AAA-Potential antioxidants and tyrosinase inhibitors from synthetic polyphenolic deoxybenzoins

合集下载

Antibody structure, instability, and formulation

MINIREVIEWAntibody Structure,Instability,and FormulationWEI WANG,SATISH SINGH,DAVID L.ZENG,KEVIN KING,SANDEEP NEMAPﬁzer,Inc.,Global Biologics,700Chesterﬁeld Parkway West,Chesterﬁeld,Missouri63017Received14March2006;revised17May2006;accepted4June2006Published online in Wiley InterScience().DOI10.1002/jps.20727 ABSTRACT:The number of therapeutic monoclonal antibody in development hasincreased tremendously over the last several years and this trend continues.At presentthere are more than23approved antibodies on the US market and an estimated200ormore are in development.Although antibodies share certain structural similarities,development of commercially viable antibody pharmaceuticals has not been straightfor-ward because of their unique and somewhat unpredictable solution behavior.This articlereviews the structure and function of antibodies and the mechanisms of physical andchemical instabilities.Various aspects of formulation development have been examinedto identify the critical attributes for the stabilization of antibodies.ß2006Wiley-Liss,Inc.and the American Pharmacists Association J Pharm Sci96:1–26,2007Keywords:biotechnology;stabilization;protein formulation;protein aggregation;freeze drying/lyophilizationINTRODUCTIONProtein therapies are entering a new era with the inﬂux of a signiﬁcant number of antibody pharmaceuticals.Generally,protein drugs are effective at low concentrations with less side effects relative to small molecule drugs,even though,in rare cases,protein-induced antibody formation could be serious.1Therefore,this category of therapeutics is gaining tremendous momentum and widespread recognition both in small and large drugﬁrms.Among protein drug therapies,antibodies play a major role in control-ling many types of diseases such as cancer, infectious diseases,allergy,autoimmune dis-eases,and inﬂammation.Since the approval of theﬁrst monoclonal antibody(MAb)product -OKT-3in1986,more than23MAb drug products have entered the market(Tab.1).The estimated number of antibodies and antibody derivatives constitute20%of biopharmaceutical products currently in development(about200).2The global therapeutic antibody market was predicted to reach$16.7billion in2008.3There are several reasons for the increasing popularity of antibodies for commercial develop-ment.First,their action is speciﬁc,generally leading to fewer side effects.Second,antibodies may be conjugated to another therapeutic entity for efﬁcient delivery of this entity to a target site, thus reducing potential side effects.For instance, Mylotarg is an approved chemotherapy agent composed of calicheamicin conjugated to huma-nized IgG4,which binds speciﬁcally to CD33for the treatment of CD33-positive acute myeloid leukemia.Another example is the conjugation of immunotoxic barnase with the light chain of the anti-human ferritin monoclonal antibody F11as potential targeting agents for cancer immuno-therapy.4Third,antibodies may be conjugated to radioisotopes for speciﬁc diagnostic purposes. Examples include CEA-Scan for detection of color-ectal cancer and ProstaScint for detection of prostate stly,technology advancement has made complete human MAb available,which are lessimmunogenic.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY20071 Correspondence to:Wei Wang(Telephone:(636)-247-2111;Fax:(636)-247-5030;E-mail:wei.2.wang@pﬁ)Journal of Pharmaceutical Sciences,Vol.96,1–26(2007)Pharmacists AssociationT a b l e 1.C o m m e r c i a l M o n o c l o n a l A n t i b o d y P r o d u c t s#B r a n d n a m e M o l e c u l eM A bY e a r C o m p a n y R o u t e I n d i c a t i o n M A b C o n c B u f f e r E x c i p i e n t s S u r f a c t a n t p H1A v a s t i n B e v a c i z u m a bH u m a n i z e d I g G 1,149k D a2004G e n e t e c h a n d B i o O n c o l o g y I V i n f u s i o nM e t a s t a t i c c a r c i n o m a o f c o l o n o r r e c t u m ,b i n d s V E G F 100m g a n d 400m g /v i a l (25m g /m L )s o l u t i o n 5.8m g /m L m o n o b a s i c N a P h o s H 2O ;1.2m g /m L d i b a s i c N a P h o s a n h y d r o u s (4m L ,16m L ﬁl l i n v i a l )60m g /m L a -T r e h a l o s e d i h y d r a t e (4m L ,16m L ﬁl l i n v i a l )0.4m g /m L P S 20(4m L ,16m L ﬁl l i n v i a l )6.22B e x x a rT o s i t u m o m a b a n d I -131T o s i t u m a b M u r i n e I g G 2l2003C o r i x a a n d G S KI V I n f u s i o nC D 20p o s i t i v e f o l l i c u l a r n o n H o d g k i n s l y m p h o m aK i t :14m g /m L M A b s o l u t i o n i n 35m g a n d 225m g v i a l s ;1.1m g /m L I 131-M A b s o l u t i o n10m M p h o s p h a t e (M A b v i a l )145m M N a C l ,10%w /v M a l t o s e ;I 131-M A b :5–6%P o v i d o n e ,1–2,9–15m g /m L M a l t o s e ,0.9m g /m L N a C l ,0.9–1.3m g /m L A s c o r b i c a c i d 7.23C a m p a t h A l e m t u z u m a bH u m a n i z e d ,I g G 1k ,150k D a2001I l e x O n c o l o g y ;M i l l e n i u m a n d B e r l e xI V i n f u s i o nB -c e l l c h r o n i c l y m p h o c y t i c l e u k e m i a ,CD 52-a n t i g e n 30m g /3m L s o l u t i o n3.5m g /3m L d i b a s i c N a P h o s ,0.6m g /3m L m o n o b a s i c K P h o s 24m g /3m L N a C l ,0.6m g /3m L K C l ,0.056m g /3m L N a 2E D T A 0.3m g /3m L P S 806.8–7.44C E A -S c a n (l y o )A c r i t u o m a b ;T c -99M u r i n e F a b ,50k D a1996I m m u n o m e d i c s I V i n j e c t i o n o r i n f u s i o nI m a g i n g a g e n t f o r c o l o r e c t a l c a n c e r1.25m g /v i a l L y o p h i l i z e d M A b .R e c o n s t i t u t e w 1m L S a l i n e w T c 99m 0.29m g /v i a l S t a n n o u s c h l o r i d e ,p o t a s s i u m s o d i u m t a r t r a t e t e t r a h y d r a t e ,N a A c e t a t e .3H 2O ,N a C l ,g l a c i a l a c e t i c a c i d ,H C l S u c r o s e5.75E r b i t u x C e t u x i m a bC h i m e r i c h u m a n /m o u s e I g G 1k ,152kD a 2004I m C l o n e a n d B M S I V i n f u s i o n T r e a t m e n t o fE GF R -e x p r e s s i n g c o l o r e c t a l c a r c i n o m a 100m g M A b i n 50m L ;2m g /m L s o l u t i o n1.88m g /m L D i b a s i c N a P h o s Á7H 2O ;0.42m g /m L M o n o b a s i c N a P h o s ÁH 2O8.48m g /m L N a C l 7.0–7.46H e r c e p t i n (l y o )T r a s t u z u m a bH u m a n i z e d I g G 1k1998G e n e t e c h I V i n f u s i o n M e t a s t a t i c b r e a s t c a n c e r w h o s e t u m o r o v e r e x p r e s s H E R 2p r o t e i n 440m g /v i a l ,21m g /m L a f t e r r e c o n s t i t u t i o n 9.9m g /20m L L -H i s t i d i n e H C l ,6.4m g /20m L L -H i s t i d i n e400m g /20m L a -T r e h a l o s e D i h y d r a t e 1.8m g /20m L P S 2067H u m i r a A d a l i m u m a bH u m a n I g G 1k ,148k D a2002C A T a n d A b b o t t S CR A p a t i e n t s n o t r e s p o n d i n g t o D M A R D s .B l o c k s T N F -a l p h a40m g /0.8m L s o l u t i o n (50m g /m L )0.69m g /0.8m L M o n o b a s i c N a P h o s Á2H 2O ;1.22m g /0.8m L D i b a s i c N a P h o s Á2H 2O ;0.24m g /0.8m L N a C i t r a t e ,1.04m g /0.8m L C i t r i c a c i d ÁH 2O 4.93m g /0.8m L N a C l ;9.6m g /0.8m L M a n n n i t o l 0.8m g /0.8m L P S 805.28L u c e n t i s R a n i b i z u m a bH u m a n i z e d I g G 1k f r a g m e n t2006G e n e n t e c h I n t r a v i t r e a l i n j e c t i o n A g e -r e l a t e d m a c u l a r d e g e n e r a t i o n (w e t )10m g /m L s o l u t i o n10m M H i s t i d i n e H C l10%a -T r e h a l o s e -D i h y d r a t e 0.01%P S 205.52WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 2007DOI 10.1002/jps9M y l o t a r g (l y o )G e m t u z u m a b o z o g a m i c i nH u m a n i z e d I g G 4k c o n j u g a t e d w i t h c a l i c h e a m i c i n2000C e l l t e c h a n d W y e t h I V i n f u s i o nH u m a n i z e d A b l i n k e d t o c a l i c h e a m i c i n f o r t r e a t m e n t o f C D 33p o s i t i v e a c u t e m y e l o i d l e u k e m i a 5m g p r o t e i n -e q u i v a l e n t l y o p h i l i z e d p o w d e r /20-m L v i a l M o n o b a s i c a n d d i b a s i c N a P h o s p h a t e D e x t r a n 40,S u c r o s e ,N a C l 10O n c o S c i n tS a t u m o m a b p e n d e t i d eM u r i n e I g G 1k c o n j u g a t e d t o G Y K -D T P A1992C y t o g e n I V i n j e c t i o nI m a g i n g a g e n t f o r c o l o r e c t a l a n d o v a r i a n c a n c e r0.5m g c o n j u g a t e /m L s o l u t i o n (2m L p e r v i a l )P h o s p h a t e b u f f e r s a l i n e 6.011O r t h o c l o n e O K TM u r o m o m a b -C D 3M u r i n e ,I g G 2a ,170k D a1986O r t h o B i o t e c h I V i n j e c t i o nR e v e r s a l o f a c u t e k i d n e y t r a n s p l a n t r e j e c t i o n (a n t i C D 3-a n t i g e n )1m g /m L s o l u t i o n2.25m g /5m L m o n o b a s i c N a P h o s ,9.0m g /5m L d i b a s i c N a P h o s 43m g /5m L N a C l 1m g /m L P S 807Æ0.512P r o s t a S c i n tI n d i u m -111c a p r o m a b p e n d e t i d e M u r i n e I g G 1k -c o n j u g a t e d t o G Y K -D T P A1996C y t o g e n I V i n j e c t i o nI m a g i n g a g e n t f o r p r o s t a t e c a n c e r0.5m g c o n j u g a t e /m L s o l u t i o n (1m L p e r v i a l )P h o s p h a t e b u f f e r s a l i n e 5–713R a p t i v a (l y o )E f a l i z u m a bH u m a n i z e d I g G 1k2003X o m a a n d G e n e n t e c h S C C h r o n i c m o d e r a t e t o s e v e r e p l a q u e p s o r i a s i s ,b i n d s t o C D 11a s u b u n i t o f L F A -1150m g M A b /v i a l ;125m g /1.25m L (100m g /m L )a f t e r r e c o n s t i t u t i o n w i t h 1.3m L S W F I 6.8m g /v i a l L -H i s t i d i n e H C l ÁH 2O ;4.3m g /v i a l L -H i s t i d i n e123.2m g /v i a l S u c r o s e 3m g /v i a l P S 206.214R e m i c a d e (l y o )I n ﬂi x i m a bC h i m e r i c h u m a n /m u r i n e M A b a g a i n s t T N F a l p h a (a p p .30%m u r i n e ,70%c o r r e s p o n d s t o h u m a n I g G 1h e a v y c h a i n a n d h u m a n k a p p a l i g h t c h a i n c o n s t a n t r e g i o n s )1998C e n t o c o r I V i n f u s i o nR A a n d C r o h n ’s d i s e a s e (a n t i T N F a l p h a )100m g /20-m L V i a l ,10m g /m L o n r e c o n s t i t u t i o n2.2m g /10m L M o n o b a s i c N a P h o s H 2O ,6.1m g /10m L D i b a s i c N a P h o s Á2H 2O 500m g /10m L S u c r o s e 0.5m g /10m L P S 807.215R e o P r o A b c i x i m a bF a b .C h i m e r i c h u m a n -m u r i n e ,48k D a 1994C e n t o c o r /L i l l y I V i n j e c t i o n a n d i n f u s i o n R e d u c t i o n o f a c u t e b l o o d c l o t r e l a t e d c o m p l i c a t i o n s 2m g /m L s o l u t i o n 0.01M N a P h o s p h a t e 0.15M N a C l 0.001%(0.01m g /m L )P S 807.216R i t u x a n R i t u x i m a bC h i m e r i c m o u s e /h u m a n I g G 1k w i t h m u r i n e l i g h t a n d h e a v y c h a i n v a r i a b l e r e g i o n (F a b d o m a i n ),145kD a1997I D E C a n d G e n e n t e c h I V i n f u s i o nN o n H o d g k i n ’s l y m p h o m a .(a n t i C D 20-a n t i g e n )10m g /m L s o l u t i o n7.35m g /m L N a C i t r a t e Á2H 2O9m g /m L N a C l 0.7m g /m L P S 806.5(C o n t i n u e d )ANTIBODY FORMULATION3DOI 10.1002/jpsJOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 200717S i m u l e c t (l y o )B a s i l i x i m a bC h i m a r i c I g G 1k ,144kD a1998N o v a r t i s I V i n j e c t i o n a n d i n f u s i o nP r e v e n t i o n o f a c u t e k i d n e y t r a n s p l a n t r e j e c t i o n ,I L -2r e c e p t o r a n t a g o n i s t10m g a n d 20m g /v i a l ,4m g /m L o n r e c o n s t i t u t i o n 3.61m g ,7.21m g M o n o b a s i c K P h o s ;0.50m g ,0.99m g N a 2H P O 40.8m g ,1.61m g N a C l ;10m g ,20m g S u c r o s e ;40m g ,80m g M a n n i t o l ;20m g 40m g G l y c i n e 18S y n a g i s (l y o )P a l i v i z u m a bH u m a n i z e d I g G 1k ,C D R o f m u r i n e M A b 1129,148k D a 1998M e d I m m u n e I M i n j e c t i o nP r e v e n t r e p l i c a t i o n o f t h e R e s p i r a t o r y s y n c y t i a l v i r u s (R S V )50m g a n d 100m g /v i a l ,100m g /m L o n r e c o n s t i t u t i o n47m M H i s t i d i n e ,3.0m M G l y c i n e 5.6%M a n n i t o l19T y s a b r i N a t a l i z u m a bH u m a i n z e d I g G 4k2004B i o g e n I D E C I V I n f u s i o nM S r e l a p s e 300m g /15m L s o l u t i o n 17.0m g M o n o b a s i c N a P h o s ÁH 2O ,7.24m g d i B a s i c N a P h o s Á7H 2O f o r 15m L 123m g /15m L N a C l3.0m g /15m L P S 806.120V e r l u m a N o f e t u m o m a b M u r i n e F a b 1996B o e h r i n g e r I n g e l h e i m a n d D u P o n t M e r c k I V i n j e c t i o n I m a g i n g a g e n t f o r l u n g c a n c e r10m g /m L s o l u t i o nP h o s p h a t e b u f f e r s a l i n e?21X o l a i r (l y o )O m a l i z u m a bH u m a n i z e d I g G 1k ,149k D aG e n e n t e c h w N o v a r t i s a n d T a n o xS CA s t h m a ,i n h i b i t s b i n d i n g o f I g E t o I g E r e c e p t o r F C e R I202.5m g /v i a l ,D e l i v e r 150m g /1.2m L o n r e c o n s t i t u t i o n w i t h 1.4m L S W F I 2.8m g L H i s t i d i n e H C l ÁH 2O ;1.8m g L H i s t i d i n e145.5m g S u c r o s e 0.5m g P S 2022Z e n a p a x D a c l i z u m a bH u m a n i z e d I g G 1,144k D a1997R o c h e I V i n f u s i o nP r o p h y l a x i s o f a c u t e o r g a n r e j e c t i o n i n p a t i e n t s r e c e i v i n g r e n a l t r a n s p l a n t s .I n h i b i t s I L -2b i n d i n g t o t h e T a c s u b u n i t o f I L -2r e c e p t o r c o m p l e x 25m g /5m L M A b S o l u t i o n3.6m g /m L M o n o b a s i c N a P h o s ÁH 2O ;11m g /m L D i b a s i c N a P h o s Á7H 2O4.6m g /m L N a C l 0.2m g /m L P S 806.923Z e v a l i nI b r i t u m o m a b -T i u x e t a nM u r i n e I g G 1k -t h i o u r e a c o v a l e n t l i n k a g e t o T i u x e t a nI D E C I V i n f u s i o nC D 20a n t i g e n .(K i t w i t h Y t t e r i u m -90i n d u c e s c e l l u l a r d a m a g e b y b e t a e m i s s i o n )3.2m g /2m L s o l u t i o n 09%N a C l 7.1T a b l e 1.