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Scientific RepoRts | 7:40318 | DOI: 10.1038/srep40318
www.nature.com/scientificreports
Herb pair Danggui-Honghua:
mechanisms underlying blood
stasis syndrome by system
pharmacology approach
Shi-Jun Yue1,2,*, Lan-Ting Xin1,2,*, Ya-Chu Fan1,2, Shu-Jiao Li3, Yu-Ping Tang3, Jin-Ao Duan3,
Hua-Shi Guan1,2 & Chang-Yun Wang1,2
Herb pair Danggui-Honghua has been frequently used for treatment of blood stasis syndrome (BSS) in
China, one of the most common clinical pathological syndromes in traditional Chinese medicine (TCM).
However, its therapeutic mechanism has not been clearly elucidated. In the present study, a feasible
system pharmacology model based on chemical, pharmacokinetic and pharmacological data was
developed via network construction approach to clarify the mechanisms of this herb pair. Thirty-one
active ingredients of Danggui-Honghua possessing favorable pharmacokinetic proles and biological
activities were selected, interacting with 42 BSS-related targets to provide potential synergistic
therapeutic actions. Systematic analysis of the constructed networks revealed that these targets such
as HMOX1, NOS2, NOS3, HIF1A and PTGS2 were mainly involved in TNF signaling pathway, HIF-1
signaling pathway, estrogen signaling pathway and neurotrophin signaling pathway. The contribution
index of every active ingredient also indicated six compounds, including hydroxysaor yellow A,
saor yellow A, saor yellow B, Z-ligustilide, ferulic acid, and Z-butylidenephthalide, as the principal
components of this herb pair. These results successfully explained the polypharmcological mechanisms
underlying the eciency of Danggui-Honghua for BSS treatment, and also probed into the potential
novel therapeutic strategies for BSS in TCM.
With the ever-increasing acceptance of combination therapy and the prevalence of chronic diseases in the world,
the use of traditional Chinese medicine (TCM) has become an emerging trend1. A TCM prescription usually con-
tains numerous ingredients synergistically and holistically acting on the diseases. Herb pairs, the simplest form
and the centralized representative of Chinese herbal compatibility, intrinsically convey the basic idea of TCM
prescriptions2. Danggui and Honghua combined as an herb pair have been frequently used in TCM prescriptions,
which could be ascended to an ancient and classical formula Danggui-Honghua Decoction. Danggui, the radix of
Angelica sinensis (Oliv.) Diels, has been used for thousands of years in the East Asia and was rstly recorded in a
classical masterpiece of TCM Shennong Bencao Jing (200–300 A.D., Han Dynasty). Danggui has also been called
“female ginseng”, predominantly renowned for treatment of intractable gynecological disorders3. Honghua, the
dried orets of Carthamus tinctorius L., has been used extensively in TCM to treat stroke, coronary heart disease
and angina pectoris. In the Compendium of Materia Medica, Honghua was described as being able to invig-
orate the circulation of blood4. Other classical formulae, such as Tao-Hong-Si-Wu Decoction, Xue-Fu-Zhu-Yu
Decoction, and Bu-Yang-Huan-Wu Decoction, also contain these two herbs5.
Danggui-Honghua is commonly used for treating clinical blood stasis, which is described as slowing or pooling
of the blood due to disruption of heart Qi of TCM6. Ancestors and modern scholars considered that “sorrow nu” of
the seven emotions and “cold evil” of the six evils were the primary causes of acute blood stasis7. Modern pathology
shows that blood stasis is generally manifested by cardio-cerebrovascular diseases such as myocardial infraction,
1Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy, Ocean
University of China, Qingdao 266003, P. R. China. 2Laboratory for Marine Drugs and Bioproducts, Qingdao National
Laboratory for Marine Science and Technology, Qingdao 266071, P. R. China. 3Jiangsu Key Laboratory for High
Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, P. R. China. *These
authors contributed equally to this work. Correspondence and requests for materials should be addressed to H.S.G.
(email: hsguan@ouc.edu.cn) or C.Y.W. (email: changyun@ouc.edu.cn)
Received: 19 April 2016
Accepted: 05 December 2016
Published: 11 January 2017
OPEN
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Scientific RepoRts | 7:40318 | DOI: 10.1038/srep40318
coronary heart disease, and high blood pressure resulting from hematological disorders including hemorrhage,
congestion, thrombosis, and local ischemia6,8. Although well-practiced in clinical medicine, the mechanisms of
how Danggui-Honghua exerts the therapeutic eects on blood stasis syndrome (BSS) remain elusive.
System biology is now considered as a holistic and ecient tool to study the role of TCM1. Combined with
pharmacology and pharmacodynamics, system biology has given birth to a promising subject, i.e., system phar-
macology9. On this basis, an integrated mode combining pharmacokinetics prediction and network pharma-
cology techniques has been developed and successfully applied to interpret the mechanisms of several Chinese
herbal medicines and formulae at molecular network level10,11. In the present study, the mechanisms of herb pair
Danggui-Honghua in treating BSS were investigated by the system pharmacology model based on chemical,
pharmacokinetic and pharmacological data together with contribution index analysis.
Results
e molecular mechanisms of the herb pair Danggui-Honghua against BSS were investigated by a network con-
struction approach with system pharmacology model based on chemical, pharmacokinetic and pharmacological
data (Fig.1). All of the ingredients from this herb pair were explored to build a compound library. Next, the
oral bioavailability (OB), Caco-2 cell permeability and drug-likeness (DL) of the ingredients were collected and
calculated to screen the potential active compounds. en the potential targets and their corresponding diseases
and pathways of the active compounds were data-mined from literature and public database/soware sources.
