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Artosimmin- A Potential Anti-Cancer Lead Compound from Artocarpus odoratissimus Gwendoline C.L. Eea*, Siow H. Teoa, Mawardi Rahmania, Chan K. Limb, Yang M. Limc and Choon F.J. Bongd a
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
b
Department of Chemical Science, Faculty of Science, Engineering & Technology, Universiti Tunku Abdul Rahman, Kampar, 31900, Perak, Malaysia c
Faculty of Engineering & Science, Universiti Tunku Abdul Rahman, Jalan Genting Klang, 53300 Kuala Lumpur, Malaysia. d
Department of Crop Science, Faculty of Agriculture & Food Sciences, Universiti Putra Malaysia Bintulu Campus, 97008, Bintulu, Sarawak, Malaysia Received January 19, 2010: Revised February 09, 2010: Accepted February 17, 2010
Abstract: Our recent study on Artocarpus odoratissimus (Moraceae) has resulted in the purification of a new prenylated pyranoflavone derivative artosimmin (1) and traxateryl acetate (2). Details of structural elucidations associated with 1 and 2 are reported by combining 1D, 2D NMR and mass spectrometric methods. Compound 2 is obtained for the first time from this species. Furthermore, the biological assay results exhibited compound 1 to be significantly cytotoxic against cancer cell lines (HL-60 & MCF-7) and also possessed antioxidant properties toward 1,1-diphenyl-2-picrylhydrazyl radical (DPPH).
Keywords: Artocarpus odoratissimus, moraceae, pyranoflavone, triterpenoid, cytotoxicity, antioxidant. INTRODUCTION Artocarpus odoratissimus (Moraceae) originated in Borneo. It is cultivated in the Philippines and is also found in the wild extensively in Brunei Darussalam and parts of Sabah and Sarawak [1]. This versatile tree has provided the needs of the rural communities by providing food, nutrition and many other applications [2]. Artocarpus odoratissimus has shown antioxidant properties, is a source of phytochemicals, as well as it inhibits the oxidation of organic molecules in food and preserves defence of living systems. This species contains polyphenol compounds such as gallic acid, ellagic acid and ferulic acid derivatives and is a promising source of natural food antioxidant [3]. During our course of research on the constituents of this Artocarpus species, we isolated and structurally elucidated a new pyranoflavone (1) and triterpenoid (2). The biological assay tests indicated compound 1 to be a potential anticancer and antioxidant lead compound. No phytochemical investigation or structural characterization on this plant species has been reported to date. RESULTS AND DISCUSSION Compound 1 is an orange amorphous powder with mp 210-213 oC. The UV spectrum exhibited absorption maxima *Address correspondence to this author at the Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia; Tel: 603-89466785, Fax: 603-89435380; E-mail:
[email protected]
1570-1786/10 $55.00+.00
at 213, 271, 340, and 446 nm in methanol indicating a flavone derivative. 1 was assigned a molecular formula of C25H24O7 on the basis of its EIMS spectrum (M+, m/z 436). The fragment ion peaks at m/z 421 [M+ – CH3] and m/z 381 [M+ – (CH3)2C=CH] were due to the loss of a methyl group and a four carbon side chain (butenyl) from the C-5’ prenyl moiety at the B-ring of the structure. Meanwhile, the fragmentation at m/z 365 [421 – H2C=C(CH3)2] demonstrated the loss of another side chain of the C-9 butenyl moiety at D-ring of the structure. Subsequent loss of one carbonyl group from each of these fragment ions gave other fragment ion peaks at m/z 393 [421 – CO] and m/z 337 [365 – CO], respectively. The comparison between the 13C NMR spectrum with the DEPT NMR spectrum of compound 1 gave a total of 25 carbon signals indicating signals for three protonated aromatic carbons which are C-2’ ( 106.8), C-6 ( 99.6) and C-8 ( 94.7) as well as eleven substituted aromatic carbons that are C-7 ( 164.7), C-5 ( 163.1), C-8a ( 158.0), C-2 ( 157.1), C-6’ ( 149.9), C-3’ ( 140.5), C-4’ ( 150.3), C-5’ ( 118.4), C-3 ( 110.1), C-1’ ( 107.1) and C-4a ( 105.2). The chelated carbonyl carbon gave a signal at 178.9 (C-4). The existence of one 2-methylpropenyl unit was revealed by the signals at 138.6 (C-11), 122.3 (C-10), 18.6 (C-12), 25.8 (C-13). Meanwhile, the presence of a 2-methyl-2butenyl unit was indicated by the signals at 131.6 (C-16), 122.9 (C-15), 25.8 (C-18), 23.0 (C-14), 17.8 (C-17). The 1H NMR spectrum indicated the presence of four singlet vinylic methyl proton signals at 1.94 (H-12), 1.72 (H-17), 1.66 (H-13), and 1.61 (H-18).The spectrum also showed
© 2010 Bentham Science Publishers Ltd.
