LI Zhiyong,ZHU Na,LI Jianliang,FENG Liang,JIANG Yanyan,LI Caifeng,LIN Ling,HUANG Xiulan

LI Zhiyong,Postdoctoral mobile station of environmental biology,Faculty of Life Science and Technology,Kunming University of Science and Technology,Kunming,Yunnan 650500,China;Center for Analytical Chemistry of Chinese Materia Medica,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China

ZHU Na,LIN Ling,HUANG Xiulan,Pharmacological Department,School of Pharmacy,Minzu University of China,Beijing,100081,China

LI Jianliang,Aerosol preparation research center,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China

FENG Liang,School of Traditional Chinese Pharmacy,China Pharmaceutical University,Nanjing,Jiangsu 211198,China

JIANG Yanyan,Department of Chinese Materia Medica Chemistry,School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing,102488,China

LI Caifeng,Academician work station,Jiangxi University of Traditional Chinese Medicine,Nanchang,Jiangxi,330004,China

Abstract OBJECTIVE:To evaluate the compatibility of Tianma(Rhizoma Gastrodiae,TM),Yanlingcao (Trillium tschonoskii Maxim,YLC) and Bingpian (Borneolum Syntheticum,BP),and their efficacy in the treatment of cerebral ischemic stroke.METHODS:Network pharmacology was used to determine the compatibility of TM,YLC,and BP,and their potential mechanism.The middle cerebral artery occlusion (MCAO) rat model was used to evaluate the curative effect of the six combinations of TM,YLC,and BP (TZB1-TZB6) on cerebral ischemia,by using the weight matching method to form.The potential component changes of TM and YLC in the blood and brains of rats were analyzed using ultra performance liquid chromatography-mass spectrometry.Finally,molecular docking linked the results of animal experiments and network pharmacology,determining the potential component contributors of TM and YLC to treating ischemic stroke.RESULTS:TZB reduced the cerebral infarct volume and protected the nerve cells in MCAO rats.The components of TM and YLC were also identified in the blood and brain homogenate,and BP can facilitate the entry of the components of TM and YLC into the blood and brain.Diosgenin,pennogenin,and gastrodin induced effective binding activities with adenosine receptor a1.CONCLUSION:We investigate an approach that improves the means of folk prescription combined with multi technology that maybe promote the transformation of Chinese medicinal prescription into component-based Chinese medicine.

Keywords:infarction,middle cerebral artery;brain ischemia;molecular docking simulation;Adenosine receptor a1;network pharmacology

1.INTRODUCTION

Cerebrovascular accidents (stroke) are the second leading cause of death and the third leading cause of disability worldwide.1Among all the stroke cases,87%are ischemic,which is caused by restricted blood flow in a specific area of the brain,resulting in an irreversibly damaged ischemic core.2The development of ischemic stroke starts with a blood flow cessation with energy depletion,followed by severe pathological changes and brain damage through a series of molecular cascades after cerebral artery occlusion.3In China,the preventive and treatment methods of stroke center on modern Western Medicine.Also,most patients with stroke undergo treatment with Chinese herbal medicine or acupuncture.Approximately 66% of patients use Chinese herb products in the first 48 h of acute ischemic stroke.4

Tianma(Rhizoma Gastrodiae,TM) has been used successfully for several centuries in China to treat various diseases caused commonly by wind invasion,modern studies show that TM has neuroprotective and anti-vertigo activities,with beneficial effects against cardio-cerebral-vascular diseases.5Yanlingcao (Trillium tschonoskiiMaxim,YLC) is one of the four well-known herbs among the Tujia people in China.It has beneficial effects,such as tranquilizing and calming the mind,expelling wind,promoting blood circulation,and relieving pain.6The compatibility of TM and YLC,which is prevalent in Tujia folk medicine in Wuling Mountains Area,are effective in expelling wind,dissipating phlegm,tonifying the brain,and calming the mind and can be used to treat dizziness and ischemic encephalopathy.7However,considering that Tujia people have no written language,the ideal doses of TM and YLCremain under development,which limit their clinical application.BP is a messenger drug in Traditional Chinese Medicine (TCM),frequently used to evaluate the compatibility of TCM prescriptions for treating brain diseases.This study focuses on the compatibility of TM,YLC,and BP,and their efficacy and appropriate dosages,for treating cerebral ischemic stroke,based on the TCM theory and the long-term medical practice of the Tujia people.