Subsequently, the acquired pharmacological data were integrated into the compound-target (C-T), target-disease
(T-D), and target-pathway (T-P) networks, respectively. Finally, a contribution index of every active ingredient
based on network based ecacy weighted by literature was calculated. Based on the above analyses, the underly-
ing mechanisms of Danggui-Honghua for treating BSS were explored.
Ingredient comparisons in Danggui and Honghua. e ingredients in Danggui and Honghua were
retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TcmSP™ ,
http://sm.nwsuaf.edu.cn/lsp/tcmsp.php)12 and were manually supplemented. e main components of Danggui
are phthalides and organic acids13, whereas the major ingredients in Honghua are quinochalcone C-glycosides
and avonoid glycosides possessing one or more glucose units4. Since those glycosides in this herb pair might be
deglycosylated by the glycosidase in the intestinal tract, 11 aglycones were also incorporated into the compound
library labeled by_qt. us, a total of 347 ingredients were retrieved for Danggui (143) and Honghua (204). e
detailed information about these molecules was provided in SupplementaryTableS1.
To investigate the molecular diversity of the ingredients from Danggui and Honghua, ingredient compari-
sons were conducted based on seven signicant properties, including the molecular weight (MW), Moriguchi
octanol–water partition coe. (LogP) (MLogP), the number of donor atoms for H-bonds (nHDon), the number
of acceptor atoms for H-bonds (nHAcc), OB, Caco-2 and DL (Fig.2). (1) From the MW, the distinct average
numbers of MWs of the constituents from Danggui (208.71) and Honghua (343.94) were observed. By two tailed
t-test, the MWs of the individual compounds from these two herbs were signicantly dierent (P = 7.36E–13).
(2) e MLogP values of the constituents from Danggui (3.16) and Honghua (2.93) were similar and displayed no
signicant dierence (P = 0.62), indicating that the majority of the ingredients in both herbs were hydrotropic.
(3) e average nHDon number of Danggui constituents (0.90) was signicantly lower than that of Honghua
constituents (3.41) (P = 1.23E–11). (4) e average nHAcc number of Danggui constituents (2.26) was also signif-
icantly lower than that of Honghua constituents (5.62) (P = 8.84E–10). (5) Compared with Honghua constituents
Figure 1. e whole framework based on an integration strategy of system pharmacology.
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Scientific RepoRts | 7:40318 | DOI: 10.1038/srep40318
(average OB value = 26.48), Danggui constituents possessed higher average OB value of 37.28 (P = 3.50E–7).
(6) For permeability, the average Caco-2 value of Danggui constituents (1.05) was signicantly higher than that
of Honghua constituents (0.18) (P = 8.99E–9). (7) Whereas for DL analysis, unlike OB and Caco-2 parame-
ters, Danggui constituents exhibited lower average DL index (0.11), which is signicantly dierent from that of
Honghua (0.30) (P = 2.44E–12).
e above analysis suggested that the constituents of Danggui and Honghua were diverse but the majority of
them satised the Lipinski’s rule of ve. Apart from the hydrotropic property, Danggui was signicantly dierent
from Honghua in other properties of the chemical components. ese dierences are not surprising due to the
distinct chemo-physical properties of the ingredients from these two herbs. e above results also showed that
the constituents from Danggui have better pharmacokinetic properties (OB and Caco-2), whereas the ingredi-
ents from Honghua possess better drug-likeness (DL). From a chemotaxonomic point of view, phthalides from
Danggui and quinochalcone C-glycosides from Honghua have only been found in the families Apiaceae and
Asteraceae, respectively. Although these major ingredients between Danggui and Honghua are obviously dif-
ferent, the two herbs exhibit the identical blood-activating and stasis-dissolving eects and the warm nature of
TCM, which may also clarify why Danggui-Honghua could produce synergistic and complementary eects.
Active ingredients in Danggui-Honghua. Although a single herb or TCM formula usually contains a
considerable number of bioactive components, maybe only a few with desirable pharmacodynamic and pharma-
cokinetic properties are responsible for its therapeutic eects. In the present work, three crucial ADME (absorp-
tion, distribution, metabolism, and excretion) parameters, including OB, Caco-2, and DL, were employed to
screen most of the active compounds from Danggui-Honghua. A few active compounds that do not meet all of
these three criteria were also selected for their high amounts and high bioactivities. Consequently, a total of 31
active compounds were selected from the 347 compounds of this herb pair (Table1).
Active ingredients from Danggui. By ADME screening, 15 out of 143 ingredients with excellent phar-
macological eects were extracted from Danggui, and the majority of them possess satisfactory pharmacoki-
netic proles. For instance, caeic acid (DG-14, OB = 54.97%, Caco-2 = 0.21 and DL = 0.19) has antioxidant,
anti-inammatory, antimutagenic, antibacterial and anti-carcinogenic eects14. Likewise, β-sitosterol (DG-15,
OB = 36.91%, Caco-2 = 1.33 and DL = 0.75) exhibits potent anti-inammatory and antipyretic activities15. It
should be point out that phthalides, the representative ingredients in Danggui, show low DL values, but they
exhibit potent antifungal, antibacterial, anti-inammatory, and antioxidant activities16. Similarly, although phe-
nolic constituents hold low DL values, they also exhibit remarkable pharmacological eects17,18. Specically,
Z-ligustilide (DG-1) and ferulic acid (DG-12) have been chosen as the marker components for quality control
of Danggui in Chinese Pharmacopoeia19. In view of the facts mentioned above, phthalides and phenolic constit-
uents were also deemed as the active ingredients for further analysis. Furthermore, the contents of the selected
constituents in Danggui were also considered. rough a wide-scale text mining of Google Scholar, the total
content of ferulic acid, Z-butylidenephthalide (DG-5), senkyunolide A (DG-2), senkyunolide I (DG-3), and
Z-ligustilide was found to be nearly 43 mg/g20, and nicotinic acid (DG-8) was up to 0.198 mg/g21. Noteworthy,
coniferyl ferulate (DG-9) is also abundant in Danggui and exhibits multiple biological activities such as antioxi-
dant, vasodilating and antibacterial eects18. Nodakenin (DG-11) has been reported to possess neuroprotective,
anti-inammatory, antibacterial, and memory-improving eects22. Based on the above considerations, it was
reasonable to believe that een compounds could be listed as potential active ingredients for Danggui (Table1).