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30
OH 29
OH HO
2' 3'4' 1' 6' 5'
O 7 8 6 5
OH
8a 4a
3
15
O
9 10
O
17
14
2 4
16 18
O
13
signals for two vinylic protons at 5.43 and 5.19 (H-10, H15), one benzylic oximethine proton at 6.16 (H-9), two methylene protons at 3.29 (H-14), a chelated hydroxyl at 12.91 (5-OH) and three aromatic protons at 7.16, 6.43 and 6.22 (H-2', H-8, H-6). The spectral data and 1H NMR decoupling experiments on 1 exhibited the presence of the partial structures of one prenyl (a) and one methyl-propenyl (b) units in the molecule (Scheme 2).
Scheme 1. HMBC long-range correlations for 1.
Scheme 2. Partial Structures of Compound 1.
The HMBC spectrum of 1 gave the connectivity of 1HC long range correlations as shown in Scheme 1. The aromatic protons showed 2 J and 3J correlations with the adjacent carbons at the A-ring. The signal for H-6 at 6.22 gave cross-peaks to signals C-7, C-4a and C-8 at 164.7, 105.2 and 94.7, respectively. Meanwhile the signal for H-8 at 6.43 gave correlations with the signals for C-8a C-7, C4a and C-6 at 158.0, 164.7, 105.2 and 99.6, respectively. These assignments confirmed the position of the two meta-coupled protons, which is consistent with a
13
2 3
12 11
1
241
O 24
1 4
19 20 21 22 12 18 17 13 28 11 26 25 16 14 15 9 8 10 27 5 7 6 23
2
5,7-dihydroxyl substitution of the A-ring. On the other hand, the aromatic protons of the B-ring also exhibited long-range couplings with the adjacent carbons at the B-ring. The signal of H-2’ ( 7.16) correlates to the signals of C-2 ( 157.1), C6’ ( 149.9) and C-3’ ( 140.5). At the same time, the benzylic methylene protons (H-14) showed correlations with the two substituted aromatic carbons, C-6’ ( 149.9) and C5’ ( 118.4) and the two olefinic carbons, C-16 ( 131.6) and C-15 ( 122.9). Thus, these correlations revealed the presence of the partial structure a, 3-methylbut-2-enyl group which should be attached to carbon C-5’. Also, the position of the isolated proton H-2’ was assigned based on 3,4’dihydroxyl substitution of B-ring. The flavonoid D-ring carried an oxygen which was due to cyclization of an initial C-3 prenyl unit to a hydroxyl group at C-6’ of the B-ring in which the proton signal for H-9 at 6.16 was correlated to C-2, C-6’, C-11, C-10 and C-3 at 157.1, 149.9, 138.6, 122.3 and 110.1. Hence, the partial structure b, the methylpropenyl unit was therefore located at C-9. The 1H-1H COSY spectrum revealed that H-9 coupled to H-10 while H-14 coupled to H-15 in the two side chains. Besides, it also exhibited a meta coupling pattern between the two aromatic protons H-6 and H-8 at the A-ring. This assignment was further confirmed by referring to the 1J and long-range correlations shown by HMQC and HMBC experiments respectively. Hence, the structure of this flavonoid was concluded to be artosimmin (1). The spectral data are summarized in Table 1. Compound 2 was isolated from the hexane extract. It has an mp of 239-241 oC (Lit. 238-240 oC, [4]). This compound was analyzed to have a molecular formula C32H52O2 according to the molecular ion peak at m/z 468. The 13C NMR assignment of 2 was made by performing DEPT NMR experiment and by comparison with the corresponding data of 2 from Atractylodes lancea. [4] The DEPT spectrum analysis further supported the structure to have a total of 9 CH3, 9 CH2, 7 CH and 7 quaternary carbons which are in agreement with the molecular formula. Based on literature search, this is the first isolation of triterpenoids from Artocarpus species. It was deduced to be traxateryl acetate (2) based on the spectral evidence and by comparison of spectral data with literature values [4]. Compound 1 was significantly bioactive in the HL-60 (human promyelocytic leukemia) and MCF-7 (human breast adenocarcinoma) cancer cell bioassays, with IC50 of 1.1 and 3.4 g/ ml using MTT method (Table 2), respectively. Meanwhile, compound 1 showed strong scavenging action against the DPPH radical. Treatment of methanolic DPPH
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Table 1.
