Secondary metabolite mapping

identifies Scutellaria inhibitors

of human lung cancer cells

Jiayu Gao, Huiying Zhao, Peter J. Hylands, Olivia Corcoran

Journal of Pharmaceutical and Biomedical Analysis,

In press, 2010

IntroductionIntroduction

Scutellaria baicalensis Georgi root, used as a adjuvant in conventional chemotherapy.

Quality of herbs, wide variability in quality amongst commercial products of identical

S. baicalensis and claimed formulation.

Functional biomarkers, chemical constituents shown to have in vitro activity relevant to

therapeutic use.

Baicalin, baicalein, wogonin, and oroxylin A, functional biomarkers of anti-cancer

activities against a wide range of cancer cells except lung cancer.

HPLC and NMR secondary metabolite model for functional biomarker identification.

0

10

20

30

40

50

60

0 0.1 0.2 0.3 0.4 0.5 0.6

Concentration of S.baicalensis (mg/ml)

Gro

wth

inh

ibo

tory

(%

)

sample from CHINA

sample from LONDON

MethodMethod

Metabolic profile (chemical fingerprint), generated by HPLC and NMR spectroscopic

data.

Principal component analysis (PCA) and Partial least squares analysis (PLS)

PCA, a tool for outlier detection and discerning patterns and trends in

multidimensional data.

PLS, a model that describes the correlation between the data matrix and the feature of

interest.

Preparation of extracts and MTT testPreparation of extracts and MTT test

25% EtOH 45% EtOH 70% EtOH 100% EtOH

100% MeOH

Methylene Chloride

Ethyl Acetate

Butanol Water

Initial analysis of HPLC and NMR dataInitial analysis of HPLC and NMR data

0

20

40

60

80

100

0 50 100 150 200 250 300Concentration of extract (μg/ml)

Gro

wth

inhi

bitio

n (%

)

25% ethanol

45% ethanol

70% ethanol

100% ethanol

Methylene chloride

Ethyl acetate

Butanol

Water

？ ？

HPLC-PCA mapHPLC-PCA map

IC50>124ug/ml

58<IC50<124ug/ml

IC50<58ug/ml

Loading and contribution plotLoading and contribution plot

Factor1

Factor2

Factor3

12.8333

12.95

16.1

12.85

12.9667

16 16.1167

12.8667

12.9833

16.0167

16.1333

12.8833

13

16.0333

16.15

12.7833

12.9

13.0167

16.05

16.1667

12.8

12.9167

13.0333

16.0667

16.1833

12.8167

12.9333

13.05

16.0833

4.43333

4.554.66667

4.783334.95.016675.133335.255.366675.483335.65.716675.83333

5.956.066676.183336.36.416676.533336.656.766676.8833377.116677.233337.357.466677.583337.7

7.816677.93333

8.058.166678.283338.48.516678.633338.758.866678.983339.19.216679.333339.45

9.566679.683339.89.9166710.033310.1510.266710.383310.510.616710.733310.8510.9667

11.0833

11.2

11.316711.433311.5511.666711.783311.912.016712.133312.2512.366712.483312.6

12.7167

13.066713.183313.313.416713.533313.6513.766713.88331414.116714.233314.3514.466714.583314.7

14.816714.933315.05

15.166715.2833

15.4

15.5167

15.6333

15.75

15.8667

15.9833

16.2167

16.333316.45

16.566716.683316.816.916717.033317.1517.266717.383317.517.616717.733317.8517.9667

18.0833

18.2

18.3167

18.433318.5518.666718.783318.919.016719.133319.2519.366719.483319.619.716719.833319.9520.066720.183320.320.416720.533320.6520.766720.883321

21.1167

21.2333

21.3521.4667

21.5833

21.721.816721.933322.0522.166722.283322.422.516722.633322.7522.866722.9833

23.1

23.216723.333323.45

23.566723.683323.823.916724.033324.1524.266724.383324.524.616724.733324.8524.966725.083325.225.316725.433325.5525.666725.783325.926.016726.133326.2526.366726.483326.626.716726.833326.9527.066727.183327.327.416727.533327.6527.766727.88332828.116728.233328.3528.466728.583328.728.816728.933329.0529.166729.283329.429.516729.633329.7529.866729.983330.130.216730.333330.4530.566730.683330.830.916731.033331.1531.266731.383331.531.616731.733331.8531.966732.083332.232.316732.433332.5532.666732.783332.933.016733.133333.2533.366733.483333.633.716733.833333.9534.066734.183334.334.416734.533334.6534.766734.88334.45

