the most important genera for herbal medicine. Besides the wide range usage of its species in many cuisines and food-manufacturing industries around the world, there has been a growing interest on new compound isolation studies due to many bioactive properties of several members of the genus (Baris et al., 2011; Duman, 2000; Harley, 1982). Some Mentha species are commonly used in Turkish folk medicine as carminative, aromatisan, and for the treatment of neural induced nausea (Baytop, 1984). They have also been traditionally used as an antiseptic for the treatment of cold, sinusitis, cholera, food poisoning, bronchitis, and tuberculosis, and also as antiflatulent, expectorant, diuretic,
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antitussive, and menstruate (Barnes et al., 2007; Zargari, 1990). Besides, many bioactive properties of Mentha species, including antioxidant (Ko¸sar et al., 2004; She et al., 2010a), analgesic (Shalaby et al., 2000; Villasenor & Sanchez, 2009), antimutagenic (Baris et al., 2011), antihistaminic (Yamamura et al., 1998), and calcium channel blocking activities (Shah et al., 2010) have been also reported. It is known that bioactive properties are mainly associated with the chemical composition of medicinal plants (GuribFakim, 2006). In this context, the genus Mentha can be thought as one of the best candidates for new bioactive compound isolation studies because bicyclic lactones (Villasenor & Sanchez, 2009), triterpenes (Monte et al., 1997), flavonoids (Ko¸sar et al., 2004; Shalaby et al., 2000), phenolic acids (Ko¸sar et al., 2004; Shalaby et al., 2000), monoterpenes (She et al., 2010b), lignans (Zheng et al., 2007), and aliphatic glycosides (Yamamura et al., 1998) were reported from its various species in previous studies. Thus, the aim of the present study was determined as isolation and structure elucidation of bioactive compounds from Mentha longifolia (L.) Hudson subsp. longifolia, by using bio-guided fractionation process, and determination of their solely antigenotoxic potential.
Materials and methods General experimental procedures 1
H and 13C NMR spectra were recorded on Varian Mercury plus 400 MHz for proton and 100 MHz for carbon by using TMS as an internal standard. The solvents were DMSO-d6 and CD3OD. HR-(pos.) and MALDI-TOF-MS (reflectron pos., mass resolution 14 700) were performed on Applied Biosystems Voyager DETM (Applied Biosystems, Foster City, CA). Optical rotations were obtained on Rudolph-Research Analytical Autopol IV automatic polarimeter. UV spectra were measured with a Biotek mQuant MQX200 microplate spectrophotometer (Applied Biosystems, Foster City, CA). IR spectra were run on a Perkin Elmer FT-IR Spectrum Bx (Applied Biosystems, Foster City, CA). CD spectra were recorded on a Jasco Model J-815 Spectropolarimeter. Silica gel 60 (0.063–0.200 mm, Merck, Darmstadt, Germany) and Sephadex LH-20 (Fluka, Thessaloniki, Greece) were used for open column chromatographic separations. Lichroprep RP-18 (25–40 mm, Merck, Darmstadt, Germany) reversed phase material was used for vacuum liquid chromatography (VLC). TLC analyses were carried out on pre-coated Kieselgel 60 F254 aluminum sheets (Merck, Darmstadt, Germany). Compounds were detected by UV fluorescence and spraying 1% vanillin–H2SO4 reagent, followed by heating at 105 C for 1–2 min. In the Ames/Salmonella test system, direct acting mutagens sodium azide (NaN3) and 9-aminoacridine (9-AA) were obtained from Sigma-Aldrich (St. Louis, MO) and Merck (Darmstadt, Germany), respectively. Other solvents and pure chemicals including magnesium sulfate (MgSO4), sodium ammonium phosphate (Na2NH2PO4), D-glucose, D-biotin, sodium chloride (NaCl), L-histidine HCl, sodium phosphatedibasic (Na2HPO4), crystal violet, citric acid monohydrate, potassium phosphate-dibasic (K2HPO4), and sodium phosphate-monobasic (NaH2PO4) were also obtained from Sigma
