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Effects of phlorizin isolated from apple leaves and its aglycone phloretin on adrenal cortisol production
P.1.g. Basic and clinical neuroscience − Neuropharmacology [3] Taylor, D., Paton, C., Kapur, S., 2012. Psychotropic-related QTc prolongation, In: Taylor, D., Paton, C., Kapur, S., The Maudsley Prescribing Guidelines in Psychiatry, 12th ed., Wiley-Blackwell, pp. 110. [4] Hottigoudar, R.U., Gopinathannair, R., 2013. ‘Inappropriate’ sinus tachycardia: does the 100 beats per min cut-off matter? Future Cardiol 9, 273–288.
P.1.g.047 Effects of phlorizin isolated from apple leaves and its aglycone phloretin on adrenal cortisol production N. Kuwabara1 ° , H. Fukuda1 , M. Aoki1 , Y. Takato1 , S. Iiduka1 , M. Yoshie1 , K. Tamura1 , A. Yokosuka2 , Y. Mimaki2 , H. Sato3 , S. Komori4 , A. Suzuki4 , M. Kutsukake5 , E. Tachikawa1 1 Tokyo University of Pharmacy & Life Sciences, Endocrine and Neural Pharmacology, Hachioji, Japan; 2 Tokyo University of Pharmacy & Life Sciences, Medicinal Pharmacognosy, Hachioji, Japan; 3 Tokyo University of Pharmacy & Life Sciences, Center for Fundamental Laboratory for Education, Hachioji, Japan; 4 Iwate University, Agro-biosciences- Faculty of Agriculture, Morioka, Japan; 5 Toyama University, Graduate School of Medicine and Pharmaceutical Sciences- Faculty of Pharmacy and Pharmaceutical Sciences for Education, Toyama, Japan Background: Apple leaves are discarded in large amounts to facilitate the growth of apple fruits. In response to adrenal corticotropic hormone (ACTH), adrenal cortex secrets cortisol and maintains homeostasis or corps with stress. However, under the long-term or the excessive stress, the oversecreted cortisol reveals a lot of adverse effects and leads to many onsets of disorders, such as immunosuppression, metabolic disorders and cancers. To beneficially utilize the apple leaves, we tried to isolate some bioactive components and examined the effects of the components on cortisol production in bovine adrenal cortical cells. Methods: Apple leaves (3 kg) were extracted with 30 L of MeOH for 1 weeks at room temperature. The MeOH extract solution was concentrated under the reduced pressure and refined by HP-20 column chromatography. The 70% MeOH fraction was further separated using various chromatography. Bovine adrenal cortical cells were prepared by collagenase digestion as described previously [1]. After 48 hr of culture, the cells were preincubated with the apple leaf component for 15 min at 37º, and then incubated with ACTH or other stimuli and the apple leaf component for 90 min at 37º. Cortisol production in the medium was extracted with dichloromethane and quantified by the sulfonic acid condensation method using a fluorescene spectrophotomer at an excitation wavelength of 470 nm and an emission wavelength of 520 nm [2]. The Ca2+ influx into the cells induced by ACTH was measured by fura-2AM which is a fluorescent indicator of Ca2+. Date were expressed as mean ± standard error of mean values. Significance was assessed using the Tukey-Kramer multiple comparisons test. A P-value <0.05 was considered statistically significant. Results: We found a dihydrochalcone glycoside phlorizin from apple leaves. Phlorizin (50–200 mM) did not affect the cortisol production in bovine adrenal cortical cells stimulated by ACTH. But phloretin, an aglycone of phlorizin inhibited the cortisol production in a dose-dependent manner (2–100 mM). Next, the inhibitory mechanism of phloretin in the cortisol production was investigated. Phloretin reduced the cortisol production induced by dibutyryl cyclic AMP. But phloretin did not inhibit the cortisol production drived by pregnenolne, a precursor of cortisol
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systhesis, and 22(R)-hydroxycholesterol, a membrane-permeable cholesterol analog. In addition, phloretin did not change the cyclic AMP elevation in the cells stimulated by ACTH. On the other hand, the aglycone blocked the ACTH-induced Ca2+ influx into the cells. Conclusions: Phloretin, an aglycone of phlorizin isolated apple leaves, reduced the production of cortisol in bovine adrenal cortical cells stimulated by ACTH. It is strongly suggested that the inhibitory effect of phloretin on the cortisol production is attributed to the blockade of conversion to pregnenolone from cholesterol and of the ACTH-induced Ca2+ influx into the cells. These results indicate the possibility that the components contained in the apple leaves are beneficial to treat and prevent the diseases due to stress. References [1] Tachikawa, E., Itho, K., Kudo, K., Harada, K., Kashimoto, T., Hirota, Y., 1999. Effects of interferons on cortisol production in bovine adrenal fasciculata cells stimulated by adrenocorticotropin. J Pharm Pharmacol 51, 465–473. [2] Hasegawa, E., Nakagawa, S., Miyate, Y., Takahashi, K., Ohta, S., Tachikawa, E., Yamato, S., 2013. Inhibitory effect of protopanaxatriol ginseng metabolite M4 on the production of corticosteroids in ACTHstimulated bovine adrenal fasciculata cells. Life Sci 92, 687–693.
