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(1% w/v) were added to single samples (100 mL) placed
into 96-well microplate. The plate was wrapped in alu-
minium foil and shaken briefly. The absorbance was
measured at 570 nm and finally the nitrite concentration
determined from a sodium nitrite standard curve (0–120
mM). The values were expressed as mM nitrite released
into the culture medium.
CLOGD7.4, pKa and RM were taken from our previous
study.17 Stepwise multivariable regression analysis was
applied to develop QSAR models using SYSTAT. The
models were presented in normalised form and assessed
by the explained variance r2, standard error of estimate
(SEE) and F ratio.
References and Notes
Determination of GAGs
1. Klose, N.; Niedbolla, K.; Schwazrtz, K.; Bottcher, I. Arch.
Pharm. 1983, 316, 941.
2. Satsangi, R. K.; Zaidi, S. M.; Misra, V. S. Pharmazie 1983,
38, 341.
3. Pignatello, R.; Mazzone, S.; Panico, A. M.; Mazzone, G.;
Penissi, G.; Castano, R.; Matera, M.; Blandino, G. Eur. J.
Med. Chem. 1991, 26, 929.
GAGs were tested in the culture medium by utilizing a
spectrophotometric assay using 1.9-dimethyl methylene
blue (DMB) at a wavelength of 535 nm.20,21 A standard
curve for determination of GAGs concentration (100–
500 mg/mL) was used. As a standard, GAG chondroitin
sulphate derived from shark cartilage, was used.
4. Hadjipavlou-Litina, D.; Geronikaki, A.; Sotiropoulou, E.
Res. Commun. Chem. Pathol. Pharmacol. 1993, 79, 355.
5. Geronikaki, A.; Hadjipavlou-Litina, D. Pharmazie 1993,
48, 948.
6. Shield, M. J. Eur. J. Rheumatol. Inflamm. 1993, 13, 7.
7. Hess, E. V.; Herman, J. H. An. J. Med. 1986, 81, 36.
8. Redini, F.; Mauviel, G. Agents Action 1990, 31, 358.
9. Rainsford, K. D. Am. J. Med. 1999, 107, S27.
10. McInnes, I. B.; Leung, P. B.; Field, M.; Huang, F. P.;
Sturrock, R. D.; Kinninm, J. J. Exp. Med. 1996, 184, 1519.
11. Misra, R.; Stephan, S.; Chander, C. L. Inflamm. Res.
1999, 48 (Suppl. 2), S119.
12. De Nanteuil, G.; Portevin, B.; Benoist, A. Farmaco 2001,
56, 107.
13. Kaur, H.; Halliwell, B. FEBS Lett. 1994, 350, 9.
14. Manfield, L.; Jang, D.; Murell, G. A. C. Inflamm. Res.
1996, 45, 254.
15. Martin Lotz, M. Osteoarthritis 1999, 25, 269.
16. Pettipher, E. R.; Higgs, G. A.; Henderson, B. Proc. Natl.
Acad. Sci. U.S.A. 1986, 83, 874.
17. Hadjipavlou-Litina, D.; Geronikaki, A.; Mgonzo, R.;
Doytcinova, I. Drug Develop. Res. 1999, 48 .
18. Cardile, V.; Panico, A. M.; Geronikaki, A.; Gentile, B.;
Ronsisvalle, G. Farmaco 2002, 57, 1009.
19. Green, L. C.; Wagner, D. A.; Glogovski, J.; Reis, D. J.
Anal. Biochem. 1982, 126, 131.
20. Farndale, R. W.; Sayers, C. A.; Barrett, A. J. Connect
Tissue Res. 1982, 9, 247.
Statistical analysis
Each experiment was repeated at least three times in
quadruplicate samples. All statistical analyses were per-
formed using statistical software package SYSTAT.
Difference were considered significant at p<0.05.
QSAR
The chemical structure of the compounds was described
by three groups of parameters: steric, electrostatic and
hydrophobic. Some of the parameters described local
properties, others referred to global ones. Molecular
volume (MV) and molecular refractivity (MR) were
used in the QSAR as global steric properties. The par-
tial charges at some of the main atoms (qNring, qS, qNco
and qNR1) were applied to describe the local electron
density. Polarizability (Pol) and parachor (P) were used
as global electrostatic descriptors. The local hydro-
phobicity was presented by the parameters fR and fR1
,
and the global one by CLOGP. CLOGD7.4 and the
TLC-derived parameter RM presented the distribution
of the compounds in the octanol/buffer pH7.4 system.
pKa values were also included in the study. An indicator
variable ICH CH was included in the study to distinguish
2
between CH22 and CH2CH2 bridges. MV, MR, Pol, P, fR
and fR1 were calculated by ACD/ChemSketch.22 The
partial atomic charges were computed by AM1 semi-
empirical method implemented in SYBYL6.7.23 after
MM optimisation of the molecules. CLOGP,
21. Stone, J. E.; Akttar, N.; Botchway, S.; Pennock, C. A.
Clin. Biochem. 1994, 31, 147.
22. Advanced Chemistry Development; ACD/Labs.: Toronto,
Canada.
23. SYBYL 6.7; Tripos Inc.: 1699 Hanley Road, St. Louis,
MO 63144, USA.