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(St. Louis, MO), Merck (Darmstadt, Germany), Difco (Oxoid, UK), and Fluka (Thessaloniki, Greece). Plant material The plant samples of M. longifolia subsp. longifolia were collected at the flowering stage from the Palando¨ken Mountain of Erzurum located in the eastern Anatolia in Turkey. The taxonomic identification of plant material was confirmed by Dr. Meryem Sengul Koseoglu, a senior plant taxonomist in Biology Department of Atatu¨rk University, Erzurum, Turkey. The voucher specimen has been deposited at the Herbarium of the Department of Biology (ATA Herbarium 9732), Atatu¨rk University. The collected plant materials were dried in the shade, then, the aerial parts of the plant samples were separated from the stem and ground in a grinder with a 2 mm in diameter mesh. Extraction, fractionation, and isolation Air-dried and powdered aerial parts of the plant (1000 g) were extracted four times with MeOH at 40 C (4 2 L). After evaporation of the combined extracts in vacuo, 140 g of MeOH extract was obtained. The crude extract was dissolved in water and subjected to liquid–liquid partitions successively with petroleum ether (25 0.5 L), CHCl3 (8 0.5 L), EtOAc (6 0.5 L), and n-butanol (15 0.5 L). Thereafter, the solvents were evaporated under reduced pressure in a rotary evaporator oven at 45 C. The residues obtained were 33.4, 14.7, 8.4, and 36.3 g. Fractionation of M. longifolia subsp. longifolia was done by using the Ames/Salmonella test system as a guide to obtain the mutagenic or antimutagenic extracts and compounds. According to the results, the n-butanol extract showed significant antimutagenic activity. Then, the n-butanol extract (36.3 g) was first subjected to a silica gel column and eluted with a solvent gradient of CHCl3:MeOH (100:0 ! 0:100) to afford five main fractions (Fr. A: 450 mg, Fr. B: 1.9 g, Fr. C: 11.8 g, Fr. D: 5.6 g, and Fr. E: 8.2 g). The mutagenic and antimutagenic potential was revaluated by using the same test system and three of them (A, C, and E fractions) were determined as antimutagenic. The fraction A (450 mg) was rechromatographed by Sephadex LH-20 CC; eluting with MeOH to give a subfraction (Fr. A1). Purification of subfraction A1 (50 mg) by Sephadex LH-20 CC using MeOH gave compound 3 (3.6 mg). The fraction C (11.8 g) was rechromatographed by Sephadex LH-20 CC; eluting with MeOH to give three subfractions (Fr. C1–3). Fraction C1 was further fractionated by vacuum liquid chromatography using reversed phase material with MeOH–H2O mixtures (0–50%) to give three subfractions (Fr. C1.1–1.3). Purification of subfractions C1.1 (253 mg) and C1.3 (35 mg) by Sephadex LH-20 CC using MeOH gave compound 1 (150 mg) and compound 5 (6.3 mg), respectively. Fraction C1.2 (207 mg) was also eluted with a gradient of CHCl3–MeOH (85:15 ! 80:20, v/v) by silica gel column chromatography to yield compound 6 (18.3 mg). On one hand, fraction C2 was subjected to vacuum liquid chromatography using reversed phase material and MeOH– H2O mixtures (0–50%) as a solvent to give Fr.C2.1 and Fr.C2.2. Purification of subfractions C2.1 (473 mg) and C2.2 (758 mg)
DOI: 10.3109/13880209.2014.948633
by Sephadex LH-20 CC using MeOH gave compound 9 (10 mg) and compound 2 (7.6 mg), respectively. On the other hand, fraction C3 directly gave pure compound 4 (7 mg). The fraction E (8.2 g) was rechromatographed by Sephadex LH-20 CC; eluting with MeOH to give two subfractions (Fr. E1–2). Repeated CC of the fraction E1 on Sephadex LH-20, silica gel, and reversed phase material gave compound 8 (8.8 mg). Finally, purification of subfraction E2 (20 mg) by Sephadex LH-20 CC using MeOH gave compound 7 (5.2 mg).