1
Ee et al.
H NMR and 13C NMR Data for 1 (400 and 100 MHz, CD3COCD3) 1
No.
13
H
C
2
-
157.1
-
3
-
110.1
-
4
-
178.9
-
4a
-
105.2
-
5
-
163.1
6 7
-
164.7
-
8
6.43 (1H, d, J= 1.8Hz)
94.7
99.6 (C-6) (3J), 164.7 (C-7) ( 2J), 105.2 (C-4a) (3J), 158.0 (C-8a) (2J)
8a
-
158.0
-
9
6.16 (1H, d, J= 9.2 Hz)
69.4
157.1 (C-2) ( J), 110.1 (C-3) ( J), 122.3 (C-10) (2J), 138.6 (C-11) (3J), 149.9 (C-6’) (3J)
10
5.43 (1H, dd, J= 9.2, 1.8 Hz)
122.3
18.6 (C-12) (3J), 25.8 (C-13) (3J)
11
-
138.6
3
2
-
12
1.94 (3H, s)
18.6
138.6 (C-11) ( J), 122.3 (C-10) (3J), 25.8 (C-13) ( 3J)
13
1.66 (3H, s)
25.8
138.6 (C-11) (2J), 122.3 (C-10) (3J), 18.6 (C-12) ( 3J)
14
3.29 (2H, m)
23.0
122.9 (C-15) (2J), 131.6 (C-16) (3J), 118.4 (C-5’) ( 2J), 149.9 (C-6’) ( 3J)
15
5.19 (1H, td, J= 7.4, 1.8 Hz)
122.9
17.8 (C-17) (3J), 25.8 (C-18) (3J)
16
-
131.6
2
-
17
1.72 (3H, s)
17.8
122.9 (C-15) ( J), 131.6 (C-16) (2J), 25.8 (C-18) ( 3J)
18
1.61 (3H, s)
25.8
122.9 (C-15) (3J), 131.6 (C-16) (2J), 17.8 (C-17) ( 3J)
1’
-
107.1
3
-
2’
7.16 (1H, s)
106.8
157.1 (C-2) ( J), 140.5 (C-3’) (2J), 118.4 (C-6’) (3J)
3’
-
140.5
-
4’
-
150.3
-
5’
-
118.4
-
6’
-
149.9
-
5-OH
12.91 (1H, s)
-
-
3
structural moiety for cancer cells inhibitory activities of flavonoids. EXPERIMENTAL General Procedures Melting points were determined on a Barnstead Electrothermal digital melting point apparatus IA9000 series. IR spectra were recorded on Perkin-Elmer 100 series
Cytotoxic Activities of 1 Against HL-60 and MCF-7 Cell Linesa b
1
HL-60
1.1 ± 0.1
1.4
d
3.4 ± 0.3
d
3.1
IC50 , g/ ml: IC50 is mean of at least 3 determinations ± S.D. positive control of HL-60 cell line. positive control of MCF-7 cell line. d not tested.
