Supplementary Information

Systematic Investigation of Ginkgo Biloba Leaves for

Treating Cardio-cerebrovascular Diseases in an
Animal Model

Yinfeng Yang1, Yan Li1, *, Jinghui Wang1, Ke Sun2, Weiyang Tao2, Zhenzhong Wang3,
Wei Xiao3, Yanqiu Pan1, *, Shuwei Zhang1 and Yonghua Wang 2, *

1. Key Laboratory of Industrial Ecology and Environmental Engineering (MOE),

Department of Materials Sciences and Chemical Engineering, Dalian University
of Technology, Dalian, Liaoning, 116024, China;
2. College of Life Sciences, Northwest University, Shaanxi, 712100, China;
3. State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process,
Jiangsu KanionParmaceutical CO. LTD, Lianyungang, Jiangsu, 222001, P. R.
China.
Table of Contents
Page
Figure S1. C-T-P network of GBLs on treating CCVDs. 2
31 bioactive compounds obtained from Ginkgo biloba leaves
Table S1. 3-4
and their corresponding ADME parameters.
Table S2. 80 potential drug targets and their corresponding diseases. 5-7
Table S3. 40 KEGG pathways associated with CCVDs. 8
Supplementary Text MATERIALS AND METHODS 9-10
References References of MATERIALS AND METHODS 11

1
Figure S1. C-T-P network of GBLs on treating CCVDs. This net presented a clear mutil-level
structure, that is, multi-components hit on muti-target genes show their pharmacological effects
through multi-pathways. TTLs: terpene trilactones.

2
Table S1. 31 bioactive compounds obtained from Ginkgo biloba leaves and their corresponding
ADME parameters.

NO. Compound Name OB (%) DL HL Type

C01 Apigenin 45.09 0.21 16.81 Flavonoids
C02 Bilobalide 86.51 0.36 5.90 TTLs
§
C03 Chrysoeriol-7-glucoside_QT 46.36 0.27 16.30 Flavonoids
C04 Diosmetin 50.41 0.27 15.98 Flavonoids
C05 Flavoxanthin 51.03 0.56 16.75 Others
C06 Ginkgolide A 42.85 0.74 4.08 TTLs
C07 Ginkgolide B 44.38 0.73 5.15 TTLs
C08 Ginkgolide C 48.69 0.73 6.74 TTLs
C09 Ginkgolide J 44.60 0.74 4.12 TTLs
C10 Ginkgolide M 47.90 0.75 7.61 TTLs
Isorhamnetin3-O-[2-O,6-O-Bis(α-L-rhamno
C11§ 46.62 0.31 14.83 Flavonoids
syl)-β-D-glucosyl]_QT
Kaempferol
C12§ 41.43 0.24 14.72 Flavonoids
3-(2″-β-D-glucosyl)-α-L-rhamnoside_QT
Kaempferol
§
C13 3-O-(6″-O-(α-L-rhamnosyl)-β-D-glucosyl)_ 41.81 0.24 14.72 Flavonoids
QT
Kaempferol
C14§ 3-O-[2-O,6-O-Bis(α-L-rhamnosyl)-β-D-gluc 67.09 0.24 14.70 Flavonoids
osyl]_QT
§
C15 Kaempferol 3-O-β-D-glucoside_QT 63.98 0.24 14.69 Flavonoids
§
C16 Kaempferol 3-O-β-D-rutinoside_QT 41.91 0.24 14.71 Flavonoids
C17 Kaempferol 69.61 0.24 14.85 Flavonoids
kaempferol-3-O-β-D-glucosyl-(1-2)-α-L-rha
C18§ 52.75 0.27 16.37 Flavonoids
mnoside_QT
C19§ Kaempferol-3-rhamnoglucoside_QT 41.79 0.24 14.73 Flavonoids
§
C20 Kaempferol-3-rhamnoside_QT 42.22 0.24 14.71 Flavonoids
§
C21 Kaempferol-7-rhamnoside_QT 62.71 0.24 14.69 Flavonoids
C22§ Luteolin-3′-glucoside_QT 53.22 0.25 15.77 Flavonoids

