The In Vitro Inhibitory Effect of Flavonoid Astilbin on 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase on Vero Cells

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1 Chi nese Med i cal Jour nal (Taipei) 2001;64: Orig i nal The In Vitro Inhibitory Effect of Flavonoid Astilbin on 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase on Vero Cells Tso-Hsiao Chen 1 Ju-Chi Liu 1,2 Jun-Jen Chang 1 Min-Fung Tsai 1 Ming-Hsiung Hsieh 1,2 Paul Chan 1,2 1 De part ment of Med i cine, Tai pei Med i cal Uni ver sity-wan Fang Hos pi tal; and 2 De part ment of Med i cine, Tai pei Med i cal Uni ver sity, Tai pei, Tai wan, R.O.C. Key Words atherosclerosis; Chinese herbs; hyperlipidemia Background. Epidemiological stud ies have shown that hy per cho les - ter ol emia is a ma jor risk fac tor for cor o nary heart dis ease. In clin i cal tri als of lipid low er ing ther apy, 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase in hib i tor has been shown to de crease car diac events and mor tal ity. Flavonoids are polyphenolic nat u ral an ti ox i dants ex ist ing in veg e ta bles, fruits and bev er ages such as tea and wine. Pre vi - ous stud ies have shown that some an ti ox i dants had hypocholesterolemic ef fect, and flavonoid in take was as so ci ated with the de crease of mor - tal ity from cor o nary ar tery dis ease. The aim of this study was to eval u - ate the in hib i tory ef fect of flavonoids on HMG-CoA reductase. Methods. The meth ods for anal y sis of spe cific in hib i tors of mevalonate biosynthesis have been well-established, using Vero cells, a cell line ob tained from kid neys of Af ri can green mon keys. Flavonoids iso lated from dif fer ent tra di tional Chi nese herbs were dis solved in DMSO and in cu bated with Vero cells with or with out the ad di tion of 1 mm me - valonate or 5 mm so dium ac e tate in or der to ob serve cell growth for 24 h. Results. Con cen tra tions of 1 mm mevalonate or 5 mm so dium ac e tate were added into cul ture me dium in or der to ob serve the ef fect on cell growth. Dif fer ent con cen tra tions of pravastatin to in hibit cell growth were used as a pos i tive con trol. About 40 flavonoid com pounds were used for study, only one com pound, astilbin (be long ing to the flavonol group), showed sig nif i cant in hi bi tion of Vero cell growth. Con clu sions. This study shows that one flavonoid com pound, iso lated from tra di tional me dic i nal herbs, may be an effective HMG-CoA re - ductase inhibitor which might be developed into a new hypo - cholesterolemic agent. [Chin Med J (Tai pei) 2001;64: ] Flavonoids are a large group of polyphenolic com - pounds pos sess ing an ti ox i dant ac tiv ity that oc cur nat u rally in a va ri ety of foods from veg e ta ble or i gin such as ap ples, on ions and bev er ages such as tea and red wine. 1,2 The most im por tant groups of flavonoids are anthrocyanins, flavonols, fla vones and flavonones. Flavonols are scav en gers of superoxide an ions, 3 sin - glet ox y gen, 4 and lipid peroxy rad i cals, 5 and they can se ques ter metal ions through liganding. 6 Ox i dized low-density lipoproteins OX-(LDL) are cytotoxic and atherogenic, and are be lieved to be an im por tant step in the for ma tion of atherosclerotic plaques. 7 A pre vi - Re ceived: July 18, Ac cepted: April 16, Cor re spon dence to: Paul Chan, MD, PhD, Di vi sion of Car dio vas cu lar Med i cine, Tai pei Med i cal Uni ver sity-wan Fang Hos pi tal, 111, Sec. 3, Hsin-Lung Road, Tai pei 117, Tai wan. Fax: ; chanpaul@wanfang.gov.tw

2 382 Tso-Hsiao Chen, et al. Chi nese Med i cal Jour nal (Taipei) Vol. 64 No. 7

