a. Chemical structures of three predominant flavonol aglycones

Figure 1: Chemical structures of three predominant flavonol aglycones (a) and ginkgolic acids (b) in G. biloba

Factors

Levels

-1

0

1

Pretreatment temperature (A)

160

180

200

liquid-to-Solid ratio (B)

50:1

55:1

60:1

Extraction temperature (C)

80

90

100

Figure 2: The rutin standard curve

Figure 3: Experimental conditions and results of single factor analysis

Figure 4: G. biloba leaves powders with or without different high temperature pretreatment: (a) without high temperature pretreatment, (b) 100 ℃, (c) 120 ℃, (d) 140 ℃, (e) 160 ℃, (f) 180 ℃, (g) 200 ℃

Number

A (Pretreatment temperature)

B (Liquid-to-solid ratio)

C (Extraction temperature)

Content (%)

1

160

60

90

3.16902

2

200

55

100

3.12114

3

160

50

90

3.16902

4

180

55

90

3.47609

5

200

60

90

3.21945

6

180

55

90

3.45183

7

160

55

100

3.32606

8

200

50

90

2.91621

9

180

60

80

3.28010

10

160

55

80

3.11731

11

180

55

90

3.51631

12

180

50

100

3.35862

13

180

60

100

3.42757

14

180

50

80

3.12624

15

180

55

90

3.45055

16

200

55

80

3.07070

17

180

55

90

3.49716

Source

Sum of Squares

Df

Mean Square

F Value

p-value Prob>F

Model

0.50

9

0.056

63.096

< 0.0001

significant

A

0.026

1

0.026

29.05

0.0010

B

0.035

1

0.035

39.01

0.0004

C

0.051

1

0.051

57.58

0.0001

AB

0.023

1

0.023

25.93

0.0014

AC

0.006

1

0.006

7.07

0.0325

BC

0.002

1

0.002

2.03

0.1970

A2

0.262

1

0.262

295.98

< 0.0001

B2

0.051

1

0.051

57.79

0.0001

C2

0.021

1

0.021

23.23

0.0019

Residual

0.006

7

0.001

Lack of Fit

0.00

3

0.00

1.19

0.4186

not significant

Pure Error

0.00

4

0.00

Cor Total

0.510

16

Figure 5: Interactive effects of pretreatment temperature, liquid-to-solid ratio, and extraction temperature on the content of G. biloba leaves flavonoids (as predicted by 3-D surfaces and contour plots)

Figure 6: UV Chromatogram at 360nm of the 60% ethanol extracts

Figure 7: The total ion current chromatogram of the 60% ethanol extracts

Id#

Compound

m/z MS

Adduct

RT

m/z MS/MS

Reference

1

3-O-[6-O-(α-L-rhamnosyl)-ß-D-glucosyl] myricetin

625.1399

M-H

5.570

316,271

23

2

3-O-[2-O-(6-O-p-coumaroyl)-β-D-glucosyl]-α-L-
rhamnosyl-7-O-(β-D-glucosyl) quercetin

917.2349

M-H

6.118

755, 609, 462, 300

23

3

3-O-[2-O, 6-O-bis (α-L-rhamnosyl)-β-D-glucosyl]
kaempferol

739.2077

M-H

6.243

284

23

4

3-O-[2-O, 6-O-bis (α-L-rhamnosyl)-β-D-glucosyl]
isorhamnetin

769.2178

M-H

6.360

314

23

5

3-O-[6-O- (α-L-rhamnosyl)-β-D-glucosyl] quercetin

609.1441

M-H

6.810

464, 300

23

6

3-O-[6-O- (α-L-rhamnosyl)-β-D-glucosyl]-3-
methylmyricetin

639.1552

M-H

7.077

331

23

7

3-O-(β-D-glucosyl] quercetin

463.0000

M-H

7.162

301

23

8

3-O-[6-O-(α-L-rhamnosyl)-β-D-glucosyl]
kaempferol

593.1490

M-H

7.823

285

23

9

kaempferol 3-O-β-D-glucoside

447.0851

M-H

8.162

284, 255

24

10

7-O-β-D-glucosyl apigenin

431.0948

M-H

8.529

268

23

11

quercetin 3-O-[2-O, 6-O-bis (α-L-rhamnosyl)-β-D-
glucoside]

755.1827

M-H

10.221

609, 300

25

12

Quercetin

301.0337

M-H

13.293

151, 107

23

13

Kaempferol

285.0383

M-H

18.509

227, 159, 117

23

14

Isorhamnetin

315.0000

M-H

19.447

300, 271, 151

23

a. Reducing Power Assay

b. DPPH Radical Scavenging Activity Assay

c. Scavenging of Hydrogen Peroxide Assay

Figure S1: The mass spectrogram of Compound 1

Figure S2: The mass spectrogram of Compound 2

Figure S3: The mass spectrogram of Compound 3

Figure S4: The mass spectrogram of Compound 4

Figure S5: The mass spectrogram of Compound 5

Figure S6: The mass spectrogram of Compound 6

Figure S7: The mass spectrogram of Compound 7

Figure S8: The mass spectrogram of Compound 8

Figure S9: The mass spectrogram of Compound 9

Figure S10: The mass spectrogram of Compound 10

Figure S11: The mass spectrogram of Compound 11

Figure S12: The mass spectrogram of Compound 12

Figure S13: The mass spectrogram of Compound 13

Figure S14: The mass spectrogram of Compound 14