Int J Clin Exp Med 2019;12(12):13504-13513 www.ijcem.com /ISSN:1940-5901/IJCEM0100588. Original Article Utilizing network pharmacology to explore the scientific connotation of processing technology about Rehmanniae Radix Praeparata. Ruoqi Li, Guowei Zhou, Tianwei Xia, Yuxuan Wang, Chaoqun Ma, Jirong Shen. Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, Jiangsu, China. Received August 5, 2019; Accepted October 8, 2019; Epub December 15, 2019; Published December 30, 2019. Abstract: Objective: Network pharmacology method was adopted in this study to establish differential component- target network of Rehmanniae Radix Praeparata before and after “steamed for nine times and dried for nine times”, explore the mechanism of the difference of pharmacological effect, reveal the scientific connotation of the process- ing method. Methods: The different components of Rehmanniae Radix Praeparata before and after “steamed for nine times and dried for nine times” were detected by consulting and screening the literature. PubChem database was used to convert all compounds to standard Canonical SMILES format. SMILES format files were imported into Swiss Target Prediction platform to predict potential targets for different components (possibility > 0). Targets with the highest confidence (score 0.900) were screened from STRING database to construct protein-protein interaction network. GO enrichment and KEGG pathway enrichment were analyzed by ClusterProfiler package in R language. Results: A total of 12 different components (catalpol, ajugol, Rehmannioside A, Acteoside, 5-hydroxymethy lfurfu- ral, Isoacteoside, stachyose, D(+)-Sucrose, raffinose, fructose, glucose and Manninotriose) were screened through literature review, and 101 potential targets were predicted. The GO analysis contained a total of 113 enrichment results. A total of 15 pathways were enriched by KEGG. Conclusion: The changes of glucoses (stachyose, raffinose and Manninotriose) in Rehmanniae Radix Praeparata may be the main reason for the enhancement of the tonifying effect (such as Notch signaling pathway, neuroactive ligand receptor interaction, etc.) after “steamed for nine times and dried for nine times”. The changes of iridoid terpenoids (catalpol, ajugol, rehmannin A, etc.) and carbohydrates (D(+)-Sucrose, raffinose, glucose, Manninotriose, etc.) may be the reasons for the loss of the hypoglycemic effect of Rehmanniae Radix Praeparata after “steamed for nine times and dried for nine times”. The change of pharmaco- logical action is the result from chemical composition interaction of Rehmanniae Radix Praeparata.. Keywords: Rehmanniae Radix Praeparata, network pharmacology, steamed for nine times and dried for nine times, differential components, efficacy. Introduction. Rehmannia glutinosa is root of Rehmannia glu- tinosa. The Rehmanniae Radix can be divided into fresh Rehmanniae Radix, Rehmanniae Ra- dix, Rehmanniae Radix Praeparata. “Rehman- niae Radix Praeparata” and its processing method are first recorded in “prepared qianjin yaofang” and “qianjin yifang” written by Simiao Sun. Although processing technology of Re- hmanniae Radix Praeparata is controversial, herbalists regard “steamed for nine times and dried for nine times” as processing criteria. Sci- entists have discovered the differences of the content and pharmacological effects of Reh-. manniae Radix Praeparata before and after “steamed for nine times and dried for nine times”, but the specific mechanism is unclear.. Network pharmacology abolish the traditional research mode of “one target, one disease, one drug” . The strategy resonates well with the holistic view of traditional Chinese medicine (TCM), which is referred to as “multi-ingredient, multi-pathway and multi-target synergy”. In re- cent years, many related research reports indi- cate that exploring the molecular mechanism of TCM efficacy will become possible. This study retrieved the difference of composition and its targets of Rehmanniae Radix Praeparata. www.ijcem.com. Processing technology and Rehmanniae Radix Praeparata. 