4.566674.68333

4.84.916675.033335.155.266675.383335.55.616675.733335.855.96667

6.083336.26.316676.433336.556.666676.783336.97.016677.133337.257.366677.483337.67.71667

7.83333

7.95

8.066678.183338.38.416678.53333

8.658.766678.8833399.116679.233339.359.46667

9.583339.79.816679.9333310.0510.166710.283310.410.516710.633310.7510.866710.9833

11.111.2167

11.333311.4511.566711.683311.811.916712.033312.1512.266712.383312.512.6167

12.7333

13.083313.213.316713.433313.5513.666713.783313.914.016714.133314.2514.366714.483314.614.716714.8333

14.9515.0667

15.183315.3

15.4167

15.5333

15.65

15.7667

15.8833

16.2333

16.3516.466716.583316.716.816716.933317.0517.166717.283317.417.516717.633317.7517.866717.9833

18.118.2167

18.333318.4518.566718.683318.818.916719.033319.1519.266719.383319.519.616719.733319.8519.966720.083320.220.316720.433320.5520.666720.783320.921.0167

21.1333

21.25

21.3667

21.4833

21.6

21.716721.833321.9522.066722.183322.322.416722.533322.6522.766722.883323

23.1167

23.233323.3523.4667

23.583323.723.816723.933324.0524.166724.283324.424.516724.633324.7524.866724.983325.125.216725.333325.4525.566725.683325.825.916726.033326.1526.266726.383326.526.616726.733326.8526.966727.083327.227.316727.433327.5527.666727.783327.928.016728.133328.2528.366728.483328.628.716728.833328.9529.066729.183329.329.416729.533329.6529.766729.88333030.116730.233330.3530.466730.583330.730.816730.933331.0531.166731.283331.431.516731.633331.7531.866731.983332.132.216732.333332.4532.566732.683332.832.916733.033333.1533.266733.383333.533.616733.733333.8533.966734.083334.234.316734.433334.5534.666734.783334.94.46667

4.583334.7

4.816674.933335.055.166675.283335.45.516675.633335.75

5.866675.98333

6.16.216676.333336.456.566676.683336.86.916677.033337.157.266677.383337.57.616677.73333

7.85

7.96667

8.083338.28.316678.433338.558.666678.783338.99.016679.133339.259.366679.48333

9.69.716679.833339.9510.066710.183310.310.416710.533310.6510.766710.883311

11.116711.2333

11.3511.466711.583311.711.816711.933312.0512.166712.283312.412.5167

12.6333

12.75

13.113.216713.333313.4513.566713.683313.813.916714.033314.1514.266714.383314.514.6167

14.733314.8514.9667

15.0833

15.215.3167

15.4333

15.55

15.666715.7833

15.916.25

16.366716.483316.616.716716.833316.9517.066717.183317.317.416717.533317.6517.766717.883318

18.116718.2333

18.3518.466718.583318.718.816718.933319.0519.166719.283319.419.516719.633319.7519.866719.983320.120.216720.333320.4520.566720.683320.820.916721.0333

21.15

21.2667

21.3833

21.5

21.6167

21.733321.8521.966722.083322.222.316722.433322.5522.666722.783322.923.016723.1333

23.2523.3667

23.483323.623.716723.833323.9524.066724.183324.324.416724.533324.6524.766724.88332525.116725.233325.3525.466725.583325.725.816725.933326.0526.166726.283326.426.516726.633326.7526.866726.983327.127.216727.333327.4527.566727.683327.827.916728.033328.1528.266728.383328.528.616728.733328.8528.966729.083329.229.316729.433329.5529.666729.783329.930.016730.133330.2530.366730.483330.630.716730.833330.9531.066731.183331.331.416731.533331.6531.766731.88333232.116732.233332.3532.466732.583332.732.816732.933333.0533.166733.283333.433.516733.633333.7533.866733.983334.134.216734.333334.4534.566734.683334.834.9167

4.366674.48333

4.64.71667

4.833334.955.066675.183335.35.416675.533335.655.76667

5.883336

6.116676.233336.356.466676.583336.76.816676.933337.057.166677.283337.47.516677.63333

7.757.86667

7.983338.18.216678.333338.458.566678.683338.88.916679.033339.159.266679.383339.59.616679.733339.859.9666710.083310.210.316710.433310.5510.666710.783310.911.0167

11.133311.25

11.366711.483311.611.716711.833311.9512.066712.183312.312.416712.5333

12.65

12.7667

13.116713.233313.3513.466713.583313.713.816713.933314.0514.166714.283314.414.516714.6333

