%REP]WMWSJ 2YXVEGIYXMGEP7S]-WSJPEZSRIW 9WMRK0'4(%

.MQ/VSP1MOI=SYRKERH1EVO&IRZIRYXM ;EXIVW'SVTSVEXMSR

1EVGL

'ST]VMKLX;EXIVW'SVT -QTSVXERGISJ-WSJPEZSRIW

# 4SXIRXREXYVEPERXMS\MHERXWJSYRHMRWS]FEWIH JSSHTVSHYGXW

# -QTPMGEXIHMRVIHYGXMSRSJ0(0GLSPIWXIVSP

# +IRMWXIMRERH(EMH^IMRMQTPMGEXIHMRVIHYGMRK GERGIVVMWO FVIEWXTVSWXVEXI GSPSR

# ;IEOIWXVSKIRMGIJJIGXQE]VIPMIZIQIRSTEYWEP W]QTXSQW 'ST]VMKLX;EXIVW'SVT -QTSVXERGISJ-WSJPEZSRIW 2YXVEGIYXMGEP

*IHIVEP6IKMWXEV3GX XLI*(%EPPS[WEPEFIPGPEMQXLEX

l|KVEQWSJWS]TVSXIMREHE] EWTEVXSJEHMIXPS[MRWEXYVEXIHJEXERHGLSPIWXIVSP QE]VIHYGIXLIVMWOSJLIEVXHMWIEWIz

'ST]VMKLX;EXIVW'SVT %REP]WMWSJ-WSJPEZSRIW

# +'17QIXLSHMWWIRWMXMZIERHEGGYVEXIFYXPEFSVMSYWERH I\TIRWMZI r WIZIVEP74)WXITWVIUYMVIHXSMWSPEXIHMJJIVIRXJSVQW r L]HVSP]WMWXSEKPYGSRI r HIVMZEXM^EXMSR

# 0'4(%LEWEREHZERXEKIFIGEYWIEPPMWSJPEZSRIJSVQW GERFIWITEVEXIHERHUYERXMXEXIHYWMRK9:(IXIGXMSR r 7IRWMXMZMX] r 'SIPYXMSR -HIRXMJMGEXMSRMRGSQTPI\QEXVMGIW?

# 0'17QIXLSHWFIMRKHIZIPSTIH 'ST]VMKLX;EXIVW'SVT %REP]WMWSJ-WSJPEZSRIW 1IXLSH(IZIPSTQIRX3FNIGXMZIW # (IWMKRIHJSV9:ERH1EWW7TIG(IXIGXMSR # 6IWSPYXMSRSJEPP7S]-WSJPEZSRIW # %REP]WMWXMQI

# PIWWXLERQMRYXIWJSVGLVSQEXSKVETL]

# PIWWXLERQMRYXIWMRNIGXMSRXSMRNIGXMSR # 'SRJMVQEXMSREP'LIQMWXVMIW # %TTPMGEXMSRXS(MIXEV]7YTTPIQIRXWERH*SSH1EXVMGIW # 9WITYFPMWLIHVIJIVIRGIWEWJSYRHEXMSRJSV # GLVSQEXSKVETL] 1YVTL]IXEP*VEROIIXEPERH&EVRIWIXEP # WEQTPITVIT 1YVTL]IXEP 'ST]VMKLX;EXIVW'SVT 'SQQSR7S]-WS*PEZSRI 7XVYGXYVIERHT/E

T/ b¥ HOO E %KPYGSRI7XVYGXYVI

(EMH^IMR 1;! O OH HOO

H3CO +P]GMXIMR 1;! O OH T/Eb¥ HOO

+IRMWXIMR 1;! OH O OH T/ b¥ E 'ST]VMKLX;EXIVW'SVT 'SQQSR7S]-WS*PEZSRI+P]GSWMHI 7XVYGXYVIERHT/E

HO OH pKa ~ 11 O (EMH^MR O O 1;! HO (EMH^IMR+PYGSWMHI OH

O OH %GIX]P(EMH^MR 1;! 1EPSR]P(EMH^MR (EMH^MR%GIX]P+PYGSWMHI O O 1;! O C (EMH^MR1EPSR]P+PYGSWMHI CH HO O 3 HO O OH pKa ~ 5.7 O O O O O O HO HO OH OH

O O OH OH

'ST]VMKLX;EXIVW'SVT 4VMGISJ-WSJPEZSRI7XERHEVHW -WSJPEZSRI%REP]XIW 5X] QK  4VMGI (EMH^IMR   (EMH^MR   %GIX]P(EMH^MR   1EPSR]P(EMH^MR   +P]GMXIMR   +P]GMXMR   %GIX]P+P]GMXMR   1EPSR]P+P]GMXMR   +IRMWXIMR   +IRMWXMR   %GIX]P+IRMWXMR   1EPSR]P+IRMWXMR   8SXEPTVMGISJ7XERHEVHW 