Active ingredients from Honghua. In Honghua, only 16 ingredients passed through the strict ltering cri-
teria, and most of them exhibit potent pharmacological activities. For examples, quercetin (HH-9, OB = 46.43%,
Caco-2 = 0.05 and DL = 0.28) presents anti-inammatory, anti-proliferative, and hepatoprotective activities23;
Figure 2. e molecular diversity of all ingredients from Danggui and Honghua. Molecular properties
consist of molecular weight (MW), Moriguchi octanol-water partition coe. log P (MLogP), number of donor
atoms for H-bonds (nHDon), number of acceptor atoms for H-bonds (nHAcc), oral bioavailability (OB),
Caco-2 permeability (Caco-2) and drug-likeness (DL). *P < 0.05 by two tailed t-test (vs. Danggui).
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No. Name Structure OB (%) Caco-2 DL Herbs
DG-1 Z-Ligustilide 51.30 1.31 0.07 A. sinensis
DG-2 Senkyunolide A 68.28 1.30 0.07 A. sinensis
DG-3 Senkyunolide I 46.80 1.00 0.08 A. sinensis
DG-4 Senkyunolide K 61.75 0.52 0.08 A. sinensis
DG-5 Z-Butylidenephthalide 42.44 1.32 0.07 A. sinensis
DG-6 3-Butylidene-7-hydroxyphthalide 62.68 1.00 0.08 A. sinensis
DG-7 Neocnidilide 83.83 1.23 0.07 A. sinensis
DG-8 Nicotinic acid 47.65 0.34 0.02 A. sinensis
DG-9 Coniferyl ferulate 4.54 0.71 0.39 A. sinensis
DG-10 Folic acid 68.96 − 1.50 0.71 A. sinensis
DG-11 Nodakenin 57.12 − 0.79 0.69 A. sinensis
DG-12 Ferulic acid 39.56 0.47 0.06 A. sinensis
DG-13 Vanillin 55.14 0.41 0.06 A. sinensis
DG-14 Caeic acid 54.97 0.21 0.19 A. sinensis
DG-15 β-Sitosterol 36.91 0.33 0.75 A. sinensis
HH-1 Carthamone 5.93 − 1.81 0.63 C. tinc torius
Continued
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No. Name Structure OB (%) Caco-2 DL Herbs
HH-2 Saor yellow A 22.75 − 2.52 0.75 C. tinctorius
HH-3 Hydroxysaor yellow A 4.77 − 2.77 0.68 C. tinctorius
HH-4 Precarthamin 22.00 − 1.40 0.67 C. t inctorius
HH-5 Saor yellow B 12.00 − 2.40 0.67 C. tincto rius
HH-6 6-Hydroxykaempferol 62.13 0.16 0.27 C. tinctorius
HH-7 Kaempferol 67.43 0.26 0.24 C. tinctorius
HH-8 Nicotiorin 3.64 − 1.77 0.73 C. tinctorius
HH-9 Quercetin 46.43 0.05 0.28 C. tinctoriu s
HH-10 Rutin 11.70 − 1.93 0.68 C. tinc torius
Continued
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luteolin (HH-15, OB = 36.16%, Caco-2 = 0.19 and DL = 0.25) shows antioxidant, anti-inflammatory and
anti-allergic eects24. Surprisingly, quinochalcone C-glycosides, the main active and characteristic compounds
in Honghua, exhibit low OB and Caco-2 values. However, HSYA (HH-3, OB = 4.77%, Caco-2 = − 2.77 and
DL = 0.68) has been chosen for the quality control of Honghua in Chinese Pharmacopoeia25 and has been devel-
oped into the intravenous injection in China to treat cardiac-cerebral vascular ailments with good clinical eects4.
Saor yellow B (HH-5, OB = 12.00%, C aco-2 = − 2.40 and DL = 0.67) has also shown potent neuroprotective
and anti-oxidative abilities on cerebral ischemic injury26. us, quinochalcone C-glycosides were also selected for
targeting. Notably, scutellarin (HH-12, OB = 2.64%, Caco-2 = − 1.08 and DL = 0.79) has been used in tablet and
injection forms in China since 1984 for treatment of acute cerebral infarction and paralysis induced by hyperten-
sion, cerebral thrombosis, and cerebral hemorrhage27. Scutellarein (HH-13), the aglycone of scutellarin which is a
candidate drug for cardio-cerebrovascular diseases was also selected for our further analysis. In addition, it is nec-
essary to incorporate nicotiorin (HH-8, OB = 3.64%, Caco-2 = − 1.77 and DL = 0.73) into further investigation
for that this compound has potent neuroprotective, analgesic, anti-hypertensive and anti-anaphylactic eects28.
Target proteins of Danggui-Honghua. Searching for the targets of candidate drugs solely by the exper-
imental approaches is overspending, labor-intensive, and time-consuming. In the present work, an integrated
in silico approach was introduced to identify the target proteins for the active ingredients of Danggui-Honghua.