Goniothalamin
c
Cell Lines
MCF-7
c
164.7 (C-7) ( J), 94.7 (C-8) ( 3J), 105.2 (C-4a) (3J)
99.6
Table 2.
b
2
6.22 (1H, d, J= 1.8Hz)
solution of 1 with serial dilutions was observed spectrophotometrically. Vitamin C was used as a positive control. 1 exhibited high potential of scavenging effect towards the DPPH radical with an IC50 value of 32.1 g/ ml (Table 3). Previous studies have shown that some flavonoids such as catechins possess protective effects against cancer diseases. These protective properties are mainly attributed to their antioxidant properties such as scavenging free radical effect [5]. This observation may lead to the assumption that isoprenyl-derived substitutents (prenyl) may be an important
a
HMBC
-
Tamoxifen -
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Antioxidant Scavenging Capacity of DPPH by 1f
Table 3.
g
1
Vitamin C
DPPH Scavenging Capacity 32.1 ± 3.1
12.2
IC50, g/ ml: IC50 is mean of at least 3 determinations ± S.D. g positive control. f
spectrophotometer. UV spectra were recorded on a Shimadzu UV-160A, UV-Visible recording spectrophotometer. EIMS spectral data were obtained using a Shimadzu GCMS-QP5050A spectrometer. NMR spectra were recorded with a JEOL JNM EX-400 FTNMR spectrometer, operating at 399.7 MHz (1H, COSY, HMQC and HMBC) and 100.4 MHz (13C, DEPT), respectively. CDCl3 and CD3COCD 3 were used as solvents. The optical density in MTT assay and the absorbent in DPPH free radical scavenging of antioxidant assay were measured by using ELISA microplate reader. Column chromatography was carried out using Merck Kieselgel 60 grade 1.09385.1000 mesh 240-400 (0.0400.063 mm), Merck Kieselgel 60 PF254 grade 1.07749.1000 and Sigma Lipophilic Sephadex LH-20. Thin layer chromatography was performed on precoated 0.25 mm thickness Merck Kieselgel 60 F254 grade 105554.0001 aluminium plates, while Centrifugal preparative thin layer chromatography was carried out on Chromatotron Model 7924 (Harison Research, Palo Alto, USA) using a 1 or 2 mm rotor with Merck Kieselgel 60 PF254 grade 1.07749.1000. Reagents RPMI-1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were obtained from SigmaAldrich. Typsin blue, microculture 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Roche Diagnostics, USA as well as dimethylsulfoxide (DMSO). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and ascorbic acid (vitamin C) were purchased from Fisher, Scientific Malaysia.
and methanol, followed by Sephadex LH20 column chromatography which gave five subfractions. Repeated radial chromatography on subfraction 5 afforded compound 1. The crude hexane extract (6.0 g) afforded traxateryl acetate (2) after repeated washing with methanol. The structures of compounds 1 and 2 were elucidated using UV, IR, NMR and MS data. Artosimmin (1) yellow solid (95 % chloroform/ 5 % methanol); mp 213215 oC; UV (MeOH) max (log ) nm: 213 (4.16), 271 (3.72), 340 (3.42); IR (KBr) max cm-1: 3527 (OH), 2853 (C-H stretching), 1734 (C=C unsaturated), 1653 (conjugated C=O), 1598, 1567, 1513, 1488, 1447 (C=C aromatic), 1306, 1239 (C-O); EIMS m/z (rel. int.): 436 (100) [M+, C25H24O7], 421 (40), 393 (15), 381 (78), 365 (20), 337 (12), 325 (62), 297 (6), 254 (5), 206 (4), 191 (12), 183 (2), 165 (4), 153 (19). 1H NMR (CD3 COCD3, 400 MHz): 12.91 (1H, s, OH5), 7.16 (1H, s, H-2’), 6.43 (1H. d, J= 1.8 Hz, H-8), 6.22 (1H, d, J= 1.8 Hz, H-6), 6.16 (1H, d, J= 9.2 Hz, H-9), 5.43 (1H, dd, J= 9.2, 1.8 Hz, H-10), 5.19 (1H, td, J= 7.4, 1.8 Hz, H-15), 3.29 (2H, m, H-14), 1.94 (3H, s, H-12), 1.72 (3H, s, H-17), 1.66 (3H, s, H-13), 1.61 (3H, s, H18); 13C NMR (CD3COCD3, 100 MHz): 178.9 (C, C-4), 164.7 (C, C-7), 163.1 (C, C-5), 158.0 (C, C-8a), 157.1 (C, C-2), 149.9 (C, C-6’), 140.5 (C, C-3’), 150.3 (C, C4’ ), 138.6 (C, C-11), 131.6 (C, C-16), 122.9 (CH, C15), 122.3 (CH, C-10), 118.4 (C, C-5’), 110.1 (C, C-3), 107.1 (C, C-1’), 106.8 (CH, C-2’), 105.2 (C, C-4a), 99.6 (CH, C-6), 94.7 (CH, C-8), 69.4 (CH, C-9), 18.6 (CH3, C-12), 25.8 (2 CH3, C-13 & C-18), 23.0 (CH2, C-14), 17.8 (CH3, C-17).
