J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Review

Volume 7, September 2019, pages 27-35

UF-LC-DAD-MSⁿ for discovering enzyme inhibitors for nutraceuticals and functional foods

Figure 1. Typical procedures of the ultrafiltration liquid chromatography-mass spectrometry (UF-LC-MS) method.

Figure 2. Ultrafiltration LC-DAD chromatograms of potential 5-LOX inhibitors in kudzu (Pueraria lobate) as compared to that of the crude extract without affinity purification. 5-LOX concentration: a, 0 ng/µL; b, 0.1 ng/µL; c, 0.2 ng/µL; d, 0.3 ng/µL; e, crude extract of P. lobata. Peaks 1, Puerarin; 2, Daidzin; 3, 3′-Methoxy-puerarin; 4, 3′-Hydroxy-puerarin; 5, Daidzein. Figure adopted from Zhao et al., 2016 with permission.

**Table 1.** Major MMP-2/5-LOX inhibitors identified in plant extracts by UF-LC-MS
Compounds	MW^a	Relative binding to enzyme^b	IC₅₀^d (µM)	Source	References^e
^aMW: molecular weight; ^bRelative binding to enzyme = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; ^cConcentration of MMP-2 and 5-LOX were 20 and 0.1ng/μL, respectively. ^dIC₅₀: Concentration required for 50% inhibition of the enzyme activity under the assay conditions. ^eRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.
Hydroxyl safflower yellow A	612	2.01	5.76	137.3	3.72	C. tinctorius	Li et al. 2014; Zhao et al. 2016
Anhydrosafflor yellow B	1,062	16.29	22.93	2.82	3.45
Astilbin	450	14.44	40.78	107.8	8.45	S. glabra	Li et al. 2015; Zhao et al. 2016
Isoastilbin	450	-	4.30	-	46.6
Engelitin	434	21.14	5.07	106.9	21.33
Isoengelitin	434	-	1.54	-	191.31
Resveratrol	228	41.59	44.26	53.1	4.38
prim-O-Glucosylcimifugin	468	4.40	9.22	108.87	9.76	S. divaricata
4′-O-β-D-Glucosyl-5-O- methylvisamminol	452	12.42	32.55	15.6	15.53
Cimifugin	307	0.80	1.35	313.25	93.66
sec-O-Glucosylhamaudol	438	0.63	35.96	344.4	7.45
Puerarin	416		-		25.8	P. lobata	Zhao et al. 2016
Daidzin	416		40.41		4.32
3′-Methoxy-puerarin	432		-		-
3′-Hydroxy-puerarin	446		18.28		7.36
Daidzein	254		19.68		6.65

Table 1. Major MMP-2/5-LOX inhibitors identified in plant extracts by UF-LC-MS

Compounds

MW^a

Relative binding to enzyme^b

IC₅₀^d (µM)

Source

References^e

MMP-2^c

5-LOX^c

MMP-2

5-LOX

^aMW: molecular weight; ^bRelative binding to enzyme = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; ^cConcentration of MMP-2 and 5-LOX were 20 and 0.1ng/μL, respectively. ^dIC₅₀: Concentration required for 50% inhibition of the enzyme activity under the assay conditions. ^eRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.