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Identification of compounds 1–9 Extraction, fractionation, and isolation steps of the present study led to the isolation of two new chalcone glycosides together with known six flavonoid glycosides and one phenolic acid (Figure 1). The structures of compounds were elucidated based on the analysis of their physical and spectroscopic data (1H NMR, 13C NMR, DEPT, 2D NMR, EIMS, and MALDI-MS). The known compounds isolated in the present study were identified by comparison of their physical and spectroscopic data with previous information in the literature. (bR)-,3,20 ,60 -Tetrahydroxy-4-methoxy-40 -O-rutinosyldihydrochalcone (1): Yellowish powder; ½30 D 139.39 (c 0.66, MeOH); IR max cm1: 3342, 1633; UV (MeOH) lmax (log ") 225 (4.03), 284 (3.92) nm; MALDI-MS: m/z 629.2068 [M + H]+ (theoretical 629.2076); CD (MeOH, c ¼ 3.18 104 M) []281 5325, []247 + 691.2; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data (Table 1). (R)-,4,20 ,60 -Tetrahydroxy-40 -O-rutinosyldihydrochalcone (2): Yellowish powder; ½30 D 40 (c 0.2, MeOH); IR max cm1: 3342, 1633; UV (MeOH) lmax (log ") 229 (4.57), 287 (4.65) nm; MALDI-MS: [M + H]+ at m/z 599.1966 (theoretical 599.1970); CD (MeOH, c ¼ 4.18 104 M) []281 6217, []250 + 516.1; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data (Table 1). Apigenin-7-O-glucoside (3): Yellow amorphous powder. EIMS m/z 269 [M Glc]+ (Calcd for C21H20O10). 1H NMR (DMSO-d6, 400 MHz): 7.93 (2H, d, J ¼ 8.8 Hz, H-20 , 60 ), 6.91 (2H, d, J ¼ 8.8 Hz, H-30 , 50 ), 6.84 (1H, s, H-3), 6.79 (1H, d, J ¼ 2.1 Hz, H-8), 6.42 (1H, d, J ¼ 2.1 Hz, H-6), 5.04 (1H, d, J ¼ 7.0 Hz, H-100 ), 3.68–3.86 (2H, m, Ha-600 , Hb-600 ), 3.14– 3.33 (4H, m, H-200 , 300 , 400 , 500 ). 13C NMR (DMSO-d6, 100 MHz): 182.7 (C-4), 164.9 (C-2), 163.6 (C-7), 162.1 (C5), 161.7 (C-40 ), 157.6 (C-9), 129.3 (C-20 , 60 ), 121.7 (C-10 ), 116.7 (C-30 , 50 ), 106.0 (C-10), 103.8 (C-3), 100.6 (C-100 ), 100.2 (C-6), 95.5 (C-8), 77.8 (C-300 ), 77.1 (C-500 ), 73.8 (C-200 ), 70.2 (C-400 ), 61.3 (C-600 ). 1H NMR and 13C NMR agree with data given in the literature for apigenin-7-O-glucoside (Baris et al., 2011; Gu¨venalp et al., 2006, 2009). Luteolin-7-O-glucoside (4): Yellow amorphous powder. EIMS m/z 285 [M Glc]+ (Calcd for C21H20O11). 1H NMR (DMSO-d6, 400 MHz): 7.42 (1H, dd, J ¼ 8.4/2.1 Hz, H-60 ), 7.39 (1H, d, J ¼ 1.8 Hz, H-20 ), 6.88 (1H, d, J ¼ 8.4 Hz, H-50 ), 6.77 (1H, d, J ¼ 1.8 Hz, H-8), 6.73 (1H, s, H-3), 6.42 (1H, d, J ¼ 1.8 Hz, H-6), 5.05 (1H, d, J ¼ 7.3 Hz, H-100 ), 3.69 (1H, dd, J ¼ 10.2/- Hz, H-600 b), 3.14–3.63 (5H, m, H-200 , 300 , 400 , 500 , 600 a). 13C NMR (DMSO-d6, 100 MHz): 182.6 (C-4), 165.1
Antigenotoxic chalcone glycosides of Mentha (L.)
3
(C-2), 163.6 (C-7), 161.8 (C-5), 157.6 (C-9), 150.6 (C-40 ), 146.5 (C-30 ), 122.0 (C-10 ), 119.9 (C-60 ), 116.7 (C-50 ), 114.2 (C-20 ), 106.0 (C-10), 103.8 (C-3), 100.5 (C-100 ), 100.2 (C-6), 95.4 (C-8), 77.8 (C-500 ), 77.0 (C-300 ), 73.8 (C-200 ), 70.2 (C-400 ), 61.3 (C-600 ). 1H NMR and 13C NMR agree with data given in the literature for luteolin-7-O-glucoside (Kuruu¨zu¨m-Uz et al., 2008). Apigenin-7-O-rutinoside (5): Yellow amorphous powder. EIMS m/z 269 [M-(Glc + Rh)]+ (Calcd for. C27H30O14). 1H NMR (CD3OD, 400 MHz): 7.86 (2H, d, J ¼ 8.7 Hz, H-20 , 60 ), 6.93 (2H, d, J ¼ 8.7 Hz, H-30 , 50 ), 6.74 (1H, d, J ¼ 1.8 Hz, H-8), 6.63 (1H, s, H-3), 6.50 (1H, d, J ¼ 1.8 Hz, H-6), 5.03 (1H, d, J ¼ 7.3 Hz, H-100 ), 4.70 (1H, d, J ¼ 1.4 Hz, H-1000 ), 4.03 (1H, d, J ¼ 9.1 Hz, H-600 b), 3.89 (1H, dd, J ¼ 3.2/1.5 Hz, H-2000 ), 3.70 (1H, dd, J ¼ 9.5/3.6 Hz, H-3000 ), 3.29–3.66 (7H, m, H-200 , 300 , 400 , 500 , 600 a, 4000 , 5000 ), 1.17 (3H, d, J ¼ 6.2 Hz, H6000 ). 