(C o n t i n u e d )#B r a n d n a m e M o l e c u l eM A bY e a r C o m p a n y R o u t e I n d i c a t i o n M A b C o n c B u f f e r E x c i p i e n t s S u r f a c t a n t p H4WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 2007DOI 10.1002/jpsDevelopment of commercially viable antibody pharmaceuticals has,however,not been straight-forward.This is because the behavior of antibodies seems to vary,even though they have similar structures.In attempting to address some of the challenges in developing antibody therapeutics, Harris et al.5reviewed the commercial-scale formulation and characterization of therapeutic recombinant antibodies.In a different review, antibody production and puriﬁcation have been discussed.2Nevertheless,the overall instability and stabilization of antibody drug candidates have not been carefully examined in the litera-ture.This article,not meant to be exhaustive, intends to review the structure and functions of antibodies,discuss their instabilities,and sum-marize the methods for stabilizing/formulating antibodies.ANTIBODY STRUCTUREAntibodies(immunoglobulins)are roughly Y-shaped molecules or combination of such molecules(Fig.1). Their structures are divided into two regions—the variable(V)region(top of the Y)deﬁning antigen-binding properties and the constant(C)region (stem of the Y),interacting with effector cells and molecules.Immunoglobulins can be divided into ﬁve different classesÀIgA,IgD,IgE,IgM,and IgG based on their C regions,respectively desig-nated as a,d,e,m,and g(ﬁve main heavy-chain classes).6Most IgGs are monomers,but IgA and IgM are respectively,dimmers and pentamers linked by J chains.IgGs are the most abundant,widely used for therapeutic purposes,and their structures will be discussed as antibody examples in detail.Primary StructureThe structure of IgGs have been thoroughly reviewed.6The features of the primary structure of antibodies include heavy and light chains, glycosylation,disulﬁde bond,and heterogeneity. Heavy and Light ChainsIgGs contain two identical heavy(H,50kDa)and two identical light(L,25kDa)chains(Fig.1). Therefore,the total molecular weight is approxi-mately150kDa.There are several disulﬁde bonds linking the two heavy chains,linking the heavy and light chains,and residing inside the chains (also see next section).IgGs are further divided into several subclasses—IgG1,IgG2,IgG3,and IgG4(in order of relative abundance in human plasma),with different heavy chains,named g1, g2,g3,and g4,respectively.The structural differences among these subtypes are the number and location of interchain disulﬁde bonds and the length of the hinge region.The light chains consist of two types—lambda(l)and kappa(k). In mice,the average of k to l ratio is20:1,whereas it is2:1in humans.6The variable(V)regions of both chains cover approximately theﬁrst 110amino acids,forming the antigen-binding (Fab)regions,whereas the remaining sequences are constant(C)regions,forming Fc(fragment crystallizable)regions for effector recognition and binding.6The N-terminal sequences of both the heavy and light chains vary greatly between different antibodies.It was suggested that the conserved sequences in human IgG1antibodies Figure1.Linear(upper panel)and steric(lower panel)structures of immunoglobulins(IgG).ANTIBODY FORMULATION5DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007are approximately95%and the remaining5% is variable and creates their antigen-binding speciﬁcity.5The V regions are further divided into three hypervariable sequences(HV1,HV2,and HV3)on both H and L chains.In the light chains,these are roughly from residues28to35,from49to59,and from92to103,respectively.6Other regions are the framework regions(FR1,FR2,FR3,and FR4).The HV regions are also called the complementarity determining regions(CDR1,CDR2,and CDR3). While the framework regions form the b-sheets, the HV sequences form three loops at the outer edge of the b barrel(also see Section2.2).Disulﬁde BondsMost IgGs have four interchain disulﬁde bonds—two connecting the two H chains at the hinge region and the other two connecting the two L chains to the H chains.6Exceptions do exist.Two disulﬁde bonds were found in IgG1and IgG4 linking the two heavy chain in the hinge region but four in IgG2.7In IgG1MAb,HC is linked to the LC between theﬁfth Cys(C217)of HC and C213on the LC.In IgG2and IgG4MAbs,it is the third Cys of HC(C123)linking to the LC.7A disulﬁde bond between HC C128and LC C214 was found for mouse catalytic monoclonal anti-bodies(IgG2a).8IgGs have four intrachain disulﬁde bonds, residing in each domain of the H and L chains, stabilizing these domains.The intrachain disul-ﬁde bonds in V H and V L are required in functional antigen binding.9Native IgG MAbs should not have any free sulfhydryl groups.7However, detailed examination of the free sulfhydryl groups in recombinant MAbs(one IgG1,two IgG2,and one IgG4)suggests presence of a small portion of free sulfhydryl group(approximately0.02mol per mole of IgG2or IgG4MAb and0.03for IgG1.7In rare cases,a free cysteine is found.A nondisulﬁde-bonded Cys at residue105was found on the heavy chain of a mouse monoclonal antibody,OKT3 (IgG2a).10OligosaccharidesThere is one oligosaccharide chain in IgGs.6This N-linked biantennary sugar chain resides mostly on the conserved Asn297,which is buried between the C H2domains.5,11For example,the oligosaccharide resides on Asn-297of the C H2 domain of chimeric IgG1and IgG3molecules12but on Asn299in a monoclonal antibody,OKT3 (IgG2a).10The oligosaccharide,often microheter-ogeneous,is typically fucosylated in antibodies produced in CHO or myeloma cell lines5and may differ in other cell lines.2,11There are many factors that dictate the nature of the glycan microheterogenity on IgGs.These include cell line,the bioreactor conditions and the nature of the downstream processing.An additional oligo-saccharide can be found in rare cases.A human IgG produced by a human-human-mouse hetero-hybridoma contains an additional oligosaccharide on Asn75in the variable region of its heavy chain.13In addition,O-linked carbohydrates could also exist in this antibody.Proper glycosylation is critical for correct functioning of antibodies.11It was demonstrated that removal of the oligosaccharide in IgGs(IgG1 and IgG3)made them ineffective in binding to C1q, in binding to the human Fc g RI and activating C; and generally more sensitive to most proteases than their corresponding wild-type IgGs(one exception).12This is because the binding site on IgG for C1q,theﬁrst component of the complement cascade,is localized in the C H2domains.11 Furthermore,the glycosylation can affect the antibody conformation.12Oligosaccharides in other regions can also play a critical role.Removal of an oligosaccharide in a Fv region of the CBGA1antibody resulted in a decreased antigen-binding activity in several ELISA systems.13In addition,this oligosaccharide might play critical role in reducing the antigenicity of the protein.14The sugar composition of the oligosaccharide is also critical in antibody functions.It has been shown that a low fucose(Fuc)content in the complex-type oligosaccharide in a humanized chimeric IgG1is responsible for a50-fold higher antibody-dependent cellular cytotoxicity(ADCC) compared with a high Fuc counterpart.15 HeterogeneityPuriﬁed antibodies are heterogeneous in struc-ture.This is true for all monoclonal antibodies (MAbs)due to differences in glycosylation pat-terns,instability during production,and terminal processing.5For example,ﬁve charged isoforms were found in recombinant humanized monoclo-nal antibody HER2as found by capillary iso-electric focusing(cIEF)and sodium dodecyl sulfate–capillary gel electrophoresis(SDS–CGE).16Six separate bands were focused under6WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007DOI10.1002/jpsIEF for two mouse monoclonal antibodies IgG2a (k)and IgG1(k).17A mature monoclonal antibody, OKT3(IgG2a),contain cyclized N-terminus (pyroglutamic acid,À17D)in both H and L chains, processed C-terminus(no Lys,À128D)of the H chains,and a small amount of deamidated form.10 Similar observation was also reported for a huma-nized IgG1(k).18In rare cases,gene cross-over may lead to formation of abnormal heavy chains.For example,a puriﬁed monoclonal anti-IgE antibody contains a small amount of a variant H chain, which had16fewer amino acid residues than the normal H chain(position is between Arg108of the L chain and Ala124of the H chain).19 Secondary and Higher-Order StructureThe basic secondary and higher-order structural features of IgGs have been reviewed.6Only a small portion of the three-dimensional structures of IgGs has been solved.20The antibody’s secon-day structure is formed as the polypeptide chains form anti-parallel b-sheets.The major type of secondary structure in IgGs is these b-sheets and its content is roughly70%as measured by FTIR.21The light chain consists of two and the heavy chain contains four domains,each about 110amino acid long.6,20All these domains have similar folded structures—b barrel,also called immunoglobulin fold,which is stabilized by a disulﬁde bond and hydrophobic interaction(pri-mary).These individual domains($12kDa in size)interact with one another(V H and V L;C H1 and C L;and between two C H3domains except the carbohydrate-containing C H2domain)and fold into three equal-sized spherical shape linked by a ﬂexible hinge region.These three spheres form a Y shape(mostly)and/or a T shape.22The less globular shape of IgGs is maintained both by disulﬁde bonds and by strong noncovalent interactions between the two heavy chains and between each of the heavy-chain/light-chain pairs.23Through noncovalent interactions,a less stable domain becomes more stable,and thus,the whole molecule can be stabilized.24A detailed study indicates that the interaction between two CH3domains are dominated by six contact residues,ﬁve of these residues(T366,L368, F405,Y407,and K409)forming a patch at the center of the interface.25These noncovalent interactions are spatially oriented such that variable domain exchange(switching V H and V L; inside-out IgG;ioIgG)induces noncovalent multimerization.26The six hypervariable regions in CDR(L1,L2, L3,H1,H2,and H3)form loops of a few predictable main-chain conformations(or canonical forms), except H3loop,which has too many variations in conformation to be predicted accurately.27,28 There is a slight difference in the loop composition and shape between the two types of light chains.20 However,no functional difference was found in antibodies having l or k chain.6Basic Functions of AntibodiesThe basic functions of antibodies have been reviewed.6There are two functional areas in IgGs—the V and C regions.The V regions of the two heavy and light chains offer two identical antigen-binding sites.The binding of the two sites (bivalent)can be independent of each other and does not seem to depend on the C region.29The exact antigen-binding sites are the CDR regions with participation of the frame work regions.30 Binding of antigens seems through the induced-ﬁt mechanism.31,32The induced-ﬁt mechanism allows multispeciﬁcity and polyreactivity.It has been suggested that about5–10residues usually contribute signiﬁcantly to the binding energy.32 The C regions of antibodies have three main effector functions(1)being recognized by receptors on immune effector cells,initiating antibody-dependent cell cytotoxicities(ADCC),(2)binding to complement,helping to recruit activated pha-gocytes,and(3)being transported to a variety of places,such as tears and milk.6In addition,C domains also modulate in vivo stability.23,29,33The function of Fc is affected by the structure of Fab. Variable domain exchange(switching V H and V L; inside-out IgG;ioIgG)affected Fc-associated func-tions such as serum half-life and binding to protein G and Fc g RI.26The hinge region providesﬂexibility in bivalent antigen binding and activation of Fc effector functions.26Two chimeric IgG3antibodies lacking a genetic hinge but with Cys residues in CH2 regions was found to be deﬁcient in their inter-molecular assembly,and both IgG3D HþCys and IgG3D Hþ2Cys lost greatly their ability to bind Fc g RI and failed to bind C1q and activate the complement cascade.34Alternative Forms of AntibodiesIn addition to species-speciﬁc antibodies,other antibody forms are generated to meet various needs.In the early development of antibody therapies,antibodies were made from murineANTIBODY FORMULATION7DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007sources.However,these antibodies easily elicit formation of human anti-mouse antibody (HAMA).Therefore,humanized chimeric antibo-dies were generated.Chimeric monoclonal anti-bodies(60–70%human)are made of mouse variable regions and human constant regions.2 Such antibodies can still induce formation of human anti-chimeric antibody(HACA).Highly humanized antibodies,CDR-grafted antibodies, are made by replacing only the human CDR with mouse CDR regions(90–95%human).2These antibodies are almost the same in immunogeni-city potential as completely human antibodies, which may illicit formation of human anti-human antibody(HAHA).Other alternative forms of antibodies have also been generated and these different forms have been reviewed.35Treatment with papain would cleave the N-terminal side of the disulﬁde bonds and generate two identical Fab fragments and one Fc fragment.Fab0s are50kDa(V HþC H1)/ (V LþC L)heterodimers linked by a single disul-ﬁde bond.Treatment with pepsin cleaves the C-terminal side of the disulﬁde bonds and pro-duces a F(ab)02fragment.The remaining H chains were cut into several small fragments.6Cleavage by papain occurs at the C-terminal side of His-H22836or His-H227.37Reduction of F(ab0)2will produce two Fab0.23Fv fragments are noncovalent heterodimers of V H and V L.Stabilization of the fragment by a hydrophilicﬂexible peptide linker generates single-chain Fv(scFvs).2Fragments without constant domains can also be made into domain antibodies (dAbs).These scFvs are25–30kDa variable domain (V HþV L)dimers joined by polypeptide linkers of at least12residues.Shorter linkers(5–10residues)do not allow pairing of the variable domains but allow association with another scFv form a bivalent dimer (diabody)(about60kDa,or trimer:triabody about 90kDa).38Two diabodies can be further linked together to generate bispeciﬁc tandem diabody (tandab).39Disulﬁde-free scFv molecules are rela-tively stable and useful for intracellular applica-tions of antibodies—‘‘intrabodies.’’38The smallest of the antibody fragments is the minimal recognition unit(MRU)that can be derived from the peptide sequences of a single CDR.2ANTIBODY INSTABILITYAntibodies,like other proteins,are prone to a variety of physical and chemical degradation path-ways,although antibodies,on the average,seem to be more stable than other proteins.Antibody instabilities can be observed in liquid,frozen,and lyophilized states.The glycosylation state of an antibody can signiﬁcantly affect its degradation rate.40In many cases,multiple degradation path-ways can occur at the same time and the degrada-tion mechanism may change depending on the stress conditions.41These degradation pathways are divided into two major categories—physical and chemical instabilities.This section will explore the possible degradation pathways of antibodies and their inﬂuencing factors.Physical InstabilityAntibodies can show physical instability via two major pathways—denaturation and aggregation. DenaturationAntibodies can denature under a variety of conditions.These conditions include temperature change,shear,and various processing steps. Compared with other proteins,antibodies seem to be more resistant to thermal stress.They may not melt completely until temperature is raised above708C,21,42,43while most other mesophilic proteins seem to melt below708C.44Shear may cause antibody denaturation.For example,the antigen-binding activity of a recombinant scFv antibody fragment was reduced with aﬁrst-order rate constant of0.83/h in a buffer solution at a shear of approximately20,000/s.45Lyophilization can denature a protein to var-ious extents.An anti-idiotypic antibody(MMA 383)in a formulation containing mannitol,sac-charose,NaCl,and phosphate was found to loose its in vivo immunogenic properties(only10–20% of normal response rate)upon lyophilization.46 Since the protein showed no evidence of degrada-tion after lyophilization,no change in secondary structure by CD(29%b-sheet,14%a-helix,and 57%‘‘other’’),the loss of activity was attributed to the conformational change.Indeed,tryptophan ﬂuorescence properties were different between the lyophilized and unlyophilized antibodies.46 AggregationAntibody aggregation is a more common manifes-tation of physical instability.The concentration-dependent antibody aggregation was considered the greatest challenge to developing protein formulations at higher concentrations.47This is8WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007DOI10.1002/jps。

