In vitro effects of policosanol (Saccharum officinarum L wax alcohols) on the 5-lipooxygenase enzyme
Efecto in vitro del policosanol (alcoholes de la cera de Saccharum officinarum L) sobre la enzima 5-lipooxigenasa
DraC. Yohani Pérez Guerra, MSc. Ambar Oyarzábal Yera, DraC. Rosa Mas Ferreiro,Téc. Sonia Jiménez Despaigne, DraC. Vivian Molina Cuevas
Centro de Productos Naturales, Centro Nacional de Investigaciones Científicas (CNIC). La Habana, Cuba.
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ABSTRACT
Introduction: policosanol, a mixture of high molecular weight aliphatic alcohols purified from sugarcane with octacosanol as the main component, shows cholesterol-lowering and antiplatelet effects in addition to an inhibitory effect on type I cicloxygenase.
Objective: to determine whether policosanol may inhibit 5-LOX enzyme activity in vitro.
Methods: effects on 5-LOX enzyme activities were assessed in rat blood polymorphonuclear leukocytes. Vehicle or Policosanol suspensions (0.6 to 6 000 µg/mL) were added to tubes containing the reaction mix and then absorbance changes at 234 nm were measured.
Results: added Policosanol inhibited in vitro 5-LOX activity by 30 %, which was not a significant figure but depended on the concentration(r= 0.992; p< 0.05); it was 1 250 µg/mL.
Conclusions: policosanol did not significantly inhibit 5-LOX enzyme activity in rat PMNL preparations, so that it does not seem to be a dual inhibitor of COX and-LOX enzymes. This result differs from that found for beeswax alcohols and underlines the different effects of the mixtures of long-chain fatty alcohols purified from the sugarcane and the beeswax.
Key words: policosanol, sugarcane wax alcohols, lipoxygenases, 5-LOX, Saccharum officinarum L.
RESUMEN
]]> Introducción: el policosanol es una mezcla de alcoholes alifáticos aislados y purificados de la caña de azúcar cuyo componente mayoritario es el octacosanol, con efecto sobre la reducción de colesterol y antiagregante plaquetario, además inhibe la ciclooxigenasa (COX) tipo 1. Palabras clave: policosanol, alcoholes de la cera de caña de azúcar, lipooxygenasas, 5-LOX, Saccharum officinarum L.
INTRODUCTION
Policosanol is a mixture of eight high molecular weight aliphatic alcohols purified from sugar cane (Saccharum officinarum, L) wax, wherein octacosanol (C28) (60-70 %) is the main component, and C24, C26, C27, C29, C30 , C32 and C34 alcohols are at lower concentrations.1 In turn, D-002 is a mixture of six high molecular weight aliphatic alcohols (C24, C26, C29, C30, C32, C34) purified from beeswax that contains triacontanol (C30) as the most abundant component.2
]]> Policosanol has been reported to inhibit cholesterol synthesis through a regulation of HMG-CoA reductase3 that involves the phosphorylation of AMP-kinase and HMG-CoA reductase, a mechanism demonstrated in hepatoma cells and in mouse liver after intragastric administration.4,5 Also, it significantly raises LDL receptor-dependent processing, increasing LDL catabolic rate.6 Recent data support that policosanol inhibits cyclooxygenase-1 (COX-1) activity in vitro,7 which could explain, at least partly, its antiplatelet effects.8,9 This inhibition of 5-LOX by D-002 should also explain, at least partly, its antioxidant effects on lipid peroxidation, but such inhibition has not been demonstrated for policosanol.In light of these issues, this study was undertaken to investigate whether policosanol may inhibit 5-LOX activity in vitro.
METHODS
ANIMALS
Male Wistar rats (180-200 g) purchased from the Centre for Laboratory Animals Production (CENPALAB, Habana, Cuba) were adapted for 7 days to laboratory conditions: controlled temperature 25 ± 2 °C, relative humidity 60 ± 5 % and 12 h light/dark cycles. Food (rodent pellets from CENPALAB) and water were provided at libitum.
After a 12 h fast rats were anaesthetized in ether atmosphere, sacrificed by exsanguinations. We studied the effect on 5-LOX enzyme activity by using the cytosolic fraction of rat polymorphonuclear leukocytes (PMNL) prepared from total blood samples.
The study was conducted in accordance with the Cuban Guidelines for the laboratory animals care and Good Laboratory Practices. An independent ethic board for animal use approved the protocol of this study.
MATERIALS
ADMINISTRATION AND DOSAGE
Policosanol was obtained from the Plant of Natural Products (National Centre for Scientific Research, Havana City, Cuba), after corroborate that they met the quality criteria for batch releases. The composition of the batch, assessed with a validated gas chromatographic method,10 was as follows: tetracosanol 0.07 %, hexacosanol 4.9 %, heptacosanol 0.8 %, octacosanol 63.8 %, nonacosanol 0.5 %, triacontanol 12.8 %, dotriacontanol 6.8 %, tetratriacontanol 2.4 %. Policosanol concentrations were prepared as suspensions in Tween 20/water (2 %) vehicle. Nordihydroguaiaretic acid (NDGA) (Merck, Germany), the 5-LOX reference inhibitor, was dissolved in carboxymethyl-cellulose (2 %).