13C NMR (CD3OD, 100 MHz): 182.9 (C-4), 165.6 (C2), 163.5 (C-7), 161.8 (C-5), 161.7 (C-40 ), 157.7 (C-9), 128.5 (C-20 , C-60 ),121.9 (C-10 ),116.0 (C-30 , C-50 ),106.0 (C-10), 103.0 (C-3), 100.9 (C-100 ),100.3 (C-1000 ), 99.9 (C-6), 95.1 (C8), 76.6 (C-500 ), 76.0 (C-300 ), 73.5 (C-4000 ), 72.9 (C-200 ), 71.2 (C-2000 ), 70.9 (C-3000 ), 70.1 (C-400 ), 68.6 (C-5000 ), 66.2 (C-600 ), 16.7 (C-6000 ). 1H NMR and 13C NMR agree with data given in the literature for apigenin-7-O-rutinoside (Baris et al., 2011). Luteolin-7-O-rutinoside (6): Yellow amorphous powder. EIMS m/z 285 [M-(Glc + Rh)]+ (calc. for. C27H30O15). 1H NMR (CD3OD, 400 MHz): 7.34 (1H, d, J ¼ 2.2 Hz, H-20 ), 7.32 (1H, dd, J ¼ 8.7/2.2 Hz, H-60 ), 6.88 (1H, d, J ¼ 8.7 Hz, H-50 ), 6.65 (1H, d, J ¼ 2.1 Hz, H-8), 6.52 (1H, s, H-3), 6.45 (1H, d, J ¼ 2.1 Hz, H-6), 5.00 (1H, d, J ¼ 7.3 Hz, H-100 ), 4.72 (1H, d, J ¼ 1.5 Hz, H-1000 ), 4.05 (1H, d, J ¼ 9.5 Hz, H-600 b), 3.91 (1H, dd, J ¼ 3.5/1.6 Hz, H-2000 ), 3.73 (1H, dd, J ¼ 9.5/ 3.3 Hz, H-3000 ), 3.29–3.66 (7H, m, H-200 , 300 , 400 , 500 , 600 a, 4000 , 5000 ), 1.18 (3H, d, J ¼ 6.2 Hz, H-6000 ). 13C NMR (CD3OD, 100 MHz): 182.8 (C-4), 165.6 (C-2), 163.5 (C-7), 161.7 (C5), 157.6 (C-9), 149.9 (C-40 ), 145.7 (C-30 ), 122.3 (C-10 ), 119.5 (C-60 ), 115.7 (C-50 ), 113.2 (C-20 ), 105.9 (C-10), 103.1 (C-3), 100.9 (C-100 ), 100.4 (C-1000 ), 100.0 (C-6), 95.0 (C-8), 76.6 (C500 ), 76.0 (C-300 ), 73.6 (C-4000 ), 72.9 (C-200 ), 71.3 (C-2000 ), 70.9 (C-3000 ), 70.2 (C-400 ), 68.6 (C-5000 ), 66.4 (C-600 ), 16.7 (C-6000 ). 1 H NMR and 13C NMR agree with data given in the literature for luteolin-7-O-rutinoside (Wang et al., 2003). Apigenin-7-O-glucuronide (7): Yellow amorphous powder. EIMS m/z 269 [M-Glu]+ (Calcd for. C21H18O11). 1H NMR (DMSO-d6, 400 MHz): 7.87 (2H, d, J ¼ 8.8 Hz, H-20 , 60 ), 6.88 (2H, d, J ¼ 8.8 Hz, H-30 , 50 ), 6.80 (1H, s, H-3), 6.77 (1H, d, J ¼ 1.8 Hz, H-8), 6.42 (1H, d, J ¼ 1.8 Hz, H-6), 5.05 (1H, d, J ¼ 7.3 Hz, H-100 ), 3.61 (1H, d, J ¼ 10.3 Hz, H-500 ), 3.13–3.40 (3H, m, H-200 , 300 , 400 ). 13C NMR (DMSO-d6, 100 MHz): 182.6 (C-4), 172.6 (C-600 ), 164.9 (C-2), 163.7 (C-7), 162.4 (C5), 161.7 (C-40 ), 157.6 (C-9), 129.4 (C-20 , 60 ), 121.3 (C-10 ), 116.7 (C-30 , 50 ), 105.9 (C-10), 103.5 (C-3), 100.3 (C-100 ), 100.2 (C-6), 95.3 (C-8), 77.2 (C-300 ), 74.4 (C-500 ), 73.6 (C-200 ), 72.6 (C-400 ). 1H NMR and 13C NMR agree with data given in the literature for apigenin-7-O-glucuronide (Baris et al., 2011; Guvenalp et al., 2009). Luteolin-7-O-glucuronide (8): Yellow amorphous powder. EIMS m/z 285 [M-GlcA]+ (Calcd for. C21H18O12). 1H NMR (DMSO-d6, 400 MHz): 7.40 (1H, d, J ¼ 2.2 Hz, H-20 ), 7.37 (1H, dd, J ¼ 8.4/2.2 Hz, H-60 ), 6.84 (1H, d, J ¼ 8.4 Hz, H-50 ),
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Figure 1. Chemical structures of compounds 1–9.
Pharm Biol, Early Online: 1–9
HO
HO
HO
4''' 6'''
H3C
2'''
3'''
O
1'''
R1
5'''
O 4''
HO
O
6'' 5''
HO
3'
O
OH
4'
1''
2''
α
1'
5'
5
1
6'
6
β O
OH
R1 OH H
1 2
4
2
2'
OH
3''
3
OH
R2 OCH3 OH
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R2 3'
OH
2'
O
R1 HO
4''
5''
HO
3''
8
O 7
1''
2''
OH
6
4
O
R1 CH2OH CH2OH COOH COOH
R2 H OH H OH R
HO
HO
3'
2'''
3'''
6'''
H3C
6'
3
10 5
3 4 7 8
HO
5'
2
OH
4'''
1'
O
9
4'
O
2'
1'''
5'''
O
HO
O
6''
4''
5''
HO
3''
OH
O
9
7
1''
2''
8
O
6
6'
3
10 4
5
O
R H OH
5 6
5' 9'
COOH
O 2 3
1
4
HO
7'
9
7 8
6 5
9
O
6'
OH 4'
3'
1'
8'
5'
1' 2
OH
HO
OH 4'
2'
OH
R2
Antigenotoxic chalcone glycosides of Mentha (L.)
DOI: 10.3109/13880209.2014.948633
Table 1. 1H and
13
C NMR spectroscopic data for compounds 1 and 2. Compound 1
No.