Hydroxyl safflower yellow A

612

2.01

5.76

137.3

3.72

C. tinctorius

Li et al. 2014; Zhao et al. 2016

Anhydrosafflor yellow B

1,062

16.29

22.93

2.82

3.45

Astilbin

450

14.44

40.78

107.8

8.45

S. glabra

Li et al. 2015; Zhao et al. 2016

Isoastilbin

450

4.30

46.6

Engelitin

434

21.14

5.07

106.9

21.33

Isoengelitin

434

1.54

191.31

Resveratrol

228

41.59

44.26

53.1

4.38

prim-O-Glucosylcimifugin

468

4.40

9.22

108.87

9.76

S. divaricata

4′-O-β-D-Glucosyl-5-O- methylvisamminol

452

12.42

32.55

15.6

15.53

Cimifugin

307

0.80

1.35

313.25

93.66

sec-O-Glucosylhamaudol

438

0.63

35.96

344.4

7.45

Puerarin

416

25.8

P. lobata

Zhao et al. 2016

Daidzin

416

40.41

4.32

3′-Methoxy-puerarin

432

3′-Hydroxy-puerarin

446

18.28

7.36

Daidzein

254

19.68

6.65

**Table 2.** Major COX-2 inhibitors identified in plant extracts by UF-LC- MS
Compounds	MW^a	Relative binding to COX-2^b	IC₅₀ (μM)^c	Source	References^d
^aMW: molecular weight; ^bRelative binding to COX-2 = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; ^cIC₅₀: Concentration required for 50% inhibition of the enzyme activity under the assay conditions; ^dRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.
10-Gingerol	350	0.17	32.0	Z. officinale	Van et al. 2011
8-Shogaol	304	0.27	17.5
10-Shogaol	332	0.35	7.5
Senkyunolide O	380	52	5	L. chuanxiong	Cao et al. 2010
Phenethyl-trans-ferulate	297	25	31	N. incisum
Cryptoanshinone	296	31	22	S. miltiorrhiza
Roburic acid	440	45	9	G. macrophylla

Table 2. Major COX-2 inhibitors identified in plant extracts by UF-LC- MS

Compounds

MW^a

Relative binding to COX-2^b

IC₅₀ (μM)^c

Source

References^d

^aMW: molecular weight; ^bRelative binding to COX-2 = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; ^cIC₅₀: Concentration required for 50% inhibition of the enzyme activity under the assay conditions; ^dRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.

10-Gingerol

350

0.17

32.0

Z. officinale

Van et al. 2011

8-Shogaol

304

0.27

17.5

10-Shogaol

332

0.35

7.5

Senkyunolide O

380

L. chuanxiong

Cao et al. 2010

Phenethyl-trans-ferulate

297

N. incisum

Cryptoanshinone

296

S. miltiorrhiza

Roburic acid

440

G. macrophylla

**Table 3.** α-Glucosidase inhibitors identified by UF-LC-MS
Compounds	MW^a	Source	References^b
^aMW: molecular weight; ^bRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.
Quercetin	448	G. biloba; A. senticosus	Liu et al. 2013; Zhou et al. 2012
Apigenin	270	G. biloba	Liu et al. 2013
Kaempferol	286
Isorhamnetin	316
Rutin	610	A. senticosus	Zhou et al. 2012
Hyperin	464
Isoquercitrin	464
1,5-Dicaffeoylquinic acid	516
1,4-Dicaffeoylquinic acid	516
4,5-Dicaffeoylquinic acid	516
5,7,3,2′,6′-Pentahydroxy flavanone	304	S. baicalensis	Wang et al. 2014
Chrysin-6-C-arabinosyl-8-C-glucoside	548
Chrysin-6-C-glucosyl-8-C-arabinoside	548
5,7,2′,5′-Tetrahydroxy-8,6′-dimethoxy flavonne	346
Baicalin	446
Oroxylin A-7-O- glucuronide	460
Wogonoside	460
Baicalein	270
Skulllcapflavon II	374
Wogonin	284
Oroxylin A	284
Quercetin-3-O-rha- (1–4)-glcosyk-rhamnoside	756	C. oxyacantha	Li et al. 2009
Vitexin-2″-O-glucoside	594
Vitexin-2″-O-rhamnoside	578
Vitexin	432

Table 3. α-Glucosidase inhibitors identified by UF-LC-MS

Compounds

MW^a

Source

References^b

^aMW: molecular weight; ^bRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.