14.7514.866714.9833

15.1

15.216715.3333

15.45

15.5667

15.683315.8

15.916716.2667

16.383316.5

16.616716.733316.8516.966717.083317.217.316717.433317.5517.666717.783317.9

18.0167

18.133318.25

18.366718.483318.618.716718.833318.9519.066719.183319.319.416719.533319.6519.766719.88332020.116720.233320.3520.466720.583320.720.816720.933321.05

21.1667

21.2833

21.4

21.5167

21.6333

21.7521.866721.983322.122.216722.333322.4522.566722.683322.822.916723.033323.15

23.266723.3833

23.523.616723.733323.8523.966724.083324.224.316724.433324.5524.666724.783324.925.016725.133325.2525.366725.483325.625.716725.833325.9526.066726.183326.326.416726.533326.6526.766726.88332727.116727.233327.3527.466727.583327.727.816727.933328.0528.166728.283328.428.516728.633328.7528.866728.983329.129.216729.333329.4529.566729.683329.829.916730.033330.1530.266730.383330.530.616730.733330.8530.966731.083331.231.316731.433331.5531.666731.783331.932.016732.133332.2532.366732.483332.632.716732.833332.9533.066733.183333.333.416733.533333.6533.766733.88333434.116734.233334.3534.466734.583334.734.816734.93334.383334.5

4.61667

4.73333

4.854.966675.083335.25.31667

5.433335.555.666675.78333

5.96.01667

6.133336.256.366676.483336.66.716676.833336.957.066677.18333

7.37.416677.533337.65

7.766677.88333

88.116678.233338.358.466678.583338.78.816678.933339.059.166679.283339.49.516679.633339.759.866679.9833310.110.216710.333310.4510.566710.683310.810.916711.0333

11.1511.2667

11.383311.511.616711.733311.8511.966712.083312.212.316712.433312.55

12.6667

13.133313.2513.366713.483313.613.716713.833313.9514.066714.183314.314.416714.533314.65

14.766714.8833

1515.1167

15.2333

15.35

15.4667

15.5833

15.7

15.8167

15.933316.2833

16.416.5167

16.633316.7516.866716.983317.117.216717.333317.4517.566717.683317.817.9167

18.0333

18.1518.2667

18.383318.518.616718.733318.8518.966719.083319.219.316719.433319.5519.666719.783319.920.016720.133320.2520.366720.483320.620.716720.833320.9521.0667

21.1833

21.321.4167

21.5333

21.65

21.766721.88332222.116722.233322.3522.466722.583322.722.816722.933323.05

23.166723.2833

23.423.516723.633323.7523.866723.983324.124.216724.333324.4524.566724.683324.824.916725.033325.1525.266725.383325.525.616725.733325.8525.966726.083326.226.316726.433326.5526.666726.783326.927.016727.133327.2527.366727.483327.627.716727.833327.9528.066728.183328.328.416728.533328.6528.766728.88332929.116729.233329.3529.466729.583329.729.816729.933330.0530.166730.283330.430.516730.633330.7530.866730.983331.131.216731.333331.4531.566731.683331.831.916732.033332.1532.266732.383332.532.616732.733332.8532.966733.083333.233.316733.433333.5533.666733.783333.934.016734.133334.2534.366734.483334.634.716734.833334.95

4.4

4.51667

4.63333

4.75

4.866674.983335.15.216675.333335.455.566675.683335.8

5.916676.03333

6.156.266676.383336.56.616676.733336.856.966677.083337.2

7.316677.433337.557.66667

7.783337.9

8.016678.133338.258.366678.483338.68.716678.833338.959.066679.183339.39.416679.533339.659.766679.883331010.116710.233310.3510.466710.583310.710.816710.933311.05

11.166711.2833

11.411.516711.633311.7511.866711.983312.112.216712.333312.4512.5667

12.6833

13.1513.266713.383313.513.616713.733313.8513.966714.083314.214.316714.433314.5514.6667

14.783314.9

15.0167

15.133315.25

15.3667

15.4833

15.6

15.7167

15.8333

15.9516.3

16.416716.5333

16.6516.766716.88331717.116717.233317.3517.466717.583317.717.816717.9333

18.05

18.1667

18.2833

18.418.516718.633318.7518.866718.983319.119.216719.333319.4519.566719.683319.819.916720.033320.1520.266720.383320.520.616720.733320.8520.9667

21.0833

21.2

21.316721.4333

21.55

21.666721.783321.922.016722.133322.2522.366722.483322.622.716722.833322.9523.0667