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1 2 3 4 5 6 A B C¼O OCH3 10 20 30 40 50 60 100 200 300 400 500 600 1000 2000 3000 4000 5000 6000
5
H (J in Hz) 6.78 d (2.1) 6.70 d (8.1) 6.94 dd (8.1, 2.1) 2.75 dd (17.0, 2.9) 3.11 dd (17.0, 12.4) 5.31 dd (11.4, 2.9) 3.85 s 6.18 d (2.2) 6.16 d (2.2) 4.93 d (7.0) 3.41–3.48 m 3.45–3.60 m 3.60–3.70 m 3.40–3.50 m 3.58–3.65 m 3.98 dd (9.5, 2.5) 4.68 d (1.83) 3.88 dd (3.48, 1.83) 3.60–3.65 m 3.30–3.40 m 3.55–3.65 m 1.18 d (6.2)
C 130.4 113.7 145.2 145.7 115.2 118.2 42.9 79.5 197.4 55.3 103.8 163.3 95.9 165.6 96.8 163.7 101.0 73.5 75.9 71.2 76.7 66.3
Compound 2
3,4,1, b 3, 1, 6 3,4,1, b C¼O C¼O, 1, b 2,6,1, a, C¼O
7.32 d (8.4) 6.81 d (8.4) 6.81 d (8.4) 7.32 d (8.4) 2.74 dd (17.1, 2.9) 3.15 dd (16.9, 12.4) 5.39 dd (12.4, 2.9)
C 129.7 128.0 115.2 157.9 115.2 128.0 42.9 79.4 197.3
HMBC 4, 1, b 4, 1, 5, b 4, 1, 3, b 4, 1, b C¼O C¼O, b C¼O,1, 2, 6
4 10 ,50 ,20 ,40 , C¼O
6.18 d (2.2)
10 ,30 ,40 ,60 , C¼O
6.16 d (2.2)
99.9 70.2 70.8 72.9 68.6 16.8
6.75 (1H, d, J ¼ 1.8 Hz, H-8), 6.68 (1H, s, H-3), 6.39 (1H, d, J ¼ 1.8 Hz, H-6), 5.06 (1H, d, J ¼ 7.3 Hz, H-100 ), 3,61 (1H, d, J ¼ 9.8 Hz, H-500 ), 3.14–3.31 (3H, m, H-200 , H-300 , H-400 ); 13C NMR (DMSO-d6, 100 MHz): 182.5 (C-4), 172.8 (C-600 ), 165.2 (C-2), 163.7 (C-7), 161.7 (C-5), 157.6 (C-9), 151.1 (C40 ), 146.6 (C-30 ), 121.6 (C-10 ), 119.7 (C-60 ), 116.7 (C-50 ), 114.1 (C-20 ), 105.9 (C-10), 103.5 (C-3), 100.4 (C-100 ), 100.3 (C-6), 95.3 (C-8), 77.1 (C-300 ), 74.5 (C-500 ), 73.6 (C-200 ), 72.6 (C-400 ). 1H NMR and 13C NMR agree with data given in the literature for luteolin 7-O-glucuronide (Gulluce et al., 2012). Rosmarinic acid (9): White-yellow amorphous solid; EIMS m/z 360 [M] (Calcd for. C18H16O8). 1H NMR (CD3OD, 400 MHz): 7.50 (1H, d, J ¼ 15.7 Hz, H-7), 7.02 (1H, d, J ¼ 1.8 Hz, H-2), 6.90 (1H, dd, J ¼ 8.0/1.8 Hz, H-6), 6.76 (1H, d, J ¼ 8.0 Hz, H-5), 6.76 (1H, d, J ¼ 1.8 Hz, H-20 ), 6.67 (1H, d, J ¼ 8.0 Hz, H-50 ), 6.62 (1H, dd, J ¼ 8.0/1.8 Hz, H-60 ), 6.26 (1H, d, J ¼ 15.7 Hz, H-8), 5.06 (1H, dd, J ¼ 9.7/ 3.4 Hz, H-80 ), 3.09 (1H, d, J ¼ 14.1/3.1 Hz, H-70 b), 2.93 (1H, d, J ¼ 14.1/9.7 Hz, H-70 a), 13C NMR (CD3OD, 100 MHz): 176.5 (C-90 ), 167.9 (C-9), 148.2 (C-4), 145.5 (C-7), 145.5 (C-30 ), 144.7 (C-40 ), 143.6 (C-3), 130.0 (C-10 ), 126.7 (C-1), 121.7 (C-60 ), 120.6 (C-6), 116.3 (C-20 ), 115.3 (C-50 ), 115.0 (C-5), 114.4 (C-2), 114.0 (C-8), 76.6 (C-80 ), 37.6 (C-70 ). 1H NMR and 13C NMR agree with data given in the literature for rosmarinic acid (Ly et al., 2006). Bacterial strains Õ
H (J in Hz)
HMBC
Salmonella typhimurium TA1535 (ATCC number: 29629) Õ and S. typhimurium TA1537 (ATCC number: 29630) strains
40
600
4.93 d (7.3) 3.41–3.48 m 3.45–3.60 m 3.60–3.70 m 3.40–3.50 m 3.58–3.65 m 3.98 dd (9.5, 2.5) 4.68 bs 3.88 dd (3.48, 1.83) 3.60–3.65 m 3.30–3.40 m 3.55–3.65 m 1.18 d (6.2)
103.8 163.2 95.9 165.7 96.8 163.3 101.0 73.5 75.9 71.2 76.7 66.2
10 , 50 , 20 , 40 10 , 30 , 40 , 60
99.9 70.1 70.9 72.9 68.6 16.7
40
600
were provided by The American Type Culture Collection – Bacteria Department of Georgetown University, Washington, DC. Both strains were stored at 80 C. Working cultures were prepared by inoculating nutrient broth with the frozen cultures, followed by an overnight incubation at 37 C with gentle agitation (Oh et al., 2008). Viability assays and determination of test concentrations Toxic levels of the test materials toward S. typhimurium TA1535 and 1537 strains were determined as described in detail elsewhere (Yu et al., 2001). These tests confirmed that there was normal growth of the background lawn, spontaneous colony numbers within the regular range, and no significant reduction in cell survival. Thus, for the concentrations and conditions reported here, no toxicity or other adverse effects were observed. The Ames/Salmonella test The bacterial mutagenicity and antimutagenicity assays were performed according to described procedures (Mortelmans & Zeiger, 2000). The known mutagens NaN3 (in distilled water – 1 mg/plate) for S. typhimurium TA1535 and 9-AA (in methanol – 10 mg/plate) for S. typhimurium TA1537 were used as positive controls and 10% DMSO was used as a negative control. The plate incorporation method was used to assess the results of mutagenicity and antimutagenicity assays (Mortelmans & Zeiger, 2000).