抗菌肽鲎素的研究进展

抗菌肽鲎素的研究进展李寒梅;唐勇军;王顺启;代建国【摘要】已报道的抗菌肽鲎素 (tachyplesins, TPs) 共分5种, 均由鲎血细胞分离产生, 是鲎先天免疫系统的重要功能物质, 为鲎机体抵抗病原物入侵提供天然防御屏障.近30年的研究表明, TPs抗菌肽显示出广谱生物活性、较低正常细胞毒性、不易产生微生物抗性等特点, 作为一种全新型的肽类药物备受关注.文中主要从TPs 的分子结构, 对微生物、藻类细胞、肿瘤细胞及一些病毒的抑杀活性, 作用机制, 以及工程表达情况等方面进行介绍, 综述TPs的应用前景, 并对当前研究瓶颈进行评述.%Antibacterial peptides tachyplesins (TPs), isolated from the hemocytes of horseshoe crabs and divid-ed into five kinds, play an important role in immunological function in the limulus immune system and pro-vide a natural defense barrier for the invasion of pathogens. Nearly 30 years of research shows that TPs ex-hibit broad-spectrum bioactivities, low cytotoxicity toward normal cells, and low levels of acquired resistance in microbes. As an entirely new type of peptide drug, TPs have become an intensely studied topic. Herein, the molecular structure of TPs, bioactivity against all kinds of microorganisms and tumor cells, action mech-anisms and engineering expression are introduced. The application and the bottleneck of current research of TPs are also described.【期刊名称】《生命科学研究》【年(卷),期】2018(022)004【总页数】7页(P338-344)【关键词】抗菌肽鲎素(TPs);生物活性;作用机制;基因工程;应用;瓶颈【作者】李寒梅;唐勇军;王顺启;代建国【作者单位】南昌大学生命科学学院, 中国江西南昌 330031;深圳职业技术学院应用化学与生物技术学院,中国广东深圳 518000;深圳职业技术学院应用化学与生物技术学院,中国广东深圳 518000;南昌大学生命科学学院, 中国江西南昌330031;深圳职业技术学院应用化学与生物技术学院,中国广东深圳 518000【正文语种】中文【中图分类】Q74鲎又称马蹄鲎(horseshoe crab),是一种非常古老的海洋动物,其血液为蓝色,出现在古生代寒武纪,且近4亿年来形态未发生变化,具有“生物活化石”的称号[1]。

培训学习资料-ICH指导原则2022年学习资料_

一、Quality guidelines-质量研究指导原则-Harmonisation achieveme ts in the Quality area-include pivotal milestones suc as the conduct-of stability studies,defining relevan thresholds-for impurities testing and a more flexibl -approach to pharmaceutical quality based on-Good Man facturing PracticeGMPrisk-management.
说明-ICH的论题主要分为四类，因此CH根据论题的类别不同而进行相应的编-码分类：-1.“Q”类论题：Q代 QUALITY,指那些与化工和医药，质量保证方面-的相关的论题。-2.“S”类论题：S代表SAFETY,指些与实验室和动物实验，临床前研-究方面的相关的论题。-3.“E”类论题：E代表EFFICACY,指那些与人临床研究相关的课题。-4.“M”类论题：M代表MU儿TIDISCIPLINARY,指那些不可单独划入以上个分类的交叉涉及的论题。同时M又细分为5个小类-M1:常用医学名词MedDRA-M2:药政信息传递之电子标 -M3:与临床试验相关的临床前研究时间的安排-M4:常规技术文件CTD-M5:药物词典的数据要素和标准
Q1A-Q1 F Stability稳定性-Q1AR2Stability Testing of New D ug Substances and Products-新原料药和制剂的稳定性试验-Q1B Stabilit Testing Photostability Testing of New Drug Substance -新原料药和制剂的光稳定性试验-Q1 C Stability Testing for New Dosage Forms新剂型的稳定性试验-Q1D Bracketing and Matrixing Designs f r Stability Testing of New-原料药和制剂稳定性试验的交叉和矩阵设计-Q1 E E aluation of Stability Data稳定性数据的评估-Q1F Stability Data Package for Registration Applications in Climatic-Zon s lll and IV-在气候带川和V，药物注册申请所提供的稳定性数据

TAT肽—阿霉素磁性脂质体的制备及初步实验研究

2013届硕士研究生学位论文TAT肽-阿霉素磁性脂质体的制备及初步实验研究PREPARATION AND INITIAL INVESTIGATION OFTAT-DOXORUBICIN MAGNETIC LIPOSOMES学科专业药剂学研究方向纳米药物导师徐维平教授研究生黄向华论文完成单位安徽中医药大学2013年5月•合肥目录中文摘要 (1)Abstract (2)英文缩略词表 (4)前言 (6)第一章阿霉素脂质体体外分析方法的建立 (11)1.仪器与试药 (11)1.1仪器 (11)1.2试药 (11)2.方法与结果 (12)2.1含量测定方法学的建立 (12)2.1.1测定波长的确定 (12)2.1.2标准曲线的绘制 (12)2.1.3精密度试验 (13)2.1.4稳定性试验 (14)2.1.5回收率试验 (14)2.2包封率检测方法的建立 (15)2.2.1阿霉素脂质体制备预实验 (16)2.2.2总药量测定方法 (16)2.2.3游离药物测定方法 (16)3. 讨论与小结 (18)3.1讨论 (18)3.2小结 (18)第二章磁性阿霉素脂质体制备方法筛选及制备工艺的研究 (19)1.仪器与试药 (19)1.1仪器 (19)1.2试药 (19)2 实验方法与结果 (20)2.1 阿霉素脂质体制备处方和制备工艺筛选 (20)2.1.1脂质体制备方法的选择 (20)2.1.2 空白脂质体制备条件的优化 (21)2.1.3单因素考察脂质体的处方和工艺 (23)2.1.4 正交设计优化脂质体处方和工艺 (24)2.2磁性阿霉素脂质体的研制 (27)2.2.1 Fe3O4的制备 (27)2.2.2 TAT肽修饰磷脂 (28)2.2.3 TAT-阿霉素脂质体和TAT-Fe3O4阿霉素脂质体的制备 (28)3 讨论与小结 (29)3.1 讨论 (29)3.2小结 (31)第三章脂质体的理化性质和体外实验 (32)1 仪器与材料 (32)1.1仪器 (32)1.2试药 (33)1.3细胞株 (33)2 方法与结果 (34)2.1 脂质体理化性质的研究 (34)2.1.1形态观察 (34)2.1.2 粒径分布与zeta电位的测定 (34)2.1.3 包封率和含量的测定 (35)2.1.4 脂质体的稳定性 (37)2.2脂质体体外释放实验 (37)2.2.1阿霉素在PBS溶液中分析方法的建立 (37)2.2.2 样品释放度测定 (40)2.3脂质体体外细胞实验 (41)2.3.1细胞培养 (42)2.3.2 MTT实验方法 (43)2.3.3 细胞摄取实验 (43)2.3.4 细胞毒性实验 (44)2.3.5体外细胞抑制率实验 (45)3 讨论与小结 (47)3.1讨论 (47)3.2 小结 (48)第四章阿霉素脂质体在小鼠体内穿透血脑屏障研究 (49)1.材料与仪器 (49)1.1实验动物与材料 (49)1.2仪器设备 (49)2.实验方法与结果 (50)2.1实验动物分组 (50)2.2动物实验方法 (50)2.2.1游离阿霉素注射液的配制 (50)2.2.2阿霉素脂质体注射液的配制 (50)2.2.3 尾静脉注射剂量的确定 (50)2.2.4给药 (51)2.2.5组织样本收集 (51)2.3高效液相检测方法的建立 (51)2.3.1色谱条件 (51)2.3.2生物组织样品处理 (51)2.3.3方法专属性 (52)2.3.4脑组织标准曲线的建立 (52)2.3.5回收率的测定 (53)2.3.6精密度试验 (54)2.3.7稳定性试验 (55)2.4高效液相法测定组织中阿霉素含量 (55)2.5脑组织中铁含量的测定 (57)2.5.1组织样品处理 (57)2.5.2含量测定 (57)3讨论与小结 (57)3.1讨论 (57)3.2小结 (59)全文总结 (60)参考文献 (61)综述：脑靶向脂质体的研究进展 (66)作者简介 (75)攻读学位期间发表的学术论文目录 (76)致谢 (77)中文摘要目的：血脑屏障的存在使得大部分药物无法进入大脑，从而加大了中枢系统疾病的治疗难度。

ScienceAdvances：在类风湿性关节炎中，肠道丁酸代谢物有助于自身抗体的产生

ScienceAdvances：在类风湿性关节炎中，肠道丁酸代谢物有助于自身抗体的产生类风湿关节炎（RA）是一种病因未明的慢性、以炎性滑膜炎为主的系统性疾病。

其特征是手、足小关节的多关节、对称性、侵袭性关节炎症，经常伴有关节外器官受累及血清类风湿因子阳性，可以导致关节畸形及功能丧失。

临床上，ACPAs（抗环瓜氨酸肽抗体）的存在表明软骨和骨骼严重受损的可能性增加，以及预后较差的强烈关节变形。

对疾病病理生理学的见解强调，各种炎症途径可导致RA中异常的全身免疫反应。

有人提出，失调的免疫反应的起源发生在具有环境重大贡献的遗传易感个体中。

在环境因素中，肠道微生物群已成为RA异常全身免疫反应的可能候选者。

来自Science Advances的名为Intestinal butyrate-metabolizing species contribute to autoantibodyproduction and bone erosion in rheumatoid arthritis的文章通过使用准配对队列策略，揭示了丁酸菌在RA发病机制中的关键作用，并表明基于丁酸菌的治疗在RA中具有潜力。

研究在比较25名未接受治疗的RA患者与29名年龄和性别匹配的健康个体时，发现两组在微生物多样性方面没有显着差异。

两组之间所有细菌物种的单方差比较没有发现任何显着偏离的物种。

研究表示处理这些高维数据集是非常困难的，因为众多的辅助因子是难以控制的。

在这项研究中，使用了一种新的准配对队列策略，该策略基于一个小型队列，有25个未接受治疗的患者和29个配对对照组。

简而言之，研究重建了一个准配对队列，其中具有相似物种特征但表型相反的样本是孪生的，然后通过配对样本的统计测试鉴定具有RA相关特征的物种。

因此，发现了186个与RA相关的物种，其中149个代表性过高，37个缺乏RA。

出乎意料的是，这些物种中有相当大比例，186个物种中的55个（29.6%）。

硒盐胁迫对拟南芥耐硒突变体和野生型抗氧化酶类活性的影响

硒盐胁迫对拟南芥耐硒突变体和野生型抗氧化酶类活性的影响许晖;李亚男【摘要】The effects of selenium salt stress on the activities of antioxidant enzymes in selenium -tolerant mutant 58(5) and wild type WT of Arabidopsis thaliana were researched .It was found that:under the stress of high concentration of selenium salt , the activities of GSH-Px, SOD and POD in the plants of Arabidopsis thaliana all increased , and the activities of these antioxidant en-zymes in 58(5) were obviously higher than those in WT .The above results indicated that selenium -tolerant mutant 58(5) of Ara-bidopsis thaliana had stronger tolerance to selenium .%研究了硒盐胁迫对拟南芥耐硒突变体和野生型抗氧化酶类活性的影响，发现在高浓度硒胁迫下，植物体内的谷胱甘肽过氧化物酶（GSH－Px）、超氧化物歧化酶（SOD）、过氧化物酶（POD）的活性均升高，而且拟南芥耐硒突变体58（5）中这几种酶的活性均明显高于野生型拟南芥WT中的。