2.MATERIALS AND METHODS

Evaluating the compatibility of TM,YLC,and BP using network pharmacology The compounds of TM,YLC,and BP were obtained using the Chemistry Database (http://www.organchem.csdb.cn),TCMID (http://www.megabionet.org/tcmid/),and TCMSP,8(http://lsp.nwu.edu.cn/tcmsp.php),DruLi-TO (http://www.niper.nic.in/pi_dev_tools/DruLiToWeb)and SwissADME (http://www.swissadme.ch/) were respectively used to calculate the drug likeness of compounds,following Lipinski’s“rule of five”9,10and to screen the compounds that are easily absorbed by the gastrointestinal (GI) tract.The properties of the compounds were determined using the BOILED-Egg.11Moreover,the potential targets of TM,YLC,and BP were predicted using the Swiss Target Prediction platform (http://www.swisstargetprediction.ch/).The disease targets of "apoplexy," "apoplexia," "stroke," and"ischemic stroke" were searched in the OMIM(https://www.omim.org/),Human Phenotype Ontology(https://hpo.jax.org/),and TCMIP (http://www.tcmip.cn).The common targets of TM,YLC,and BP (named as TZB) were determined,observed to interact with the targets of stroke,and then processed using the STRING database (https://www.string-db.org/) to obtain the protein-protein interaction (PPI) and perform a pathway enrichment analysis.The compound-candidate target networks of TM,YLC,and BP respectively,and the"TZB-compound-target-pathway" networks were constructed using Cytoscape 3.6.1.In contrast,the core targets related to the active compounds of TZB treating stroke were correlated and included in a mechanism network enrichment analysis using the STRING database.

2.1.Composition design of TM,YLC,and BP

The limited doses of TM and BP were based on theChina Pharmacopoeia(2015 edition),the YLC dose was based on theTujia medical formulae12andstandards for Chinese medicinal materials in Hubei Province(2009),and determined by numerous research studies and preliminary experiments.The upper limited doses of TM,YLC,and BP (15 g/kg,5 g/kg,and 39 mg/kg,respectively) were determined using the weight matching method and set to six equal gradient level dosages.Every component in the TCM formula could be measured according to the relationship between the dose and the combined effect (the dose-response relationship).13Moreover,the dose distribution principle of weight matching method is shown in Table S1,whereas Table 1 shows the dose distribution of TM,YLC,and BP.

Table 1 Dose distribution of TM,YLC,and BP (g/kg)

2.2.Preparation of the ethanol extracts of TM,YLC,and BP

TMand YLC were purchased from the Anguo medicinal material market in Hebei Province,whereas BP was purchased from the Tongrentang Pharmacy (Beijing,China).All voucher specimens were identified by Prof.Yaojun Yang from the School of Chinese Matacia Medica of Beijing University of Chinese Medicine.Ethanol extracts of TZB1-TZB6 were then obtained according to the following protocol:TM and YLC were weighted according to the dosages used in different compatibility groups (TZB1-TZB6),ground into powders (filtered through a 2 mm steel sieve),refluxed in 10 times the volume of ethanol (70% v/v),and heated twice using the reflux extraction method for 45 min each.The extracts were filtered,and the two filtrates were combined,evaporating to the required concentration under reduced pressure.BP was then dissolved with a small amount of alcohol and added to the ethanol extracts of TZB1-TZB6.

2.3.Effect of TZB1-TZB6 on rats with cerebral ischemia-reperfusion injury

2.3.1.Care and use of animals

Male Sprague-Dawley rats [Approval No.SCXK(Beijing) 2012-0001],300-320 g,were provided by Beijing Vital River Laboratory Animal Technology Co.Ltd.(Beijing,China).The animals were free fed with standard chow and water under a 12/12 h dark/light cycle and specific-pathogen-free conditions,adaptive feeding for 3 d.The Animal Experimental Ethical Inspection Form of Beijing University of Chinese Medicine,China approved the animal study protocol (Approval Number:BUCM-4-2017040816-2085).