13505 Int J Clin Exp Med 2019;12(12):13504-13513. before and after “steamed for nine times and dried for nine times”, used bioinformatics meth- ods to explore the scientific connotation of pro- cessing methods, “steamed for nine times and dried for nine times”, in order to provide a basis for further research and wide application in clinic. The workflow of this study was shown in Figure 1.. Methods. Screening of different components of Rehmanniae Radix Praeparata before and af- ter “steamed for nine times and dried for nine times”. We consulted the literature related to the meth- od “steamed for nine times and dried for nine times” about Rehmanniae Radix Praeparata (by the end of June 1, 2019, and removed the non-“steamed for nine times and dried for ni- ne times” processing methods) in the China National Knowledge Infrastructure (Http:// www.cnki.net/) and PubMed (Https://www.. erty was set to “Homo Sapiens” to obtain the predicted targets (Possibility > 0). The results are visualized using Cytoscape 3.2.1.. Construction of protein-protein interaction (PPI) network. STRING database (http://string-db.org) can as- sess and integrate direct (physical) and indirect (chemical) associations among proteins . The target data were uploaded to the STRING database, the attribute was set to “Homo Sa- piens”, the confidence score was set to the highest confidence (score 0.900), and the pro- tein-protein interaction network (PPI) was con- structed.. Enrichment analysis of the GO function and KEGG pathway. We used the org.Hs.eg.db packet in the R lan- guage to convert the gene name to Entirez IDs, then used the ClusterProfiler packet to enrich the network with GO enrichment and KEGG enrichment pathways . The pathways whose. Figure 1. Flowchart of utilizing network pharmacology to explore the scien- tific connotation of “steamed for nine times and dried for nine times” about Rehmanniae Radix Praeparata. . ncbi.nlm.nih.gov/pu), and fil- tered out the components th- at have been verified the con- tent of Rehmanniae Radix Pr- aeparata before and after “st- eamed for nine times and dri- ed for nine times”. All acti- ve compounds in Rehmanni- ae Radix Praeparata were input to the Pubchem data- base (https://pubchem.ncbi. nlm.nih.gov/)  to obtain the Canonical SMILES.. Prediction of potential targets of different components and construction of compound- target network. Swiss Target Prediction (ht- tp://www.swisstarge tpredic- tion.ch/) is a network platform that can predict small mole- cule targets on the basis of the 2D and 3D similarity mea- surements of ligands . Fir- stly, the Canonical SMILES of compounds were submitted to the platform. Then, the prop-. Processing technology and Rehmanniae Radix Praeparata. 13506 Int J Clin Exp Med 2019;12(12):13504-13513. P-values were less than 0.05 were considered statistically significant.. Results. Screening of different components of Rehmanniae Radix Praeparata before and af- ter “steamed for nine times and dried for nine times”. The results showed the content of Catalpol, Acteoside, ajugol, stachyose, D(+)-Sucrose, Raf- finose decreased, and the content of Rehman- nioside_A, 5-hydroxymethylfurfural, Isoacteosi- de, Fructose, glucose, Manninotriose in Rehm- anniae Radix Praeparata increased after “stea- med for nine times and dried for nine times” [8-10]. Cycloenedoid terpenoids D was not included in the study due to the content re- mained controversial.. Prediction of potential targets of different components and construction of composition- target network. After obtaining the Canonical SMILES format of the compound, the Swiss Target Prediction da- tabase predicted a total