Quercetin

448

G. biloba; A. senticosus

Liu et al. 2013; Zhou et al. 2012

Apigenin

270

G. biloba

Liu et al. 2013

Kaempferol

286

Isorhamnetin

316

Rutin

610

A. senticosus

Zhou et al. 2012

Hyperin

464

Isoquercitrin

464

1,5-Dicaffeoylquinic acid

516

1,4-Dicaffeoylquinic acid

516

4,5-Dicaffeoylquinic acid

516

5,7,3,2′,6′-Pentahydroxy flavanone

304

S. baicalensis

Wang et al. 2014

Chrysin-6-C-arabinosyl-8-C-glucoside

548

Chrysin-6-C-glucosyl-8-C-arabinoside

548

5,7,2′,5′-Tetrahydroxy-8,6′-dimethoxy flavonne

346

Baicalin

446

Oroxylin A-7-O- glucuronide

460

Wogonoside

460

Baicalein

270

Skulllcapflavon II

374

Wogonin

284

Oroxylin A

284

Quercetin-3-O-rha- (1–4)-glcosyk-rhamnoside

756

C. oxyacantha

Li et al. 2009

Vitexin-2″-O-glucoside

594

Vitexin-2″-O-rhamnoside

578

Vitexin

432

**Table 4.** XOD inhibitors identified by UF-LC-MS
Compounds	MW^a	Source	References^b
^aMW: molecular weight; ^bRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.
Amentoflavone	538	S. tamariscina	Wang et al. 2014
Robustaflavone	538
Tanshinone II B	310	S. miltiorrhizae	Liu et al. 2013
Tanshindiol B	312
Tanshindiol A	312
15,16-Dihydrotanshinone I	278
1,2-Dihydrotanshinone I	278
Danshenxinkun B	280
Cryptotanshinone	296
Tanshinone I	276
3-Hydroxy methylenetanshinquinone	294
Mmethylene tanshinquinone	278
Tanshinone II A	294

Table 4. XOD inhibitors identified by UF-LC-MS

Compounds

MW^a

Source

References^b

^aMW: molecular weight; ^bRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.

Amentoflavone

538

S. tamariscina

Wang et al. 2014

Robustaflavone

538

Tanshinone II B

310

S. miltiorrhizae

Liu et al. 2013

Tanshindiol B

312

Tanshindiol A

312

15,16-Dihydrotanshinone I

278

1,2-Dihydrotanshinone I

278

Danshenxinkun B

280

Cryptotanshinone

296

Tanshinone I

276

3-Hydroxy methylenetanshinquinone

294

Mmethylene tanshinquinone

278

Tanshinone II A

294

**Table 5.** Screening the NA inhibitors from *R. scutellaria* extract by ultrafiltration LC-MS
Compounds	MW^a	Relative binding to NA^b	IC₅₀ (μM)^c	Source	References^d
^aMW: molecular weight; ^bRelative binding to Neuraminidase = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; ^cIC₅₀: Concentration required for 50% inhibition of the enzyme activity under the assay conditions; ^dRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.
Baicalin	446	20.32	49.34	R. scutellaria	Liu et al. 2011
oroxylin A-7-O-gluacid	460	18.05
wogonin	460	20.01	29.83
Baicalein	270	25.16	30.52
wogonoside	284	26.08
oroxylin A	284	28.23

Table 5. Screening the NA inhibitors from R. scutellaria extract by ultrafiltration LC-MS

Compounds

MW^a

Relative binding to NA^b

IC₅₀ (μM)^c

Source

References^d

^aMW: molecular weight; ^bRelative binding to Neuraminidase = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; ^cIC₅₀: Concentration required for 50% inhibition of the enzyme activity under the assay conditions; ^dRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.

Baicalin

446

20.32

49.34

R. scutellaria

Liu et al. 2011

oroxylin A-7-O-gluacid

460

18.05

wogonin

460

20.01

29.83

Baicalein

270

25.16

30.52

wogonoside

284

26.08

oroxylin A

284

28.23

**Table 6.** Screening the QR-2 inhibitors from extracts of marine sediment bacterial cultures by ultrafiltration LC-MS
Compounds	MW^a	IC₅₀ (μM)^c	source	References^d
^aMW: molecular weight; ^bRelative binding to Quinone reductase-2 = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; ^cIC₅₀: Concentration required for 50% inhibition of the enzyme activity under the assay conditions; ^dRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.
xanthohumol	354	196	H.lupulus	Choi et al. 2011
xanthohumol D	370	110
tetrangulol methyl ether	318	0.16	Actinomycessp

Table 6. Screening the QR-2 inhibitors from extracts of marine sediment bacterial cultures by ultrafiltration LC-MS

Compounds

MW^a

Relative binding To QR-2^b

IC₅₀ (μM)^c

source

References^d

^aMW: molecular weight; ^bRelative binding to Quinone reductase-2 = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; ^cIC₅₀: Concentration required for 50% inhibition of the enzyme activity under the assay conditions; ^dRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.

xanthohumol

354

196

H.lupulus

Choi et al. 2011

xanthohumol D

370

110

tetrangulol methyl ether

318

0.16

Actinomycessp