Plant Materials
Traxateryl Acetate (2)
The stem bark of Artocarpus odoratissimus was collected in May 2007 from Sri Aman, Sarawak, Malaysia. The specimen was identified by Dr. Rusea Go from the Department of Biology, Faculty of Science, UPM, Serdang, Selangor in Malaysia.
White needles (90 % hexane/ 10 % ethyl acetate); mp 239-241 oC (Lit. 238-240 oC [4]) IR (KBr) max cm-1: 2921, 2821 (C-H stretching), 1733 (C=O ester), 1451, 1367 (CH 3 bending), 1241 (C-O); EIMS m/z (rel. int.): 468 (6) [M+, C32H52O2], 453 (1), 408 (1), 250 (1), 249 (2), 218 (100), 190 (7), 189 (19), 175 (5) . 1H NMR (CDCl3, 400 MHz): 5.12 (1H, t, J= 3.68 Hz), 4.51 (1H, m, H-3), 1.07 (3H, s H26), 1.01 (3H, s, H-23), 0.98 (3H, s, H-27), 0.92 (3H, s, H-29), 0.88 (3H, s, H-30), 0.87 (3H, s, H-24), 0.80 (6H, s, H-23 & 28) ; 13C NMR (CDCl3, 100 MHz): 170.9 (C, CH3-CO), 139.6 (C, C-20), 124.3 (CH, C-21), 80.9 (CH, C-3), 59.0 (CH, C-18), 55.2 (CH, C-5), 23.6 (CH2, C-2), 38.4 CH2, C-1), 47.6 (CH, C-9), 42.0 (C, C-8), 41.5 (CH2, C-22), 40.0 (C, C-14), 39.6 (2 CH, C-13 &19), 37.7 (C, C-4), 36.8 (C, C-10), 33.7 (C, C17), 32.8 (CH2, C-16), 31.2 (CH2, C-7), 28.7 (CH3, C23), 28.1 (CH3, C-30), 26.6 (CH2, C-15), 23.3 (CH2, C12), 23.2 (CH3, C-26), 21.4 (CH3, C-29), 21.3 (CH3, CH3-CO), 18.2 (CH2, C-6), 17.5 (CH3, C-28), 16.8 (CH3, C-25), 16.7 (CH3, C-24), 15.7 (CH3, C-27).
Extraction and Isolation Solvent extractions on the dried powdered stem bark of Artocarpus odoratissimus (3.5 kg) yielded the crude hexane, chloroform, ethyl acetate and ethanol extracts, respectively. The crude ethyl acetate extract (42.5 g) was fractionated into fractions by using silica gel vacuum liquid chromatography (VLC) with a stepwise gradient system (hexane, hexanechloroform, chloroform-ethyl acetate, ethyl acetate-methanol and methanol) to give five major fractions which were combined based on the TLC profile. Fraction 9-13 (6.1 g) was subjected to a series of column chromatography, over silica gel with gradient system such as hexane, hexanechloroform, chloroform-ethyl acetate, ethyl acetate-methanol
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Cancer Cell-Lines Culture Two human cancer cell lines were used. They were HL60 and the MCF-7 cell lines. Both cell lines were obtained from the National Cancer Institute, Maryland, USA. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 5 % Fetal Bovine Serum (FBS), 100 IU ml-1 penicillin and 100g/ml streptomycin by using 25 ml flask in a 37 oC incubator with 5 % CO2. Cytotoxicity and MTT Assay Cytotoxic assay was carried out using the microculture 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously [6, 7]. The exponential growth of cells was performed by suspending the cells in 100 L of stock culture (1 105 cells mL-1) per well which were plated in 96-well plate and incubated for 24 hours at 37 o C under a humidified 5 % CO2. The stock solution was prepared by dissolving the sample in absolute ethanol to a final concentration of 1 mg/mL. Serial dilutions of the stock solution were carried out to produce seven sample solutions. The growth medium was removed from the wells. The cells in each well were treated with 100 L of varying concentrations of test specimens in their respective medium. The positive control was made containing only untreated cell population in 100 L of growth medium. Each concentration of sample was tested in triplicate and allowed to proceed for 72 hours at 37 o C in 90 % humidified 5 % CO2 atmosphere. Then, 100 L of MTT stock solution (5 mg in 1 mL PBS) was added to each well to determine the fraction of adherent cells relative to the untreated cell population. The plates were further incubated for 4 hours at 37 o C. 100 L of ethanol was added to each well to solubilize the waterinsoluble purple formazan crystal. After 30 minutes, the absorbance (OD) of the samples and the reference were measured by using ELISA spectrophotometer microplate reader at wavelength 550 nm. The inhibition concentration of 50 % reduction (killed cells) in cell number, IC50 was appraised visually to determine the absorbance (OD) against concentration curve.