of 280 potential tar- gets and 101 potential targets (Table 1) after removing duplication. All 101 potential targets were used to build a component-target network (Figure 2). There were a total of 113 nodes and 280 edges in the network. The green rectangle represented the difference between the ingre- dients before and after “steamed for nine times and dried for nine times”, the yellow oval repre- sented the predicted targets, and the edges between nodes represented the relationships between the components and the targets.. Construction of protein-protein interaction (PPI) network. 101 targets were uploaded to STRING data- base, and the confidence score was set to the highest confidence (score 0.9) to construct the PPI network (Figure 3A). There were 69 nodes, 173 edges, with an average node degree of 3.43 in the PPI network. The network node rep- resented the target, and the line represent- ed the interaction among the targets. Key tar- gets were (in order): APP, ADORA1, ADRA2A, ADRA2B, ADRA2C, PTAFR, ADORA3, DRD2, DRD3 and HTR1B according to the importance (Figure 3B).. Enrichment analysis of the GO function and KEGG pathway. A total of 113 enrichment results were ana- lyzed in the GO analysis. 15 results were relat- ed to cellular component (CC), 23 results were related to molecular function (MF), 75 results were related to biological process (BP). The top 20 results were listed in Table 2 according to P value from low to high. Most of them were relat- ed to the biological processes, such as Notch receptor processing, adenylate cyclase-activat- ing adrenergic receptor signaling pathway, ade- nosine receptor signaling pathway, release of sequestered calcium ion into cytosol, one-car- bon metabolic process, glycogen catabolic pro- cess, and so on. Cell composition included inte- gral component of plasma membrane, gamma- secretase complex, extracellular matrix, and so on. G-protein coupled adenosine receptor activity, adrenergic receptor activity, endopepti- dase activity, neurotransmitter receptor activi- ty, etc. were related to molecular functions.. A total of 15 pathways were enriched by KEGG (Figure 4). Most of them were related to metab- olism, such as starch and sucrose metabolism, galactose metabolism, fructose and mannose metabolism, nitrogen metabolism, and so on. In addition, many genes were enriched in ner- vous system-related pathways, such as neuro- active ligand-receptor interaction, Notch signal- ing pathway, calcium signaling pathway, and so on.. Discussion. Rehmannia glotinosa is widely used in tradi- tional Chinese medicine. Many active ingredi- ents of Rehmannia glotinosa have been stud- ied in recent years. For example, catalpol has been reported to have anti-inflammatory, anti- oxidative and other pharmacological activities, such as hypoglycemic [11, 12], anti-tumor [13, 14], protecting nervous system and cardiovas- cular system [15-18]. Rehmannioside A has a significant effect on treating leukopenia model mice . Acteoside can protect brain, and has anti-hypertension, anti-oxidation, anti-apopto- sis abilities [26-30]. Sugar is an important nutrient and efficacy component of Rehmanniae Radix, including Rehmannia glotinosa oligosac- charide (RGO) and Rehmannia glotinosa po- lysaccharide (RGP). The highest content of RGO in Rehmanniae Radix is salicose, while in. Processing technology and Rehmanniae Radix Praeparata. 