说明突变体58（5）具有较强的耐硒特性。

【期刊名称】《江西农业学报》【年(卷),期】2014(000)012【总页数】3页(P63-65)【关键词】硒盐胁迫;拟南芥;耐硒突变体;抗氧化酶;活性【作者】许晖;李亚男【作者单位】湖北省荆州农业科学院，湖北荆州 434010;长江大学农学院，湖北荆州434023【正文语种】中文【中图分类】Q945.78当植物受辐射、干旱、低温、病虫害等逆境伤害时,植物体内会产生大量的自由基,这些自由基可被谷胱甘肽过氧化物酶(GSH-Px)、过氧化物酶(POD)、多酚氧化酶(PPO)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)等抗氧化酶系统所清除,因而,增强植物的抗氧化能力能够提高植物对环境胁迫的抗性[1]。

1-s2.0-S0196978112003671-main[1]

Peptides38(2012)13–21Contents lists available at SciVerse ScienceDirectPeptidesj o u r n a l h o m e p a g e:w w w.e l s e v i e r.c o m/l o c a t e/p e p t i d esand characterization of novel antioxidant peptides from enzymatic hydrolysates of tilapia(Oreochromis niloticus)skin gelatinYufengZhang,Xiu Duan,Yongliang Zhuang∗1.IntroductionReactive oxygen species(ROS)and free radicals are very unsta-ble and react rapidly with other groups or substances in the body, leading to cell or tissue injury[18,32].Under normal conditions, ROS is effectively eliminated by the antioxidant defense system, such as antioxidant enzymes and non-enzymatic factors.However, under pathological conditions,the balance between the gener-ation and the elimination of ROS is broken.ROS could modify DNA,proteins,and small cellular molecules,and play a signiﬁcant role in the occurrence of diseases,such as cardiovascular diseases, diabetes mellitus,neurological disorders,and even Alzheimer’s dis-ease[17,29].Therefore,it is important to inhibit the oxidation and formation of ROS and free radicals occurring in the living body[10].Synthetic antioxidants like butylated hydroxytoluene (BHT)and butylated hydroxyanisole(BHA)are generally used for radical scavenging in biological systems,but these antioxidants pose potential risks to human health,and their use as food addi-tives is restricted[3,13].Thus,more studies focused on natural antioxidants,such as tocopherols,catechin,phenolic compounds and peptides[36].Recently,many peptides,such as corn peptides,∗Corresponding author.E-mail address:kmylzhuang@(Y.Zhuang).soybean peptides and chickpea peptides and so on,are reported to possess antioxidant activities against ROS and free radicals[2,37]. Moreover,several peptides derived fromﬁsh skin gelatin have also shown potent antioxidant activities in different oxidative systems and proven to act as potent antioxidants[7,34].Tilapia(Oreochromis niloticus)is an important specie in fresh-water aquaculture.It is the third most widely culturedﬁsh,after carp and salmonids[6].In the past years,the production of tilapia has increased steadily and has become one of the leading export-ing aquatic products.The increase of processing means that more skins and other wastes are produced.It has been reported that 70%of the dry matter ofﬁsh skin is collagen.When heated above 40◦C,collagen is converted into gelatin[38].Therefore,the tilapia skin is a good resource for production of gelatin,which is expected to prepare nature protein hydrolysates with high ROS scavenging activities[21].Therefore,in this paper,the enzymatic condition of tilapia skin gelatin(TSG)was optimized by orthogonal experiment.The hydrolysates of TSG were chosen as a potential antioxidant pep-tide resource,and peptides with high hydroxyl radical scavenging activity were separated using gelﬁltration chromatography,ion exchange chromatography,and RP-HPLC.Furthermore,the amino acid sequences of the antioxidant peptides were identiﬁed using nano-LC-ESI mass spectrometry,which were important,especially when a therapeutic effect was expected.0196-9781/$–see front matter©2012Elsevier Inc.All rights reserved. /10.1016/j.peptides.2012.08.01414Y.Zhang et al./Peptides 38(2012)13–21Table 1The range analysis of properase E on DH obtained from the L 9(43)orthogonal experiment.No.(A)E /S (%)(B)Hydrolysis temperature (◦C)(C)pH(D)Hydrolysis time (h)DH (%)1111112.46±0.262122215.63±1.413133315.67±0.364212316.23±0.415223113.74±0.226231214.89±0.687313216.39±0.858321315.68±0.229332116.17±0.14K 114.58715.02714.34314.123K 214.95315.01716.01015.637K 316.08015.57715.26715.860Best level A 3B 3C 2D 3R a1.4930.5601.6671.737R orderD >C >A >BaRefers to the result of extreme analysis.2.Material and methods2.1.Materials and reagentsThe tilapia skin was provided by New Ocean Food(Kunming,China).Multifect neutraland properaseE chasedfrom GenencorInternational Co.,China.SP Sephadex C-25and Sephadex G-15were purchased from GE Healthcare.Acetonitrile (HPLC grade)was purchased from Merck KGaA (Darmstadt,Germany).Other chemicals and reagents used were of analytical grade commercially available.skin gelatin from skin was rinsed solution (1:8,with water to pH 7,and then ric acid solution (1:8,w/v)for 30with water to pH 7.Finally,warring type blender (DS-1,BIAO,China)and extracted with dis-tilled water (1:10,w/v)for 8h at 60◦C with continuous stirring.The resulting viscous solution was clariﬁed by centrifugation at 5200×g for 20min (BR4i,Jouan,France)at room temperature and then lyophilized using freeze drying equipment (Alpha1-2,Christ,Germany).the enzymatic hydrolysis conditions of two enzymestilapia skin gelatin hydrolysates,enzymatic hydrol-performed using two different enzymes (properase E and neutral).An orthogonal L 9(43)test design was used the optimal hydrolysis condition of each enzyme.Four controllable variables,including enzyme-to-substrate ratio (E /S ),hydrolysis temperature (T ),pH and hydrolysis time (t )were selected for optimization.The selected variables and their levels were listed in Tables 1and 2.All hydrolysis assays were done in hydrolysis and preparation of tilapia skinhydrolysates the conditions of two were hydrolysis was applied to Table 3,the double-hydrolysis includes the progressive and mixed hydrolysis selected enzymes.The progressive hydrolysis is a singleenzyme hydrolysis at its optimum,following the other hydrolysis at its optimum;the mixed hydrolysis is a double-enzyme hydrolysis at either optimum [40].After the hydrolysates in boiling water for 10min and centrifuged at 3000min.was collected to measure their activities.Table 2The range No.(A)E /S (%)(B)Hydrolysis temperature (◦C)(C)pH(D)Hydrolysis time (h)DH (%)111118.51±0.67212229.74±0.413133310.52±0.464212310.71±0.05522319.87±0.52623129.16±0.757313210.34±0.56832139.87±0.07933219.66±0.25K 19.5909.8539.1809.342K 29.9139.82710.0379.747K 39.9579.78010.24310.367Best level A 3B 1C 3D 3R a0.3670.0731.0631.020R orderC >D >A >BaRefers to the result of extreme analysis.Y.Zhang et al./Peptides 3815Table 3Thedegreeof hydrolysis and hydroxyl radical scavenging activity ofthe hydrolysatesfrom different enzymatictreatments.EnzymeDH(%)Hydroxyl radicalscavenging ability(%)Properase E18.01±0.3562.47±4.15Multifect neutral 12.60±0.4852.21±2.05M 119.65±0.6858.05±2.56M 215.24±0.0351.01±1.82P 120.61±0.1060.38±0.06P 222.11±0.1999.24±0.40M 1was hydrolyzedwith the mixtureof properase E and multifect neutral at the optimum of Properase E,M 2was hydrolyzed with the mixture of properase E and multifect neutral at the optimum of multifect neutral,P 1was hydrolyzed with prop-erase E at its optimum then multifect neutral at its optimum,P 2was hydrolyzed with multifect neutral at its optimum then properase E at its optimum.2.5.of the degree of Degree of hydrolysis (DH)was evaluated according method [8,16].Each triplicate.DH was deﬁned as:DH (%)=h (mmol/g)h tot (mmol/g)×where h is the number of broken h tot is the total number of peptide bonds per unit weight.The h tot for TSG was 8.41mmol per gram protein.2.6.DPPH free-radical scavenging The DPPH free radical vious method with some solution was added to 2.0mL of mixture was left in dark for 30absorbance of the resulting experiments were carried out in DPPH radical was expressed as Scavenging activity (%)=A control −A sampleA control×100(1)where A control was the absorbance of the control without sampleand A sample was the absorbance with sample.The IC 50value wasthat is required toSuperoxide according to .The reaction mixture,mL 4.5mL mixture of 50mM 8.2)was incubated at 25◦C for 10min.Then (3mM,prepared by 10mM HCl)was added to the reaction mixture immediately and measured at 320nm every 30s in 5min.All samples were run in triplicate and each sample and control.The superoxide activity was calculated using the following Scavenging activity (%)=S control −S samplecontrol×100(2)where S control was the slope of absorbance variation of the con-trol and S sample was the slope of absorbance variation of sample.2.8.scavenging activity assayThe hydroxyl radical scavenging activity skin hydrolysates (TSGH)was assayed by with modiﬁcations [8].The reaction mixture,mL of was incubated with 0.3mL of FeSO 4(8mM),1mL of (3mM)and 0.25mL of H 2O 2(20mM)at 37◦C for 30min.The reac-tion was cooled by ﬂowing water to room temperature.Then adding 0.45mL distilled water into the mixture to make the end be 3.0mL and centrifuging at 3000×g for 10min.The of the supernatant was measured at 510nm,and 1mL of solution was used instead of TSGH solution as a control.bility of scavenging the hydroxyl radical was calculated to Eq.(1).The TSGH above in dis-tilled water G-25gel ﬁltration column cm)distilled water at 0.5mL min.was then the same solution and mon-nm.The fraction showing the highest antioxidant collected and concentrated.This fraction was thencationic exchange column (Ф2.6cm ×50cm)with aC-25equilibrated with 20mM sodium acetate buffer (pH 4.0).The column was washed with the same buffer and eluted with a linear gradient of NaCl concentrations from 0to 1.0M ata ﬂow rate of 0.8mL min −1and monitored at 220nm.The frac-the highest antioxidant activity was concentrated on Sephadex G-15eluted with distilled water at a rate of 0.5mL min −1and monitored at 220nm.The frac-the highest antioxidant activity was further high performance liquid chromatography on a Zorbax semi-preparative C18(Ф9.4mm ×250(Agient Technologies,USA),using a linear gradient of containing 0.1%TFA (5–30%,in 30min)at a ﬂow rate min −1.The fractions showing the high antioxidant activi-rechromatographed on the Zorbax semi-preparative C18(Ф9.4mm ×250mm)column (Agient Technologies,USA)at a ﬂow rate of 2.0mL min −1with a linear gradient of acetonitrile contain-ing 0.1%TFA (5–20%,in 30min).Finally,the fractions showing high antioxidant activities were measured on the Zorbax analysis C18(Ф4.6mm ×250mm)column (Agient Technologies,USA)at a ﬂow rate of 1.0mL min −1with a linear gradient of acetonitrile contain-ing 0.1%TFA (5–25%,in 20procedures were repeated until enough for the activity assay 2.10.Molecular mass and amino acid sequence of mass and amino acid sequence of puriﬁed peptides were determined using a Q-TOF mass eter (Micromass,Altrincham,UK)coupled with an electrospray All results were expressed as means ±and analyzed by the SPSS 11.5statistical software.Data were analyzed using one-way analysis of variance (ANOVA).P <0.05indicated sta-tistical signiﬁcance.16Y.Zhang et al./Peptides 38(2012)13–213.Results and discussion3.1.Optimization of hydrolysis conditions for two different enzymesIt is well known that various parameters,such as enzyme-to-substrate ratio (E /S ),hydrolysis temperature (T ),pH and hydrolysis time (t ),signiﬁcantly affect the degree of hydrolysis (DH)of proteinbioactivitiesof hydrolysates.Orthogonal testwas drawnthe inﬂuence of different hydrolysis factors on DH of thehydrolysates of TSG.Theexperimental designand resultswere shown in Tables 1and 2.In view of orthogonal analysis,we adopt statistical software to calculate the values of a in Tables 1and 2,the range analysis tial extent of the four factors to DH for neutral were:D (t )>C (pH)>A (E /S )>B (T )and C (pH)>D (t )>A (E /S )>B (T ).So the maximum DH of TSG could be obtained when the hydrolysis conditions for properase E and multifect neutral were D 3C 2B 3A 3(4.5h,pH 9.0,E /S 5%and 55◦C)and C 3D 3A 3B 1(pH 8.0,E /S 5%and 35◦C),ing the optimal results,we the DH of two enzymatic processes were 18.01%and 12.60%(Table 3),respectively.The DH and hydroxyl radical scav-enging activity of hydrolysates of properase E was higher than that of multifect neutral.3.2.Double-enzyme hydrolysis and preparation of tilapia skingelatin hydrolysates (TSGH)The enzymatic conditions of TSG for properase E and multifectneutral were optimized by orthogonal experiment,and double-enzyme hydrolysis of TSG was further applied.As shown in Table 3,P 2had the highest DH and the hydroxyl radical scavenging ability hydrolysis.Previous studies showedinﬂuence the resulting hydrolysates,since DH length as well as the of products obtained [1,31].DH showed stronger antioxidant some previous studies that low ﬁsh protein hydrolysates made a dant activity [20,31].So P 2was gelatin hydrolysates (TSGH),and then ing equipment.3.3.Antioxidant activities of tilapia skin (TSGH)In order to evaluate the values on scavenging cal,superoxide anion radical and hydroxyl radical activities were determined,compared with reduced glutathione (GSH).DPPH is a relatively stable organic radical and is widely used as a substrate to evaluate the efﬁcacy of antioxidants.DPPH •-scavenging activity of TSGH was evaluated and compared with GSH.As shown in Fig.1a,the DPPH-scavenging activity of TSGH increased with increasing concentration used.The IC 50values of TSGH and GSH were 3.66and 0.69mg mL −1,respectively.You et al.