Botanical Studies (2012) 53: 301-306.
BIOCHEMISTRY
Semicarbazide-sensitive amine oxidase inhibitory and hydroxy! radical scavenging activities of aspartic acid
β-hydroxamate and glutamic acid γ-monohydroxamate
Yuh-Hwa LIU1,2, Yin-Shiou LIN3, Tai-Lin LEE4, and Wen-Chi HOU4'5'*
1Division of Gastroenterology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan 2Department of Primary Care Medicine, Taipei Medical University, Taipei, Taiwan
3School of Pharmacy, Taipei Medical University, Taipei, Taiwan
4Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan
(Received December 5, 2011; Accepted February 20, 2012)
ABSTRACT. It was reported that semicarbazide-sensitive amine oxidase (SSAO) in plasma was elevated in diabetes mellitus and heart failure. SSAO catalyzed the oxidative deamination of various amines to produce hydrogen peroxide which is one possible source of oxidative stresses. In this research, two amino acid hydrox-amates, namely aspartic acid p-hydroxamate (AAH) and glutamic acid y-monohydroxamate (GAH), showed dose-dependently inhibitory against SSAO from bovine plasma. The IC50 of AAH and GAH was 0.7 mM and 0.023 mM, respectively, in the comparisons with 0.035 mM of semicarbazide (positive controls). The AAH showed mixed noncompetitive inhibition against bovine SSAO in the respect to benzylamine (substrate) and benzylamine-SSAO (substrate-enzyme complex). It was found that the V'max value was reduced and K'm value was increased in the presence of AAH (0.4 mM). Using electron spin resonance to investigate the hydroxyl radical scavenging activities, AAH and GAH showed effectively scavenging activities and IC50 was 0.66 mM and 1.21 mM, respectively.
Keywords: Aspartic acid p-hydroxamate (AAH); Electron spin resonance; Hydroxyl radical; Glutamic acid y-monohydroxamate (GAH); Semicarbazide-sensitive amine oxidase (SSAO).
INTRODUCTION
ganisms, and the organs of mammals (vasculature, dental pulp, eye, and plasma) (Boomsma et al., 2000).
Amine oxidases (AOs) have been divided into two main groups (Jalkanen and Salmi, 2001), one is the flavin adenine dinucleotide-containing enzymes (monoamine oxidase A (MAO-A), -B, and polyamine oxidase) (Shih et al., 1999) and the other contains a cofactor possessing one or more topaquinones and copper ions [diamine oxidase, lysyl oxidase, or semicarbazide-sensitive AO (SSAO)],(Klinman and Mu, 1994; Klinman, 1996; Lyles, 1996). MAO-A and MAO-B were well known mitochondria en-zymes (EC 1.4.3.4) that have firmly established roles in the metabolism of neurotransmitters (Shih et al., 1999). They can be distinguished selectively by specific inhibi-tors of clorgyline and deprenyl, respectively, and MAO inhibitors were recently used to treat anxiety disorders and Alzheimer's disease (Yamada and Yasuhara, 2004). The SSAO (EC 1.4.3.6), arises from the inhibition by semi-carbazide, is a name for a group of heterogenous enzymes widely distributed in nature, including plants, microor-
AO converts amines into the corresponding aldehydes, generating hydrogen peroxide and ammonia. Benzylamine appears to be a good substrate for all SSAOs and MAO-B (Lyles, 1996), and a variety of other amines (e.g., sero­tonin, tyramine, tryptamine, polyamine, and dopamine) have been reported to be substrates for some but not all SSAOs. It has been found that endogenous compounds, aminoacetone and methylamine, are good substrates for most SSAOs (Precious et al., 1988; Lyles and Chalmers, 1992; Jalkanen and Salmi, 2001). In recent research, it was found that plasma SSAO was elevated in diabetes mel-litus and heart failure, and is implicated in atherosclerosis, endothelial damage, and glucose transport into adipocytes (Boomsma et al., 1995, 1997, 1999; Yu and Zuo, 1993, 1996, 1997).