3
To be continued
NO. Compound Name OB (%) DL HL Type
§
C23 Myricetin 3-O-β-D-rutinoside_QT 51.08 0.31 13.87 Flavonoids
Quercetin
C24§ 50.46 0.28 14.37 Flavonoids
3-(2″-β-D-glucosyl)-α-L-rhamnoside_QT
C25§ Quercetin 3-rhamnoside_QT 46.17 0.28 14.22 Flavonoids
C26§ quercetin-3-O-glucoside_QT 46.49 0.28 14.24 Flavonoids
§
C27 Rutin_QT 47.39 0.28 14.23 Flavonoids
C28 Stigmasterol 43.83 0.76 5.68 Others
C29 Isorhamnetin 7.02 0.31 14.64 Flavonoids
C30 Myricetin 9.80 0.31 14.05 Flavonoids
C31 Quercetin 9.46 0.28 14.42 Flavonoids
§
Aglycones of corresponding molecules

4
 Table S2. 80 potential drug targets and their corresponding diseases.
NO. Target Name Uniprot ID Gene Name Classification Related Diseases
T01 Prothrombin P00734 F2 Enzymes Cardiovascular Diseases
T02 Estrogen receptor alpha P03372 ESR1 Nuclear receptors Cardiovascular Diseases
T03 Mineralocorticoid receptor P08235 NR3C2 Nuclear receptors Cardiovascular Diseases
T04 Matrix metalloproteinase-9 P14780 MMP9 Enzymes Cardiovascular Diseases
Transcription
T05 Transcription factor p65 Q04206 NFKB3 Cardiovascular Diseases
factors
Glycosaminoglyc
T06 Vascular endothelial growth factor A P15692 VEGFA an binding Cardiovascular Diseases
proteins
Transcription
T07 Transcription factor AP-1 P05412 JUN Cardiovascular Diseases
factors
Glycosaminoglyc
T08 Tumor necrosis factor P01375 TNF an binding Cardiovascular Diseases
proteins
G protein-coupled
T09 Muscarinic acetylcholine receptor M2 P08172 CHRM2 Cardiovascular Diseases
receptors
Vascular endothelial growth factor
T10 P35968 KDR Enzymes Cardiovascular Diseases
receptor 2
3-hydroxy-3-methylglutaryl-coenzyme
T11 P04035 HMGCR Enzymes Cardiovascular Diseases
A reductase
G protein-coupled
T12 Beta-2 adrenergic receptor P07550 ADRB2 Cardiovascular Diseases
receptors
T13 Prostaglandin G/H synthase 2 P35354 COX-2 Enzymes Cardiovascular Diseases
T14 Nitric-oxide synthase, endothelial P29474 NOS3 Enzymes Cardiovascular Diseases
T15 Glucocorticoid receptor P04150 NR3C1 Nuclear receptors Cardiovascular Diseases
G protein-coupled
T16 Platelet-activating factor receptor P25105 PTAFR Cardiovascular Diseases
receptors
Musculoskeletal
T17 Phospholipase A2 P04054 PLA2G1B Enzymes
Diseases
T18 Mitogen-activated protein kinase 14 Q16539 MAPK14 Enzymes Cardiovascular Diseases
T19 Nitric oxide synthase, inducible P35228 NOS2 Enzymes Cardiovascular Diseases
T20 Prothrombin P00734 F2 Enzymes Cardiovascular Diseases
T21 Matrix metalloproteinase-3 P08254 MMP3 Enzymes Cardiovascular Diseases
Gamma-aminobutyric-acid receptor
T22 P14867 GABRA1 Ion channels Cardiovascular Diseases
subunit alpha-1
T23 Retinoic acid receptor RXR-beta P28702 RXRB Nuclear receptors Cardiovascular Diseases
T24 Retinoic acid receptor RXR-alpha P19793 RXRA Nuclear receptors Cardiovascular Diseases
G protein-coupled
T25 Muscarinic acetylcholine receptor M3 P20309 CHRM3 Cardiovascular Diseases
receptors
Peroxisome proliferator-activated
T26 P37231 PPARG Nuclear receptors Cardiovascular Diseases
receptor gamma