[36]reported the IC 50value of DPPH ·-scavenging activity of loach protein hydrolysate was 2.64mg mL −1.It was similar to our result.Superoxide anion radical cannot directly initiate lipid oxidation,but it is potential precursors of stronger reactive oxidative species such as hydroxyl radical,so it is signiﬁcant to scavenge this rad-ical.Fig.1b showed superoxide anion radical-scavenging activity of TSGH as function of concentration used,and the IC 50values of TSGH and GSH were 0.56and 0.27mg mL −1,respectively.The hydroxyl radical scavenging activity of TSGH was shown in Fig.1c.00.01.02.03.04.05.0Concentration (mg.mL -1)S c a v e n g i n g e f f e c t o f D P P H (%)0204060801000.0 1.0 2.03.04.0Concentration (mg.mL -1)S c a v e n g i n g e f f e c t o f •O 2 (%)0.00.51.01.52.0Concentration (mg.mL -1)abFig.1.Scavenging reactive oxygen effect of TSGH as function of concentrations used,(a)DPPH,(b)•O 2and (c)•OH.GSH:reduced standard from The scavenging activity was also dependent on the concentration used.TSGH showed high hydroxyl radical scavenging activity with the IC 50value being 0.74mg mL −1.This indicated that TSGH might contain peptides which are more easily accessible to the hydroxyl radicals and allows these peptides to trap the radicals more easily.Hydroxyl radical is the most reactive radical and can be formed from superoxide anion and hydrogen peroxide in the presence of metal ions,such as copper or iron.The hydroxyl radical has beenY.Zhang et al./Peptides 38(2012)13–21170.000.200.400.600.801.00306090Tube numberA b s o r b a n c e (220 n m ),24681012A B C D EFractionI C 50 v a l u e /m g ·m L -1abFig.2.Sephadex G-25gel chromatography (a)and IC 50value (mg mL −1)of each fraction (b).Elution was performed at ﬂow rate of 0.5mL min with distilled water and monitored at 220nm.0.000.050.100.150.200.250510152025303540Tube numberA b s o r b a n c e (220 n m ),200400600800C1C2C3FractionsI C 50 v a l u e /μg .m L-1abFig.3.Fraction C was further separated by SP Sephadex C-25gel chromatography (a),and IC 50value (mg mL −1)of each fraction was measured by hydroxyl radical scavenging activity (b).Elution was performed at ﬂow rate of 0.8mL min with 20mmol/L sodium acetate buffer (pH 4.0)with a linear gradient of NaCl concentrations from 0to 1.0mol/L and monitored at 220nm.to be highly damagingspecies in freeradical pathol-attacking almost every molecule in living cells.Therefore,the of hydroxyl radical is probably one of the most effective defense of a living body against various diseases.Base on the rea-son,the scavenging hydroxyl radical activity was selected as the peptides in the study.3.4.peptidesThe water and loaded onto a Sephadex G-25gel ﬁltration column (1.6cm ×80cm),which had been previously equilibrated with distilled water.Five fractions,named A–E,were collected separately (Fig.2a).Each fraction was0.000.200.400.600.801.00102030405060Tube numberA b s o r b a n c e (220 n m ),C3a C3b C3c C3dFractionsI C 50 v a l u e /μg .m L -1abFig.4.Fraction C 3was separated by Sephadex G-15column (a),and IC 50value (mg mL −1)of each fraction was measured by hydroxyl radical scavenging ability (b).Elution was performed at ﬂow rate of 0.5mL min with distilled water and monitored at 220nm.18Y.Zhang et al./Peptides 38(2012)13–21pooled,concentrated,and measured their hydroxyl radical scav-enging activities.As shown in Fig.2b,fraction C had the highest antioxidant ability with the IC 50value being 0.38mg mL −1.SP Sephadex C-25(main functional group:sulfopropyl)was one of the strong cation exchangers and it was widely utilized for separating bioactive peptides.So,the fraction C obtained from Sep-ahadex G-25was further separated by SP Sephadex C-25column into three fractions (C 1,C 2and C 3)and their antioxidant activities were also measured (Fig.3).Obviously,comparing with other two fractions,fraction C 3enging activitywith IC 50wasselected for the further In order to remove salt 15column was used.The and measured for their hydroxyl radical scavenging abilities(Fig.4).Obviously,the antioxidant activity of the fraction C 3c wassigniﬁcantly higher than IC 50110.80␮g mL −1(Table 4).The fraction C 3c was preparative C18column using a linear gradient of (5–30%in 30min)in Fig.5,15Fig.5.separated by semi-preparing RP-HPLC.Elution wasperformed with the linear gradient of acetonitrile (5–30%in 30min)containing 0.1%TFA at a ﬂow rate of 2mL min and monitored at 220nm.Numbers 1–15represented the elution peaks of C 3c1–C 3c15.Fig.6.Chromatography of C 3c1and C 3c14separated by semi-preparing RP-HPLC.Elution was performed with the linear gradient (5–20%in 30min)containing0.1%TFA and monitored at 220nm.Then they analytical column into C 3c1-P and C 3c14-P .Elution was performed with the linear gradient of acetonitrile (5–25%in 20min)Y.Zhang et al./Peptides 38(2012)13–2119WT-8-318.33100WT-13-646.21Fig.7.Identiﬁcation of molecular mass and amino acid sequences of the puriﬁed peptides (C 3c1-P and C 3c14-P )by Nano-LC-ESI-Q-TOF MS/MS.designated as C 3c1–C 3c15,were collected separately.Each fraction was pooled and concentrated.After measuring the hydroxyl radi-cal scavenging of the 15fractions,we found that C 3c1and C 3c14had higher antioxidant activities than that of others at the same con-centration.The IC 50values of C 3c1and C 3c14were 6.98␮g mL −1and 8.71␮g mL −1(Table 4),respectively.Then C 3c1and C 3c14were further puriﬁed on the same semi-preparative C18column with a different linear gradient of acetonitrile (5–20%in 30min)containing 0.1%TFA (Fig.6).This step was repeated for several times and collecting the same peak.Finally,they were further puriﬁed in an RP-HPLC analytical column to conﬁrm their purity and the chromatogram was shown in Fig.6.The two fractions with one single peak,named C 3c1-P and C 3c14-P ,were obtained and their IC 50values of hydroxyl radical scavenging activities were 4.61and 6.45␮g mL −1,respectively.3.5.Characterization of puriﬁed peptidesAntioxidant activity of peptides is remarkably dependent on molecular weight,amino acid composition and their sequencesTable 4Puriﬁcation of C 3c1-P and C 3c14-P from TSGH and their IC 50values.FractionStepIC 50(␮g mL −1)TSGH –1084.65±7.60C Sephadex G-25380.13±1.66C 3SP Sephadex C-25135.95±4.27C 3c Sephadex G-15110.80±19.80C 3c-1RP-HPLC semi-preparative column6.98±0.22C 3c-148.71±0.26C 3c1-P RP-HPLC analytical column4.61±0.15C 3c14-P6.45±0.1920Y.Zhang et al./Peptides 38(2012)13–21[1,28].The molecular mass and amino acid sequences of C 3c1-P and C 3c14-P were determined using nano-LC-ESI mass spectrometry (Fig.7).The molecular weights of the two peptides were 317.33Da and 645.21Da,and they were composed of three and ﬁve aminoacids with the sequencesrespectively.It wasreported thatantioxidantthe 20amino acid residues perlar weight,the higher their chance to exert biological effects [25].The size of two peptides was similarto the peptides isolated from horse mackerel,loach and sardinella(518.5,464.2Da and 263Da)[4,26,35],but smaller than the pep-tides,which isolated from the protein hydrolysates of alaska pollakconger eel (928Da)and hoki (1801Da)[15,23,32].been reported that hydrophobic amino acids have a signiﬁ-on radical scavenging [22,24].For peptides,high contentamino acids could increase their antioxidant activ-ity.Similar result was also reported by Ranathunga et al.[23],which reported hydrophobic amino acid residues,like Leu,couldincrease the presence of the peptides at the water–lipid interface and access to scavenge free radicals generated at the the present study,C 3c1-P was found to havethe high activity with IC 50value of 4.61␮g mL −1,this Leu at C-terminal.Furthermore,the amino acid peptide might play an important role in its,and Gly-Leu and Gly-Pro sequence wereimportant role in [5,21].It to C 3c1-P had the Gly-Leu.3c14-P without hydrophobic amino had a high hydroxyl radicalscavenging activity.Apart from its agreement with the reportedantioxidant peptide size,this could be mainly explained by the existence of Tyr at C-terminal and N-terminal.Some peptides con-taining aromatic amino acid residues (Trp or Tyr)showed strongantioxidative effects [14,33].The antioxidant activity of Tyr may due to the special capability of phenolic groups to serve as hydrogen donors,which is one mechanism of inhibiting the radical-mediatedperoxidizing chain reaction [12,27].Guo et al.studied the pep-tides (Arg-Tyr,Lys-Tyr,Tyr-Tyr and Tyr-Asp-Tyr)containing Tyr residues of royal jelly protein had strong hydroxyl-radical andhydrogen-peroxide scavenging activity [9].It was similar to ourresults.Moreover,hydroxyl radical could be formed from super-oxide anion and hydrogen peroxide in the presence of transitionmetal ions,such as Fe 2+and inhibit the formation of the amino acid residues,like Glu sequence of C 3c14-P ,which had ions through their charged activity of metal ions [19,39].4.ConclusionsIn this study,the enzymatic conditions of properase E and mul-tifect neutral for tilapia skin gelatin (TSG)were optimized.The optimum hydrolysis conditions for properase E and multifect neu-tral were 4.5h,pH 9.0,E /S 5%,55◦C and pH8.0,4.5h,E /S 5%,35◦C,respectively.The hydrolystate obtain by progressive hydrolysis with multifect neutral at its optimum then properase E at its opti-mum had the highest DH and hydroxyl radical scavenging activity.Two antioxidant peptides (C 3c1-P and C 3c14-P )were puriﬁed using gel chromatography,ion exchange chromatography and phase HPLC,and their sequences were identiﬁed as (317.33Da)and Tyr-Gly-Asp-Glu-Tyr (645.21Da).They displayed high hydroxyl radical scavenging activity with IC 50value of 4.61and 6.45␮g mL −1.Based on these results,two peptides have the potential to be developed into new health foods.AcknowledgmentWe gratefully thank the National Natural Science Foundation of China (Grant No.31101392)for the ﬁnancial support on thisresearch.References [1]Alemán A,Giménez B,Pérez-Santin E,Gómez-Guillén MC,Montero P.Contri-bution of Leu and Hyp residues to antioxidant and ACE-inhibitory activitiesof peptide sequences isolated from squid gelatin hydrolysate.Food Chem2011;125:334–41.[2]Andrés M,Herminia D,Juan CP.Antioxidant properties of ultraﬁltration-recovered soy protein fractions from industrial efﬂuents and their hydrolysates.Process Biochem 2006;41:447–56.[3]Barlow S,Schlatter J.Risk assessment of carcinogens in food.Toxicol ApplPharm 2010;2:180–90.[4]Bougatef A,Nedjar-Arroume N,Manni L,Ravallec R,Barkia A,Guillochon D,et al.Puriﬁcation and identiﬁcation of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinella aurita )by-products proteins.Food Chem2010;118:559–65.[5]Byun HG,Lee JK,Park HG,Jeon JK,Kim SK.Antioxidant peptides isolated from the marine rotifer,Brachionus rotundiformis .Process Biochem 2009;44:842–6.[6]El-Sayed AM.Tilapia culture.Oxford:CABI publishing;2006.p.1–24.[7]Giménez B,Alemán A,Montero P,Gómez-Guillén MC.Antioxidant and func-tional properties of gelatin hydrolysates obtained from skin of sole and squid.Food Chem 2009;114:976–83.[8]Guo ZY,Liu HY,Chen XL,Ji X,Li PC.Hydroxyl radicals scavenging activityof N-substituted chitosan and quaternized chitosan.Bioorg Med Chem Lett 2006;16:6348–50.[9]Guo H,Kouzuma Y,Yonekura M.Structures and properties of antioxidativepeptides derived from royal jelly protein.Food Chem 2009;113:238–45.[10]Halliwell B,Murcia MA,Chirico S,Aruoma OI.Free radicals and antioxidants in food and in vivo:what they do and how they work.Crit Rev Food Sci 1995;35:7–20.[11]Jamilah B,Harvinder KG.Properties of gelatins from skins of ﬁsh:black tilapia (Oreochromis mossambicus )and red tilapia (Oreochromis nilotica ).Food Chem 2002;77:81–4.[12]Jung MY,Kim SK,Kim SY.Riboﬂavin-sensitized photo oxidation of ascorbicacid:kinetics and amino acid effects.Food Chem 1995;53:397–403.[13]Kaur C,Kapoor HC.Antioxidants in fruits and vegetables –the millennium’shealth.Int J Food Sci Technol 2001;36:703–25.[14]Kawashima K,Itoh H,Miyoshi M,Chibata I.Antioxidant properties of branched-chain amino acid derivatives.Chem Pharm Bull (Tokyo)1979;27:1912–6.[15]Kim SY,Je JY,Kim SK.Puriﬁcation and characterization of antioxidant peptidefrom hoki (Johnius belengerii )frame protein by gastrointestinal digestion.J Nutr Biochem 2007;18:31–8.[16]Li XD,Niu ZX,Zhang BL.Various methods available for the determination ofhydrolyzed degree of whey protein.China Dairy Ind 2006;34:59–62.[17]Moskovitz J,Yim KA,Choke PB.Free radicals and disease.Arch Biochem Biophys 2002;397:354–9.Nazeer RA,Sampath Kumar NS,Jai Ganesh R.In vitro and in vivo studies on theantioxidant activity of ﬁsh peptide isolated from the croaker (Otolithes ruber )muscle protein hydrolysate.Peptide 2012;35:261–8.Park PJ,Jung WK,Nam KS,Shahidi F,Kim SK.Puriﬁcation and characterization of antioxidative peptides from protein hydrolysate of lecithin-free egg yolk.JAm Oil Chem Soc 2001;78:651–6.Raghavan S,Kristinsson HG,Leeuwenburgh C.Radical scavenging and reducing ability of tilapia (Oreochromis niloticus )protein hydrolysates.J Agric Food Chem 2008;56:10359–67.[21]Rajapakse N,Mendis E,Byun HG,Kim SK.Puriﬁcation and in vitro antioxida-tive effects of giant squid muscle peptides on free radical-mediated oxidative systems.J Nutr Biochem 2005;16:562–9.[22]Rajapakse N,Mendis E,Jung WK,Je JY,Kim SK.Puriﬁcation of a radical scaveng-ing peptide from fermented mussel sauce and its antioxidant properties.Food Res Int 2005;38:175–82.[23]Ranathunga S,Rajapakse N,Kim SK.Puriﬁcation and characterization of antiox-idative peptide derived from muscle of conger eel (Conger myriaster ).Eur Food Res Technol 2006;222:310–5.[24]Ren JY,Zhao MM,Shi J,Wang JS,Jiang YM,Cui C,et al.Puriﬁcation andidentiﬁcation of antioxidant peptides from grass carp muscle hydrolysates by consecutive chromatography and electrospray ionization-mass spectrometry.Food Chem 2008;108:727–36.[25]Roberts PR,Burney JD,Black KW,Zaloga GP.Effect of chain length on absorp-of biologically active peptides from the gastrointestinal tract.Digestion[26]Sampath Kumar NS,Nazeer RA,Jaiganesh R.Puriﬁcation and biochemical char-acterization of antioxidant peptide from horse mackerel (Magalaspis cordyla )viscera protein.Peptides 2011;32:149–56.。