The galactosamine, mannosamine, and glucosamine were showed to have dose-dependent inhibitory activi­ties against SSAO (O'Sullivana et al., 2003). In our pub­lished results, several purified natural products, synthetic compounds, and crude extracts showed SSAO inhibitory activities. For examples, the myricetin galloylglycoside
*Corresponding author: E-mail: wchou@tmu.edu.tw; Fax: +886-2-2378-0134.
302
Botanical Studies, Vol. 53, 2012
with 10 mL deionized water), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), ferrous sulfate, L-glutamic acid y-monohydroxamate (GAH, G2253), horseradish peroxi-dase (148 units/mg solid), and semicarbazide were pur­chased from Sigma Chemical Co. (St. Louis, MO, USA). Hydrogen peroxide (33%) was from Wako Pure Chemical Industry (Osaka, Japan). Other chemicals and reagents were from Sigma Chemical Co. (St. Louis, MO, USA).
Determination of SSAO inhibitory activity
SSAO inhibitory activities were determined by spectro-photometric method according to Szutowicz et al. (1984) with modifications (Liu et al., 2011a,b) using hydrogen peroxide to plot the SSAO standard curve. The total 200 jaL reaction solution (containing 50 joL of 200 mM phos­phate buffer, pH 7.4, 50 joL of 8 mM benzylamme, SSAO, and different amounts of AAH or GAH, and semicarba-zide) was placed at 37°C for one hour and then heated at 100°C boiling water for 5 min to stop reaction. After cool­ing and brief centrifugation, the 90 j L reaction solution was isolated and added to 710 j L of spectrophotometric solution containing 200 j L of 200 mM phosphate buffer (pH 7.4), 100 joL of 2 mM ABTS solution, and 25 joL of horseradish peroxidase (10 j g/mL). Means of triplicates were measured. Deionized water was used instead of AAH or GAH or semicarbazide as a blank experiment. One unit of SSAO activity was defined as amounts of enzyme to produce one nanomole of hydrogen peroxide during one hour under above-mentioned reaction condi­tion. The changes of absorbance at 420 nm were recorded during 1 min and expressed as AA420nm/min. The relative SSAO activity (%) was calculated with the equation:
(ΔA420nm/min,blank- ΔA420nm/min,sample)÷ΔA420nm/min,blank*100%
Figure 1. The effects of AAH and GAH on SSAO from bovine plasma. (A) The structure of AAH and GAH, and (B) inhibi­tion of SSAO activities by AAH, GAH, and semicarbazide, respectively. The SSAO inhibition (%) was calculated with the equation: (ΔA420 nm/minblank- ΔA420 nm/minsample)÷ΔA420 nm/minblank*100%
isolated from leaves of Acacia confusa (Lee et al., 2006) and geraniin isolated from Phyllanthus urinaria (Lin et al., 2008) showed dose-dependent inhibitory activities against SSAO. The hydroxamic acid derivative of hydroxyurea showed MAO and SSAO inhibitory activities (Liu et al., 2010). The synthesized alginic acid hydroxamate (Liu et al., 2007), synthesized galacturonic acid hydroxamate (Liu et al., 2011a) and glucuronic acid hydroxamate (Liu et al., 2011b) were reported to have SSAO inhibitory activities. The extracts of Taiwanofungus camphoratus (Chang-Chih) showed dose-dependent inhibitory activities against SSAO (Wang et al., 2007).