P.1.g.048 Anti-inflammatory effect of alpha-lipoic acid in carrageenan- and lipopolysaccharideinduced models of inflammation in rats N. Doncheva1 ° , D. Getova2 , M. Ivanovska3 , A. Petrova3 , M. Murdjeva3 1 Medical University, Pharmacology and Drug Toxicology, Plovdiv, Bulgaria; 2 Medical University Plovdiv, Pharmacology and clinical pharmacology, Plovdiv, Bulgaria; 3 Medical University Plovdiv, Department of Microbiology and Immunology, Plovdiv, Bulgaria Purpose: Alpha-lipoic acid (ALA) has been shown to have antioxidant and anti-inflammatory properties. It is commonly used to treat diabetic neurovascular and metabolic problems [1]. Carrageenan-induced paw edema is widely used for determining the acute phase of inflammation [2]. Lipopolysaccharide (LPS), a Gram-negative bacterial outer membrane component, triggers the inflammatory response, leading to the release of large number of endogenous inflammatory mediators, including tumor necrosis factor alpha (TNF-a), interleukins (IL-4, IL-6, IL-10, IL-13, IL-1), chemokines and reactive oxygen species (ROS) [3]. The aim of our study was to evaluate the anti-inflammatory effect of ALA in carrageenan- and LPS-induced models of inflammation in rats. Methods: To study the effect of ALA on carrageenan-induced paw edema, forty male Wistar rats were divided in five groups (n = 8): control, positive control group (diclofenac 25 mg/kg) and three experimental groups treated intraperitoneally (i.p.) with 30, 60 and 90 mg/kg alpha-lipoic acid for 14 days, respectively. The paw volume of the animals were measured on 60, 120, 180 minutes with a plethysmometer (Ugo Basile, Italy). To study the effect of chronic administration of ALA in LPS-induced inflammation model forty male Wistar rats were divided in five groups (n = 8): control, model group and three experimental groups treated i.p. with 30, 60 and 90 mg/kg alpha-lipoic acid. In LPS-induced model of inflammation single dose of 250 mg/kg LPS from Escherichia coli 055: B was injected i.p.. The serum level of two pro-inflammatory cytokines including tumor necrosis
P.1.g.047 Effects of phlorizin isolated from apple leaves and its aglycone phloretin on adrenal cortisol production N. Kuwabara1 ° , H. Fukuda1 , M. Aoki1 , Y. Takato1 , S. Iiduka1 , M. Yoshie1 , K. Tamura1 , A. Yokosuka2 , Y. Mimaki2 , H. Sato3 , S. Komori4 , A. Suzuki4 , M. Kutsukake5 , E. Tachikawa1 1 Tokyo University of Pharmacy & Life Sciences, Endocrine and Neural Pharmacology, Hachioji, Japan; 2 Tokyo University of Pharmacy & Life Sciences, Medicinal Pharmacognosy, Hachioji, Japan; 3 Tokyo University of Pharmacy & Life Sciences, Center for Fundamental Laboratory for Education, Hachioji, Japan; 4 Iwate University, Agro-biosciences- Faculty of Agriculture, Morioka, Japan; 5 Toyama University, Graduate School of Medicine and Pharmaceutical Sciences- Faculty of Pharmacy and Pharmaceutical Sciences for Education, Toyama, Japan Background: Apple leaves are discarded in large amounts to facilitate the growth of apple fruits. In response to adrenal corticotropic hormone (ACTH), adrenal cortex secrets cortisol and maintains homeostasis or corps with stress. However, under the long-term or the excessive stress, the oversecreted cortisol reveals a lot of adverse effects and leads to many onsets of disorders, such as immunosuppression, metabolic disorders and cancers. To beneficially utilize the apple leaves, we tried to isolate some bioactive components and examined the effects of the components on cortisol production in bovine adrenal cortical cells. Methods: Apple leaves (3 kg) were extracted with 30 L of MeOH for 1 weeks at room temperature. The MeOH extract solution was concentrated under the reduced pressure and refined by HP-20 column chromatography. The 70% MeOH fraction was further separated using various chromatography. Bovine adrenal cortical cells were prepared by collagenase digestion as described previously [1]. After 48 hr of culture, the cells were preincubated with the apple leaf component for 15 min at 37º, and then incubated with ACTH or other stimuli and the apple leaf component for 90 min at 37º. Cortisol production in the medium was extracted with dichloromethane and quantified by the sulfonic acid condensation method using a fluorescene spectrophotomer at an excitation wavelength of 