US 20140004214A1

(19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0004214 A1

Kedrowski et al. (43) Pub. Date: Jan. 2, 2014

(54) METHODS TO ISOLATE ANTI-MICROBIALS Publication Classi?cation FROM FRUIT OR SEED EXTRACTS

(51) Int. Cl. (71) Applicant: Wisconsin Alumni Research A61 K 36/45 (2006.01)

Foundation, Madison, WI (US) C07D 493/14 (2006.01)

(72) Inventors' Brant Lawrence KedroWski Oshkosh (52) US‘ Cl‘ ' ’ ’ CPC ............ .. A61K36/45 2013.01 ' C07D 493/14

WI (US); Teri Shors, Madison, WI (US) ( )’ (2013 01)

Assigneej Wisconsin Alumni Research ......................................... .. Foundation, Madison, WI (US)

(21) Appl.No.: 13/918,070 (57) ABSTRACT

(22) Filed: Jun. 14, 2013

Related U's' Apphcatlon Data The invention provides a method of isolating one or more

(60) Provisional application No. 61/660,332, ?led on Jun. compounds present in fruit juice or seed extracts that have 15, 2012. anti-microbial activity.

Patent Application Publication Jan. 2, 2014 Sheet 1 0f 22 US 2014/0004214 A1

Figure 1. Separation scheme for ‘BOMX cranberry powder.

Ocean Spray Gruc'a QGMX Cranberiy

Powder

Disscive in water, 045 micron iiiter

insoiubie residue + Fiétered Juice

1 cm Sciid Phase Extraction

Fraction Fraction Fraction Fraction F6 + F1 + F2 4- F3

ion exchange chromatography ion exchange

Fractio n Fraction Fraction Fracnon Fsa + Fab

‘ cs prep~HPLC " ca prep-HPLC

Figure 2. Separation scheme for pressed cranberry seed powder.

Botanic Oil innovations Pressed Cranberry Seed Powder

Suspend in methanoi/wa’zer, 0,45 micron ?lter

insoiubie residue + Seed Extraci

G8 prep-HPLC

Fraction Fraciion Fractio n Fra ction E0 + E1 + E2 + E3

Patent Application Publication Jan. 2, 2014 Sheet 2 0f 22 US 2014/0004214 A1

Figure 3. Overlaid chromatograms of fractions F1 (blue), {green} and F3 {red} from the ?rst purification using C18 solid phase exti'actinn.

350

325

300

mAL)

0 5 1a 15 20 2s s5 40 A5 55 so as m 75 sh 8'5 90 Minutes

Overiaid HPLC chromatogmms of pigment fractions F1 (b1ue),F2 {green}, and F3 (red) from QOMX cranberry powder separated by C18 solid phase extraction. All peaks were detected at 340 mm

Patent Application Publication Jan. 2, 2014 Sheet 3 0f 22 US 2014/0004214 A1

reen], before and after the second puri?catinn step with strong catiun exchange chromatography, respecti‘v'ely. Fi ure 4-. Overhzid aiwmmntovrnmsz offractions F2 "red‘ and F23 o J g

m m ,w a

MA

iii-egg» / m w a“ r / J A, A\\\ M ff». ,2. x A A?iik W, It. ,

a w A 2. m .

g

u a 0 u 7 0 0 0 ,w 7 1

m w w w 0

50

Minutes

HPLC chromatograms of‘J‘OMX pigment fractions F2 {red} and F'Za (green), before and after strong Cation exchange chromatography, respectively.

A A1} beaks were detected between 250~600 nm.

Patent Application Publication Jan. 2, 2014 Sheet 4 0f 22 US 2014/0004214 A1

Figure 5. Overland chromatograms nffractions F232 {red}, F233 (green). F234 (blue) and FZaS (mamon) after the {bird puri?cation step with prep scale CS HPLC.

:agcsww. my Ho!

Alb '

0.6 l

Minutes

Overlaid i-IPLC chroinatograinss of‘BOMX pigment fractions F2212 ue} and 13 (red), F2a3 {green}, F2514 (b after the third puri?cation step with prep scale (18 HPLC. All pea ls; were detected etw-zen 250600 (maroo nm.