Predictive models were used including Similarity Ensemble Approach (SEA, http://sea.bkslab.org/)29, STITCH
(http://stitch.embl.de/)30 and PharmMapper sever (http://59.78.96.61/pharmmapper)31, and databases were
mined including Herbal Ingredients’ Targets database (HIT, http://lifecenter.sgst.cn/hit/)32, erapeutic Targets
Database (TTD, http://bidd.nus.edu.sg/group/ttd/)33, DrugBank (http://www.drugbank.ca/) and Google Scholar.
Finally, 42 targets related to BSS were determined, interacting with the selected 31 active ingredients of this herb
pair (Table2).
Target proteins of Danggui. irty-three targets were identied for 15 active ingredients of Danggui with 108
interactions. Multiple therapeutic targets concerning BSS were mediated by the active ingredients of Danggui, such as
ESR1, ESR2, PTGS1, PTGS2, JUN, ICAM1, NOS2, NOS3, and MAPK1. Most of these targets are involved in vascular
and central neural systems. For instances, ESR1 and ESR2 mediate the vascular system to promote the functional
recovery of vascular injury and provide neuroprotective eects in central neural system34; PTGS1 and PTGS2 con-
tribute to atherosclerosis and thrombosis by regulating the production of eicosanoids that modulate physiological
processes in the vessel wall35; JUN modulates smooth muscle cell proliferation in response to vascular angioplasty36;
additionally, ICAM1 mediates the adhesion of neutrophils and monocytes to vascular endothelium37. Specically,
Z-ligustilide, senkyunolide I, ferulic acid and coniferyl ferulate may mediate NOS2 or NOS3 to increase the nitric
oxide biosynthesis, thereby possibly exerting blood-vessel dilation, neuronal signal transmission, coordination of heart
No. Name Structure OB (%) Caco-2 DL Herbs
HH-11 Eriodictyol 71.79 0.17 0.24 C. tinctoriu s
HH-12 Scutellarin 2.64 − 1.08 0.79 C. tinctorius
HH-13 Scutellarein 18.97 0.31 0.24 C. tinctor ius
HH-14 Acacetin 34.97 0.67 0.24 C. tinctor ius
HH-15 Luteolin 36.16 0.19 0.25 C. tinctorius
HH-16 Rosmarinic acid 48.60 0.82 0.46 C. tinctor ius
Table 1. Active ingredients and ADME parameters of Danggui-Honghua. Note: DG-: the active compound
from Danggui (the radix of A. sinensis), HH-: the active compound from Honghua (the orets of C. tinctorius).
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rhythm and regulation of cellular respiration activities38. Vanillin may interact with four potential targets including
PTGS2, JUN, MMP9 and MAPK1 which are also relevant to nervous system and vascular diseases39.
Except for vanillin, nicotinic acid and β-sitosterol, other active ingredients from Danggui also interacted
with the targets related to inammation, abdominal pain, dysmenorrhea, embolism, thrombosis and ischemia.
Six potential targets including F2, F7, F10, F11, TBXA2R and MAPK14 are relevant to disorders of thrombosis,
which could clarify why Danggui exhibits strong anticoagulant function16. Additionally, there are also several tar-
gets such as PPARG, SOD1, RELA and GSK3B are involved in the ischemic and inammation processes. It should
ID Target UniProt ID Gene name Related diseases
T-01 Estrogen receptor P03372 ESR1 Nervous system diseases, Cardiovascular disease, Brain injury, Hyperlipidemia
T-02 Estrogen receptor beta Q92731 ESR2 Cardiovascular disease, Vascular injury response, Neoplasms
T-03 Peroxisome proliferator activated receptor gamma P37231 PPARG Inammation, Ischemic heart disease
T-04 Superoxide dismutase [Cu-Zn] P00441 SOD1 Ischemic injury, Neoplasms
T-05 Prostaglandin G/H synthase 1 P23219 PTGS1 Cardiovascular disease, Chronic inammatory diseases
T-06 Prostaglandin G/H synthase 2 P35354 PTGS2 Inammation, Nervous system diseases, Myocardial infarction, Stroke, Pain
T-07 Transcription factor AP-1 P05412 JUN Vascular disease, Immune system diseases, Cancer
T-08 Intercellular adhesion molecule 1 P05362 ICAM1 Inammation, Cardiovascular disease, Asthma, Autoimmune diseases
T-09 Signal transducer and activator of transcription
1-alpha/beta P42224 STAT 1 Ischemic injury, Inammatory disorders, Myocardial ischemia and reperfusion
injury
T-10 Hypoxia-inducible factor 1-alpha Q16665 HIF1A Stroke, Cardiovascular diseases
T-11 Vascular endothelial growth factor A P15692 VEGFA Neoplasms, Ischemic heart disease, Coronary artery disease
T-12 Nitric oxide synthase, inducible P35228 NOS2 Ischemia reperfusion injuries
T-13 Nitric oxide synthase, endothelial P29474 NOS3 Cardiovascular disease, Inammation, Coronary artery disease, Angina
T-14 Prothrombin P00734 F2 Coagulative disorders, romboembolic disorders, Coronary atherosclerosis,
rombosis, Myocardial infarction
T-15 Coagulation factor VII P08709 F7 Coagulative disorders, Cardiovascular disease, romboembolism
T-16 Coagulation factor Xa P00742 F10 Cardiovascular disease, Coagulative disorders
T-17 Coagulation factor XI P03951 F11 Clotting Disorders
T-18 romboxane A2 receptor P21731 TBXA2R Platelet adhesion