2.3.2.MCAO model

The MCAO model was performed,as explained previously.14When those rats that underwent MCAO awakened,their neurological functional scores were determined within 24 h after reperfusion using Zea longa’s scoring scale;15the rats with a score of 1-3 were included in the next experiments.Furthermore,the MCAO and sham-operated rats were administered with penicillin for three days to prevent infection after operation.

2.4.Drug administration in MCAO and sham-operated rats

The extracts of TZB1-TZB6 were prepared with distilled water,whereas the positive control drug,Butylphthalide(NBP,CSPC NBP Pharmaceutical.Co.Ltd.,Shijiazhuang,China) was prepared with olive oil.We randomized 64 MCAO rats into nine different treatment groups as follows:vehicle group,TZB1-TZB6 groups,NBP group,and sham control group (another nine shamoperated rats).The rats were treated with the vehicle(distilled water):the NBP-treated rats were given 70 mg/kg,whereas the TZB-treated rats were given 7.05,10.14,9.31,12.8,13.31,and 17.56 g/kg (TZB1-TZB6),respectively.All treated rats received oral doses of the specific drugs for seven days (once a day) after the end of modeling.Also,the sham-operated rats were treated with distilled water.

2.5.TTC staining and the calculation of the cerebral infarct volume

After 24 h of reperfusion,two rats were randomly selected from the MCAO group and the sham-operated group,respectively.TTC staining was performed.After the last administration,all of the animals were euthanized.Furthermore,the TTC staining was repeated,and the cerebral infarct volume in each group was measured usingImage-Pro Plus 6.0.The calculation formula is as follows:

Infarct volume=([x-(y-z)] × 2 mm)/(x × 2 mm) × 100%x,total brain area of the non-ischemic side;y,total brain area of the ischemic side;z,infarcted area of the brain.

2.6.Histopathological evaluation

To evaluate the effects of TZB1-TZB6 on cerebral I/R injury,histopathology examinations of cortex neurons were performed in each group through hematoxylineosin (HE) staining and Nissl staining.All histological sections were scanned using NanoZoomer-SQ(Hamamatsu Photonics,Japan).The nerve cells of the functional area of the cerebral cortex and the corpus striatum in the ischemic side of the brain were counted by selecting six visions randomly.Furthermore,the histopathological changes in the ischemic cortex were analyzed according to Kato’s method.16Table S2 lists the grading criteria.

2.7.Statistical analysis

The results were presented as means with a standard error of the mean.The significance of the between-group differences was evaluated using the SPSS Statistics version 17.0 (SPSS Inc.,Chicago,IL,USA).ThePvalues were calculated using the Student'st-test or oneway analysis of variance.P＜ 0.05 indicates a statistical significance,andP＜ 0.01 a significant difference.

2.8.Analysis of the components of TZBn in the blood and brain using Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

The extract of TZBn(obtained based on the efficacy results) was frozen to the lyophilized powder with lyophilizer (ALPHA1-2,CHRIST,Germany) and dissolved with 10 % methanol;the supernatant fluid was then prepared after centrifugation at 10000 rpm for 5min(5804R,EPPENDORF,Germany).The male rats (n=18,6 rats per group) were divided randomly into three groups as follows:control group,TZBngroup,and TZngroup (the composition of TM and YLC prepared in proportion to TZBn).The rats of TZBnand TZngroups were administered with TZBnand TZn,respectively,whereas the rats of the control group were administered with distilled water twice a day for seven days.The blood samples were then collected from the orbital plexus of the eyes at 0.5,1,2,3,4,and 5 h after last part of the administration and immediately transferred into heparinized tubes and centrifuged at 3500 rpm for 10 min to prepare the plasma supernatant.The cerebrums of the rats were stripped off and prepared in homogenates (1∶1 with normal saline).All the samples were transferred into clean tubes and stored at -80 ℃.Furthermore,the plasma and brain homogenates (200 μL) were treated with acetonitrile (3 times the volume);the supernates were extracted after centrifugation and dried with nitrogen.