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For the mutagenicity assays, the mutagenic index was calculated for each concentration, which is the average number of revertants per plate divided by the average number of revertants per plate with the negative (solvent) control. A sample was considered as mutagenic when a dose– response relationship and a two-fold increase in the number of revertants with at least one concentration were observed (Gulluce et al., 2010). For the antimutagenicity assays, the inhibition rate of mutagenicity was calculated by using the following equation (M: the number of revertants/plate induced by mutagen alone, S0: the number of spontaneous revertants, S1: the number of revertants/plate induced by the extract plus the mutagen): inhibition% ¼ {1 [(M S1)/(M S0)]} 100 About 25–40% inhibition was defined as moderate antimutagenicity; 40% or more inhibition as strong antimutagenicity; and less than 25% inhibition as no antimutagenicity (Gulluce et al., 2010). Statistical analysis The results are presented as the average and standard error of three experiments with duplicate plates/dose experiment. The data were further analyzed for statistical significance using analysis of variance (ANOVA), and the difference among means was compared by high-range statistical domain using Tukey’s test. A level of probability was taken as p50.05 indicating statistical significance (Gulluce et al., 2010).
Results and discussion Chemical results Compound 1 was obtained as a yellowish powder. The molecular formula was established as C28H36O16, by positiveion and reflectron MALDI-MS, which showed molecular ion [M + H]+ at m/z 629.2068 (theoretical 629.2076). The IR spectrum displayed intense absorbtion bands for hydroxyl (3342 cm1), and carbonyl (1633 cm1) functionalities. Analysis of the 13C NMR and DEPT spectra indicated the presence of 28 carbon signals of which 15 carbon atoms were assigned to be aglycon, 12 carbons to the sugar moiety, and one carbon to the methoxy group. 1H NMR spectrum indicated the presence of an ABX-type aromatic proton system appearing at 6.70 (1H, d, J ¼ 8.1 Hz), 6.94 (1H, dd, J ¼ 8.1/2.1 Hz), and 6.78 (1H, d, J ¼ 2.1 Hz) due to a ring protons as well as a methoxyl proton signal at 3.85, a pair of meta-coupled aromatic protons at 6.18 and 6.16 (1H each, both d, J ¼ 2.2 Hz) and a set of three peculiar aliphatic protons 2.75 (1H, dd, J ¼ 17.0/2.9 Hz), 3.11 (1H, dd, J ¼ 17.0/ 12.4 Hz), and 5.31 (1H, dd, J ¼ 11.4/2.9 Hz) together with one glucopyranosyl part and one rhamnopyranosyl part. The large coupling constant (7.0 Hz) of the anomeric proton 4.93 indicated the presence of the b-glucose moiety. The small coupling constant (1.83 Hz) of the anomeric proton 4.68 indicated the presence of the a-rhamnose moiety. The 1H and 13 C NMR signals of compound 1 were completely assigned by a combination of HMQC, HMBC, and 1H–1H COSY experiments. The 1H–1H COSY correlation between H2-a (H 2.75, 3.11) and H-b (H 5.31) and the HMBC correlations of H2-a to C¼O (C 197.4), C-b (C 79.5), and C-1 (C 130.4);
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H-b to C¼O (C 197.4), C-a (C 42.9), C-1, C-2 (C 113.7), and C-6 (C 118.2); H-100 (H 4.93) to C-40 (C 165.6); H-1000 (H 4.68) to C-600 (C 66.3), and H-OMe to C-4 (C 145.7) were determined (Figure 2). The absolute configuration at the b position was assigned as R from the CD spectrum that showed a positive Cotton effect at 247 nm and a negative Cotton effect at 281 nm (Nel et al., 1999). Thus, the structure of compound 1 was characterized as (bR)-b,3,20 ,60 -tetrahydroxy-4-methoxy-40 -O-rutinosyldihydrochalcone. Compound 2 was also isolated as a yellowish powder. The molecular formula was established as C27H34O15, by positiveion and reflectron MALDI-MS, which showed molecular ion [M + H]+ at m/z 599.1966 (theoretical 599.1970). IR spectra of 2 showed similar bands compared to those of 1. The 13C NMR and DEPT spectra indicated the presence of 27 carbon signals of which 15 carbon atoms were assigned to be aglycon, and 12 carbons to the sugar moiety. The 1H NMR spectrum showed a pair of A2B2 type signals at 7.32 and 6.81 (2H each, both d, J ¼ 8.4 Hz) attributed to a parasubstituted phenyl group, a pair of meta-coupled aromatic protons at 6.18 and 6.16 (1H each, both d, J ¼ 2.2 Hz) and a set of three peculiar aliphatic protons 2.74 (1H, dd, J ¼ 17.1/ 