T-19 Heme oxygenase 1 P09601 HMOX1 Cardiovascular disease, Ischemic injury of the liver, Inammation, Vascular
disease, Cerebral vasospasm
T-20 Phosphatidylinositol-4,5-bisphosphate 3-kinase
catalytic subunit, gamma isoform P48736 PIK3CG Heart failure, Myocardial infarction, Cancer, Angioedema
T-21 Platelet-activating factor acetylhydrolase Q13093 PLA2G7 Atherosclerosis, Cardiovascular disorders
T-22 Type-1 angiotensin II receptor P30556 AGTR1 Cardiovascular disease, Heart failure, Ischemic stroke, Hypertension
T-23 Apoptosis regulator Bcl-2 P10415 BCL2 Immune system diseases, Cardiovascular diseases
T-24 Transcription factor p65 Q04206 RELA Embolic focal cerebral ischemia, Ischemic renal injury, rombosis,
Inammation, Atherosclerosis
T-25 Matrix metalloproteinase-2 P08253 MMP2 Atherosclerosis, Cancer, Multiple sclerosis, Coronary artery disease
T-26 Matrix metalloproteinase-9 P14780 MMP9 Atherosclerosis, Cancer, Multiple sclerosis, Coronary artery disease, Heart
failure
T-27 Acetylcholinesterase P22303 AChE Brain ischemia, Nervous system diseases, Cognitive decits
T-28 Caspase-3 P42574 CASP3 Venous thrombosis
T-29 Caspase-8 Q14790 CASP8 Nervous system diseases
T-30 Caspase-9 P55211 CASP9 Nervous system diseases, Brain injury
T-31 Alpha-1A adrenergic receptor P35348 ADRA1A Urogenital system, Hypertrophic vascular disease
T-32 Beta-1 adrenergic receptor P08588 ADRB1 Cardiac arrhythmias, Cardiovascular disease, Coronary heart disease, Immune
system disorders
T-33 Muscarinic acetylcholine receptor M2 P08172 CHRM2 Autoimmune cardiomyopathy, Chronic obstructive pulmonary disease, Pain
T-34 Neprilysin P08473 NEP Congestive Heart Failure, Hypertension, Prostate cancer
T-35 Glycogen synthase kinase-3 beta P49841 GSK3B Brain injury, Immunodeciency, Ischemia, Alzheimer’s disease
T-36 3-Hydroxy-3-methylglutaryl-coenzyme A reductase P13702 HMGCR Atherosclerosis, Cardiovascular disease, Coronary heart disease, Myocardial
infarction
T-37 Mitogen-activated protein kinase 1 P28482 MAPK1 Neurodegenerative diseases, Proliferative diseases
T-38 Mitogen-activated protein kinase 10 P53779 MAPK10 Ischemic stroke, Neurological diseases
T-39 Mitogen-activated protein kinase 14 Q16539 MAPK14 rombosis, Inammation, Alzheimer’s disease
T-40 Angiotensin-converting enzyme P12821 AC E Heart failure, Hypertension, Vascular disease
T-41 Platelet activating factor receptor P25105 PTAFR Ocular allergy, Hypertension, Inammation
T-42 Fibroblast growth factor receptor 1 P11362 FGFR1 Peripheral vascular disease, Coronary heart disease, Cancer
Table 2. Target information of Danggui-Honghua.
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be pointed out that some major targets, such as PTGS2, NOS3 and CHRM2, are also closely concerned with the
pain-related diseases, which may contribute to the blood-activating and stasis-dissolving eects of Danggui.
Target proteins of Honghua. For Honghua, by target shing, 16 active ingredients were validated to
bind with 36 target proteins related to BSS. ere are also plenty targets mediated by the active ingredients from
Honghua that are involved in vascular and central neural systems. For example, HSYA may have the poten-
tial to act on 18 targets including HIF1A, VEGFA, HMOX1, PTGS2, CASP3, CASP9, AGTR1, PTAFR, and
GSK3B. Actually, HSYA has been identied as an inhibitor of HIF1A, which might contribute to its therapeutic
application in vascular diseases40. Beyond that, it also has a strong antagonistic eect on the PTAFR protein,
which may explain its function in both inammatory and neuropathic pain responses41. Analogously, scutellarin
exhibits strong neuroprotective eect because it may interact with STAT1, NOS2, NOS3, VEGFA and FGFR1
simultaneously42.
Inammatory response is of enormous signicance in BSS. Six active compounds of Honghua, including
6-hydroxykaempferol (HH-6), eriodictyol (HH-11), rosmarinic acid (HH-16), kaempferol, quercetin, and luteo-
lin, may have interactions with PPARG which is expected to control inammation associated with gut, myocar-
dial, lung and cerebral ischemia43. Other ve active ingredients, HSYA, rutin (HH-10), saor yellow A (HH-2),
saor yellow B and eriodictyol, were identied to interact with PTGS1, which is a potential target for the next
generation of anti-inammatory drugs44.
Of note, we have implemented molecular docking combined with the in vitro experiments to explore the
structure-activity relationships of the active ingredients among Si-Wu-Tang series containing the herb pair
Danggui-Honghua45,46. Flavonoids including quercetin (IC50 = 0.035 mM), luteolin (IC50 = 0.052 mM), kaemp-
ferol (IC50 = 0.109 mM), and acacetin (IC50 = 0.140 mM) showed F2 inhibition activity in vitro. By the soware of
Molegro Virtual Docker (http://www.molegro.com), quercetin (− 89.72 kJ/mol) exhibited high average MolDock
score value and interacted with the active site residues Ala230, His79, Lys88, Gly258, Ser256, and Trp86 of F245.