3 July 2001 Flavonoid Astilbin on HMG-CoA Reductase 383 ous re port showed that flavonols and fla vones had anti platelet ef fect and re duced thrombogenic ten den - cies, and the mech a nism was prob a bly due to in hi bi - tion of cyclo-oxygenase. 8 Flavonoids have also been stud ied in re la tion to their im prove ment of vas cu lar fragility. 9 Mul ti ple ep i de mi o log i cal stud ies have shown that in creased veg e ta ble and fruit con sump tion has in verse re la tion ship to the oc cur rence of stroke and CHD in dif fer ent pop u la tions The ex pla na tion of this phe nom e non is prob a bly due to an in crease of flavonoid in take. For ex am ple, the Zutphen Study has shown that flavonoid in take was sig nif i cantly in - versely as so ci ated with mor tal ity from CHD and showed an in verse re la tion with in ci dence of myo - car dial in farc tion. 10 The rel a tive risk of cor o nary heart dis ease mor tal ity in the high est ver sus the low - est tertile of fla vonoid in take was Be sides ben e fi cial an ti ox i dant ef fect of flavonoid, we pre - sume that the pos i tive re sults of the above memtioned ep i de mi o log i cal stud ies were prob a bly due to hypocholesterolemic ef fect. Pre vi ous stud ies have dem on strated that some an ti ox i dants, such as beta-carotene, vi ta min C and vi ta min E have hypocholesterolemic ef fect, which is re lated to the in hi bi tion of HMG-CoA reductase This study was un der taken to eval u ate whether flavonoids have HMG-CoA reductase in hib i tory ef fect. Methods Flavonoid com pounds (flavonols, fla vones, fla - vanones) were iso lated in the lab o ra tory of Dr. Kuo of China Med i cal Col lege, Tai-Chung, Tai wan. HMG- CoA, DL-mevalonate (lactone form), pen i cil lin, strep - to my cin, methylrosaniline chlo ride and trypsin were pur chased from Sigma (Sigma Chemical Co., MO, USA). Pravastatin was a gen er ous gift from Sankyo Com pany (To kyo, Ja pan). Ea gle s min i mum es sen tial me dium (MEM) and calf se rum were ob tained from Gibco Co. Mu nich, Ger many. Micro plates (96 wells) were ob tained from Corning Co. California, USA. Other ma te ri als were com mer cially avail able. All fla - vonoid com pounds were dis solved in 100% DMSO. Cell cul ture me dium had 0.01% DMSO (cells ). Af ter in cu ba tion for 24 h, cell growth and mor phol ogy was ex am ined by light mi cro scope (Olym pus, Ja pan). Con cen tra tions of 1 mm mevalonate or 5 mm so dium ac e tate were added into cul ture me dium in or der to ob serve the ef fect on cell growth. Dif fer ent con cen tra - tions of pravastatin to in hibit cell growth were used as a pos i tive con trol. Vero cells Vero cells, an es tab lished cell line from kid ney cells of Af ri can green mon key, were grown in a hu - mid i fied in cu ba tor (5% CO 2 ) at 37 C in a 1-1 glass flask con tain ing 200 ml of Ea gle s min i mum es sen - tial me dium (MEM) sup ple mented with pen i cil lin (100 units/ml), strep to my cin (100 µg/ml), 0.075% (w/v) NaHCO 3, 0.03% (w/v) glutamine, and calf se - rum at 5% (v/v) (5% CS-MEM). Cells were subcultured ac cord ing to standand trysinization pro - ce dures. Vero cells ( cells) in 100 µl of the above mix ture, but now with calf se rum at 2% (v/v), were in oc u lated in each well of microplates and in cu - bated at 37 C for 1 h. Then, var i ous amounts of flavonoid com pounds (flavonols, fla vones, flavonones) 100 µg/ml with and with out 1 mm mevalonate or 5 mm so dium ac e tate were added to the wells. Vero cells were grown in a hu mid i fied in cu ba tor (5% CO 2 ) at 37 C for 24 h. Mea sure ment of cell growth Cell growth was mea sured by the method of Armstrong. 16 Cells grown on each well of 96-well microplates were washed twice with 100 µl of cal - cium- and mag ne sium-free phos phate-buffered sa - line (PBS) and stained with 50 µl of stain ing so lu - tion (methylrosaniline 0.5%, NaCl 0.85%, for - mamide 5% and eth a nol 50%) for 20 min utes. Then the stain ing so lu tion was re moved and the cells were washed with wa ter. The absorbance at 540 nm was mea sured by microplate pho tom e ter (Titertek Co., Osaka, Ja pan).