Figure 6!). Analytical HPLC used for final purificatian of fraction FZaB at 50 min.

Peaks detected at 355 11

Figure 63. Analytical HPLC used for final purification offi'actin‘n at 56 min.

Separateil using a pen [alluomphenyl (2} phase column (from Phenmm'mex). Peaks detected at 277 nm.

Patent Application Publication Jan. 2, 2014 Sheet 5 0f 22 US 2014/0004214 A1

Figure 7. Antiviral activity of fractions FZaZ and F2213 against vaccinia virus;

‘31; inhibitinn O . '

£00 mgfL ii) mg/L i mg/L

Concentration

Figure 8. Antiviral activiiy offi'aciitms F2a2 and F233 against 100 'E‘CIDSO Pnliovirus 1

80

i: a

'33 60

53 4t- -

55’ .20

M90 nag/L i0 Jug/i. 1 1113/1)

Concentration

Figure 9. Antiviral activity of fractions; F2212 and F2213 against. ‘10 TCIDSO in?uenza A/HlNl

“QM F2 :12

====E - F226 "/5 inhibition

iO-J mg/L 1-) mg/L 1 mg/L Concentration

Patent Application Publication Jan. 2, 2014 Sheet 6 0f 22 US 2014/0004214 A1

Figure 10, Overlaid chromatograms 0f cranberry seed puwdes' fractions E1 {blue}, E2 {green}, and E3 ,6. scale (maroon) after purification prep

40.0 52.5 .5 30.0 25.0 BUG

2 .. 2.

2 z _

1

G O 5 0 m. 22a 0

4 s 2 2 1 1

:<E

25 225

Minutes 101."

Overiaid HPLC chromatograms of cranberry seed powder fractions E1 {blue}, E2 {green}, and E3 (maroon) after purification with prep scale C8 HPLC. All peaks were detected between 250660 nm.

Figure 11, Antiviral activity of fractions E0, E1, E2 and. E3 vaccinia ‘13115

2 2 2 2 2 2 2 2 2 2 2 2

.2 2

ha» 22

.2 22

2 2 2 Z 2

.2 2 w 2

2

2... 22 ... 22 L2 2

2

. 22

. 2

2 2 2 2 2

Hu 0 Hu 0 ,- nU ?v 0 on 6 A. 2 2 222252222 ,

i 111:; L 072: mg/L 1-1) m :00 mg/L

Concentraimn

Patent Application Publication Jan. 2, 2014 Sheet 7 0f 22 US 2014/0004214 A1

Figure 12. Example of vaccinia virus plaque reduction assay plates after staining

(i) Vaccinia virus

control

Figure 13,

Rep. 3

Rep. 4

Control Plate Test Sample Plate 200 H) nig/L

rug/L 3 wells 3 W911’;

(~) Vaccima virus

Example of Poliovirus 1 cytopathic effect inhibition plates aftEr staining

POHGVH‘US 1 Back Titer Plate 10-7 w3 10-19 [>1

F iii

'i‘esti Sampkl. Plate 10 1 0.1 g-) r-)

1 0O ‘PC5950

Polio

IHT'U TCEDEO

Poiio

iii-0 TCEEJSO

P0110

1 0O TCEDSO

PoiiC

Patent Application Publication Jan. 2, 2014 Sheet 8 0f 22 US 2014/0004214 A1

Figure 14. Example of hemaggluainaticm test. used to confirm inhibition of'in?uenzca A/HiNi cvtupathic e?‘ect

atriccim'mmim {Hbzmugglutimiiun

51mm: fm'malion= (--) licmrgg'unlnation

Patent Application Publication Jan. 2, 2014 Sheet 9 0f 22 US 2014/0004214 A1

Result: 104-5 Parain?wznza 3 : 1G6 T613350) 10's‘5 E’araim'luenza 3 r 10 TCIDSO

FZaZ % inhibition Comenti'atiwn ‘100 TIIIFDSO Pzu'aim’luerrza 3 1G TCZBSG Parzain?uema ?