Plasma SSAO activity stains on 10% native polyacrylamide gels
Our previous report showed that aspar-tic acid p-hydroxamate (AAH) and glutamic acid y-monohydroxamate (GAH) (Figure 1A) exhibited angio-tensin converting enzyme inhibitory activities, and scav­enging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide radicals (Liu et al., 2004). The AAH exhibited antitumor activity on L5178Y leukemia (Thomasset et al., 1991), therapeutic effect on friend eryth-roleukemia (Tournaire et al., 1994a) and antiproliferative activity on friend virus-infected erythropoietic progenitor cells (Tournaire et al., 1994b). In this research, AAH and GAH were effectively to inhibit SSAO activities from bovine plasma in a dose-dependent manners, and also showed effectively scavenging activities against hydroxyl radicals.
The SSAO activity staining on a 10% polyacrylam-ide gel was according to the method of Lee et al. (2002). Bovine plasma (23.45 units) was premixed with AAH or GAH (0, 1.5, 1.75, 2, 2.25, and 2.5 mM) overnight and prepared for electrophoresis. When native PAGE was finished, the gels were balanced for 20 min twice in 50 mM Tris-HCl buffer (pH 7.9) before activity staining. The process of plasma SSAO activity staining was as below. Eighty mg benzylamine and 40 mg AEC was dissolved in 10 mL dimethylformamide and then added to 40 mL, 50 mM Tris-HCl buffer (pH 7.9) as the substrate solution, in which the gel was submerged and shaked for 5 min. Then, 200 j L horseradish peroxidase (5 mg/mL) was added. The gel was gentle shaking at room temperature. The gel was destained with 10% acetic acid and then washed with dis­tilled water.
MATERIALS AND METHODS
Materials
Determination of the kinetic constant of bovine SSAO in the presence of AAH
L-Aspartic acid p-hydroxamate (AAH, A6508), ben-zylamine, 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, ABTS), bovine plasma (P-4639, reconstitute
The Km and Vmax of bovine SSAO (18.76 units) and the
LIU et al. 一 Anti-SSAO and hydroxyl radical scavenging activities of Asp-NHOH and Glu-NHOH
303
Figure 2. Effects of different concentrations of AAH and GAH on SSAO activity from bovine plasma using activity staining in 10%
native-PAGE gels. Lane 1 to lane 5 were 1.5, 1.75, 2, 2.25, and 2.5 mM, respectively.
Km' and Vmax' of bovine SSAO in the presence of AAH (0.4 mM) were calculated from Lineweaver-Burk plots using different concentrations of benzylamine as substrate (0.67, 0.83, 1, 1.33, 2, and 4 mM).
Scavenging activity of hydroxyl radicals as measured by electron spin resonance (ESR) spectrometry
Hydroxyl radicals were generated by means of the Fenton reaction (Liu et al., 2010). The reaction mixture (500 j L) included AAH (0.5 mM and 2.5 mM) or GAH (1.0, 1.5, and 2.5 mM), 5 mM DMPO, and 0.05 mM fer-rous sulfate. After mixing, the solution was transferred to an ESR quartz cell and placed at the cavity of the ESR spectrometer. Hydrogen peroxide was added to give a fi-nal concentration of 0.25 mM. Deionized water was used instead of the sample solution as the blank. After 40 s, the relative intensity of the signal of the DMPO-OH spin adduct was measured. All ESR spectra were recorded at ambient temperature (25°C) on a Bruker EMX-6/1 ESR spectrometer equipped with WIN-ESR SimFonia software version 1.2. The conditions of ESR spectrometry were as follows: center field, 345.4 5.0 mT; microwave power, 8 mW (9.416 GHz); modulation amplitude, 5 G; modulation frequency, 100 kHz; time constant, 0.6 s; and scan time, 1.5 min.
Figure 3. The kinetic properties of bovine SSAO (18.76 units) in the absence and presence of AAH (0.4 mM) in Lineweaver-Burk plots using different concentrations of benzylamine as sub­strate (0.67, 0.83, 1, 1.33, 2, and 4 mM).
but not clorgyline or deprenyl, therefore, it was confirmed that the AO activity from bovine plasma was belonged to SSAO (Liu et al., 2011a). Figure 1B showed the effects of AAH and GAH on bovine plasma SSAO inhibitory activities. It was found that AAH or GAH showed dose-dependly inhibitory against SSAO from bovine plasma. In calculations, the IC50 of AAH and GAH was 0.7 mM and 0.023 mM, respectively, in the comparisons with 0.035 mM of semicarbazide (positive controls).