PREPARATION OF THE POLYMORPHONUCLEAR LEUKOCYTES (PMNL) CYTOSOLIC FRACTION
The effects on 5-LOX activity were assessed by using enzyme preparations from the cytosolic fraction of rat blood polymorphonuclear leukocytes (PMNL) freshly isolated.11 In brief, venous blood samples were collected in tubes containing EDTA (10 %) and diluted to 10 mL with the same volume of 0.9 % NaCl (saline) solution. Then 6 ml of diluted blood were gently layered over 3 ml of 14.1 % Nycodenz (density 1.077 g/ml, 20 oC) prepared in 0.44 % NaCl and 5 mmol/L Tris HCl buffer (pH 7.2), and centrifuged at 800 x g for 30 min. at 20 °C. After centrifugation, the mononuclear cells formed as band at the Nycodenz-plasma interface were removed with a Pasteur pipette, washed with 50 mmol/L phosphate buffer/1 mmol/L EDTA (pH 7.4), and centrifuged at 400 × g for 10 min. The pellet was washed again with the same buffer, re-suspended in the same buffer and then used as the crude enzyme preparation.
PMNL were then sonicated (3 cycles of 30 s, sub-maximal potency), centrifuged at 2000 × g for 10 min at 0 °C, and the supernatant centrifuged at 100 000 × g for 1 h at 4 °C. The cytosolic fraction was then frozen at -20 °C to use.
EFFECTS ON 5-LOX ENZYME ACTIVITY
Parallel tubes containing the vehicle (2 % Tween-20/H2O), policosanol (0.6, 4.8, 19.5, 78.1, 312.5, 1 250, 2 500, 5 000, 5 500, 6 000 µg/mL), or NDGA (50 µg/mL) were run. Once the substrate was added, the increase of absorbance at 234 nm was measured every min for 10 min in the spectrophotometer. The enzyme activity was expressed as mmol of conjugated dienes/min/mg protein.
STATISTICAL ANALYSES
Data were expressed as the mean ± SD. Comparisons between treated and control groups were performed with the Mann-Whitney U tests. Statistical significance was chosen for a= 0.05. Dose-effect relationship was assessed by using a linear regression and correlation test. Regression analysis was used to calculate IC50, defined as the concentration of inhibitor necessary for 50 % inhibition of the enzyme reaction. Data were processed with the Statistics Software for Windows (Release 4.2 Stat Soft Inc, Tulsa OK, US).
RESULTS
Table summarizes the effects on 5-LOX activity. The addition of the 5-LOX inhibitor (NDGA 50 mg/ml) inhibited significantly and markedly the enzyme activity by 88 %.
Policosanol addition dose-dependently (r= 0.992; p< 0.05), but not significantly, inhibited in vitro 5-LOX activity to approximately 30 %, achieving this mild ceiling effect with a concentration of 1 250 µg/mL.
]]> DISCUSSION
This study demonstrates that the addition of policosanol (0.6-6 000 µg/mL) did not inhibit significantly or meaningfully in vitro 5-LOX activity in rat PMNL.
As expected, the addition of NDGA 50 µg/mL, a 5-LOX inhibitor used as reference, inhibited significantly and markedly (88 %), a result that confers validity to our experimental conditions for the testing of 5-LOX enzyme activity and to the present results obtained in such conditions.
Although policosanol produced a concentration-dependent reduction of 5-LOX activity up to 30 %, which suggests some action on this target, it failed to inhibit it significantly. This result diverges from that found for D-002, a mixture of beeswax alcohols that contains octacosanol, which inhibited both 5-LOX and COX enzyme activities, with a greater affinity for 5-LOX.12 In fact, since recent studies have demonstrated that octacosanol, the main component of policosanol,13 produces antinflammatory effects in vivo,14,15 and policosanol inhibits COX-1 activity,8 but it does not produce gastrotoxicity,1,14-16 we expected that policosanol should inhibit 5-LOX enzyme, so that this effect could minimize the gastrotoxicity induced by COX inhibition.16
In fact, inflammation involves high levels of arachidonic acid (AA) from damaged cell membrane phospholipids generated through the activity of phospholipase A2 enzyme, which is then metabolized through the COX and LOX pathways to produce mediators like prostaglandins, thromboxanes, prostacyclins, and highly inflammatory leukotrienes (LT). COX inhibitors curtail prostaglandins formation, which turn the AA metabolism towards the LOX pathway, increasing the production of LT and enhancing the toxicity due to the prostaglandins deficit, as demonstrated the high concentrations of LTB4 seen in gastric ulcers induced by COX inhibitors, which attract leukocytes to the stomach and contributes to produce the ulceration.17,18
Nevertheless, this study was conducted in vitro, so that we cannot exclude that policosanol may inhibit 5-LOX in vivo. In such regard, we should remember that policosanol and octacosanol administered orally,19,20 but not added in vitro,21 exhibits antioxidant effects. Then, may be such effect requires, as the activation of AMP kinase and subsequent inhibition of HMGCoA reductase,3,4 the metabolism of fatty alcohols to acids. Further studies, therefore, should explore the possibility that policosnol can inhibit 5-LOX in vivo.
CONCLUSIONS
In vitro addition of Policosanol (0.6 to 6 000 µg/mL) did not inhibit significantly 5-LOX enzyme activity in rat PMNL preparations, so that it does seem to be a dual inhibitor of COX and-LOX enzymes. This result diverges of that found for beeswax alcohols and underlines the different effects of the mixtures of long-chain fatty alcohols purified from sugarcane and bees waxes.
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Recibido: 27 de septiembre de 2013.
Aprobado: 13 de noviembre de 2013.
Yohani Pérez Guerra. Centro de Productos Naturales, Centro Nacional de Investigaciones Científicas (CNIC). Calle 198 entre 19 y 21, Atabey, municipio Playa, Apartado Postal 6414, La Habana, Cuba. Correo electrónico: yohani.perez@cnic.edu.cu
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