It was also found that ferulic acid (60.45%), quercetin (70.00%), kaempferol (45.15%), HSYA (45.28%), rutin
(90.83%), and scutellarin (53.46%) in 20 μ g/mL showed higher estrogenic activity in vitro than caeic acid
(24.73%) in 20 μ g/mL. And ferulic acid (− 72.8 kJ/mol), quercetin (− 76.3 kJ/mol) and rutin (− 87.2 kJ/mol)
exhibited high average MolDock score values and strong binding anity to ESR146. It could be concluded that the
predicted targets were in agreement with our previous experiments. erefore, the integrated in silico approach
should be feasible and convincible to explore the compound-target interactions of Danggui-Honghua.
Target and contribution index analysis to decipher the combination rule of Danggui-Honghua.
To facilitate the visualization and interpretation of the complex relationships between all active ingredients of
Danggui-Honghua and their targets, a bipartite graph of C-T network was constructed (Fig.3). Two character-
istics of Danggui-Honghua were observed from network analysis: (1) the promiscuous properties of its active
ingredients and (2) the existence of highly interconnected compounds. e average number of potential targets
per active ingredient was 6.5. And all active ingredients in this herb pair were potential multiple-kinase inhib-
itors or activators. Amongst them, those ones with high interconnection degrees were responsible for the high
interconnectedness of the C-T network, especially HSYA (degree = 18), Z-ligustilide (degree = 15), quercetin
(degree = 12), luteolin (degree = 11), ferulic acid (degree = 11) and Z-butylidenephthalide (degree = 10). From
the topological features of this network and the functional properties of the proteins (SupplementaryTableS2
and Fig.S1A), there were dierent modes of actions between compounds and targets in Danggui-Honghua. As
shown in the C-T network (Fig.3), the ecacy of this herb pair not only concentrated on modulating the crucial
targets involving in the vascular and central neural systems (ESR1, ICAM1, HMOX1 and NOS3), but also, more
essentially, focused on the regulation of the other proteins mediating inammation, thrombosis, ischemia, dys-
menorrhea and abdominal pain (F7, TBXA2R, PTGS2, CHRM2, and NOS2) to relieve the pathological changes
and prolong the ecient curing process. For example, in animal models, increased expression of HMOX1 has
been shown to protect tissues and cells against ischemia-reperfusion injury, oxidative stress, inammation and
hypoxia-induced vascular stasis47. e protein HMOX1 was found to have interactions with 9 ingredients, and
senkyunolide A, kaempferol and eriodictyol synergistically increase HMOX1 expression48–50. In addition, the neu-
rotoxic proinammatory mediator PTGS2 was also connected with several active ingredients. Especially, HSYA,
Z-ligustilide and ferulic acid were conrmed to enhance anti-inammatory eects via signicantly attenuating
the expression level of PTGS24,51,52.
e T-D network was further constructed based on all the targets and their corresponding diseases. As shown
in the graphical network (Fig.4), both Danggui and Honghua could regulate the proteins related to vascular and
central neural system diseases as well as inammation and pain. e target scope of Honghua was broader than
that of Danggui. Several targets mediated by the active ingredients of Honghua including PTAFR, PTGS2, NOS3
and STAT1, may alleviate the accompanying symptoms of BSS such as inammation and pain. As to Danggui,
its active ingredients mediated several targets including ADRB1, CHRM2, GSK3B, BCL2 and ICAM1, which
may be helpful for improving the immune system. Modern research has demonstrated that ischemic processes
release mediators activating the innate immune system that may be induced by the formation of thrombi inside
blood vessels53. Fortuitously, several targets associated with cancer were also discerned in the T-D network. HSYA
from Honghua was validated to antagonize tumor angiogenesis by inhibiting the protein expression of VEGFA,
MMP9 and HIF1A4. Additionally, Z-butylidenephthalide, senkyunolide A and Z-ligustilide from Danggui were
conrmed to exhibit anti-proliferative potential and signicant synergy on colon cancer cells54.
As mentioned earlier, a contribution index of every active ingredient was proposed based on network based
ecacy weighted by literature. According to calculated results (Fig.5 and SupplementaryTableS3), six com-
pounds emerged from the active ingredients, including HSYA, saor yellow A, saor yellow B, Z-ligustilide,
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ferulic acid, and Z-butylidenephthalide. They displayed the most contribution to the blood-activating and
stasis-dissolving eects of Danggui-Honghua with a sum of CIs of 88.13%. erefore, the above discussion may
fully clarify why Danggui-Honghua could produce synergistic and complementary eects.
Pathway analysis to explore the underlying mechanisms of Danggui-Honghua. Signaling path-
ways, as an important component of the system pharmacology, link receptor-ligand interactions to pharma-
codynamics outputs55. e canonical pathways associated with BSS treatment and prophylaxis were extracted
from Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) database, which ends
up with 20 KEGG pathways, including cAMP signaling pathway, calcium signaling pathway, NF-κ B sig naling
pathway and sphingolipid signaling pathway (Fig.6). e NF-κ B signaling pathway is crucial for focal cerebral
ischemia/reperfusion induced inammatory injury56. Previous research has shown that HSYA, saor yellow A,
and caeic acid could inhibit the NF-κ B signaling pathway contributing to the cross-talk of multiple targets in
anti-inammation57. e sphingolipid signaling pathway was proven to play a critical role in the ischemic precon-
ditioning and the pathophysiology of stroke58.