UPLC-MS analysis was performed using the UltiMate 3000 (Thermo Fisher,MA,USA) and Q Exactive Plus(Thermo Fisher,MA,USA),coupled with ESI and Xcalibur 4.0.Chromatographic separation was performed using the ACQUITY UPLC? BEH C18 Column (1.7 μm,2.1 mm × 100 mm,Waters,MA,USA)with the injection volume being 5 μL and the column temperature being kept at 40 ℃.Table S3 shows the elution gradient condition.The mass spectral conditions included electrospray positive and negative ion modes.The voltage of the positive ion spray was 3300 V,whereas the voltage of the negative ion spray was 3500 V.The capillary temperature was 320 ℃,the sheath gas flow rate was 40 arb,and the auxiliary gas flow rate was 15 arb.The S-lens RF level was 60.0,and the auxiliary gas heating temperature was 350 ℃.The MS data were collected in the full scan mode from m/z 100 to 1200 amu.

2.9.Molecular docking

The identified components of TZBnin the blood and brain would correspond to the compounds of Tianma(Rhizoma Gastrodiae) and Yanlingcao (Trillium tschonoskii Maxim) in the network pharmacological research,and the core targets related to the components of TZBnwere mapped in the important signal pathway of TZB against stroke.The target proteins were downloaded from the RCSB Protein Data Bank(http://www.rcsb.org) and saved in pdb format.The 3D structures of these small ligand molecules were drawn with Chem 3D 18.0;these small molecules were then kept in mol2 format.Furthermore,an accurate docking with the components of TZBnand the target proteins was performed using iGEMDOCK v2.1.

3.RESULTS

3.1.New formula consisting of TM,YLC,and BP having a potential curative effect on ischemic stroke

A total of 1314 targets related to the active compounds ofTM (21 compounds),YLC(5 compounds),and BP (12 compounds) were predicted,including 273 targets related to TM,205 targets associated with YLC,and 246 targets related to BP.A total of 370 stroke-related targets were screened using the OMIM and HPO disease databases.A Venn diagram showed the common predictive targets of TM,YLC,and BP and stroke targets(Figure 1).The common targets of TM,YLC,and BP were then mapped to the stroke target network.The Kyoto Encyclopedia of Genes and Genomes (KEGG)pathway enrichment analysis of these targets showed the potential mechanism of TZB on stroke (Figure 2),including the neuroactive ligand-receptor interaction,AGE-RAGE signaling pathway in diabetic complications,cAMP signaling pathway,TNF signaling pathway,serotonergic synapse,arachidonic acid metabolism,etc.

We determined 72 common targets between the potential targets of TZB and known targets of stroke.We identified 37 core targets based on the cluster analysis if the degree value was greater than 10.Among the 37 targets,21 were related to TM and YLC,respectively,and 26 targets were related to BP.Figure 3 shows TZB’s potential anti-stroke mechanism.Moreover,the results showed that BP may improve the beneficial effects of TM and YLCon stroke.

3.2.Effect of TZB on the cerebral infarction in MCAO rats

TTC staining was then used to identify the infarcted area of the brain.In MCAO rat's brain,the infarcted area was in the cerebral cortex and striatum;however,there was no white infarctions and brain edema in the shamoperated group.Compared with model group,the cerebral infarct volumes of the rats in the TZB1,TZB3,TZB5,and NBP groups decreased significantly (P＜0.05).Furthermore,in the brain of TZB5 group,the cerebral infarction only existed in the striatum.Also,no infarctions in the functional area of the cerebral cortex were observed,with almost no signs of edema (Figure 4).