5
 To be continued
NO. Target Name Uniprot ID Gene Name Classification Related Diseases
Voltage-dependent calcium channel
T27 P54289 CACNA2D1 Ion channels Cardiovascular Diseases
subunit alpha-2/delta-1
T28 Estrogen receptor beta Q92731 ESR2 Nuclear receptors Cardiovascular Diseases
T29 Heat shock protein HSP 90-alpha P07900 HSP90AA1 Others Cardiovascular Diseases
T30 Cell division protein kinase 2 P24941 CDK2 Enzymes Cardiovascular Diseases
Sodium channel protein type 5 subunit
T31 Q14524 SCN5A Ion channels Cardiovascular Diseases
alpha
cGMP-inhibited 3',5'-cyclic
T32 Q14432 PDE3A Enzymes Cardiovascular Diseases
phosphodiesterase A
G protein-coupled
T33 Alpha-1B adrenergic receptor P35368 ADRA1B Cardiovascular Diseases
receptors
T34 Beta-lactamase P00811 ampC Enzymes Cardiovascular Diseases
T35 Androgen receptor P10275 AR Nuclear receptors Cardiovascular Diseases
T36 Coagulation factor X P00742 F10 Enzymes Cardiovascular Diseases
T37 Renin P00797 REN Enzymes Cardiovascular Diseases
cGMP-specific 3',5'-cyclic
T38 O76074 PDE5A Enzymes Cardiovascular Diseases
phosphodiesterase
T39 Heat shock protein HSP 90-beta P08238 HSP90AB1 Others Cardiovascular Diseases
Phosphatidylinositol-4,5-bisphosphate
T40 3-kinase catalytic subunit gamma P48736 PIK3CG Enzymes Cardiovascular Diseases
isoform
Nervous System
T41 Beta-secretase 1 P56817 BACE1 Enzymes
Diseases
T42 Glycogen synthase kinase-3 beta P49841 GSK3B Enzymes Cardiovascular Diseases
T43 Caspase-3 P42574 CASP3 Enzymes Cardiovascular Diseases
G protein-coupled Nervous System
T44 Muscarinic acetylcholine receptor M1 P11229 CHRM1
receptors Diseases
Nervous System
T45 Acetylcholinesterase P22303 ACHE Enzymes
Diseases
T46 human pregnane X receptor O75469 PXR Nuclear receptors Cardiovascular Diseases
Nervous System
T47 Glial fibrillary acidic protein P14136 GFAP Others
Diseases
T48 P-selectin P16109 SELP Enzymes Cardiovascular Diseases
Nervous System
T49 Cathepsin D P07339 CTSD Enzymes
Diseases
T50 Butyrylcholine esterase P81908 BChE Enzymes Cardiovascular Diseases
Glutamate [NMDA] receptor subunit
T51 Q13224 GRIN2B Ion channels Cardiovascular Diseases
epsilon-2
Neuronal acetylcholine receptor Nervous System
T52 P36544 CHRNA7 Ion channels
subunit alpha-7 Diseases

6
 To be continued
NO. Target Name Uniprot ID Gene Name Classification Related Diseases
Gamma-aminobutyric-acid receptor
T53 P34903 GABRA3 Ion channels Mental Disorders
subunit alpha-3
Gamma-aminobutyric-acid receptor
T54 P47869 GABRA2 Ion channels Mental Disorders
subunit alpha-2
Gamma-aminobutyric-acid receptor
T55 P31644 GABRA5 Ion channels Nervous System Diseases
subunit alpha-5
Sodium-dependent noradrenaline
T56 P23975 SLC6A2 Transporters Cardiovascular Diseases
transporter
T57 Sodium-dependent dopamine transporter Q01959 SLC6A3 Transporters Nervous System Diseases
T58 Sodium-dependent serotonin transporter P31645 SLC6A4 Transporters Nervous System Diseases
G protein-coupled
T59 D(2) dopamine receptor P14416 DRD2 Nervous System Diseases
receptors
T60 Aldo-ketoreductase family 1 member C3 P42330 AKR1C3 Enzymes Neoplasms
T61 Progesterone receptor P06401 PGR Nuclear receptors Neoplasms
T62 Epidermal growth factor receptor P00533 EGFR Enzymes Neoplasms
T63 Serine/threonine-protein kinase Chk1 O14757 CHEK1 Enzymes Nervous System Diseases
T64 Retinoic acid receptor RXR-gamma P48443 RXRG Nuclear receptors Cardiovascular Diseases
T65 Dipeptidyl peptidase 4 P27487 DPP4 Enzymes Cardiovascular Diseases
T66 Penicillin-binding protein 1A Q8DR59 pbpA Enzymes Bacterial infections
T67 DNA topoisomerase 2-alpha P11388 TOP2A Enzymes Bacterial infections
Corticosteroid 11-beta-dehydrogenase
T68 P28845 HSD11B1 Enzymes Cardiovascular Diseases
isozyme 1
Tyrosine-protein phosphatase Nutritional and Metabolic
T69 P18031 PTPN1 Enzymes
non-receptor type 1 Diseases
G protein-coupled
T70 Mu-type opioid receptor P35372 OPRM1 Nervous System Diseases
receptors
T71 Cyclin-A2 P20248 CCNA2 Enzymes Nervous System Diseases
T72 Cathepsin K P43235 CTSK Enzymes Musculoskeletal Diseases
Neuronal acetylcholine receptor subunit
T73 Q15822 CHRNA2 Ion channels Anesthesia and Analgesia
alpha-2
T74 Trypsin-1 P07477 PRSS1 Enzymes Digestive System Diseases
Gamma-aminobutyric-acid receptor
T75 Q16445 GABRA6 Ion channels Mental Disorders
subunit alpha-6
cAMP-dependent protein kinase
T76 P76577 PRKACA others Neoplasms
catalytic subunit alpha
T77 Genome polyprotein P26663 None Enzymes Genetic Phenomena
T78 FK506-binding protein 1A P62942 FKBP1A Enzymes Immune System Diseases
Proto-oncogene tyrosine-protein kinase
T79 P06239 LCK Enzymes Immune System Diseases
LCK
T80 Epidermal growth factor receptor P00533 EGFR Enzymes Neoplasms