For the purpose of systematically dissecting the underlying mechanisms of Danggui-Honghua, all of the tar-
gets interacting with the active ingredients were mapped onto the 20 KEGG pathways and the T-P network was
generated (Fig.7). e TNF signaling pathway exhibited the highest number of target connections (degree = 11),
followed by HIF-1 signaling pathway with 9 targets, estrogen signaling pathway and neurotrophin signaling
pathway with 8 ones, respectively. ese high-degree pathways were closely related to the vascular and central
neural systems and inammation. e TNF signaling pathway plays an important role in the ischemic stroke
and the vascular injury involved in multiple targets including JUN, PTGS2 and ICAM159,60. e HIF-1 signaling
pathway was an underlying mechanism of neuroprotection and anti-ventricular cell apoptosis61,62. Fortunately,
22 out of 31 active compounds from Danggui-Honghua, especially HSYA, quercetin, Z-ligustilide and luteolin,
were implicated in regulating the major targets of HIF-1 signaling pathway, such as HMOX1, NOS2, NOS3,
RELA, PIK3CG and MAPK1. Recently, we successfully discovered the underlying blood-activating mechanisms
of Danggui-Honghua by metabolomics analysis. Four potential metabolic pathways were speculated, including
phenylalanine metabolism, sphingolipid metabolism, arachidonic acid metabolism, and arginine and proline
metabolism63. In the present study, arginine and proline metabolism, arachidonic acid metabolism, and sphingo-
lipid signaling pathway (including sphingolipid metabolism) were also deciphered, which were consistent with
our previous metabolomics results63.
Figure 3. Compound-target network of Danggui-Honghua. irty-one active compounds (HH-/DG-,
rectangle) map 42 potential protein targets (short name referred in Table2, circle). e 15 bright green
rectangles are active compounds from Danggui and the 16 red ones represent those from Honghua. e 27
orange circles are the proteins targeted by those compounds screened out of both herbs. e 6 dark pink circles
on the upper side are the potential proteins hit by the compounds of Danggui and 9 tea green ones on the
bottom side are the potential proteins only targeted by the compounds of Honghua.
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Overall, it could be speculated that the herb pair Danggui-Honghua exert the blood-activating and
stasis-dissolving eects mainly through the regulation of TNF, HIF-1, estrogen, and neurotrophin signaling
pathways. As a holistic medicine, this herb pair may also be implicated in arginine and proline metabolism,
arachidonic acid metabolism, VEGF signaling pathway, MAPK signaling pathway, calcium signaling pathway,
and sphingolipid signaling pathway to regulate the vascular and nervous systems, as well as the inammation
and pain.
Discussion
Herb pairs, the smallest compatible units in TCM formulae, have become a prominent concern during the past
decade2. Danggui and Honghua, combined as a classical herb pair, have been frequently used in TCM prescrip-
tions5. rough data mining and pharmacological approaches, this herb pair was found to produce synergis-
tic and complementary eects to treat BSS64–69. However, the complexity of the chemical components of this
herb pair and their corresponding multiple targets in vivo led to extreme diculty to elucidate the molecular
mechanisms.
Nowadays, system pharmacology provides a powerful avenue for compatible and mechanistic exploration
of TCM10,11. In our work, an integrated system pharmacology approach, combined a number of network-based
computational methods and algorithm-based approaches, was used to select active compounds, predict targets,
construct networks, and illuminate the molecular synergy of Danggui-Honghua on BSS. irty-one active ingre-
dients with favorable bioactivities and contents were selected from the 347 compounds of Danggui-Honghua
by ADME ltering, providing foundational clues for thorough investigation on this herb pair. It was found that
some biological activities against BSS of these active ingredients have been reported previously26–28,39–42,70–72,
highlighting the credibility of our ADME ltering criteria. en, an integrated in silico approach was applied
to decipher the 42 targets for these active ingredients related to BSS including 27 common targets, which dis-
tinctly explained the action modes and biological processes that active ingredients achieve their synergistic and
complementary curative eects. It is worth mentioning that the results from several compound-target interac-
tion experiments by us and other groups indicated the reasonability of our integrated in silico approach45–51,73.
Subsequently, 20 signaling pathways associated with BSS treatment and prophylaxis by Danggui-Honghua were
retrieved. A recent study on the anti-inammatory eects of volatile oils from Danggui (78.61% Z-Ligustilide and
7.99% Z-Butylidenephthalide) using GC-MS-based metabolomics revealed that it may be involved in regulating
the arachidonic acid metabolism74, which is comparable to our ndings. Finally, the C-T, T-D and T-P networks
clearly elucidated the molecular synergistic actions of Danggui-Honghua in a holistic context. By network sys-
tematic analysis and contribution index calculation, HSYA, saor yellow A and saor yellow B in Honghua,
together with Z-ligustilide, ferulic acid and Z-butylidenephthalide in Danggui, displayed the most contribution to
the blood-activating and stasis-dissolving eects of Danggui-Honghua. Meanwhile, this herb pair could regulate
the proteins related to vascular and central neural system diseases as well as inammation and pain implicated
Figure 4. Target-disease network of Danggui-Honghua. e 6 purple circles and 9 red circles represent the
targets of Danggui and Honghua, respectively. e 27 pink circles represent the targets shared by Danggui and
Honghua. e 7 yellow diamonds represent the disorders related to those 42 targets.
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in TNF, HIF-1, estrogen, and neurotrophin signaling pathways, arginine and proline metabolism, arachidonic
acid metabolism, and VEGF signaling pathway. Noteworthy, based on our research, several targets and signaling
pathways of Danggui-Honghua acting on BSS have been found for the rst time. All of these results are expected
to help identify novel curative ecacy and take full clinical advantage of Danggui-Honghua.