2.9 Hz), 3.15 (1H, dd, J ¼ 16.9/12.4 Hz), and 5.39 (1H, dd, J ¼ 12.4/2.9 Hz) together with one glucopyranosyl part and one rhamnopyranosyl part. The b-configuration of the glucopyranosyl and a-configuration of the rhamnopyranosyl moieties were deduced from the coupling constants of the anomeric protons and the 13C NMR data of the sugar unit. The 1H–1H COSY spectrum of compound 2 showed correlations between H2- (H 2.74, 3.15) and H-b (H 5.39) and the HMBC spectrum of compound 2 indicated correlations of H2-a to C¼O (C 197.3), and C-b (C 79.4); H-b to C¼O (C 197.3), C-1 (C 129.7), and C-2,6 (C 128.0); H-100 (H 4.93) to C-40 (C 165.7), and H-1000 (H 4.68) to C-600 (C 66.2) (Figure 2). The CD spectrum showed a positive Cotton effect at 250 nm and a negative one at 281 nm which suggested R-configuration at the b carbon (Nel et al., 1999). Thus, the structure of compound 2 was established as (bR)-b,4,20 ,60 tetrahydroxy-40 -O-rutinosyldihydrochalcone. Genotoxic and antigenotoxic activity evaluation Chalcone derivatives have a great importance for medicinal studies due to their various bioactive properties (Chen et al., 2012; Kaur et al., 2009). Antigenotoxic activity is a good example for the most important biological effects of these molecules, which is mainly related to their antioxidant capabilities (Kaur et al., 2009). Recent studies have focused on isolation of new natural compounds with antigenotoxic potential from plant components owing to increasing concerns about mutagenesis and related disorders such as cancer (Baris et al., 2011; Gulluce et al., 2012). In the present study, bacterial tester strains for determination of mutagenic and antimutagenic potentials of bioactive chalcone derivatives were S. typhimurium TA1535 and TA1537. Salmonella typhimurium TA1535 is known as a tester strain capable for detection of mutagenic chemicals cause base-substitution type of mutagenesis. In contrast, S. typhimurium TA1537 is known as a tester strain capable for
Antigenotoxic chalcone glycosides of Mentha (L.)
DOI: 10.3109/13880209.2014.948633
HO
4'''
HO
HO 2'''
3'''
6'''
H3C
7
O
1'''
OH
5'''
O 4''
HO
6'' 5''
HO
O 2''
3'
O 4'
1''
α
1'
5' 6'
5
1 6
β O
OH
OCH3
4
2
2'
OH
3''
3
OH
OH
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1
HO
4'''
HO
6'''
H3C
HO 2'''
3'''
O
1'''
5'''
O
HO
6''
4''
5''
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3''
O 2''
3'
O 1''
3
OH
4'
2'
OH
α
1'
5' 6'
4
2
5
1 6
β
OH
O
OH
OH
2 Figure 2. HMBC correlations of compounds 1 and 2.
detection of mutagenic chemicals cause frame-shift mutagenesis. Furthermore, these strains also can be used for detection of antimutagenic chemicals which inhibit hazardous activities of mutagenic substances cause base-substitution and frameshift mutagenesis (Mortelmans & Zeiger, 2000). According to the results of viability assays, the concentrations up to 2 mM were determined as applicable for both chalcone glycosides without any adverse effect or toxicity on bacterial tester strains. Thus, 0.4, 0.8, 1.2, 1.6, and 2 mM concentrations were chosen to determine mutagenic and antimutagenic potential of the test compounds. In the mutagenicity assays performed with S. typhimurium TA1535 and TA1537 strains, both test compounds did not give any mutagenic activity on the tester strains (Table 2). In the antimutagenicity assays performed with the same strains, both compounds did not show an antimutagenic effect against sodium azide (NaN3) induced mutagenesis in S. typhimurium TA1535. NaN3 is known as a powerful mutagen, which affects several organisms including bacteria, plants, and animals, and generally causes base-substitutions. Previous studies showed that the mutagenicity of NaN3 is related to the production of L-azidoalanine (Sadiq & Owais, 2000). One reason for observing no antimutagenicity against NaN3 may be that both the tested chalcones do not have sufficient capability to inhibit formation of L-azidoalanine. However, these compounds showed significant antimutagenic activity against 9-aminoacridine (9-AA)-induced mutagenesis in S. typhimurium TA1537. Inhibition rates of mutagenesis in TA1537 ranged from 49.14% to 65.41% for
Table 2. The mutagenicity assay results of the novel chalcone derivatives for S. typhimurium TA1535 and TA1537 bacterial tester strains. Number of revertants
Test items
Concentration (mM/plate)