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 Table S3. 40 KEGG pathways associated with CCVDs.
No. Pathway Name KEGG_ID Category
P01 Neuroactive ligand-receptor interaction hsa04080 Signaling molecules and interaction
P02 Cytokine-cytokine receptor interaction hsa04060 Signaling molecules and interaction
P03 Cell adhesion molecules (CAMs) hsa04514 Signaling molecules and interaction
P04 PI3K-Akt signaling pathway hsa04151 Signal transduction
P05 TNF signaling pathway hsa04668 Signal transduction
P06 MAPK signaling pathway hsa04010 Signal transduction
P07 NF-kappa B signaling pathway hsa04064 Signal transduction
P08 HIF-1 signaling pathway hsa04066 Signal transduction
P09 VEGF signaling pathway hsa04370 Signal transduction
P10 Wnt signaling pathway hsa04310 Signal transduction
P11 TGF-beta signaling pathway hsa04350 Signal transduction
P12 Calcium signaling pathway hsa04020 Signal transduction
P13 Jak-STAT signaling pathway hsa04630 Signal transduction
P14 Complement and coagulation cascades hsa04610 Immune system
P15 Chemokine signaling pathway hsa04062 Immune system
P16 Toll-like receptor signaling pathway hsa04620 Immune system
P17 NOD-like receptor signaling pathway hsa04621 Immune system
P18 T cell receptor signaling pathway hsa04660 Immune system
P19 Cholinergic synapse hsa04725 Nervous system
P20 Serotonergic synapse hsa04726 Nervous system
P21 Dopaminergic synapse hsa04728 Nervous system
P22 Neurotrophin signaling pathway hsa04722 Nervous system
P23 Estrogen signaling pathway hsa04915 Endocrine system
P24 Renin-angiotensin system hsa04614 Endocrine system
P25 PPAR signaling pathway hsa03320 Endocrine system
P26 Insulin signaling pathway hsa04910 Excretory system
P27 Endocrine and other factor-regulated
hsa04961 Excretory system
calcium reabsorption
P28 Aldosterone-regulated sodium reabsorption hsa04960 Excretory system
P29 Adrenergic signaling in cardiomyocytes hsa04261 Circulatory system
P30 Vascular smooth muscle contraction hsa04270 Circulatory system
P31 Regulation of actin cytoskeleton hsa04810 Cell motility
P32 p53 signaling pathway hsa04115 Cell growth and death
P33 Apoptosis hsa04210 Cell growth and death
P34 Alzheimer's disease hsa05010 Neurodegenerative diseases
P35 Type II diabetes mellitus hsa04930 Endocrine and metabolic diseases
P36 Hypertrophic cardiomyopathy (HCM) hsa05410 Cardiovascular diseases
P37 Dilated cardiomyopathy hsa05414 Cardiovascular diseases
P38 Transcriptional misregulation in cancer hsa05202 Cancers
P39 Metabolic pathways hsa01100 Metabolism
P40 Arachidonic acid metabolism hsa00590 Lipid metabolism