细胞抗氧化英语

细胞抗氧化英语《Cellular Antioxidants: The Key to Fighting Oxidative Stress》Oxidative stress is a process that occurs in the body when there is an imbalance between free radicals and antioxidants. Free radicals, which are highly reactive molecules, can cause damage to cells and tissues, leading to various health issues such as inflammation, aging, and chronic diseases. Antioxidants play a crucial role in combating oxidative stress by neutralizing free radicals and protecting the body from their harmful effects.Within the body, cells are constantly exposed to oxidative stress due to various factors such as pollution, UV radiation, and poor diet. As a result, cells rely on antioxidants to protect themselves and maintain proper functioning. Cellular antioxidants, which include enzymes like superoxide dismutase and catalase, as well as nutrients like vitamins C and E, are essential in preventing cellular damage caused by free radicals.One of the key functions of cellular antioxidants is to scavenge free radicals and prevent them from causing oxidative damage. When cells are exposed to oxidative stress, antioxidants work to neutralize free radicals and reduce their harmful effects. This not only protects the cells from damage but also helps to maintain proper cellular function and overall health.In addition to their scavenging properties, cellular antioxidants also play a role in repairing oxidative damage. When cells are damaged by free radicals, antioxidants can help to repair the damage and restore the cell to its normal state. This process is crucial for maintaining the integrity of cellular structures and preventing further damage.Furthermore, cellular antioxidants are involved in regulating the body's response to oxidative stress. This includes activating various cellular defense mechanisms and signaling pathways that help to mitigate the effects of oxidative stress. By doing so, antioxidants help to protect cells from oxidative damage and maintain their overall integrity.In conclusion, cellular antioxidants play a crucial role in fighting oxidative stress and protecting the body from the harmful effects of free radicals. By scavenging free radicals, repairing oxidative damage, and regulating cellular responses to oxidative stress, antioxidants are essential for maintaining cellular function and overall health. Therefore, it is important to consume a diet rich in antioxidants and to engage in healthy lifestyle practices that can help to support the body's natural antioxidant defenses.。

