On the Interaction of the Anthraquinone Barbaloin with Negatively Charged DMPG Bilayers Evandro L. Duarte,* ,² Tiago R. Oliveira, ² Daiane S. Alves, ‡ Vicente Micol, ‡ and M. Teresa Lamy ² Instituto de Fı ´sica, UniVersidade de Sa ˜ o Paulo, CP 66318, CEP 05315-970, Sa ˜ o Paulo, SP, Brazil, and Instituto de Biologia Molecular y Celular, UniVersidad “Miguel Herna ´ ndez”, Alicante, Spain ReceiVed August 7, 2007. In Final Form: January 16, 2008 Barbaloin is a bioactive glycosilated 1,8-dihydroxyanthraquinone present in several exudates from plants, such as Aloe Vera, which are used for cosmetic or food purposes. It has been shown that barbaloin interacts with DMPG (dimyristoylphosphatidylglycerol) model membranes, altering the bilayer structure (Alves, D. S.; Pe ´rez-Fons, L.; Estepa, A.; Micol, V. Biochem. Pharm. 2004, 68, 549). Considering that ESR (electron spin resonance) of spin labels is one of the best techniques to monitor structural properties at the molecular level, the alterations caused by the anthraquinone barbaloin on phospholipid bilayers will be discussed here via the ESR signal of phospholipid spin probes intercalated into the membranes. In DMPG at high ionic strength (10 mM Hepes pH 7.4 + 100 mM NaCl), a system that presents a gel-fluid transition around 23 °C, 20 mol % barbaloin turns the gel phase more rigid, does not alter much the fluid phase packing, but makes the lipid thermal transition less sharp. However, in a low-salt DMPG dispersion (10 mM Hepes pH 7.4 + 2 mM NaCl), which presents a rather complex gel-fluid thermal transition (Lamy-Freund, M. T.; Riske, K. A. Chem. Phys. Lipids 2003, 122, 19), barbaloin strongly affects bilayer structural properties, both in the gel and fluid phases, extending the transition region to much higher temperature values. The position of barbaloin in DMPG bilayers will be discussed on the basis of ESR results, in parallel with data from sample viscosity, DSC (differential scanning calorimetry), and SAXS (small-angle X-ray scattering). Introduction The gel-fluid thermal transition of the anionic lipid dimyris- toylphosphatidylglycerol (DMPG), at high ionic strength, is rather cooperative, happening in a narrow temperature interval, around 23 °C, similar to that of the zwitterionic lipid dimyristoylphos- phatidylcholine (DMPC) (Marsh, D. Handbook of Lipid Bilayers; CRC Press: Boston, 2000). However, at physiological pH but low ionic strength, DMPG presents a very interesting and unusual thermal profile related to the gel-fluid transition. It exhibits a rather complex differential scanning calorimetry (DSC) ther- mogram, with several calorimetric peaks between the beginning (T m on ∼ 17 °C) and the end (T m off ∼ 35 °C) of the melting process. 1-4 Although this transition region is not structurally well understood, some of its characteristics are known: low turbidity; 2,5,6 high viscosity and conductivity; 2,5 a Bragg peak in small-angle X-ray scattering (SAXS), at about 400 Å; 7 and a composite 16-PCSL electron spin resonance (ESR) spectrum, associated with the coexistence of rather fluid and hydrated domains, possibly high curvature regions, with patches that are more rigid and hydrophobic. 8 The transition region narrows significantly as the ionic strength increases, until a single main phase transition at T m ∼ 23 °C is observed around 100 mM NaCl. 1,4 On the basis of the available data, it has been suggested that in the gel-fluid transition region DMPG could be structured as perforated vesicles, 7 which could evolve in tattered bilayer fragments. 9 Though it is well-established that no vesicle fusion takes place in DMPG low salt dispersions, at any temperature, 4,9 the presence of loose aggregates in the gel and fluid phases and their disaggregation in the gel-fluid transition region have been proposed 5,10 and cannot be ruled out. 9 In studying the interaction of the anthraquinone barbaloin (10- glucopyranosyl-1,8-dihydroxy-3-(hydorxymethyl)-9(10H)-an- thracenone, Aloin A) with DMPG membranes at high ionic strength (100 mM NaCl, close to the physiological condition), it was found that some of the attributes of the transition region of DMPG at low ionic strength were present, like a decrease in turbidity and a somewhat complex DSC profile. 11 Barbaloin is a known active ingredient extracted from leaves of different Aloe plants 12,13 and an effective herbal component traditionally used in China for treating various ailments. 14,15 For instance, it has the ability to diminish inflammation and infection 16 and has been widely used in light industry, especially in cosmetics and food products. 17 * Corresponding author: Phone: +55 11 3091-6662. Fax: +55 11 3813- 4334. E-mail: elduarte@if.usp.br. ² Universidade de Sa ˜o Paulo. ‡ Universidad “Miguel Herna ´ndez”. (1) Salonen, I. S.; Eklund, K. K.; Virtanen, J. A.; Kinnunen, P. K. J. Biochim. Biophys. Acta 1989, 982, 205-215. (2) Heimburg, T.; Biltonen, R. L. Biochemistry 1994, 33, 9477-88. (3) Riske, K. A.; Amaral, L. Q.; Lamy-Freund, M. T. Biochim. Biophys. Acta 2001, 1511, 297-308. (4) Lamy-Freund, M. T.; Riske, K. A. Chem. Phys. Lipids 2003, 122, 19-32. (5) Riske, K. A.; Politi, M. J.; Reed, W. F.; Lamy-Freund, M. T. Chem. Phys. Lipids 1997, 89, 31-44. (6) Riske, K. A.; Nascimento, O. R.; Peric, M.; Bales, B. L.; Lamy-Freund, M. T. Biochim. Biophys. Acta 1999, 1418, 133-146. (7) Riske, K. A.; Amaral, L. Q.; Dobereiner, H. G.; Lamy, M. T. Biophys. J. 2004, 86, 3722-33. (8) Riske, K. A.; Fernandez, R. M.; Nascimento, O. R.; Bales, B. L.; Lamy- Freund, M. T. Chem. Phys. Lipids 2003, 124, 69-80. (9) Alakoskela, J. M. I.; Kinnunen, K. J. Langmuir 2007, 23, 4203-4213. 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