RESULTS AND DISCUSSION
A variety of hydroxamic acid derivatives have been proven to have biological activities toward cancer, cardio­vascular diseases, Alzheimer's disease and tuberculosis (Muri et al., 2002). The hydroxamate moiety were re­ported to chelate metal ions and therefore to inhibit met-alloenzyme activities (Neilands, 1967; Hou et al., 2003; Liu et al., 2004). SSAO (EC 1.4.3.6) is a common name for a group of copper-containing metalloproteins widely distributed in nature, included plants, microorganisms, and organs of mammals (vasculature, dental pulp, eye and plasma) (Boomsma et al., 2000). Therefore, the amino acid hydroxamates of AAH and GAH (Figure 1A) were used to evaluate the SSAO inhibitory activities in the comparison with positive control of semicarbazide.
Using AO activity staining in 10% native-PAGE gels, the intensity of brown color showed AO activity. It was found that GAH higher than 1.75 mM (lane 2) in tested concentrations showed inhibitory activities against SSAO from bovine plasma (Figure 2) compared to the SSAO only (lane B in Figures 2), and the AAH in tested concen­trations showed minor inhibitory activities against SSAO from bovine plasma (Figure 2).
Figure 3 showed the kinetic parameter of SSAO from bovine plasma with or without 0.4 mM AAH additions. Without AAH additions, the Km of bovine SSAO was calculated to be 1.84 mM which was closed to 2.23 mM
(Liu et al., 2007) and 2.01 mM (Liu et al., 2011a) of the
previous report, and the Vmax was 0.1368 (AA/min). In the presence of 0.4 mM of AAH, the calculated Km' was 2.13
In our previous report, it was proved that bovine plasma contained SSAO activities which the AO activity in bo­vine plasma was specifically inhibited by semicarbazide,
304
Botanical Studies, Vol. 53, 2012
Figure 4. Scavenging activities of (A) AAH (0.5 mM and 2.5 mM), and (B) GAH (1 mM, 1.5 mM and 2.5 mM) against hydroxyl radi­cals as measured by electron spin resonance spectrometry.
mM (Km' > Km) and calculated Vmax' was 0.0833 (AA/ min) (Vmax' < Vmax). From the results of kinetic data, the AAH showed mixed noncompetitive inhibition against bo­vine SSAO in the respect to benzylamine (substrate) and benzylamine-SSAO (substrate-enzyme complex). It was suggested that the hydroxamic acid moiety in AAH might partially compete with benzylamine in the SSAO active site or benzylamine-SSAO complex and then change the SSAO kinetic properties.
ing activites of both amino acid hydroxamates were evalu­ated by ESR. Figure 4 showed the scavenging activities of AAH (Figure 4A) and GAH (Figure 4B) against hydroxyl radicals. From the changes of DMPO-OH adduct intensi­ties, it was clear that both amino acid hydroxamates exhib­ited hydroxyl radical scavenging activities. In calculations, IC50 of AAH and GAH was 0.66 mM and 1.21 mM, re-spectively.
In conclusion, SSAO catalyzed the oxidative deamina-tion of various amines to produce the corresponding alde­hydes, ammonia and hydrogen peroxide. AAH and GAH exhibited SSAO inhibitory and anti-hydroxyl-radical ac­tivities in vitro. It might be beneficial for anti-aging prop­erties, and requires further investigation.