Figure 1 BOILED-Egg of compounds;Venn diagram of targets and PPI network of stroke

Figure 2 KEGG pathways of the core targets relevant to TZB treating stroke

Figure 3 Interaction map between the known targets of stroke and the potential targets related with TZB

Figure 4 TTC staining of the coronal brain sections and infarct volume of MCAO rats

Figure 5 Effect of TZB1-TZB6 on the histopathological changes of cerebral infarction in MCAO rats (n =6)

3.3.Effect of TZB on the brain histopathology of MCAO rats

Based on the HE staining and Nissl staining of shamoperated group,the brain structures were complete and clear,and the nerve cells were arranged closely,with many complete cell structures.Because the loose changes and brain edema presented in the cerebral infarcted area of MCAO rats,it was difficult to find complete nerve cells.In the TZB1,TZB3,TZB5,and NBP groups,the number of intact cells increased significantly compared with the model group,while the severity of brain edema was reduced.Moreover,the pathological grading results showed that in model group,there were many neuron deaths in the cerebral ischemic cortex,and the infarcted area was more than 1/3 of the total area of the ischemic cortex.In the TZB3,TZB5,and NBP groups,the number of neuron deaths was relatively low,and the grading score was significantly lower (P＜0.05) (Figure 5).

3.4.Identification of the absorbed bioactive components of TZB5 in the blood and brain of rats

In the TZB1,TZB3,and TZB5 groups,the pathological state of the cerebral infarcted area improved,and the number of normal neurons increased compared with the model group.The survival state of MCAO rats treated with TZB1-TZB6 for 7 d were 45.45%,18.18%,60.00%,10.00%,80.00%,and 20.00%,respectively.TZB5 improved the survival state of rats with cerebral ischemia;also,it effectively reduced the cerebral infarct volume,improved the structure of brain tissue,and protected the nerve cells.

Figure S1 shows the total ion flow diagrams of the extracts of TM and YLCin the positive and negative ion modes of the UPLC-MS mass spectrometry.In the primary mass spectrum,the data on the RT and accurate molecular weight of each compound were obtained,and the quality error was limited in ± 5 ppm.In contrast,according to the secondary fragment ion information based on the target mass spectrum peak,the chemical structure and possible cleavage pathways of the components were identified.The chromatographic peaks were analyzed using the Thermo Xcalibur Qual Browser(Thermo Fisher Scientific,MA,USA) and based on the related studies.We identified 52 peaks in the ion chromatogram,26 of which from TM and 26 from YLC,as shown in Table S4.Based on the relative molecular mass,RT,and mass spectrometry information,the components of TZB5 and TZ5 in the blood and brain homogenate were identified.In the group without BP,36 TZB5 components were identified in the plasma sample and 17 in the homogenate samples,Table S5 shows the results.

3.5.Molecular docking results

To verify the data of the serum medicinal chemistry and network pharmacological research,we conducted a molecular docking study using the iGEMDOCK program.As shown in Table S5,20 components were identified in the blood and brains of rats in the TZB5 group from TM andYLC.In the previous network pharmacological research,we found that gastrodin,caproic acid,and parishin,β-ecdysterone,diosgenin and pennogenin,have similar chemical structures to polypodine B,polyphyllin VI,polyphyllin VII,and pennogenin 3-O-β-chacotrioside,respectively.The core target proteins related to these components mainly concentrated in the neuroactive ligand-receptor interaction pathway including PLG (PDB ID:4cik),adenosine receptor a1 (ADORA1) (PDB ID:5n2s),AGTR1 (PDB ID:4zud),F2R (PDB ID:3vw7),and NR3C1 (PDB ID:4csj),to implement molecular docking with gastrodin,caproic acid,parishin,β-ecdysterone,diosgenin,and pennogenin.Table 2 lists the Docking scores of the best docking conformation between proteins and candidate compounds.As shown in Figure 6,diosgenin,pennogenin,and gastrodin were bound to the active pocket formed by ADORA1 residues through van der Waals force and formed a stable structure through the interaction with the hydrophobic residues.