1EPSR]P)WXIVWSRFEGOSVHIVJSVWIZIVEP[IIOW 'ST]VMKLX;EXIVW'SVT 'LVSQEXSKVETLMG'LEVEGXIV 6IZIVWI4LEWI'SPYQRWEVI2SX%PMOI

7S]-WSJPEZSRIW *PS[6EXIQ0QMR &EGO4VIWW∼TWM -RNIGXMSR:SP®0 4 3 (IXIGXMSRRQ)\XVEGXIH 6 1SFMPI4LEWI%,3%GT, 5 <8IVVE¬17' ®Q 1SFMPI4LEWI&%G'2,3%G  7 89 \QQ 1 2 30% B 22% B

12% B ®KQ0 3 4 (EMH^MR Q%9 6 +P]GMXMR 2 5 7]QQIXV]' ®Q +IRMWXMR  7 8 9 %GIX]P(EMH^MR \QQ 30% B 1 %GIX]P+P]GMXMR 22% B (EMH^IMR +P]GMXIMR 12% B %GIX]P+IRMWXMR +IRMWXIMR 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 1MRYXIW 'ST]VMKLX;EXIVW'SVT -WSJPEZSRI7TIGXVE

MR%G'2%GIXMG%GMHT,

259.4

TTQ)EGL-WSJPEZSRI

248.8 259.4

255.9 248.8 4SXIRXMEP7IGSRHEV]

255.9259.4 (IXIGXMSR;EZIPIRKXLW 248.8 Q%9*YPP7GEPI RQ

%ZIλ1E\ RQ

200 220 240 260 280 300 320 340 360 380 RQ 'ST]VMKLX;EXIVW'SVT 7S]-WS*PEZSRI7IPIGXMZMX]

7]QQIXV]'®Q \QQ 30.00 pH of DI < 7, at ~ 5.5

25.00 AcetylGenistin Aglucone 20.00 AcetylGlycitin AcetylGlucosides AcetylDaidzin

k' Retention 15.00 Isoflavone Glucosides 10.00

pH 3.4 with 0.1% Acetic Acid pH 6.8 with 10 mM Na Acetate

5.00 345678pH T,FEWIHYTSR[SVOF] T,FEWIHYTSR[SVOF] 4EX1YVTL]IXEP-S[E7XEXI9RMZ 7XIZI&EVRIWIXEP9RMZSJ%PEFEQE 'ST]VMKLX;EXIVW'SVT 7S]-WS*PEZSRI7IPIGXMZMX] %POEPMRIT,[MXL%QQSRMYQ&M'EVFSREXI <8IVVE¬17' 30.00  Genistein pH of DI <7, ~5.5 25.00 AcetylGenistin Glycitein Daidzein 20.00 AcetylGlycitin Isoflavone AcetylGlucosides AcetylDaidzin 15.00 Genistin 6IXIRXMSR  Ok Glycitin 10.00 Isoflavone Daidzin Glucosides

5.00 Aglucone IsoFlavones 0.00 345678910 T, pH 9.1 'ST]VMKLX;EXIVW'SVT 7S]-WS*PEZSRIW6IWSPYXMSR %POEPMRIT,[MXL%QQSRMYQ&M'EVFSREXI 7IPIGXMZMX]'LERKI

¬ 'SPYQR<8IVVE 17'®Q ®KQ0 \QQ (EMH^MR *PS[6EXIQ0QMR +P]GMXMR ∼ &EGO4VIWW TWM +IRMWXMR 9 -RNIGXMSR:SP®0 %GIX]P(EMH^MR 7 3 (IXIGXMSRRQ)\XVEGXIH %GIX]P+P]GMXMR 1SFMPI4LEWI%Q12,,'3T, (EMH^IMR 1SFMPI4LEWI&%G'2 +P]GMXIMR Q12,,'3 %GIX]P+IRMWXMR 1 T, 6 +IRMWXIMR 2 30% B

Q%9 4 22% B 5 8 12% B

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 1MRYXIW 'ST]VMKLX;EXIVW'SVT -WSJPEZSRI7TIGXVE

MR%G'22,,'3T,

259.4

255.9 TTQ)EGL-WSJPEZSRI

248.8 268.8

4SXIRXMEP7IGSRHEV] 253.5258.2 259.4 (IXIGXMSR;EZIPIRKXLW 258.2 248.8 RQ Q%9*YPP7GEPI

%ZIλ1E\ RQ RQ9WIHJSV7MRKPI;EZIPIRKXL(IXIGXMSR

          RQ 'ST]VMKLX;EXIVW'SVT 7S]-WS*PEZSRIW6IWSPYXMSR %POEPMRIT,[MXL%QQSRMYQ&M'EVFSREXI 3TXMQM^IH6IWSPYXMSR

¬ 'SPYQR<8IVVE 17'®Q ®KQ0 \QQ (EMH^MR *PS[6EXIQ0QMR +P]GMXMR &EGO4VIWW∼TWM +IRMWXMR -RNIGXMSR:SP®0 %GIX]P(EMH^MR (IXIGXMSRRQ)\XVEGXIH %GIX]P+P]GMXMR 0S[IVMRMXMEPGSRHMXMSRWF] 1SFMPI4LEWI%Q12,,'3T, (EMH^IMR )\XIRHXMQIQMRJSVWXITERH 1SFMPI4LEWI&%G'2 +P]GMXIMR