1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。

Potential antioxidants and tyrosinase inhibitors from synthetic polyphenolic deoxybenzoinsLean-Teik Ng a ,Horng-Huey Ko b ,Tzy-Ming Lu c,*aDepartment of Agricultural Chemistry,National Taiwan University,Taipei,TaiwanbDepartment of Fragrance and Cosmetic Science,Kaohsiung Medical University,Kaohsiung,Taiwan cDepartment of Pharmacy,Tajen University,20Wei-Shin Rd,Yanpu,Pingtung 907,Taiwana r t i c l e i n f o Article history:Received 2April 2009Revised 8May 2009Accepted 9May 2009Available online 15May 2009Keywords:Antioxidant Deoxybenzoin Phytoestrogen Polyphenol Tyrosinasea b s t r a c tDeoxybenzoins (DOBs)are one-pot synthetic precursors of isoﬂavones with feature analogous to those beneﬁcial polyphenols such as resveratrol (stilbene)and phloretin (dihydrochalcone).In this study,sev-enteen polyphenolic DOBs were synthesized and evaluated by various antioxidant assays and tyrosinase inhibitory effect in vitro.Results displayed that these DOBs are powerful antioxidants;for example,2,3,4-trihydroxy-30,40-dimethoxydeoxybenzoin possesses an excellent anti-lipid peroxidation activity (IC 50=0.72±0.16l M),whilst 2,4,40,5-tetrahydroxydeoxybenzoin showed good DPPH radical scavenging activity (IC 50=0.69±0.04l M),which were better than Trolox and vitamin C.Besides exhibiting a weak metal chelating effect,these DOBs were effective in scavenging ABTS Å+and superoxide anion (O 2ÅÀ)rad-icals.DOBs also exhibited potent mushroom tyrosinase inhibitory activity;for example 2,3,40-trihydroxy-4-methoxydeoxybenzoin displayed stable and signiﬁcant inhibitory effect on tyrosinase activity,with IC 50values 43.37,43.10and 46.10l M at incubation intervals of 0.5,1.5,and 2.5h,respectively.These results suggest that,with the advantage of being readily synthesizable small molecules,DOBs can be potentially developed into clinical and industrial antioxidants.Crown Copyright Ó2009Published by Elsevier Ltd.All rights reserved.1.IntroductionDeoxybenzoins (DOBs)are precursors for one-pot synthesis of isoﬂavones 1–3and can be recovered from the later by alkali hydrolysis.4These compounds closely related to angolensinoids (a -methyldeoxybenzoins),which are known to be reductive metabolites of isoﬂavones by intestinal microﬂora both in vitro and in vivo.5–9Structurally,DOBs can be described as the abridged dihydrochalcone (keto form)and a -hydroxystilbene (enol form)(Fig.1).Soy isoﬂavones and resveratrol (stilbene)are well known phy-toestrogens with antioxidant,cardiovascular protection,cancer chemopreventive and other properties.10,11Furthermore,dihydr-ochalcones,such as phloretin 12,13and synthetic 20,50-dihydroxydihydrochalcone,14and synthetic polyphenolic chal-cones 15have been demonstrated to be potential antioxidants and/or anti-inﬂammatory agents.Although DOBs have been reported to have various interesting pharmacological properties,they have long been neglected until recently.The 2,4,40-trihydroxydeoxy-benzoin (2),synthetic precursor of daidzein,has been reported asa phytoestrogen with more potent estrogenic receptor (ER)binding capability than daidzein,and was shown to be a selective ERb ago-nist.16Another notable compound,2,4,6-trihydroxy-40-methoxyde-oxybenzoin (15),was reported to be a potential agent for treating severe allergic and inﬂammatory disorders,as it exhibited potent inhibitory effects on tyrosine kinases SYK and BTK,IgE receptor/Fc e RI-mediated mast cell degranulation and leukotriene C 4release,and UVB-induced human keratinocyte prostaglandin E 2release.17Other related derivatives of DOBs have also recently been studied for anti-bacterial activities.18,19Antioxidant therapies have been increasingly recognized to be a potential strategy for preventing acute CNS injury,20cardiovascular diseases 21and asthma.22In addition,applications of antioxidants as preservative in food industry 23and skin-protective ingredients in cosmetics are also receiving increasing attention and interests.24DOBs are small molecules with great potential for development as antioxidants due to their structural analogousness to known natu-ral products with therapeutic beneﬁts (Fig.1).In this study,17polyphenolic DOBs were synthesized and eval-uated for their antioxidant activities by various assays including anti-lipid peroxidation,metal chelating,reducing power,and free radical scavenging such as ABTS Å+,DPPH and superoxide anion.In addition,selected DOBs were also evaluated for mushroom anti-tyrosinase inhibitory activity since antioxidants with whitening ef-fect can be potentially used as cosmetic ingredients.0968-0896/$-see front matter Crown Copyright Ó2009Published by Elsevier Ltd.All rights reserved.doi:10.1016/j.bmc.2009.05.019*Corresponding author.Tel.:+88687624002x320;fax:+88687625308.E-mail address:cmlu@.tw (T.-M.Lu).Bioorganic &Medicinal Chemistry 17(2009)4360–4366Contents lists available at ScienceDirectBioorganic &Medicinal Chemistryj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /b mc2.Results and discussion 2.1.ChemistryThe synthesis of DOBs was convenient and facile by following the methods of Wähäläand Hase 1and/or Balasubramanian et al.2,3as shown in Scheme 1.Seventeen including four newly synthesized (4–6and 13)poly-oxygenated DOBs were obtained and isolated in gram quantity (yields:43.4–92.4%)as depicted (Table 1).2.2.Biological assaysDOBs were evaluated for antioxidant activities including anti-li-pid peroxidation,25metal chelating,26reducing power,27and sev-eral free radical scavenging assays such as ABTS Å+,28DPPH Å,26and O 2ÅÀ.29These assays are commonly applied in the preliminary eval-uation of natural product antioxidants,food additives,and cos-metic ingredients.Based on their structural analogousness to resveratrol,which is an effective tyrosinase inhibitor,30selected DOBs were subjected to the mushroom tyrosinase inhibitory assay.2.2.1.Anti-lipid peroxidation activity of DOBsTable 2(column 2)showed the anti-lipid peroxidation activity of various DOBs.It was found that DOB 12possessed the most po-tent activity (IC 50=0.72±0.16l M),which was about ﬁve times more potent than Trolox (IC 50=3.68±0.64l M).This result ex-plains that the poly-hydroxylated ring A of DOB can be an active anti-lipid peroxidation skeleton.However,the substitution of ring B favored the more lipophilic 40-methoxylated,30,40-dimethoxylat-ed or unsubstituted phenyl,than the hydrophilic 40-hydroxy-phenyl.It is possible that the lipophilic ring B can enhance the contact of these antioxidants with lipid,and consequently result-ing in an efﬁcient termination of the chain reaction.2.2.2.Metal ion chelating activity of DOBsAll DOBs tested showed a weak metal chelating effect (IC 50val-ues >1500l M),with the exception of DOB 10,of which the IC 50value was 1246.79±5.66l M (Table 2,column 6).This suggests that the anti-lipid peroxidation effects of these compounds were achieved via radical scavenging rather than the metal chelating property.2.2.3.Reducing power of DOBsResults showed that DOBs 5,7–11and 13possessed the great-est reducing power,especially DOB 13(IC 50=43.72±2.66l M)Table 1Substitution pattern of polyphenolic DOBs 1–17OR 1R 3R 2R 4R 5R 7R 62463'4'ABEntry Formula R 1R 2R 3R 4R 5R 6R 71C 15H 14O 4OH H OH H H H OMe 2C 14H 12O 4OH H OH H H H OH 3C 16H 16O 5OH H OH H H OMe OMe 4a C 15H 14O 4H OH OH H H H OMe 5a C 16H 16O 5H OH OH H H OMe OMe 6a C 17H 18O 4OH Me Me OH H H OMe 7C 15H 14O 4OH H OMe OH H H H 8C 15H 14O 5OH H OH OH H H OMe 9C 14H 12O 5OH H OH OH H H OH 10C 15H 14O 5OH OH OH H H H OMe 11C 15H 12O 5OH OH OH H H H OH 12C 16H 16O 6OH OH OH H H OMe OMe 13a C 15H 14O 5OH OH OMe H H H OH 14C 14H 12O 4OH H OH H OH H H 15C 15H 14O 5OH H OH H OH H OMe 16C 14H 12O 5OH H OH H OH H OH 17C 16H 16O 6OHHOHHOHOMeOMeaNew compounds.L.-T.Ng et al./Bioorg.Med.Chem.17(2009)4360–43664361which was about three times more potent than ascorbic acid (IC50=131.65±0.97l M,Table2column7).It is found that DOBs with ortho dihydroxyl ring A,except DOB4,exert an excellent fer-ric reducing power.It was also observed that DOBs with the same ring A displayed a better reducing activity when ring B was substi-tuted with C-40OH.2.2.4.ABTSÅ+radical scavenging activity of DOBsIt is surprising to note that all DOBs1–17(IC50ranging from 12.88±1.69l M to87.24±2.11l M)exhibited a more efﬁcient ABTSÅ+radical scavenging than Trolox(IC50=116.3±0.72l M)(Ta-ble2,column3).DOB11with an IC50value of12.88±1.69l M was shown to be nearly10times more potent than the positive control. This result suggests that DOBs with C-40hydroxylated ring B such as DOBs2,11,and16,would exert a greater potency than those DOBs with the same substitution in ring A.It was shown that DOBs with polyphenol feature possessed promising ABTSÅ+radical scav-enging activity.Though ABTSÅ+was a suitable assay for both hydro-philic and lipophilic antioxidants,26the more hydrophilic(poly-hydroxylated)DOBs are likely to be better ABTSÅ+radical scaveng-ers.Since phenol is known to be an active functional group for free radical scavenging,suggesting that its property can be one of the main determinants in this activity.It is found that the existence of electron withdrawing ketyl of DOB leading to a more acidic C-4phenol than that of Trolox.The better free radical scavenging activity of DOBs can be attributed to the capability of ion-pairing that enhanced the contact of phenolic anion with ABTSÅ+.Though Trolox can be dissociated into a carboxylic anion(COOÀ),it was shown to be a lesser effective radical scavenger(Fig.2).2.2.5.DPPH radical scavenging activity of DOBsTable2(column4)showed the DPPH radical scavenging activity of DOBs.Besides DOB4,all other DOBs showed a more active DPPH radical scavenging than ascorbic acid(IC50=73.69±1.76l M). Generally,when the catechol,pyrogallol or phloroglucinol ring A of DOBs was substituted by a30,40-dimethoxylated ring B(i.e., DOBs5,12and17),they would exert a greater potency in DPPH radical scavenging power.Among the different DOBs tested,DOB 9exhibited the most potent DPPH radical scavenging activity (IC50=0.69±0.04l M),which was about13times more potent than Trolox(IC50=9.20±0.44l M).2.2.6.Superoxide radical scavenging activity of DOBsAs shown in Table2(column5),DOBs displayed a potent super-oxide anion radical scavenging activity.Based on the IC50values,Table3IC50values of selected DOBs in mushroom tyrosinase inhibitory activityTreatment IC50(l M)30min90min150min178.88160.37181.452178.46135.91172.413112.06209.14239.704>300167.97177.68884.35144.19197.3610>300203.32211.9211>300>300>50012228.79131.53139.991343.3743.1046.101571.05213.86230.2416>300>300261.26Kojic acid a132.83225.14438.35Values are the average of three analyses(n=3).a Positive control;50%inhibition(IC50)values are expressed as l M.Table2IC50values of DOBs in antioxidant activitiesDOBs IC50(l M)POV a ABTS DPPH SOD-like Metal chelation Reducing power 17.33±0.3162.64±4.1957.40±5.0835.19±1.32>1500>1000225.53±2.8730.49±3.5225.20±2.21113.65±2.58>1500499.55±74.303 1.84±0.5941.94±1.4935.10±1.1189.34±1.98>1500742.99±14.204 6.47±0.5417.83±2.0275.93±6.36121.43±3.72>1500>1000524.93±2.4042.67±1.04 2.53±0.17110.59±4.79>1500112.95±1.02 620.84±1.6841.68±2.4821.92±1.4039.02±2.03>1500147.45±3.297 6.20±0.1036.01±1.9020.27±1.2454.57±3.14>150081.16±6.05 810.69±0.5539.60±1.3521.46±1.1768.18±4.45>1500102.88±10.29 988.38±5.1546.42±1.350.69±0.0458.62±1.77>150068.54±2.38 1013.28±1.8250.69±2.2621.64±1.5328.25±0.581246.79±5.66120.22±6.13 11178.38±10.0212.88±1.69 5.69±0.69115.46±3.81>1500115.27±3.96 120.72±0.1641.58±1.12 4.34±0.9582.83±3.62>1500320.95±5.36 1385.00±7.5250.51±0.5525.66±1.8296.82±3.28>150043.72±2.66 1413.36±0.7046.43±1.4812.25±0.90106.15±6.43>1500275.34±21.14 15 2.12±0.1561.61±2.8110.84±1.5731.61±0.99>1500231.93±9.53 1637.42±2.5427.35±2.6222.77±1.46109.15±5.08>1500146.77±3.23 1727.04±1.3287.24±2.11 1.35±0.1380.72±3.65>1500147.66±6.25 Trolox b 3.68±0.64116.28±0.729.20±0.4427.76±2.36—240.56±4.80 Ascorbic acid c48.52±1.42—73.69±1.7624.15±0.85—131.65±0.97 EDTA d————185.64±0.34Value are presented as mean±SD(n=3).a POV:anti-lipid peroxidation activity.b,c,d Positive controls;50%inhibition(IC50)values are expressed as l M.4362L.-T.Ng et al./Bioorg.Med.Chem.17(2009)4360–4366DOBs1,10and15exhibited a comparable activity as Trolox and vitamin C.This result suggests that DOBs with a p-methoxylated ring B coupled with resorcinol,pyrogallol,or phloroglucinol ring A(DOBs1,10,and15)could be an effective superoxide anion rad-ical scavenger.However,the anionic expulsion effect of DOBs with O2ÅÀcan limit the radical scavenging power,which might contrib-ute to the contrasting results from that of the ABTSÅ+assay.2.2.7.Anti-tyrosinase activity of DOBsMushroom tyrosinase is the common inhibitory target for the evaluation of new whitening agents of cosmetics.Results revealed that most DOBs possessed a greater anti-tyrosinase activity than kojic acid(a commercial whitening ingredient)(Table3).DOBs1, 8,13and15showed a signiﬁcant and immediate inhibition atﬁrst 30min of incubation,of which were found to have a meta-dihy-droxylated ring A with the exception of DOB13.This phenomenon is consistent with the demonstrated structural inhibition of resor-cinol-containing1,3-dihydroxyphenylpropionic acid deriva-tives.31–33In this study,the most active structure was DOB13, 2,3,40-tihydroxy-4-methoxydeoxybenzoin,which showed an al-most constant inhibitory effect on tyrosinase at all three incuba-tion intervals.Though40-hydroxylated ring B of DOB possesses a similar feature to that of the common enzyme substrate,that is, L-tyrosine,a similar inhibition manner was not observed in DOBs 2,11and16.It is therefore suggested that lipophilic C-4methoxyl of DOB13may play an important role in the inhibition of mush-room tyrosinase activity.3.ConclusionTaken together,the present study concludes that DOBs with feature analogous to isoﬂavone,dihydrochalcone and stilbene dis-played powerful antioxidant and anti-tyrosinase activities,sug-gesting that they possess potentials to be developed into clinical therapeutic antioxidants and as new ingredients of cosmetics.Fur-thermore,the fact that DOBs could be synthesized readily and eas-ily would be another advantage of these compounds in the antioxidant market.4.Experimental4.1.Synthesis of DOBsThe synthesis of DOBs with resorcinol(1–3),catechol(4–5),2,3-dimethylhydroquinone(6),2-methoxyhydroquinone(7),pyrogallol (10–12)or3-methoxycatechol(13)ring A(each0.01mole)was per-formed by condensing respective phenols with equivalent quanti-ties of various phenylacetic acids in BF3–Et2O at80–90°C under nitrogen conditioning for2–3h(Scheme1,method a).The reaction mixtures were poured into a large quantity of icy NaOAc solution, and then extracted by EtOAc according to Wähäläet al.1However, DOBs with1,2,4-trihydroxybenzene(8–9)and/or phloroglucinol (14–17)ring A were prepared according to the method as described by Balasubramanian et al.2,3with modiﬁcation.These phenols (0.02mol each)and related phenylacetonitriles(0.01mol)together with ZnCl2(0.01mol)were pre-suspended in40mL of ethyl ether at 0°C,then saturated by HCl(gas,caution!)and stirred for2h.After stirring,the reaction mixtures were left to stand over night at ambi-ent temperature.The coagulated products(imines,not isolated) were hydrolyzed by reﬂuxing in100mL MeOH–H2O(1:1;v/v)for 3–5h.DOBs(8–9and14–17)can be readily crystallized after cooling (Scheme1,method b).Seventeen polyphenolic DOBs(1–17)were obtained(Table1)after purifying with column chromatography and/or recrystallized to have satisfying yields(8–9and14–17scaled by respective phenylacetonitriles).4.2.General proceduresPhenylacetic acids,phenylacetonitriles,boron triﬂuoride ether-ate(BF3–Et2O)and related phenols were purchased from Tokyo Chemical Industry(Tokyo,Japan)or Lancaster Synthesis(More-cambe,England).Melting point(uncorrected)was determined by Electrothermal9100melting point apparatus(Electrothermal Engineering Ltd,Landon,UK).Mass spectra were recorded on a Bruker Ft Mass Spectrometer(Bruker Daltonics,Bremen,Ger-many).1H(400MHz)and13C(100MHz)NMR spectra were re-corded on a Varian Mercury-400spectrometer(Varian Inc.,Palo Alto,USA).Mushroom tyrosinase,20-azino-bis(3-ethylbenzthiazo-line-6-sulfonic acid)(ABTS),1,1-diphenyl-2-picryl-hydrazyl (DPPH),nitro-blue tetrazolium(NBT),dihydronicotinamide ade-nine dinucleotide(NADH),ethylenediaminetetraacetic acid (EDTA),Tween-20,ascorbic acid,kojic acid and Trolox were pur-chased from Sigma Chemical Co.