8
Supplementary Text
MATERIALS AND METHODS
In silico ADME Screening Models
Ideally, a drug candidate should possess both good pharmacological activity and
good ADME (absorption, distribution, metabolism and excretion) properties.
Undesirable pharmacokinetic properties such as poor oral bioavailability, short
half-life, etc., are one of the major reasons for terminating the drug development.1
Application of in silico ADME models at the early stages of drug discovery process
offers considerable potential for reducing the number of experimental studies, which
are required for compound selection and for improving the success rate.2 Presently,
three ADME-related models, including PreOB (predict oral bioavailability), PreDL
(predict drug-likeness), and PreHL (predict half-life) were employed to explore the
candidate compounds in Ginkgo biloba leaves.
PreOB
The predominant and most convenient way in various routines for drug delivery
is the oral route, so estimating human oral bioavailability is of great importance and
necessary for lead selection and further optimization. At present, the PreOB model,
developed on the basis of a robust in-house system OBioavail 1.1,3 was adopted to
predict the OB of the constituents of this herb. This model can be used as a rapid
screening filter for candidate compounds with good OB, thus significantly reducing
the original components to a smaller set for a TCM receipt.
PreDL
For the purpose of reducing attrition in drug discovery/development, the concept
of drug-likeness (DL) has been widely applied in combinatorial chemistry. Currently,
the PreDL model, a database-dependent model used for the prediction of
drug-likeness has been developed. All DL indices were calculated by using the
Tanimoto coefficient,4 a kind of similarity score for measuring the structural similarity
between herbal compounds and the drugs in Drugbank database
(http://www.drugbank.ca/).
PreHL
Usually, a drug’s metabolic stability is expressed as in vitro half-life and intrinsic
clearance. Half-life (t½) of a drug is the time taken for the concentration of a drug in
the plasma to reduce by 50%, which involves the clearance and volume of distribution,
and determines how often a drug must be administered to maintain a therapeutic
concentration.5 Herein, based on the multiple linear regression (MLR) analysis, a
PreHL model was created to screen bioactive compounds with a better performance of
correlation coefficient R2 = 0.65, Q2 = 0.62.
Taken together, the threshold values for the ADME screening models as

9
mentioned above are set to OB ≥ 40%, DL ≥ 0.20, and HL ≥ 4, respectively. The
compounds which successfully satisfy all the criteria are treated as candidate
molecules.
Predicting Target Profiles for Candidate Compounds
To predict the most likely protein targets of these bioactive compounds, an
integration of various approaches is essential. Firstly, a Systems Drug Targeting
(SysDT) model6 incorporating a large scale of chemical, genomic and
pharmacological data was developed. The procedure was based on two powerful
methods, i.e., Random Forest (RF) and Support Vector Machine (SVM) and the
generated results were represented by probability of interactions between each
molecule and 3999 targets. According to the following steps, the protein targets were
obtained: (a) The top 30 high-ranking proteins in both RF and SVM models were
extracted; (b) The overlap targets between them were chosen as candidate targets.
Secondly, to obtain the potential target proteins of herbal ingredients, a chemical
fingerprint similarity based Similarity Ensemble Approach (SEA,
7
http://sea.bkslab.org/search/) was employed. Thirdly, as a complementary method for
target fishing and a kind of target validation, data mining was performed to extract the
information involved in the molecular targets. All obtained targets were further
subjected to Therapeutic Targets Database,8 PharmGkb9 and the Comparative
Toxicogenomics Database10 to detect the relationships between drugs, targets and
diseases.
Experimental Procedures
MCAO Model
Using a previous described method11 with minor modification, the MCAO
surgery served as an in vivo Ischemia and Peperfusion (I/R) injury was performed.
Before surgery, animals were anesthetized with isoflurane. In brief, the right common
carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA)
were exposed through a mid line cervical incision. In order to occlude blood flow
through the middle cerebral artery (MCA), a 4-0 monofilament nylon suture was
prepared by rounding its tip slightly by heat and inserted from the external into the
internal carotid artery until mild resistance was encountered. The distance between the
CCA and the resistive point was approximately 19~20 mm. After 120 min, MCA
reperfusion was initiated by withdrawing the filament.
TTC Staining and Measurement of the Infarct Volume
After 22 h of reperfusion, the brains of animals anesthetized with isoflurane were
quickly dissected out and sectioned into five coronal brain slices in 2 mm thickness.
Then, these brain slices were stained with 1% 2,3,5-triphenyltetrazolium chloride
(TTC; Sigma, St. Louis, MO, USA) for 30 min in a 37 °C incubator to visualize the
infarct. The TTC-stained sections were captured with a digital camera and the
lesioned areas of each section were quantitatively analyzed by the image analysis
software ImageJ 1.42. The measured area of lesion was expressed as a percentage of
10
the total volume.
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