Polyphenols Communications 2008 Volume 2 / T5.23 ....................................................................................................................................................
Photochemically transformed resveratrol derivatives featured as biologically active agents Juraj Harmatha1*, Karel Vokáč1, Jan Šmidrkal2, Eva Kmoníčková3 and Zdeněk Zídek3 1
Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Lab. of Natural Products, Flemingovo nám. 2, 16610 Prague, Czech Republic; 2 Institute of Chemical Technology Prague, Faculty of Food and Biochemical Technology, Technická 3, 16628 Prague, Czech Republic; 3 Institute of Experimental Medicine, Academy of Sciences, Dept. of Pharmacology, Vídenská 1083, 14220 Prague, Czech Republic; *corresponding author:
[email protected]
Abstract. Effects of natural and structurally transformed stilbenes or stilbenoids on production of nitric oxide (NO) triggered by lipopolysaccharide (LPS) and interferon- (IFN-) were tested under in vitro conditions using murine resident peritoneal macrophages. Relation between the molecular structure and immunobiological activity was investigated, and implication of substituents, double bond stereochemistry, or cyclic attachment (double bond geometry fixation) was assessed. Introduction. Resveratrol and his structural analogues belong to a small, but important, group of plant secondary metabolites with variously substituted stilbene unit. They possess extensive range of biological activities. Such compounds are widely abundant in nature, but also can be prepared in various synthetic ways. Originally, we were impressed with their assumed antagonistic influence reflecting their binding affinity to the ligand-binding site of the insect ecdysteroid receptor [1]. Now, we have focused on their possible immunobiological activity in mammalian cells, especially when comparing to biogenetically related lignans [2]. OH
R
HO
HO
CH3O
HO
OH
OH OH
OCH3
OH 1. R=OH, trans-resveratrol 2. R=OCH3, methyl-resveratrol 3. R=H, pinosylvin
CH3O
4. pterostilben
5. cis-resveratrol
CH3O
OCH3 COOH
6. 3,5,7-trihydroxyphenantrene
OCH3 COOH
OCH3
OCH3
7. 2,4,4´-trimethoxy--stilbenecarboxylic acid
HO
O
8. 2,4,4´-trimethoxy--dihydrostilbenecarboxylic acid
HO
O
OH
9. 7-hydroxy-3-(4-hydroxyphenyl)coumarin
OH
O
O
OH
10. 7-hydroxy-3-(4-hydroxyphenyl)dihydrocoumarin
Materials and methods. trans-Stilbenes: trans-resveratrol (1), methyl-resveratrol (2), pinosylvin (3), and pterostilben (4), as well as their homologues: trimethoxy-stilbenecarboxylic acid 7, its dihydro-derivative 8, and their lactones: 7-hydroxy-3-(4-hydroxyphenyl)coumarin (9) and its dihydro-derivative 10 were prepared by chemical synthesis using original approach [3]. Their structural differences are obvious. cis-Resveratrol (5) and isosteric 3,5,7-trihydroxyfenantren (6) were prepared by photo-transformation in standard photoreactor [4]. The test compounds were dissolved in 251dimethylsulfoxide (DMSO). Its final amount in cell cultures was devoid of any effects on production of NO.
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Polyphenols Communications 2008 Volume 2 / T5.23 .................................................................................................................................................... Nitric oxide (NO) bioassay. Isolation and cultivation of the murine peritoneal macrophages for the bioassay was performed in a routine way [5]. Macrophages were cultured 24 hrs in presence of test compounds, applied in the presence of NO-priming immune stimuli, i.e. murine recombinant IFN- plus LPS. The concentration of nitrites in supernatants of cells was detected by Griess reagent. The absorbance at 540 nm was recorded using a micro-plate spectrophotometer. Results and discussion. We focused on trans-resveratrol (1), pinosylvin (3), and pterostilben (4), which usually occur as phytoalexins after bacterial infections (or during other environmental stress conditions) in their source organisms, e.g., grapevines (Vitis vinifera), pines (Pinus sylvestris), or other herbs, frequently used as foodstuffs. For structure-activity relationship study, we prepared a series of structurally transformed analogues, such as 2, 5-10. The trimethoxy-stilbenecarboxylic acids 7 and 8 did not inhibit NO production (Fig. 1), and from their lactones 9 and 10 only the dihydro derivative 10 displayed activity comparable to the basic trans-stilbenes 1-4. However, the cis-resveratrol (5) and its isosteric 3,5,7-trihydroxyfenantren (6), conserving the same Z oriented stereochemistry, displayed significant NO-inhibitory effect at concentrations higher than 50 M. Inhibition of NO was associated with the cytotoxicity of compounds (Fig. 2). Interestingly, pterostilben (4) inhibited NO without affecting the viability of cells. Fig. . Effects of test compounds on viability of mouse peritoneal macrophages
100
1000
80
800 OD x 1000
NO (% of control, i.e. IFN+LPS)
Fig. 1. Inhibitory effects of test compounds on LPS+IFN- -induced nitric oxide production in mouse peritoneal macrophages
60
40
600
400 Untreated
20
Untreated 3 h
200
IFN+LPS
All dead (Triton)
DMSO
DMSO 0
0 10
10
100 Compounds (µM)
100 Compounds µM
(1) trans-resveratrol EC 50 = 32.05 µM
(1) trans-resveratrol
(2) methyl-resveratrol EC 50 = 29.97 µM
(2) methyl-resveratrol
(3) pinosylvin EC 50 = 26.77 µM
(3) pinosylvin
(4) pterostilben EC 50 = 24.13 µM
(4) pterostilben
(5) cis-resveratrol EC 50 = 64.9 µM (6) 3,5,7-trihydroxyphenantrene EC
(5) cis-resveratrol 50
= 73.76 µM
(7) 2,4,4´-trimethoxy- -stilbenecarboxylic acid EC
(6) trihydroxy-phenantrene 50
>> 200 µM
(8) 2,4,4´-trimethoxy- -dihydrostilbenecarboxylic acid EC (9) 7-hydroxy-3-(4-hydroxyphenyl)coumarin EC
50
50
>> 200 µM
>> 200 µM
(10) 7-hydroxy-3-(4-hydroxyphenyl)dihydrocoumarin EC
50
= 24.36 µM
(7) trimethoxy-stilbenecarboxylic acid (8) trimethoxy-dihydrostilbenecarboxylic acid (9) hydroxyphenyl-coumarin (10) hydroxyphenyl-hydroxy-dihydrocoumarin
Stilbenoid activities could be compared with activities of biogenetically related lignans, which exhibited similar effects in the same test [2]. Our results extended the field of bioactivity of both, lignans and stilbenoids, along with the opportunity of their potential therapeutic or nutraceutic utilization. References: [1]. Harmatha J. and Dinan L. (2003) Phytochemistry Rev. 2: 321-330. [2]. Harmatha J. et al. (2004) Polyphenols communications 2004, p. 217, Gummerus printing, Helsinki. [3]. Šmidrkal J. et al. (2008) this issue. [4]. Harmatha J. et al. (2002) Steroids 67: 127-135. [5]. Harmatha J. et al. (2008) Steroids 73: 466-471. Supported by GAČR, grant No. 203/07/1227 Reprinted from: Polyphenols Communications 2008, Vol. 2 Eds.: M.T. Escribano-Bailón et al. Printing: Globalia Artes Gráficas,SL ISBN: 978-84-691-4334-6
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