Ee et al.
by dissolving 4 mg of DPPH (Sigma, USA) in 1 ml of methanol. The solution was kept in the dark at 4 o C after shaking in a sonicator for 5 minutes. The sample was dissolved in methanol to give 2 mg/mL of stock solutions. Each stock solution was diluted two fold (serial dilution) in micro centrifugal tubes to make seven sample solutions at concentrations 1.9, 3.8, 7.5, 15.0, 30.0, 60.0 and 120.0 g/ mL. The standard control used in this assay was vitamin C (Sigma, USA). The plate was wrapped with aluminium foil and placed at 37 o C. The reaction was allowed to proceed for 30 minutes. The absorbance (OD) of each well was measured spectrophotometrically at 517 nm using an ELISA microplate reader (Quant, Bio-Tek Instrument USA). Each sample was assayed in triplicate. A dose response curve presented in a graph of inhibition rate versus concentration of samples was plotted to determine the IC50 value. ACKNOWLEDGEMENTS The authors wish to thank Associate Professor Dr Jegak Uli for collection of plant samples and the Ministry of Higher Education for financial support under the FRGS research fund. REFERENCES [1] [2] [3]
[4]
[5] [6]
[7]
DPPH Assay The assay was performed according to the protocol obtained from a previous researcher with modification by using 96- well plate [8]. A solution of DPPH was prepared
[8]
Subhadrabandhu, S. Under-utilized tropical fruits of Thailand (English): Thailand. Regional Office for Asia and the Pacific, 2001. Serudin, H.; Tinggal, D.S. Tarap (Artocarpus odoratissimus): potential tropical fruits for food product opportunities: ISHC Acta Horticulture, 1992. Fadzelly, M.A.B.; Maryati, M.; Asmah, R.; Jeffrey, F. Phytochemicals and antioxidant activity of differant parts of bambangan (Mangifera pajang) and tarap (Artocarpus odoratissimus). Food Chem., 2008, 113(2), 479-483. Duan, J.A.; Wang, L.Y.; Qian, S.H.; Su, S.L.; Tang, Y.P. A new cytotoxic prenylated dihydrobenzofuran derivative and other chemical constituents from the rhizomes of Atractylodes lancea DC. Arch. Pharm. Res., 2008, 8, 965-969. Kondo, K.; Kurihara, M.; Miyata, N.; Suzuki, T.; Toyota, M. Arch. Biochem. Biophys., 1999, 362, 79-86. Ee, G.C.L.; Lim, C.K.; Rahmat, A. Structure-activity relationship of xanthones from Mesua daphnifolia and Garcinia nitida towards Human estrogen receptor negative breast cancer cell line. Nat. Prod. Sci., 2005, 11(4), 220-224. Liu, Y.; Peterson, D.A.; Kimura, H.; Schubert, D. Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J. Neurochem., 1997, 69, 581-593. Kabouche, A.; Kabouche, Z.; ÖztÜrk, M.; Kolak, U.; Topcu, G. Antioxidant abietane diterpenoids form Salvia barrelieri. Food Chem., 2007, 102, 1281-1287.