Figure 6 Molecular docking of diosgenin,pennogenin,gastrodin and ADORA1

Table 2 Best docking conformation between proteins and candidate compounds (kcal/mol)

4.DISCUSSION

The reasonable compatibilities of TM,YLC,and BP in the treatment of ischemic stroke were evaluated by network pharmacological methods,which can be used to clarify the interactive relationship between multiple components,multiple targets,multiple pathways,and complex diseases,and is popular used to understand the mechanism of the addition and subtraction theory of TCM.17Based on the findings,the TCM formula (TZB)of TM,YLC,and BP might have specific therapeutic effects on stroke.Many important TZB pathways were also predicted in stroke treatment:neuroactive ligandreceptor interaction,TNF signaling pathway,etc.

The weighting method,based on the optimized or superreplaced Latin square design and uniform design principles,is used to achieve a compatibility optimization of the TCM formulae,based on the principle of the dose-response relationship,which is beneficial for the multi-factor and multi-level optimization experimental research of the prescription.13In the experiment that used the weighting method,only six compatibility groups with different doses need to beset at most to find the best compatibility in the experiments and determine the importance of each component in the formula.18The results showed that TZB5 was the optimal combination,which could reduce the brain damage caused by cerebral ischemia/reperfusion in MCAO rats,decrease the cerebral infarct volume effectively and protect the brain cells,and improve the survival state of MCAO rats.Based on the dosage composition of TM,YLC,and BP(12 ∶1.31 ∶0.061 g/kg) in the TZB5 group.We identified 52 compounds in TM and YLC.43 and 20 chemical components of TZB5 were identified in the blood and brain of rats,respectively.To exclude the difference in the quantity of the chemical components in the blood and brain,which is caused by the different proportions of TM and YLC in the TZB5 group,the rats treated with TZ5 (without BP) were assigned as the control group.The number of chemical components identified from TZ5 (36 in blood and 17 in the brain) was lower than that from TZB5 in the blood and brains of rats.The results indicated that BP might enhance the components of TM and YLC in the blood and brain of rats,promote the drug absorption in the gastrointestinal tract and drug distribution to the brain.19

Neuroactive ligand-receptor interaction was considered as the primary signal pathway of TZB5 in the treatment of cerebral stroke,as predicted by the network pharmacological research.Adenosine is perceived as a potential neuroprotective agent in the context of stroke,although no adenosine-based therapies to treat cerebral ischemia and reperfusion are currently available.20In the brain,ADORA1 is the most abundant and widespread adenosine receptor,21having the highest affinity for adenosine of all the subtypes.22Moreover,ADORA1 expression is high in the hippocampus,cerebral cortex,basal ganglia,and some thalamic nuclei in humans and various animals.21The activation of ADORA1 is generally considered protective in the context of noxious stimuli.In our study,diosgenin and pennogenin and gastrodinshowed good binding activities with ADORA1,indicating that TZB5 may treat cerebral ischemia through neuroactive ligand-receptor interaction,and gastrodin,diosgenin,pennogenin,β-ecdysterone,and caproic acid may be the potential bioactive components.

In conclusion,combining TM,YLC,and BP as a prescription is effective in the treatment of cerebral ischemic stroke.This finding is also consistent with the principles of the Tujia folk medicine practice and TCM theory.In our study,we proved the compatibility of TM,YLC,and BP based on the network pharmacological research and pharmacological experiments.The compatible doses of TM,YLC,and BP,which are conducive to the treatment of cerebral ischemia in MCAO rats,were also determined.Moreover,serum pharmacochemistry and molecular docking contributed to the identification of the potential component contributors of TM and YLC,including gastrodin,diosgenin,pennogenin,β-ecdysterone,and caproic acid,which induce good docking activities with the proteins of the neuroactive ligand-receptor interaction pathway.However,this study has several limitations,such as an insufficient number of pharmacological indexes included in the animal experiments and the mechanism of the compatibility TM,YLC,and BP in cerebral ischemia that was not proven,especially in the results of the molecular docking.Furthermore,such limitations must be addressed accordingly in future studies.

5.ACKNOWLEDGEMENTS

We acknowledge Prof.Yang Yaojun from Beijing University of Chinese Medicine for the identification of Tianma (Rhizoma Gastrodiae),Yanlingcao (Trillium tschonoskiiMaxim).