S. typhimurium S. typhimurium TA1535 TA1537 Mean ± S.E. Mean ± S.E.
Compound 1 0.4 0.8 1.2 1.6 2.0
25.00 ± 01.29 25.83 ± 01.17 25.00 ± 01.53 24.00 ± 00.58 25.67 ± 01.23
25.50 ± 01.28 24.50 ± 01.26 23.50 ± 01.54 23.50 ± 01.98 27.33 ± 00.92
0.4 0.8 1.2 1.6 2.0
24.50 ± 00.92 25.67 ± 01.20 24.33 ± 00.67 26.17 ± 01.35 23.67 ± 01.02
23.83 ± 00.95 25.33 ± 01.67 24.33 ± 01.54 23.17 ± 01.35 25.50 ± 01.31
364.17 ± 08.66 26.17 ± 01.17
444.67 ± 10.37 23.33 ± 01.28
Compound 2
Controls NaN3a 9-AAa DMSOa (ml/plate) a
NaN3 (1 mg/plate) and 9-AA (40 mg/plate) were used as positive controls for S. typhimurium TA1535 and TA1537 strains, respectively. DMSO (dimethylsulfoxide; 100 ml/plate) was used as a negative control.
compound 1 and from 30.06 to 75.94% for compound 2 (Table 3). 9-AA, a member of acridine family, is known as a model frame-shift mutagen (Ferguson & Denny, 1991). In the frame-shift mutagenesis mechanism, it binds to DNA non-
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Table 3. The antimutagenicity assay results of the novel chalcone derivatives for S. typhimurium TA1535 and TA1537 bacterial tester strains. Number of revertants
Test items
Concentration (mM/plate)
S. typhimurium TA1535 Mean ± S.E.
S. typhimurium TA1537 Mean ± S.E.
Compound 1 0.4 0.8 1.2 1.6 2.0
367.50 ± 10.51 359.00 ± 12.35 355.67 ± 12.28 349.00 ± 08.24 372.00 ± 07.86
458.83 ± 12.61 226.17 ± 08.86y 199.50 ± 08.55y 177.00 ± 07.43y 153.83 ± 07.70y
0.4 0.8 1.2 1.6 2.0
369.00 ± 12.84 348.83 ± 08.99 368.00 ± 07.21 367.83 ± 08.08 357.83 ± 12.77
447.33 ± 07.39 311.00 ± 09.96y 266.00 ± 06.06y 171.67 ± 04.42y 107.00 ± 05.11y
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Compound 2
Controls NaN3a 9-AAa DMSOa (ml/plate)
364.17 ± 08.66 444.67 ± 10.37 26.17 ± 01.17 23.33 ± 01.28
a
NaN3 (1 mg/plate) and 9-AA (40 mg/plate) were used as positive controls for S. typhimurium TA1535 and TA1537 strains, respectively. DMSO (dimethylsulfoxide; 100 ml/plate) was used as a negative control. yp50.05 to positive control.
covalently by intercalation. Through this way, 9-AA induces frameshift mutations at hot spots where a single base, especially guanine, is repeated (Hoffmann et al., 2003). They performed experiments to determine antimutagenic properties of the test materials by using S. typhimurium TA1537 strain and 9-AA depend on inhibition of the intercalation mechanism. The results showed that both chalcone derivatives used in this study have antimutagenic activity against 9-AA. This activity may be due to their inhibition capabilities by blocking 9-AA binding to DNA. In contrast, it is known that antimutagenic activity of phytophenols is mainly dependent on their antioxidant activity (Nagy et al., 2009). The antimutagenic potencies of these molecules mainly depend on the number and the position of the polar hydroxyl groups (OH) within the molecule (Edenharder & Gru¨nhage, 2003). Chalcone derivatives are also known as antioxidative molecules with polar hydroxyl groups. Both chalcone derivatives have the same numbers of OH groups in their structures. Probable reasons for the difference between antimutagenic potentials of the test compounds may be explained by the different positions of OH between R1 and R2 groups, and sterical effect of OMe group in the structure of compound 1.
Conclusions Both chalcone derivatives isolated from M. longifolia subsp. longifolia can be considered as genotoxically safe at tested concentrations owing to the fact that they did not show any mutagenic activity on the mutant tester strains. Both of them also provided significant antimutagenic activity against known mutagen 9-AA in the same test system. These activities are valuable for further investigations focusing on protective strategies development against 9-AA induced
genotoxicity. The data obtained from the present study can also be supported by performing studies with more complex test systems resulting in more reliable results for human health applications.
Declaration of interest This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK: 107T203).
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