4 384 Tso-Hsiao Chen, et al. Chi nese Med i cal Jour nal (Taipei) Vol. 64 No. 7 Re sults Con cen tra tions of cells Vero cells were seeded in each well of a 96-well microplate at 1.0 ~ cells in 100 µl of 2% CS-MEM. Con flu ent growth was ob served af ter a 24-hour in cu ba tion when the cells were seeded at 3.0 ~ cells/well. Thus, a con cen tra tion of cells/well was adopted in sub se quent ex per i ments. Ef fect of var i ous chemicals on growth of Vero cells Fig. 1 shows the ef fect of pravastatin on growth of Vero cells. Pravastatin at the dos age of 100 µm in hib - ited cell growth com pletely. How ever, when 1 mm mevalonate was added to the cul ture me dium, both mor pho log i cal changes and growth in hi bi tion were over come and the cells grew nor mally. Ad di tion of 5 mm so dium ac e tate had no ef fect. Mor pho log i cal changes and growth in hi bi tion of var i ous flavonoid com pounds on Vero cells were ob served at a uni form dose of 100 µg/ml. These data in di cated that this meth od was highly sen si tive to mevalonate bio - synthesis in hib i tors. The re sults in di cated that most of the flavonoid com pounds could not in hibit mevalonate biosynthesis in cul tured cells (Ta bles 1-3). Al though some fla vonoid com pounds had the in hib i tory ef fect on growth of Vero cells, the ef fect was not over come af ter add ing mevalonate into the cul ture me dium. Only the astilbin (Ta ble 1) showed po tent in hib i tory ef fect on growth of Vero cells, just like the ef fect of prav astatin. Discussion The Zutphen El derly Study sug gested that lower mor tal ity from CHD and lower in ci dence of myo car - dial in farc tion was due to in creased flavonoid in - take. 10 The pre dom i nant flavonoid in foods is quer - Table 1. Effects of flavonoids (flavonols) on the growth of Vero cells with or without mevalonate Compound Conc. (µg/ml) Absorbance at 540 nm None Mevalonate (1 mm) Control 1% DMSO Galangin 100 N N Fisetin 100 N N Quercetin Quercitrin Rutin 100 N N Quercetin-3-0- (arabinopyanosyl)- 100 N N gactopyranoside Myricetin Myricitrin Kaempferol 100 N N Rhamnustrioside 100 N N Astilbin N = non-detectable. Fig. 1. Ef fects of pravastatin (o) alone or in com bi na tion with 1mM mevalonate ( ) or 5 mm so dium ac e tate ( ) on the growth of Vero cells. Table 2. Effects of flavonoids (flavanones) on the growth of Vero cells with or without mevalonate Compound Conc. Absorbance at 540 nm (µg/ml) None Mevalonate (1 mm) Control 1% DMSO Naringenin 100 N N Naringin 100 N N Hespretin Hesperidin 100 N N (+) Taxifolin 100 N N (+) Catechin 100 N N ( ) Epicatechin 100 N N N = non-detectable.