The problem of aging-related diseases, such as neu-rodegenerative diseases (ex. Alzheimer, Parkinson, and Huntington disease), have been emphasized recently. The intricate causes of the aging process are still a matter of extensive speculation, giving rise to many theories. In particular, the role of reactive oxygen species is a prereq­uisite nowadays for understanding this process (Muller et al., 2007). SSAO catalyzed the oxidative deamination of various amines to produce hydrogen peroxide which is one possible source of oxidative stresses. Ferrer et al. (2002) reported that SSAO activity was higher in the cerebral blood vessels of subjects with Alzheimer's disease. Jiang et al. (2008) reported that products of aldehyde in SSAO-catalyzed deamination might be correlated with cerebral amyloid angiopathy in Alzheimer's disease througt beta amyloid aggregation and advanced glycation end-product formation. Active hydrogen peroxide might interact with ferrous ions in vivo to produce hydroxyl radicals (the Fenton reaction), which is a possible source of oxidative stresses. Therefore, except from SSAO inhibition by AAH and GAH above-mentioned, the hydroxyl radical scaveng-
Acknowledgments. The authors want to thank the finan­cial support (SKH-TMU-101-09) from Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
LITERATURE CITED
Boomsma, F., F.H.M. Derkx, A.H. van den Meiracker, A.J. Manint Veld, and M.A.D.H. Schalekamp. 1995. Plasma se-micarbazide-sensitive amine oxidase is elevated in diabetes mellitus and correlates with glycosylated haemoglobin. Life Sci. 88: 675-679.
Boomsma, F., D.J. van Veldhuisen, P.J. de Kam, A.J. Manint Veld, A. Mosterd, K.I. Lie, and M.A.D.H. Schalekamp. 1997. Plasma semicarbazide-sensitive amine oxidase is el-
LIU et al. 一 Anti-SSAO and hydroxyl radical scavenging activities of Asp-NHOH and Glu-NHOH
305
evated in patients with congestive heart failure. Cardiovasc. Res. 33: 387-391.
Boomsma, F., A.H. van den Meiracker, S. Winkel, H.J. Aanstoot, M.R. Batstra, A.J. Manint Veld, and G.J. Bruining. 1999. Circulating semicarbazide-sensitive amine oxidase is raised both in Type I (insulin-dependent), in Type II (non-insulin-dependent) diabetes mellitus and even in childhood Type I diabetes at first clinical diagnosis. Diabetologia 42: 233-237.
Boomsma, F., J. van Dijk, U.M. Bhaggoe, A.M.B. Bouhuizen, and A.H. van den Meiracker. 2000. Variation in semicarbaz-ide-sensitive amine oxidase activity in plasma and tissues of mammals. Comp. Biochem. Physiol. Part C 126: 69-78.
Ferrer, I., J.M. Lizcano, M. Hernandez, and M. Unzeta. 2002. Overexpression of semicarbazide-sensitive amine oxidase in the cerebral blood vessels in patients with Alzheimer's disease and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. Neurosci. Lett. 32: 21-24.
Hou, W.C., M.H. Lee, F.L. Hsu, and Y.H. Lin. 2003. Inhibitory activities of semicarbazide-sensitive amine oxidase and an-giotensin converting enzyme of pectin hydroxamic acid. J. Agric. Food Chem. 51: 6362-6366.
Jalkanen, S. and M. Salmi. 2001. Cell surface monoamine oxi-dases: enzymes in search of a function. EMBO J. 20: 3893­3901.
Jiang, Z.J., J.S. Richardson, and P.H. Yu. 2008. The contribution of cerebral vascular semicarbazide-sensitive amine oxidase to cerebral amyloid angiopathy in Alzheimer's disease. Neuropathol. Appl. Neurobiol. 34: 194-204.
Klinman, J.P. and D. Mu. 1994. Quinoenzymes in biology. Annu. Rev. Biochem. 63: 299-344.
Klinman, J.P. 1996. New quinocofactors in eukaryotes. J. Biol. Chem. 271: 27189-27192.