23.183323.323.4167

23.533323.6523.766723.88332424.116724.233324.3524.466724.583324.724.816724.933325.0525.166725.283325.425.516725.633325.7525.866725.983326.126.216726.333326.4526.566726.683326.826.916727.033327.1527.266727.383327.527.616727.733327.8527.966728.083328.228.316728.433328.5528.666728.783328.929.016729.133329.2529.366729.483329.629.716729.833329.9530.066730.183330.330.416730.533330.6530.766730.88333131.116731.233331.3531.466731.583331.731.816731.933332.0532.166732.283332.432.516732.633332.7532.866732.983333.133.216733.333333.4533.566733.683333.833.916734.033334.1534.266734.383334.534.616734.733334.8534.96674.41667

4.53333

4.65

4.766674.8833355.116675.233335.355.466675.583335.75.81667

5.933336.056.166676.283336.46.516676.633336.756.866676.983337.17.216677.333337.457.566677.68333

7.87.91667

8.033338.158.266678.383338.58.616678.733338.858.966679.083339.29.316679.43333

9.559.666679.783339.910.016710.133310.2510.366710.483310.610.716710.833310.9511.0667

11.183311.3

11.416711.533311.6511.766711.88331212.116712.233312.3512.466712.5833

12.7

13.166713.283313.413.516713.633313.7513.866713.983314.114.216714.333314.4514.566714.6833

14.814.916715.0333

15.1515.2667

15.3833

15.5

15.6167

15.7333

15.85

15.9667

16.2

16.3167

16.433316.55

16.666716.783316.917.016717.133317.2517.366717.483317.617.716717.833317.95

18.0667

18.1833

18.3

18.416718.533318.6518.766718.88331919.116719.233319.3519.466719.583319.719.816719.933320.0520.166720.283320.420.516720.633320.7520.866720.9833

21.1

21.2167

21.333321.45

21.5667

21.683321.821.916722.033322.1522.266722.383322.522.616722.733322.8522.9667

23.0833

23.223.316723.4333

23.5523.666723.783323.924.016724.133324.2524.366724.483324.624.716724.833324.9525.066725.183325.325.416725.533325.6525.766725.88332626.116726.233326.3526.466726.583326.726.816726.933327.0527.166727.283327.427.516727.633327.7527.866727.983328.128.216728.333328.4528.566728.683328.828.916729.033329.1529.266729.383329.529.616729.733329.8529.966730.083330.230.316730.433330.5530.666730.783330.931.016731.133331.2531.366731.483331.631.716731.833331.9532.066732.183332.332.416732.533332.6532.766732.88333333.116733.233333.3533.466733.583333.733.816733.933334.0534.166734.283334.434.516734.633334.7534.866734.9833

NMR-PCA mapNMR-PCA map

IC50>124ug/ml

58<IC50<124ug/ml

IC50<58ug/ml

Contribution plotContribution plot

BaicalinBaicalein Wogonin

PLS prediction of inhibition by PLS prediction of inhibition by S. baicalensisS. baicalensis crude extracts crude extracts

Extract IC50 measuredvalue (µg/ml)

IC50 predicted by NMR model (µg/ml)

IC50 predicted by HPLC model (µg/ml)

a: 25% aqueous ethanol 249.47 165.27 199.03

b: 45% aqueous ethanol 175.35 199.67 160.51

c: 70% aqueous ethanol 82.46 124.41 100.21

d: ethanol 57.18 33.98 95.79

e:dichloromethane 42.23 87.68 43.87

f: ethyl acetate 82.76 60.32 94.08

g: butanol 107.11 101.75 45.34

h: water 123.59 165.70 190.78

Validation of Validation of ScutellariaScutellaria inhibitors inhibitorsA

0

20

40

60

80

100

100502512.56.253.125

Grw

oth

Inhi

bitio

n (%

) a

A549

SK-LU-1

SK-MES-1

HLF

B

0

20

40

60

80

100

100502512.56.253.125

Grw

oth

Inhi

bitio

n (%

) a

A549

SK-LU-1

SK-MES-1

HLF

C

0

20

40

60

80

100

100502512.56.253.125

Grw

oth

Inhi

bitio

n (%

) a

A549

SK-LU-1

SK-MES-1

HLF

A: baicalin

B: baicalein

C: wogonin

ConclusionConclusion

Baicalin, baicalein and wogonin are determined as functional biomarkers for S.

baicalensis inhibition on human lung cancer cells.

Secondary metabolite mapping quickly identified functional compounds from

crude plant extract.

This approach can be used as an important first step to qualify herbal

prescriptions.

The potential of NMR/HPLC-PLS analysis as a tool for the prediction of

growth inhibitory activities of plant extracts.