9 3 Q12,,'3T, %GIX]P+IRMWXMR 7 +IRMWXIMR 4

5 30% B 6 1 2 22% B 8 Q%9

11% B

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

1MRYXIW 'ST]VMKLX;EXIVW'SVT 7S]-WS*PEZSRIW (IXIGXMSR)WXMQEXI %POEPMRIT,[MXL%QQSRMYQ&M'EVFSREXI 2 µg Each in 50% MeOH 4IEOXLVYEVIFVSEHIRIHHYIXS (EMH^IMR 1I3,MR7XERHEVH 6 7 +P]GMXIMR (EMH^MR -WSGVEXMG[MXL%G'2 8 +IRMWXIMR +VEHMIRXMRMXMEXMSREXQMRYXIW 9 +P]GMXMR +IRMWXMR %GIX]P(EMH^MR %GIX]P+P]GMXMR %GIX]P+IRMWXMR 2 3

5

Q%9 1 4

(IXIGXMSR0MQMX ®KSRGSPYQR

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 1MRYXIW 'ST]VMKLX;EXIVW'SVT 7S]-WS*PEZSRIW 7EQTPI4VITEVEXMSR

&EWIHYTSR1YVTL] WTVSGIHYVI[MXLWS]FEWIHI\XVEGXWERHMRJERXJSVQYPE .%KVMG*SSH'LIQ

# )\XVEGXFIX[IIRERHKSJ7EQTPI[MXL

# Q0%G'2Q0Q1,'PQ0(-

# WXMVSZIVRMKLXEXEQFMIRXXIQTIVEXYVI # 'IRXVMJYKII\XVEGXXSVIQSZIWYWTIRHIHWSPMHW # )ZETSVEXII\XVEGXYRHIV2 # 6IWYWTIRHMRQ0SJ1I3, # %HHMXMSREPHMPYXMSRWEWETTVSTVMEXI[MXL(-[EXIV

'ST]VMKLX;EXIVW'SVT 7S]-WSJPEZSRIW 'SRGIRXVEXIH)\XVEGX 7EQTPI7S]0MJI-WSJPEZSRIW 'SPYQR

Q%9 'LVSQEXSKVEQW

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 1MRYXIW 'ST]VMKLX;EXIVW'SVT 7S]-WSJPEZSRIW'SRJMVQEXMSR 'SRGIRXVEXIH)\XVEGX

  'SPYQR7]QQIXV] '®Q 7EQTPI7S]0MJI -WSJPEZSRIW \QQ EW+P]GSWMHIW *PS[6EXIQ0QMR KQ0 1I3, &EGO4VIWW∼TWM -RNIGXMSR:SP®0 (EMH^IMR (IXIGXMSRRQ)\XVEGXIH +P]GMXIMR 1SFMPI4LEWI%,3%GT, (EMH^MR 1SFMPI4LEWI&%G'2,3%GT, +IRMWXIMR 7 +P]GMXMR 3 5 +IRMWXMR 6 %GIX]P(EMH^MR 8 %GIX]P+P]GMXMR %GIX]P+IRMWXMR 3ZIVPEMH 9 'LVSQEXSKVEQW 1 2 6 1500 mAU 1500

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

Minutes 'ST]VMKLX;EXIVW'SVT 7S]-WSJPEZSRIW (MIXEV]7YTTPIQIRX

'SPYQR

2 7 9 4 5 8

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 1MRYXIW 'ST]VMKLX;EXIVW'SVT 7S]-WSJPEZSRIW (MIXEV]7YTTPIQIRX

=SYV0MJI 'SPYQR

(MPYXIH 1SFMPI4LEWI%G'2Q12,,'3T,

'SRXEMRMRKQK (EMH^IMR! 7S]-WSJPEZSRI)\XVEGX +P]GMXIMR! (EMH^MR! 6

Q%9 +IRMWXIMR 3 +P]GMXMR!