(St.Louis,MO,USA).4.2.1.2,4-Dihydroxy-40-methoxydeoxybenzoin(1)2.27g,88%(scale:8.80mmol).R f=0.42(EtOAc–n-hexane–ace-tone=2.5:7:0.5).Colorless needles(acetone–n-hexane),mp 162°C.1H NMR(400MHz,acetone-d6):d=3.76(3H,s,OMe),4.12 (2H,s,CH2), 6.33(1H,d,J=2.8Hz,H-3), 6.45(1H,dd,J=2.4, 8.8Hz,H-5),6.88(2H,d,J=8.8Hz,H-30,50),7.26(2H,d,J=8.8Hz, H-20,60),7.96(1H,J=8.8Hz,H-6),9.48(1H,s,C-4OH),12.74(1H, s,C-2OH).13C NMR(100MHz,acetone-d6):d=44.4(CH2),55.6 (OMe),103.8(C-3),109.1(C-5),113.6(C-1),114.9(C-30,50),128.1 (C-10),131.5(C-20,60),159.8(C-40),165.9(C-2),166.9(C-4),203.9 (C@O).EIMS m/z:258[M]+,137[ring A+C@O]+.1,164.2.2.2,4,40-Trihydroxydeoxybenzoin(2)1.32g,54.1%(scale: 5.41mmol).R f=0.38(EtOAc–n-hexane–acetone=2.5:7:0.5).Colorless powders(MeOH-H2O),mp194°C. 1H NMR(400MHz,acetone-d6):d=4.16(2H,s,CH2),6.32(1H,d, J=2.4Hz,H-3), 6.43(1H,dd,J=2.4,8.8Hz,H-5), 6.79(2H,d, J=8.8Hz,H-30,50),7.16(2H,d,J=8.8Hz,H-20,60),7.94(1H,d, J=8.8Hz,H-6),12.75(1H,br s,C-2OH).13C NMR(100MHz,ace-tone-d6):d=45.0(CH2),104.3(C-3),109.5(C-5),114.1(C-1), 116.9(C-30,50),127.4(C-10),131.9(C-20,60),135.0(C-6),157.9 (C-40),166.4(C-2),167.4(C-4),204.5(C@O).EIMS m/z:244[M]+, 137[ring A+C@O]+.1,164.2.3.2,4-Dihydroxy-30,40-dimethoxydeoxybenzoin(3)2.05g,71.2%(scale,7.12mmol).R f=0.29(EtOAc–n-hexane–acetone=2.5:7:0.5).Colorless needles(MeOH),mp177–178°C.1H NMR(400MHz,CDCl3):d=3.85(3H,s,OMe), 3.86(3H,s, OMe),4.16(2H,s,CH2),6.37(1H,d,J=2.0Hz,H-3),6.39(1H,dd, J=2.4,8.8Hz,H-5),6.78(1H,d,J=2.0Hz,H-20),6.80(1H,dd, J=2.0,8.4Hz,H-60), 6.84(1H,d,J=8.4Hz,H-50),7.76(1H,d, J=8.4Hz,H-6),12.67(1H,s,C-2OH).13C NMR(100MHz,CDCl3): d=44.7(CH2),56.1(OMeÂ2),103.9(C-3),108.1(C-5),111.6(C-50),112.6(C-20),113.7(C-1),121.8(C-60),126.9(C-10),133.0(C-6),148.3(C-40),149.3(C-30),163.0(C-2),165.9(C-4),202.5 (C@O).EIMS m/z:288[M]+,137[ring A+C@O]+.34.2.4.3,4-Dihydroxy-40-methoxydeoxybenzoin(4)1.72g,66.7%(scale: 6.67mmol).R f=0.66(EtOAc–n-hexane–acetone=3:6.5:0.5).Colorless to yellowish needles(MeOH–H2O), mp164-165°C.1H NMR(400MHz,acetone-d6):d=3.75(3H,s, OMe),4.15(2H,s,CH2),6.85(2H,d,J=8.8Hz,H-30,50),6.90(1H, d,J=8.4Hz,H-5),7.20(2H,d,J=8.8Hz,H-20,60),7.54(1H,d, J=1.2Hz,H-2),7.55(1H,dd,J=1.2,8.0Hz,H-6),8.36(1H,br s, phenolic OH),8.71(1H,br,OH).13C NMR(100MHz,acetone-d6): d=45.2(CH2),56.1(OMe),115.3(C-30,50),116.3(C-2),116.8(C-5),123.8(C-6),129.2(C-1),131.0(C-10),132.0(C-20,60),146.5 (C-3),151.7(C-4),160.0(C-40),197.3(C@O).ESIMS(+)m/z:281L.-T.Ng et al./Bioorg.Med.Chem.17(2009)4360–43664363[M+Na]+;HRESIMS(+)m/z:281.0792(calcd for C15H14O4Na= 281.0790).4.2.5.3,4-Dihydroxy-30,40-dimethoxydeoxybenzoin(5)2.66g,92.4%(scale:9.24mmol).R f=0.51(EtOAc–n-hexane–acetone=2.5:7:0.5).Yellowish needles(MeOH-H2O),mp256-257°C.1H NMR(400MHz,acetone-d6):d=3.76,3.77(each3H,s, OMe),4.14(2H,s,CH2),6.80(2H,d,J=2.0,8.8Hz,H-60),6.85 (1H,d,J=8.0Hz,H-50),6.90(2H,d,J=8.0Hz,H-5),6.91(1H,d, J=2.0Hz,H-20),7.54(1H,d,J=2.0Hz,H-2),7.56(1H,dd,J=2.0, 8.0Hz,H-6),8.49(2H,br,OH).13C NMR(100MHz,acetone-d6): d=44.3(CH2),55.4(OMe),55.4(OMe),112.2(C-50),113.8(C-20), 115.0(C-2),115.5(C-5),121.8(C-60),122.6(C-6),128.5(C-1), 129.8(C-10),132.0(C-20,60),145.2(C-4),148.5(C-40),149.6(C-30),150.3(C-3),195.9(C@O).ESIMS(+)m/z:311(M+Na);HRESMS (+)m/z:311.0893(calcd for C16H16O5Na=311.0895).4.2.6.2,5-Dihydroxy-40-methoxy-3,4-dimethyldeoxybenzoin(6)1.24g,43.4%(scale: 4.34mmol).R f=0.60(EtOAc–n-hexane–acetone=2.5:7:0.5).Colorless needles(MeOH),mp177–178°C. 1H NMR(400MHz,CDCl3):d=2.19(3H,s,Ar-Me),2.22(3H,s, Ar-Me), 3.78(3H,s,OMe), 4.11(2H,s,CH2), 6.86(2H,d, J=8.4Hz,H-30,50),7.07(1H,s,H-6),7.14(2H,d,J=8.4Hz,H-20, 60),12.32(1H,s,C-2OH).13C NMR(100MHz,CDCl3):d=11.5 (Ar-Me),13.0(Ar-Me),44.1(CH2),55.3(OMe),111.6(C-6),114.2 (C-30,50),115.6(C-1),126.2(C-3),127.1(C-10),130.4(C-20,60), 134.5(C-4),145.5(C-5),155.7(C-2),158.6(C-40),203.4(C@O). EIMS m/z:286[M]+,165[ring A+C@O]+;HRESIMS(+)m/z: 309.1101(calcd for C15H14O5Na=309.1103).4.2.7.2,5-Dihydroxy-4-methoxydeoxybenzoin(7)2.24g,86.8%(scale:8.68mmol).R f=0.45(EtOAc–n-hexane–acetone=2.5:7:0.5).Yellowish powders(MeOH–H2O),mp154–155°C.1H NMR(400MHz,CDCl3):d=3.91(3H,s,OMe),4.19 (2H,s,CH2),6.44(1H,s,H-3),7.26–7.34(total5H,m,H-20–60), 7.33(1H,s,H-6),12.47(1H,s,C-2OH).13C NMR(100MHz,CDCl3): d=45.2(CH2),56.4(OMe),100.2(C-3),112.0(C-1),113.8(C-6), 127.3(C-40),129.0(C-30,50),129.6(C-20,60),134.5(C-10),138.3 (C-5),154.1(C-2),159.8(C-4),202.2(C@O).EIMS m/z:258[M]+, 167[ring A+C@O]+.344.2.8.2,4,5-Trihydroxy-40-methoxydeoxybenzoin(8)2.18g,79.6%(scale:7.96mmol).R f=0.24(EtOAc–n-hexane–acetone=3:6.5:0.5).Pinkish needles(MeOH–H2O),mp188–189°C.1H NMR(400MHz,acetone-d6):d=3.77(3H,s,OMe),4.15(2H,s,CH2),6.36(1H,s,H-3),6.88(2H,d,J=8.8Hz,H-30, 50),7.24(2H,d,J=8.8Hz,H-20,60),7.42(1H,s,H-6),12.42(1H,s, C-2OH).13C NMR(100MHz,acetone-d6):d=43.8(CH2),54.8 (OMe),103.3(C-3),111.4(C-1),114.1(C-30,50),115.8(C-6),127.4 (C-10),130.6(C-20,60),138.1(C-5),154.3(C-4),158.9(C-2),159.8 (C-40),202.6(C@O).EIMS m/z274[M]+,153[ring A+C@O]+.34 4.2.9.2,4,5,40-Tetrahydroxydeoxybenzoin(9)1.96g,75.4%(scale:7.54mmol).R f=0.26(EtOAc–n-hexane–acetone=4:5.5:0.5).Colorless needles(MeOH–H2O),mp297–299°C.1H NMR(400MHz,acetone-d6):d=4.10(2H,s,CH2),6.35 (1H,s,H-3), 6.79(2H,d,J=8.8Hz,H-30,50),7.15(2H,d, J=8.8Hz,H-20,60),7.41(1H,s,,H-6),8.40(3H,br,OH),12.44 (1H,s,C-2OH).13C NMR(100MHz,acetone-d6):d=43.9(CH2), 103.3(C-3),111.4(C-1),115.6(C-30,50),115.8(C-6),126.2(C-10), 130.6(C-20,60),138.1(C-5),154.4(C-4),156.5(C-2),159.8(C-40), 202.7(C@O).EIMS m/z:260[M]+,153[ring A+C@O]+.344.2.10.2,3,4-Trihydroxy-40-methoxydeoxybenzoin(10)2.40g,87.6%(scale:8.76mmol).R f=0.46(EtOAc–n-hexane–acetone=3:6.5:0.5).Colorless needles(MeOH–H2O),mp191–192°C.1H NMR(400MHz,acetone-d6):d=3.76(3H,s,OMe),4.22(2H,s,CH2), 6.48(1H,d,J=8.8Hz,H-5), 6.87(2H,d, J=8.8Hz,H-30,50),7.25(2H,d,J=8.8Hz,H-20,60),7.55(1H,d, J=8.4Hz,H-6),7.80(1H,s,OH),8.66(1H,s,OH),12.74(1H,s,C-2OH).13C NMR(100MHz,acetone-d6):d=44.9(CH2),56.1 (OMe),109.1(C-5),114.4(C-1),115.4(C-30,50),124.8(C-6), 128.7(C-10),132.0(C-20,60),133.8(C-3),153.4(C-2),154.1(C-4), 160.3(C-40),205.1(C@O).ESIMS(+)m/z:275[M+H]+.1,24.2.11.2,3,4,40-Tetrahydroxydeoxybenzoin(11)1.42g, 5.46%(scale:54.6mmol).R f=0.35(EtOAc–n-hexane–acetone=4:5.5:0.5).Colorless needles(MeOH–H2O),mp281–282°C.1H NMR(400MHz,acetone-d6):d=4.18(2H,s,CH2),6.48 (1H,d,J=8.8Hz,H-5),6.78(2H,d,J=8.8Hz,H-30,50),7.16(2H, d,J=8.8Hz,H-20,60),7.55(1H,d,J=8.8Hz,H-6),8.27(3H,br, OH),12.77(1H,br s,C-2OH).13C NMR(100MHz,acetone-d6): d=43.6(CH2),107.7(C-5),113.0(C-1),115.5(C-30,50),123.5(C-6),126.1(C-10),130.7(C-20,60),132.5(C-3),152.0(C-2),152.8 (C-4),156.5(C-40),203.9(C@O).EIMS m/z:260[M]+,153[ring A+C@O]+.1,2,164.2.12.2,3,4-Trihydroxy-30,40-dimethoxydeoxybenzoin(12)2.42g,79.6%(scale:7.96mmol).R f=0.43(EtOAc–n-hexane–acetone=4:5.5:0.5).Colorless needles(MeOH–H2O),mp166–167°C.1H NMR(400MHz,acetone-d6):d=3.76(3H,s,OMe), 3.77(3H,s,OMe),4.20(2H,s,CH2),6.48(1H,d,J=8.8Hz,H-5), 6.84(2H,dd,J=2.0,8.4Hz,H-60),6.87(2H,d,J=8.4Hz,H-50), 6.96(1H,d,J=2.0Hz,H-20),7.55(1H,d,J=8.8Hz,H-6),7.96 (1H,br,OH),8.78(1H,br,OH),12.74(1H,s,C-2OH).13C NMR (100MHz,acetone-d6):d=45.3(CH2),56.7(OMeÂ2),109.1(C-5),113.5(C-50),114.4(C-1),115.0(C-20),123.1(C-60),124.7(C-6),129.2(C-10),133.9(C-3),150.0(C-2),151.0(C-40),153.4(C-41),154.1(C-4),205.0(C@O).ESIMS(+)m/z:305[M+H]+.354.2.13.2,3,40-Trihydroxy-4-methoxydeoxybenzoin(13)2.15g,78.5%(scale:7.85mmol).R f=0.23(EtOAc–n-hexane–acetone=3:6.5:0.5).Colorless powders(MeOH–H2O),mp249–250°C.1H NMR(400MHz,acetone-d6):d=3.91(3H,s,OMe),4.21(2H,s,CH2), 6.65(1H,d,J=8.8Hz,H-5), 6.79(2H,d, J=8.4Hz,H-30,50),7.16(2H,d,J=8.4Hz,H-20,60),7.63(1H,d, J=8.4Hz,H-6),8.24(1H,br,OH),12.46(1H,br,C-2OH).13C NMR(100MHz,acetone-d6):d=43.9(CH2),55.8(OMe),103.5(C-5),114.2(C-1),115.5(C-30,50),122.9(C-6),126.1(C-10),130.6 (C-20,60),134.5(C-3),151.9(C-2),153.2(C-4),156.5(C-40),204.2 (C@O).ESIMS(+)m/z:297[M+Na]+;HRESIMS(+)m/z:297.0741 (calcd for C15H14O5Na=297.0739).4.2.14.2,4,6-Trihydroxydeoxybenzoin(14)1.77g,72.5%(scale:7.25mmol).R f=0.26(EtOAc–n-hexane–acetone=2.5:7:0.5).Yellowish granules(MeOH-H2O),mp164°C. 1H NMR(400MHz,acetone-d6):d=4.42(2H,s,CH2),5.95(2H,s, H-3,5),7.21(1H,m,J=8.4Hz,H-40),7.28(4H,m,H-20,30,50,60), 9.28(1H,br s,phenolic OH),11.74(2H,br s,C-2,6OH).13C NMR (100MHz,acetone-d6):d=49.5(CH2),95.3(C-3,5),104.5(C-1), 126.5(C-40),128.3(C-30,50),130.0(C-20,60),136.4(C-10), 164.8(C-2,6),164.9(C-4),203.0(C@O).EIMS m/z:244[M]+,153 [ring A+C@O]+.344.2.15.2,4,6-Trihydroxy-40-methoxydeoxybenzoin(15)2.02g,73.7%(scale:7.37mmol).R f=0.38(EtOAc–n-hexane–acetone=3:6.5:0.5).Reddish needles(MeOH–H2O),mp184–185°C.1H NMR1H NMR(400MHz,CD3OD):d=3.73,(3H,s, OMe),7.30(2H,s,CH2), 5.82(2H,s,H-3,5), 6.81(2H,d, J=8.8Hz,H-30,50),7.13(2H,d,J=8.8Hz,H-20,60).13C NMR (100MHz,CD3OD):d=49.6(CH2),55.6(OMe),95.8(C-3,5), 105.2(C-1),114.6(C-30,50),129.3(C-10),131.7(C-20,60),159.74364L.-T.Ng et al./Bioorg.Med.Chem.17(2009)4360–4366(C-40),165.8(C-4,166.3(C-2,6),204.9(C@O).EIMS m/z:274[M]+, 153[ring A+C@O]+.2,344.2.16.2,4,40,6-Tetrahydroxydeoxybenzoin(16)1.61g,61.9%(scale:6.19mmol).R f=0.34(EtOAc–n-hexane–acetone=4:5.5:0.5).Yellowish powders(MeOH–H2O),mp272°C. 1H NMR(400MHz,acetone-d6):d=4.31(2H,s,CH2),5.93(2H,s, H-3,5),6.75(2H,d,J=8.4Hz,H-30,50),7.11(2H,d,J=8.4Hz,H-20,60),8.13(1H,br s,phenolic OH),9.25(1H,br,phenolic OH), 11.74(1H,br s,C-2OH).13C NMR(100MHz,acetone-d6):d=48.6 (CH2),95.2(C-3,5),104.4(C-1),115.1(C-30,50),127.0(C-40), 130.9(C-20,60),156.2(C-40),164.8(C-4),164.8(C-2,4),203.6 (C@O).EIMS m/z260[M]+,153[ring A+C@O]+.2,164.2.17.2,4,6-Trihydroxy-30,40-dimethoxydeoxybenzoin(17)2.03g,66.8%(scale: 6.68mmol).R f=0.31(EtOAc–n-hexane–acetone=4:5.5:0.5).Colorless needles(H2O–MeOH),mp188–190°C;1H NMR(400MHz,pyridine-d5):d=3.76,(3H,s,OMe), 3.77(3H,s,OMe),4.37(2H,s,CH2),5.34(2H,s,H-3,5),6.79(1H, dd,J=2.0,8.0Hz,H-60),6.84(2H,d,J=8.0Hz,H-50),6.91(1H,d, J=2.0Hz,H-20),9.29(1H,br s,phenolic OH),11.75(2H,br s,C-2,6 OH).13C NMR(100MHz,pyridine-d5):d=49.0(CH2),55.4(OMe), 55.5(OMe),95.2(C-3,5),104.4(C-1),112.1(C-50),114.3(C-20), 122.1(C-60),128.8(C-10),148.5(C-40),149.4(C-30),164.8(C-2,6), 164.8(C-4),203.3(C@O).EIMS m/z:304[M]+,153[ring A+C@O]+.344.3.Bilogical assays4.3.1.Anti-lipid peroxidation assayThis assay was determined by the thiocyanate method as de-scribed by Mitsuda et al.25In brief,2mL of different concentrations of DOBs and positive controls(Trolox:a water soluble form of tocopherol;vitamin C:ascorbic acid)were taken and well mixed with3mL of linoleic acid emulsion consisting of2.5g Tween-20, 2.5g linoleic acid,and0.04M of potassium phosphate buffer(pH 7.0),followed by incubating at37°C.After reacting with FeCl2 and thiocyanate at several time intervals,the peroxide value was measured at wavelength500nm.4.3.2.Metal chelating assayThe chelating effect of ferrous ions by the DOBs was determined as described previously.27In brief,1mL of DOBs at different con-centrations was mixed with3.7mL of methanol and0.1mL of 2mM FeCl2.The reaction was initiated by the addition of0.2mL of5mM ferrozine,followed by shaking vigorously and left to react at room temperature for10min.The absorbance was measured at 562nm.EDTA,a strong metal chelator,was used as a standard me-tal chelating agent.4.3.3.Reducing power assayThe reducing power of DOBs was determined according to the method of Oyaizu.29In brief,2.5mL of various concentrations of DOBs or positive controls(i.e.,Trolox and vitamin C)was mixed with2.5mL of0.2M phosphate buffer(pH6.6)and2.5mL of1% potassium ferric cyanide.After incubating the mixture at50°C for20min,2.5mL of10%trichloroacetic acid was added,followed by centrifuging at3000rpm for10min.Five(5)milliliters of the upper layer solution were taken and mixed with5mL distilled water and1mL of0.1%FeCl3.The mixture was then measured at absorbance700nm.4.3.4.ABTSÅ+cation radical scavenging assayThe scavenging activity of ABTSÅ+was measured according to the method described by Re et al.26with some modiﬁcations. Brieﬂy,ABTS was dissolved in deionized water to7mM in concen-tration,which was then mixed with2.45mM potassium persul-fate.The scavenging activity was determined by mixing with 180l L of ABTS and40l L of DOBs or negative control(PBS;phos-phate buffered saline)or positive controls(i.e.,Trolox and vitaminC),followed by measuring at absorbance734nm.4.3.5.DPPH radical scavenging assayThis assay was conducted according to the method as described by Wu and Ng.27Brieﬂy,1mL of0.1mM DPPH radical solution was mixed with3ml of various concentrations of DOBs or Trolox or vitamin C dissolving in methanol.The mixture was then vortexed vigorously and left for30min at40°C in the dark.For the baseline control,3mL of methanol was used.The absorbance was measured at517nm.4.3.6.Superoxide anion radical scavenging assayThe assay of DOBs was conducted according to the method de-scribed by Gülçin.28In brief,superoxide radicals were generated in 3mL of phosphate buffer(0.1M,pH7.4)containing1mL of nitro-blue tetrazolium(NBT,300l M),1mL of PMS(120l M),1mL of NADH(968l M)and1mL of DOBs at various concentrations.The mixture was spectrophotometrically measured at560nm.Trolox and vitamin C were used as positive controls.4.3.7.Mushroom tyrosinase inhibitory assayDOBs selected for this assay are presented in Table3and were determined according to the method reported previously.36DOBs and kojic acid(positive control)were dissolved in DMSO/methanol and diluted to various concentrations using potassium phosphate buffer(pH6.8).The96-well plate containing80l L of L-tyrosine (2.0mM)and20l L of various concentrations of DOBs or kojic acid were diluted with buffer to200l L after adding20l L of mushroom tyrosinase(1000U/mL,EC1.14.18.1).Inhibitory percentage of the test compounds was determined at wavelength490nm after0.5, 1.5and2.5h of incubation.AcknowledgmentsWe are grateful to the partialﬁnancial support from the Tajen University and the NMR spectral measurements by Mr.P.L.Lin from the Instrumental Centre of Tajen University.The computer time and facilities provided by the National Center of High-perfor-mance Computing are also greatly appreciated.Supplementary dataSupplementary data(synthetic procedures and the NMR spectra of all the DOBs)associated with this article can be found,in the on-line version,at doi:10.1016/j.bmc.2009.05.019.References and notes1.Wähälä,K.;Hase,T.A.J.Chem.Soc.,Perkin Trans.11991,3005.2.Balasubramanian,S.;Nair,mun.2000,30,469.3.Balasubramanian,S.;Ward,D.L.;Nair,M.G.J.Chem.Soc.,Perkin Trans.12000,567.4.Wong, E.In The Flavonoids;Harborne,J. B.,Mabry,T.J.,Mabry,H.,Eds.;Academic Press:New York,1975;p750.Chapter14.5.Wong, E.In The Flavonoids;Harborne,J. B.,Mabry,T.J.,Mabry,H.,Eds.;Academic Press:New York,1975;p786.Chapter14.6.Heinonen,S.;Hoikkala,A.;Wähälä,K.;Adlercreutz,H.J.Steroid Biochem.Mol.Biol.2003,87,285.7.Heinonen,S.;Wähälä,K.;Adlercreutz,H.Anal.Biochem.1999,274,211.8.Coldham,N.G.;Darby,C.;Hows,M.;King,L.J.;Zhang,A.Q.;Sauer,M.J.Xenobiotica2002,32,45.9.Coldham,N.G.;Howells,L.C.;Santi,A.;Montesissa,C.;Langlais,C.;King,L.J.;Macpherson,D.D.;Sauera,M.J.J.Steroid Biochem.Mol.Biol.1999,70,169. 10.Athar,M.;Back,J.H.;Tang,X.;Kim,K.H.;Kopelovich,L.;Bickers,D.R.;Kim,A.L.Toxicol.Appl.Pharmacol.2007,224,274.11.Ososki,A.L.;Kennelly,E.J.Phytother.Rev.2003,17,845.L.-T.Ng et al./Bioorg.Med.Chem.17(2009)4360–43664365。