Due to the above ndings mainly relied on theoretical analyses, more experiments are anticipated to support
these ndings as well as potential clinical applications. It should be noted that the majority of the ingredients in
both Danggui and Honghua were hydrotropic, and the OB values of a third of the selected 31 active ingredients
were less than 30%. erefore, the availability of these active constituents by gut microbiota especially under the
disease state may be a critical step towards the emergence of their bioactivities in vivo75.
Methods
Chemical ingredients database building. All of the constituent data of Danggui (the radix of A. sinensis)
and Honghua (the orets of C. tinctorius) were retrieved from TcmSP™ , a unique system pharmacology platform
designed for herbal medicines12, and then manually supplemented through a wide-scale text-mining method.
Meanwhile, four important pharmacology-related properties were also obtained from TcmSP™ , including MW,
MLogP, nHDon and nHAcc.
Figure 5. e CI and accumulative CI of active ingredients in Danggui-Honghua.
Figure 6. Distribution of partial targets of Danggui-Honghua on the compressed pathway. e orange
nodes are potential targets. And the light blue nodes are relevant targets in the pathway.
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Active ingredients screening. e active ingredients from Danggui-Honghua were ltered by integrating
three indexes including OB, Caco-2, and DL. A robust in silico model OBioavail 1.1 (Yangling, Shaanxi, China)
that integrated the metabolism and transport information was employed to calculate the OB values of all herbal
ingredients76. ose ingredients with OB ≥ 30% were selected. e VolSurf built-in Caco-2 permeability model
(Tripos, St. Louis, America)77 was implemented to screen active compounds. Given that molecule with Caco-2
value less than − 0.4 is not permeable, the threshold of Caco-2 permeability was set to − 0.4. Database-dependent
DL evaluation approach based on Tanimoto coecient78 was applied and shown as follows.
=
×
+−×
TAB
AB
ABAB
(, )
(1)
22
In this eq.(1), A represents the molecular descriptors of herbal compounds, and B displays the average molecular
properties of all compounds in Drugbank. ose ingredients with DL ≥ 0.18 were preserved. e ingredients
were adopted as the candidate compounds for further analysis when they met all of these three criteria.
Targets shing. To identify the corresponding targets of the active ingredients of Danggui-Honghua, several
approaches combined with chemometric method, information integration and data-mining were implemented.
First of all, the biological targets of active ingredients were obtained from SEA29, STITCH30, and PharmMapper
sever31. Amongst them, the PharmMapper gives the best mapping poses by comparing with available targets
in PharmTargetDB (a large, in-house repertoire of pharmacophore database belongs to PharmMapper) and
the respective N-best t poses are generated. All active compounds were also sent to HIT32, TTD33, DrugBank
and Google Scholar to mine compound-target interactions supported by literature. en, to better dissect the
role of Danggui-Honghua in BSS treatment, all targets obtained from the previous two steps were sent to TTD,
Comparative Toxicogenomics Database (CTD, http://ctdbase.org/) and PharmGKB (http://www.pharmgkb.org)79
to mine target-related diseases. Finally, those targets which were implicated in the aforementioned pathophysiol-
ogy and clinical manifestations of BSS were retained, and the others were eliminated.
Figure 7. Target-pathway network of Danggui-Honghua where light green nodes represent the targets and
purple nodes the pathways.
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Networks construction. ree networks were constructed: (1) Compound-target network (C-T network).
Active ingredients of Danggui-Honghua and their corresponding targets were employed to generate the C-T
network. (2) Target-disease network (T-D network). All targets and their corresponding diseases were employed
to build a bipartite graph of T-D network. (3) Target-pathway network (T-P network). e pathway information
of targets were extracted from the database of KEGG, and then a bipartite T-P network composed of targets and
their corresponding putative pathways was built. All visualized network graphs were constructed by Cytoscape
3.2.1 (http://www.cytoscape.org/), an open soware package project for visualizing, integrating, modeling and
analyzing the interaction networks80.
Contribution indexes calculation. In order to estimate the contribution of each active ingredient to the
blood-activating and stasis-dissolving eects of Danggui-Honghua, a contribution index (CI) based on network
based ecacy (NE) weighted by literature was proposed and calculated by eqs(2) and (3):
∑
=
=
NE jd()
(2)
i
n
i
1
=
×
∑××
=
CI j
cNEj
cNEi
()
()
() 100%
(3)
j
i
m
i
1
where n is the number of targets associated with ingredient j; di is the degree of target i associated with ingredient
j; ci is the number of BSS-related literature of ingredient i; m is the number of ingredients.
If the sum of CIs for the top N ingredients was more than 85%, these relevant N ingredients were considered
to contribute the most to the blood-activating and stasis-dissolving eects.
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Acknowledgements
We thank Dr. Zhang L. and Liao H.X. (School of Medicine and Pharmacy, Ocean University of China) for helping
with material collection. We also thank Dr. Feng W.W. (Beijing Shijitan Hospital, China) for English improvement
and constructive comments. This work was supported by the National High Technology Research and
Development Program of China (863 Program) (No. 2013AA093001), the National Natural Science Foundation
of China (81274058), and the Taishan Scholars Program, China.
Author Contributions
C.Y.W. and H.S.G. conceived of and proposed the idea. S.J.Y. and L.T.X. designed the study. S.J.Y., L.T.X., Y.C.F.
and S.J.L. performed the experiments. L.T.X., Y.C.F., S.J.L. participated in data analysis. C.Y.W., Y.P.T. and J.A.D.
contributed to writing, revising and proof-reading the manuscript. All authors read and approved the nal
manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Yue, S.-J. et al. Herb pair Danggui-Honghua: mechanisms underlying blood stasis
syndrome by system pharmacology approach. Sci. Rep. 7, 40318; doi: 10.1038/srep40318 (2017).
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