470 nm and an emission wavelength of 520 nm [2]. The Ca2+ influx into the cells induced by ACTH was measured by fura-2AM which is a fluorescent indicator of Ca2+. Date were expressed as mean ± standard error of mean values. Significance was assessed using the Tukey-Kramer multiple comparisons test. A P-value <0.05 was considered statistically significant. Results: We found a dihydrochalcone glycoside phlorizin from apple leaves. Phlorizin (50–200 mM) did not affect the cortisol production in bovine adrenal cortical cells stimulated by ACTH. But phloretin, an aglycone of phlorizin inhibited the cortisol production in a dose-dependent manner (2–100 mM). Next, the inhibitory mechanism of phloretin in the cortisol production was investigated. Phloretin reduced the cortisol production induced by dibutyryl cyclic AMP. But phloretin did not inhibit the cortisol production drived by pregnenolne, a precursor of cortisol
S241
systhesis, and 22(R)-hydroxycholesterol, a membrane-permeable cholesterol analog. In addition, phloretin did not change the cyclic AMP elevation in the cells stimulated by ACTH. On the other hand, the aglycone blocked the ACTH-induced Ca2+ influx into the cells. Conclusions: Phloretin, an aglycone of phlorizin isolated apple leaves, reduced the production of cortisol in bovine adrenal cortical cells stimulated by ACTH. It is strongly suggested that the inhibitory effect of phloretin on the cortisol production is attributed to the blockade of conversion to pregnenolone from cholesterol and of the ACTH-induced Ca2+ influx into the cells. These results indicate the possibility that the components contained in the apple leaves are beneficial to treat and prevent the diseases due to stress. References [1] Tachikawa, E., Itho, K., Kudo, K., Harada, K., Kashimoto, T., Hirota, Y., 1999. Effects of interferons on cortisol production in bovine adrenal fasciculata cells stimulated by adrenocorticotropin. J Pharm Pharmacol 51, 465–473. [2] Hasegawa, E., Nakagawa, S., Miyate, Y., Takahashi, K., Ohta, S., Tachikawa, E., Yamato, S., 2013. Inhibitory effect of protopanaxatriol ginseng metabolite M4 on the production of corticosteroids in ACTHstimulated bovine adrenal fasciculata cells. Life Sci 92, 687–693.
P.1.g.048 Anti-inflammatory effect of alpha-lipoic acid in carrageenan- and lipopolysaccharideinduced models of inflammation in rats N. Doncheva1 ° , D. Getova2 , M. Ivanovska3 , A. Petrova3 , M. Murdjeva3 1 Medical University, Pharmacology and Drug Toxicology, Plovdiv, Bulgaria; 2 Medical University Plovdiv, Pharmacology and clinical pharmacology, Plovdiv, Bulgaria; 3 Medical University Plovdiv, Department of Microbiology and Immunology, Plovdiv, Bulgaria Purpose: Alpha-lipoic acid (ALA) has been shown to have antioxidant and anti-inflammatory properties. It is commonly used to treat diabetic neurovascular and metabolic problems [1]. Carrageenan-induced paw edema is widely used for determining the acute phase of inflammation [2]. Lipopolysaccharide (LPS), a Gram-negative bacterial outer membrane component, triggers the inflammatory response, leading to the release of large number of endogenous inflammatory mediators, including tumor necrosis factor alpha (TNF-a), interleukins (IL-4, IL-6, IL-10, IL-13, IL-1), chemokines and reactive oxygen species (ROS) [3]. The aim of our study was to evaluate the anti-inflammatory effect of ALA in carrageenan- and LPS-induced models of inflammation in rats. Methods: To study the effect of ALA on carrageenan-induced paw edema, forty male Wistar rats were divided in five groups (n = 8): control, positive control group (diclofenac 25 mg/kg) and three experimental groups treated intraperitoneally (i.p.) with 30, 60 and 90 mg/kg alpha-lipoic acid for 14 days, respectively. The paw volume of the animals were measured on 60, 120, 180 minutes with a plethysmometer (Ugo Basile, Italy). To study the effect of chronic administration of ALA in LPS-induced inflammation model forty male Wistar rats were divided in five groups (n = 8): control, model group and three experimental groups treated i.p. with 30, 60 and 90 mg/kg alpha-lipoic acid. In LPS-induced model of inflammation single dose of 250 mg/kg LPS from Escherichia coli 055: B was injected i.p.. The serum level of two pro-inflammatory cytokines including tumor necrosis