5 July 2001 Flavonoid Astilbin on HMG-CoA Reductase 385 Table 3. Effects of flavonoids (flavones) on the growth of Vero cells with or without mevalonate Compound Conc. Absorbance at 540 nm (µg/ml) None Mevalonate (1Mm) Control 1% DMSO Hydroxyflavone 100 N N 7-Hydroxyflavone 100 N N Baicalcin 100 N N Chrysin Apigenin 100 N N Apigenin N N Luteolin glucoside Linarin 100 N N Pectolinarin 100 N N Cirsimarin N = non-detectable. cetin, and higher in take of quercetin re vealed in verse re la tion ship with CHD mor tal ity. The pres ent study also in cluded the com pound quercetin, but the re sults showed that only astilbin pos sessed sig nif i cant HMG- CoA reductase in hi bi tion. Quercetin also in hib its the cytotoxicity of oxi - dised-ldl in vi tro. 17 It is pos si ble that quercetin and other flavonoids re duce the rate of for ma tion of oxi - dised-ldl and thus in hibit the growth of ather o scle - rotic plaques. In ad di tion, flavonoids in hibit cyclooxygenases, which may re duce throm bo sis. 8 Hy per cho les ter ol emia con trib utes sub stan tially to the de vel op ment and clin i cal ex pres sion of cor o nary and other forms of ath ero scle ro sis Con sid er able ev i dence sug gested that cho les terol low er ing sta bi - lizes plaques and re duces car dio vas cu lar events, in - clud ing all-cause mor tal ity Ma jor clin i cal tri als have dem on strated sig nif i cant re duc tions in car dio - vas cu lar events in pa tients with a wide range of cho - les terol lev els. 24 These find ings sug gest that ag gres - sive lipid man age ment is ben e fi cial to pa tients and that man u fac tur ing new lipid-lowering drugs is still a worth while pur suit in phar ma ceu ti cal sci ence. It is ex pected that this study s meth od ol ogy would be ad van ta geous over the con ven tional in vi tro en - zyme as say for a pri mary screen ing sys tem for the fol - low ing rea sons: 1) The intracellular en vi ron ment where an en zyme ex ists is main tained. It would be im - por tant to keep the en zyme en vi ron ments as they are in liv ing cells for screen ing en zyme in hib i tors; 2) primary as says can be done with out ra dio ac tive sub - strates; 3) a pro-drug type of in hib i tor which is ac ti - vated af ter be ing in cor po rated into liv ing cells might be dis cov ered. On the other hand, some dis ad van tages also ex ist. To cul ture cells is rather trou ble some and the run ning cost is high for rou tine screen ing work. It will take more time to eval u ate the in hib i tory po tency by this method. So, un sta ble in hib i tors might be over - looked. Mevalonate is a key in ter me di ate in cho les terol biosynthetic path way. It is pro duced from acetyl-coa by three en zymes, namely, acetoacetyl-coa thiolase, HMG-CoA synthase and HMG-CoA reductase. These en zymes are ex pected to pro vide prom is ing tar get sites for the phar ma ceu ti cal in ter ven tion of hypo - cholesterolemic agents. LDL par ti cles con tain en dog e - nous an ti ox i dants which are con sumed dur ing LDL ox - idation, 25,26 and ad di tion of ex og e nous an ti ox i dants can pro tract the lag pe riod or even pre vent LDL oxidation, as shown with α-tocopherol, butylhydroxytoluene, urate, ascorbate, flavonoids, and probucol. 27,28 Fla - vonoids (de riv a tives of phenylchromone ring) are a large group of com pounds nat u rally oc cur ring in higher and lower plants. 29 Flavonoids have been shown to be able to af fect var i ous bi o log i cal func - tions: cap il lary per me abil ity, cel lu lar se cre tory pro - cesses in volved in the in flam ma tory re sponse and in - hi bi tion of en zymes, re cep tors and car ri ers. 29 Some flavonoids are strong ox y gen free rad i cal scav en gers and good metal chelators ef fec tive in pre vent ing lipid peroxidation. 30 Al though flavonoid com pounds have been shown to have an ti ox i dant ef fect or prob a bly vas cu lar pro tec - tion from ath ero scle ro sis, some of these com pounds may also in hibit the growth of Vero cells, like prav - astatin. In con clu sion, the flavonoid com pound as - tilbin may have po tent HMG Co-A reductase in hib i - tory ef fect, and whether it has cho les terol-lowering ef - fect needs fur ther study. References 1. Hertog MGL, Hollman PCH, Katan MB. Con tent of po ten -

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