Lee, T.H., D.Z. Liu, F.L. Hsu, W.C. Wu, and W.C. Hou. 2006. Structure-activity relationships of five myricetin galloylg-lycosides from leaves of Acacia confusa. Bot. Stud. 47: 37-43.
Lin, S.Y., C.C. Wang, Y.L. Lu, W.C. Wu, and W.C. Hou. 2008. Antioxidant, anti-semicarbazide-sensitive amine oxidase, and anti-hypertensive activities of geraniin isolated from Phyllanthus urinaria. Food Chem. Toxicol. 46: 2485-2492.
Liu, D.Z., Y.S. Lin, and W.C. Hou. 2004. Monohydroxamates of aspartic acid and glutamic acid exhibit antioxidant and an-giotensin converting enzyme inhibitory activities. J. Agric. Food Chem. 52: 2386-2390.
Liu, D.Z., W.C. Wu, H.J. Liang, and W.C. Hou. 2007. Antioxi-dant and semicarbazide-sensitive amine oxidase inhibitory activities of alginic acid hydroxamates. J. Sci. Food Agric. 87: 138-146.
Liu, Y.H., W.C. Wu, Y.L. Lu, Y.J. Lai, and W.C. Hou. 2010. Antioxidant and amine oxidase inhibitory activities of hy-droxyurea. Biosci. Biotechnol. Biochem. 74: 1256-1260.
Liu, Y.H., W.J. Huang, Y.L. Lu, C.C. Lee, Y.F. Tsai, W.C. Wu,
and W.C. Hou. 2011a. Semicarbazide-sensitive amine oxi-dase inhibitory activity of galacturonic acid hydroxamate. Bot. Stud. 52: 35-40.
Liu, Y.H., W.L. Liang, C.C. Lee, Y.F. Tsai, and W.C. Hou. 2011b. Antioxidant and semicarbazide-sensitive amine oxidase inhibitory activities of glucuronic acid hydroxamate. Food Chem. 129: 423-428.
Lyles, G.A. and J. Chalmers. 1992. The metabolism of amino-acetone to methylglyoxal by semicarbazide-sensitive amine oxidase in human umbilical artery. Biochem. Pharmacol. 43: 1409-1414.
Lyles, G.A. 1996. Mammalian plasma and tissue-bound semicar-bazide-sensitive amine oxidases: biochemical, pharmaco­logical and toxicological aspects. Int. J. Biochem. Cell Biol. 28: 259-274.
Muri, E.M.F., M.J. Nieto, R.D. Sindelar, and J.S. Williamson. 2002. Hydroxamic acids as pharmacological agents. Cur. Med. Chem. 9: 1631-1653.
Neilands, J.B. 1967. Hydroxamic acids in nature. Science 156:1443-1447.
O'Sullivana, J., M. O'Sullivana, K.F. Tiptona, M. Unzetab, M.D.M. Hernandezb, and G.P. Daveya. 2003. The inhibition of semicarbazide-sensitive amine oxidase by aminohexoses. Biochim. Biophys. Acta 1647: 367-371.
Precious, E., C.E. Gunn, and G.A. Lyles. 1988. Deamination of methylamine by semicarbazide-sensitive amine oxidase in human umbilical artery and rat aorta. Biochem. Pharmacol. 37: 707-713.
Shih, J.C., K. Chen, and M.J. Ridd. 1999. Monoamine oxidase: from genes to behavior. Annu. Rev. Neurosci. 22: 197-217.
Szutowicz, A., R.D. Kobes, and P.J. Orsulak. 1984. Colorimetric assay for monoamine oxidase in tissues using peroxidase and 2',2-azinodi(3-ethylbenzthiazoline-6-sulfonic acid) as chromogen. Anal. Biochem. 138: 86-94.
Thomasset, N., F. Hamedi-Sangsari, R. Tournaire, C. Na-varro, S. Malley, L. Goetsch, J. Grange, and J. Vila. 1991. Anti-tumoral activity of L and D isomers of aspartic acid p-hydroxamate on L5178Y leukemia. Int. J. Cancer 49:421-424.