440 +IRMWXMR! %GIX]P(EMH^MR! %GIX]P+P]GMXMR! %GIX]P+IRMWXMR! 5 7 9 1 2 8

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 1MRYXIW 'ST]VMKLX;EXIVW'SVT 1IXLSH(IZIPSTQIRX *YXYVI'SRWMHIVEXMSRW

# 1IXLSHRIIHWXSMRGSVTSVEXIERHVIWSPZIXLIQEPSR]P KP]GSWMHIIWXIVWJVSQXLI-WSJPEZSRIWYWIHMRXLMWWXYH]

# %ZEMPEFMPMX]SJWXERHEVHW

# )\TIGXIHXSIPYXIEJXIVXLIMWSJPEZSRIKPYGSWMHIWERHFIJSVIXLI EGIX]PIWXIV[MXLEGMHMGQSFMPITLEWI

# )\TIGXIHXSIPYXIFIJSVIMWSJPEZSRIKPYGSWMHIWMRXLIVIKMSRSJ EKPYGSRIIPYXMSR[MXLFEWMGQSFMPITLEWI

# 9WIT,XSSTXMQM^IWIPIGXMZMX]ERHVIWSPYXMSR

# 9WI1EWW7TIGXSLIPTPSSOJSVEREP]XIW[MXLSYXXLIYWISJ WXERHEVHW

'ST]VMKLX;EXIVW'SVT 1IXLSH(IZIPSTQIRX *YXYVI'SRWMHIVEXMSRW

# 9XMPMX]SJXLIQIXLSHJSVSXLIVMWSJPEZSRIWERHJPEZSRI WXVYGXYVIWWYGLEW

# *SVQSRSRIXMR&MSGLERMR%)UYSP(ML]HVSHEMH^IMR0YXISPMR 5YIVGIXMR%TMKIRMR/EQTJIVSP1]VMGIXMR2EVMRKIRMR ,IWTIVIXMR'EXIGLMR

# 3XLIVTSP]TLIRSPMGWXVYGXYVIW

'ST]VMKLX;EXIVW'SVT 1IXLSH(IZIPSTQIRX *YXYVI'SRWMHIVEXMSRW 7EQTPI4VITEVEXMSR # )PMQMREXIXLIRIIHXSIZETSVEXIXLII\XVEGXERH VIWYWTIRHMRXS1I3,

# -QTVSZIIEVP]IPYXMRKTIEOWLETIYWMRKXS1I3,

# 4VIJIVXSI\XVEGXMR%G'22,,'3QSFMPITLEWIXLIR HMPYXI[MXLQSFMPITLEWI

# 7MRKPIWEQTPITVITJSVLMKLPMTMHWEQTPIWWYGLEW HEMV]TVSHYGXW

# 4SXIRXMEPJSV7SPMH4LEWI)\XVEGXMSR

'ST]VMKLX;EXIVW'SVT 1IXLSH(IZIPSTQIRX *YXYVI'SRWMHIVEXMSRW 'LVSQEXSKVETL] # 3TXMQM^IVIWSPYXMSRERHXLIKVEHMIRXWLETIJSVQEPSR]P KPYGSWMHIW[MXLFSXLEGMHMGERHFEWMGQSFMPITLEWIW # (IZIPSTEVIXIRXMSRXMQIMRHI\SJORS[RMWSJPEZSRI EREP]XIW # 3TXMQM^I4(%MHIRXMJMGEXMSRMRGSQTPI\QEXVMGIW

# TIEOTYVMX]ERHPMFVEV]WTIGXVYQQEXGLMRK

# RSMWIWTIGXVYQ # 9WISJ1EWW7TIGXVSQIXV]JSVMHIRXMX]GSRJMVQEXMSR

# 1SPIGYPEV;IMKLXEMHXSWIEVGLJSVERHMHIRXMJ]YRORS[RW'ST]VMKLX;EXIVW'SVT %REP]WMWSJ7S]-WSJPEZSRIW 9WMRK4(%ERH1EWW7TIG

# 8LMW[SVOWIXWXLIJSYRHEXMSRJSVGLVSQEXSKVETLMG GSRHMXMSRWXSJYRGXMSREWGSQTPMQIRXEV]ERHGSRJMVQEXMSREP GLIQMWXVMIW'LVSQEXSKVETLMGWIPIGXMZMX] # -REGSQTPI\QEXVM\WYGLEWJSSHSVREXYVEPTVSHYGXWXLIVI MWEP[E]WXLIVMWOSJ # UYERXMXEXMSRFMEWHYIXSEREP]XIGSIPYXMSR[MXLYRORS[RGSQTSRIRXW # JEPWITSWMXMZIMHIRXMJMGEXMSR # 9WI4(%ERH1EWW7TIGMHIRXMJMGEXMSRGERIPMQMREXIXLMW TVSFPIQ(IXIGXMSRWIPIGXMZMX] # %QIRHEFPIXS5'ERH6 (

'ST]VMKLX;EXIVW'SVT