6.REFERENCES

1.Johnson W,Onuma O,Owolabi M,Sachdev S.Stroke:a global response is needed.Bull World Health Organ 2016;94:634A.

2.Thompson BJ,Ronaldson PT.Drug delivery to the ischemic brain.Adv PharmacolB 2014;71:165-202.

3.Chen XM,Chen HS,Xu MJ,Shen JG.Targeting reactive nitrogen species:a promising therapeutic strategy for cerebral ischemiareperfusion injury.Acta Pharmacol Sin 2013;34:67-77.

4.Liu M,Wu B,Wang WZ,Lee LM,Zhang SH,Kong LZ.Stroke in China:epidemiology,prevention,and management strategies.Lancet Neurol 2007;6:456-64.

5.Zhan HD,Zhou HY,Sui YP,et al.The rhizome of Gastrodia elata Blume-An ethnopharmacological review.J Ethnopharmacol 2016;189:361-85.

6.Rahman SU,Adhikari A,Ismail M,et al.Beneficial effects of Trillium govanianum Rhizomes in pain and inflammation.Molecules 2016;21:1095.

7.Yuan DP,Peng FS.Chinese Tujia medicine.Beijing:Science Press of China,2014:72.

8.Yang M,Chen JL,Xu LW,Ji G.Navigating Traditional Chinese Medicine network pharmacology and computational tools.Evid-Based Compl Alt 2013;2013:731969.

9.Bickerton GR,Paolini GV,Besnard J,Muresan S,Hopkins AL.Quantifying the chemical beauty of drugs.Nat Rev Chem 2012;4:90-8.

10.Baell J,Congreve M,Leeson P,Abad-Zapatero C.Ask the experts:past,present and future of the rule of five.Future Med Chem 2013;5:745-52.

11.Daina A,Michielin O,Zoete V.SwissADME:a free web tool to evaluate pharmacokinetics,drug-likeness and medicinal chemistry friendliness of small molecules.Sci Rep 2017;7:42717.

12.Peng FS.Tujia medical formulae.Beijing:Traditional Chinese Medicine Classics Press,2007:430.

13.Ge YL,Song HY,Zhang L,Li DF,Liu P,Ji G.Research on optimizing components compatibility of Jiangzhi granula in NAFLD treatment.Zhong Xi Yi Jie He Gan Bing Za Zhi 2011;21:363-65.

14.Ren CH,Sy C,Gao JH,Ding YC,Ji XM.Animal stroke model:ischemia-reperfusion and intracerebral hemorrhage.Methods Mol Biol 2016;1462:373-90.

15.Longa EZ,Weinstein PR,Carlson S,Cummins R.Reversible middle cerebral artery occlusion without craniectomy in rats.Stroke 1989;20:84-91.

16.Kato H,Liu Y,Araki T,Kogure K.Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil:cumulative damage and protective effects.Brain Res 1991;553:238-42.

17.Li BH,Tao WY,Zheng CL,et al.Systems pharmacology-based approach for dissecting the addition and subtraction theory of Traditional Chinese Medicine:an example using Xiao-Chaihu-Decoction and Da-Chaihu-Decoction.Comput Biol Med 2014;53:19-29.

18.Zheng QS,Sun RY.Quantitative design of drug compatibility by weighted modification method.Acta Pharmacol Sin 1999;20:1043-51.

19.Cai Z,Lei X,Lin Z,et al.Preparation and evaluation of sustainedrelease solid dispersions co-loadi ng gastrodin with borneol as an oral brain-targeting enhancer.Acta Pharm Sin B 2014;4:86-93.

20.Williams-Karnesky RL,Stenzel-Poore MP.Adenosine and stroke:maximizing the therapeutic potential of adenosine as a prophylactic and acute neuroprotectant.Curr Neuropharmacol 2009;7:217-27.

21.Cunha RA.Neuroprotection by adenosine in the brain:from a receptor activation to A (2A) receptor blockade.Purinerg Signal 2005;1:111-34.

22.Dunwiddie TV,Masino SA.The role and regulation of adenosine in the central nervous system.Annu Rev Neurosci 2001;24:31-55.