Tournaire, R., S. Malley, F. Hamedi-Sangsari, N. Thomasset, J. Grange, J.F. Dore, and J. Vila. 1994a. Therapeutic effects of D-aspartic acid p-hydroxamate (DAH) on friend erythroleu-kemia. Int. J. Cancer. 58: 420-425.
Tournaire, R., S. Arnaud, F. Hamedi-Sangsari, S. Malley, J. Grange, J.P. Blanchet, J.F. Dore, and J. Vila. 1994b. Anti-proliferative effect of D-aspartic acid p-hydroxamate (DAH) on friend virus-infected erythropoietic progenitor cells. Leukemia 8: 1703-1707.
Wang, G.J., S.Y. Lin, W.C. Wu, and W.C. Hou. 2007. DPPH radical scavenging and semicarbazide-sensitive amine oxi-dase inhibitory and cytotoxic activities of Taiwanofungus camphorates (Chang-Chih). Biosci. Biotech. Biochem. 71: 1873-1878.
Yamada, M. and H. Yasuhara. 2004. Clinical pharmacology of
306
Botanical Studies, Vol. 53, 2012
MAO inhibitors: Safety and future. Neurotoxicology 25:
215-221.
Yu, P.H. and D.M. Zuo. 1993. Oxidative deamination of meth-ylamine by semicarbazide-sensitive amine oxidase leads to cytotoxic damage in endothelial cells. Diabetes 42: 594­603.
Yu, P.H. and D.M. Zuo. 1996. Formaldehyde produced endoge-
neously via deamination of methylamine. A potential risk factor for initation of endothelial injury. Atherosclerosis 120: 189-197.
Yu, P.H. and D.M. Zuo. 1997. Aminoguanidine inhibits semicar-bazide-sensitive amine oxidase activity: implications for ad­vanced glycation and diabetic complications. Diabetologia
40: 1243-1250.
天門冬胺酸羥肟酸與麩胺酸羥肟酸具有抑制
semicarbazide-敏感型胺酶與清除氫氧自由基活性
劉玉華1,2 林音秀3 李泰霖4 侯文琪4,5
1新光醫院肝膽腸胃科
2臺北醫學大學一般醫學科
3臺北醫學大學藥學系
4臺北醫學大學生藥學研究所
5臺北醫學大學附設醫院中草藥研究中心
文獻報導，semicarbazide-敏感型胺酶(SSAO)在糖尿病患與心臟受損病患的血漿中有增加。SSAO 催化氧化去胺反應，過程中產生過氧化氫，可能是細胞内產生氧化逆境來源之一。因此，本研究想探討 兩種胺基酸羥肟酸衍生物對於SSAO抑制活性與其清除氫氧自由基活性。結果顯示，天門冬胺酸羥肟酸 (AAH)與麩胺酸羥肟酸（GAH)，具有有效且濃度相關抑制商品化牛血漿來源之SSAO ，抑制SSAO活性 一半所需濃度（IC50)分別為0.7及0.023 mM ，其對照比對為semicarbazide (IC50為0.035 mM) 。 AAH在 0.4 mM下探討抑制牛血漿SSAO之動力學變化，結果顯示發現其V'max數值降低及K'm數值增加。顯示 AAH對於牛血漿SSAO的抑制反應屬於為混合型非競爭型抑制（mixed noncompetitive inhibition)，其競 爭對象為SSAO反應基質與SSAO-基質複合體。利用電子自旋共振探討氫氧自由基清除實驗，AAH與 GAH都具有濃度相關清除活性，其IC;。分別為0.66 mM與1.21 mM 。
關鍵詞：天門冬胺酸羥肟酸；電子自旋共振；氫氧自由基；麩胺酸羥肟酸；Semicarbazide-敏感型胺酶 (SSAO)。