Test 1. Test 2 Test. 3 Ave Test 1. Test 2 Test 3 Ave 100 ggfmi, 106 100 ‘100 106 100 10(1 100 10G 10 §.ag/mL 58 100 75 75 100 100 100 100 1 pg} m1’ 0 0 {3 G 100 1G0 104i 1G0 190 ngjmL 0 0 I} 0 ‘.75 7/5 ‘75 7'5

@192”

Patent Application Publication Jan. 2, 2014 Sheet 11 0f 22 US 2014/0004214 A1

I§.1i':.ibi‘§iun. of 311353 'ii'i'l {515;} iiiji‘v’éjé

it,

Patent Application Publication Jan. 2, 2014 Sheet 12 0f 22 US 2014/0004214 A1

FZaZ % Inhibition

Concentratim-x 100 TCEDSG in?uenza AKHBNZ 1.0 TCIDSO In?uenza AJIBNE

Test Test 2 T125133 {we Test 1 Test 2 Test 3 Ave

100 mg/L 1G0 100 160 100 100 180 100 100 10 nag/‘L 0 50 55) 33 ‘106 1.00 100 160 1 mg/L 0 U U 0 5O 50 50 5i) ()1 mgfi. O (1 O 0 0 O (3 (I

M i‘ZeaZé (‘/0 Inhibition

Concentration 100 TCEDSO Influenza A/HENZ i0 TCIDSQ in?uenza AJHHNP;

Test Test .2 Test 3 Ave Test 1 Test 2 Test 3 Ave

100 mgy'L 100 100 160 10G 106 160 109 100 10 mgfi, G {J O 0 50 100 56 6’? 1 mg/L [1 {J O 0 5O 0 O 17 ()1 mg/L 0 0 O O 0 O U 0

255; my?

Patent Application Publication Jan. 2, 2014 Sheet 13 0f 22 US 2014/0004214 A1

w m.

... 8M 8M 8N aw?

.....e............$..$5$....... .25.»? .2. 32:53:25; ~~~~~~~~~ 5......’ v5...- 2252a

M

mm cam magma W EQQBMWEQEO cow xmmd mwmm w

Patent Application Publication Jan. 2, 2014 Sheet 14 0f 22 US 2014/0004214 A1

k H

. \\ gm!” _ y . & , , ~ . ~ ~ . . . , 4 _ . ~ ~ ' . g .. 4 ~ ~ . .

w .w 25$ 2%

mm Q9 gmm? am am m

smm 6. “Wm “mum? w

~.... mm com m?mmwoa mam

Patent Application Publication Jan. 2, 2014 Sheet 15 0f 22 US 2014/0004214 A1

Patent Application Publication Jan. 2, 2014 Sheet 16 0f 22 US 2014/0004214 A1

% , 5 SN. .9»? .

8m 7 5m . QB . can“ an.“ _ Sm Sm . EN

2

é. n2. i 3.“ mm c9 @émmmz E?mBwEoEU c2 xwwa mmmm 83 Qua oQm mmh an; M. .IL. TVL‘VTITI:

and

. 9,1

mm 5 95mg ESmQmEoEQ c2 xwwm wwwm

g

mi: EQMMBM W.

Patent Application Publication

ramntian time = 3.38 min matecuiar formula = ?mHmii‘iia moiacuar wsight : $89.68 amu name : myricetin?'gaiamosiée

Jan. 2, 2014 Sheet 17 0f 22

reteminn time = 3.9? min moiecuim fmmuia : ?nHw?s maiacuar weight = 224.87 amu name = ainapinic acid

retention 'xime a 4.67 min maiecuiar ?mmuia : Gag-£2,523,5 moiesuar weight w 360.51) amu name = tripm?de

US 2014/0004214 A1

US 2014/0004214 A1

Preparad at 40 xtrzact E nice 10G dc:

Jan. 2, 2014 Sheet 18 0f 22

ni'bithn of Vaccinia Virus with “ranben'y J C, 60 deg j and

in?

i 2

Patent Application Publication

Figure 19

a1‘ 1' j'uicts Extract F22! Prepared. at 40 C, 60 deg C and 100 deg C

Vaccinia Virus with Crank dag

100i) 7

000

Inhibitien of

70m?

60.00

500G

40.0‘;

2 .(JL

100C

Patent Application Publication Jan. 2, 2014 Sheet 19 0f 22 US 2014/0004214 A1