13507 Int J Clin Exp Med 2019;12(12):13504-13513. Table 1. Main compounds of Rehmanniae Radix Praeparata before and after “steamed for nine times and dried for nine times”. Category Compound Pubechem. CID Canonical SMILES Targets. Cyrene ether terpenoids Catalpol 91520 C1=COC(C2C1C(C3C2(O3)CO) O)OC4C(C(C(C(O4)CO)O)O)O. MGAM, SI, TREH, FOLH1, CA2, CA1, CA12, CA9, NAALAD2, ADA, HSP90AA1, AMY2A, OGA, GGH, ADORA1, ADORA2A, CA14, PYGM, HK2, HK1, SLC6A2. Cyrene ether terpenoids ajugol 6325127 CC1(CC(C2C1C(OC=C2)OC3C(C(C(C(O3) CO)O)O)O)O)O. ADORA1, ADORA2A, MGAM, SI, TREH, FUCA1, CDA, FOLH1, LGALS3, LGALS9, CA2, CA1, CA12, CA9, TYR, CA14, HPRT1. Cyrene ether terpenoids Rehmannioside_A 78407230 C1=COC(C2C1C(C3C2(O3)CO)O) OC4C(C(C(C(O4)COC5C(C(C(C(O5)CO)O) O)O)O)O)O. MGAM, SI, FOLH1, AMY2A, AMY1A, GAA, TREH, CA2, CA1, CA12, CA9, SLC6A2, CDK1, TYR, CA14, FUCA1, NAALAD2, FPGS, ADORA2A, FDFT1. Phenylethanol glycosides Acteoside 5281800 CC1C(C(C(C(O1)OC2C(C(OC(C2OC(=O) C=CC3=CC(=C(C=C3)O)O)CO) OCCC4=CC(=(C=C4)O)O)O)O)O)O. PRKCA, MMP2, MMP12, HSP90AA1, APP, AKR1B1, AKR1B10, CYP19A1. Phenylethanol glycosides 5_hydroxymethylfurfural 237332 C1=C(OC(=C1)C=O)CO FUCA1, HSP90AA1, GBA. Phenylethanol glycosides Isoacteoside 6476333 CC1C(C(C(C(O1)OC2C(C(OC(C2O) OCCC3=CC(=C(C=C3)O)O)COC(=O) C=CC4=CC(=C(C=C4)O)O)O)O)O)O. PRKCA, MMP2, MMP12, HSP90AA1, AKR1B1, MMP13, AKR1B10, APP, IMPDH1, IMPDH2, MMP7, MMP8, ADORA1, ADORA2A, ADORA2B, ADORA3, EPHX2, TYR, IL2, MMP1, CYP19A1, TNF, TOP1, SLC5A1, PDE5A. Carbohydrate stachyose 439531 C(C1C(C(C(C(O1)OCC2C(C(C(C(O2) OCC3C(C(C(C(O3)OC4(C(C(C(O4)CO)O)O) CO)O)O)O)O)O)O)O)O)O)O. CDK1, VEGFA, FGF1, FGF2, HPSE, HSP90AA1, LGALS4, LGALS3, LGALS8, PSEN2, PSENEN, NCSTN, APH1A, PSEN1, APH1B, HTR2B, ADRA2A, ADRA2B, ADRA2C, DRD1, DRD2, ADRA1D, HTR2A, HTR2C, DRD3, CYP2D6, HTR6, ADRA1A, HTR1B, RORC, TRPV1, AMY2A, STAT3. Carbohydrate D(+)-Sucrose 5988 C(C1C(C(C(C(O1)OC2(C(C(C(O2)CO)O)O) CO)O)O)O)O. CDK1, HSP90AA1, VEGFA, PSEN2, PSENEN, NCSTN, APH1A, PSEN1, APH1B, FGF1, HPSE, FGF2, LGALS4, LGALS3, LGALS8, CA2, CA1, CA12, CA9, HTR2B, ADRA2A, ADRA2B, ADRA2C, DRD1, DRD2, ADRA1D, HTR2A, HTR2C, DRD3, CYP2D6, HTR6, ADRA1A, HTR1B, FOLH1, RORC, MGAM, SI, TRPV1, STAT3, PYGL, PYGM, PYGB. Carbohydrate Raffinose 439242 C(C1C(C(C(C(O1)OCC2C(C(C(C(O2) OC3(C(C(C(O3)CO)O)O)CO)O)O)O)O)O)O)O. CDK1, VEGFA, FGF1, FGF2, HPSE, HSP90AA1, LGALS4, LGALS3, LGALS8, PSEN2, PSENEN, NCSTN, APH1A, PSEN1, APH1B, HTR2B, ADRA2A, ADRA2B, ADRA2C, DRD1, DRD2, ADRA1D, HTR2A, HTR2C, DRD3, CYP2D6, HTR6, ADRA1A, HTR1B, RORC, TRPV1, AMY2A, MGAM, AMY1A, GAA, STAT3. Carbohydrate Fructose 24310 C1C(C(C(C(O1)(CO)O)O)O)O GBA, HSP90AA1, VEGFA, PSEN2, PSENEN, NCSTN, APH1A, PSEN1, APH1B, FGF1, FGF2, HPSE, CDK1, LGALS4, LGALS3, LGALS8. Carbohydrate Glucose 107526 C(C(C(C(C(C=O)O)O)O)O)O CHRNA7, FDFT1, ACLY, NPC1L1, GBA, PTGER2, PTGER, PYGB, G6PD, HMGCR, SLC22A6, PYGM. Carbohydrate Manninotriose 5461026 C(C1C(C(C(C(O1)OCC2C(C(C(C(O2) OCC(C(C(C(C=O)O)O)O)O)O)O)O)O)O)O)O. HTR2B, ADRA2A, ADRA2B, ADRA2C, DRD1, DRD2, ADRA1D, HTR2A, HTR2C, DRD3, CYP2D6, HTR6, ADRA1A, HTR1B, RORC, CDK1, VEGFA, STAT3, GLRA1, GLRA2, PSEN2, PSENEN, NCSTN, APH1A, PSEN1, APH1B, PPM1A, LGALS4, LGALS3, LGALS8, VDR, PTAFR, HSP90AA1, MGAM, HSD11B2, HSD11B1, AMY2A, OPRK1, AMY1A, GAA, CA2, CA1, CA12, CA9. Processing technology and Rehmanniae Radix Praeparata. 13508 Int J Clin Exp Med 2019;12(12):13504-13513. Rehmanniae Radix Praeparata is Mannino- triose. RGP is mainly composed of glucose, ga- lactose, mannose and rhamnose, and has anti- anxiety, anti-aging, anti-oxidation, immunity en- hancement, and anti-tumor effects. Studies have shown that the content of cycloenether- glycoside components such as catalpol, Reh- mannioside_D, and ajugol is decreased signifi- cantly after “steamed for nine times and dried for nine times”. The ester bond of the compo- nent is fractured by heating. The content of mandiosin, a component of phenylethanolins, is also decreased, and the content of mandio- sin increased. Some scholars believed that this was due to the reversible esterification reaction that occurs during the processing process . 5-HMF is often considered to be a marker for quickly distinguishing between Rehmanniae Radix and Rehmanniae Radix Praeparata. However, whether 5-HMF is a toxic ingredient or a special active ingredient has not been con- firmed yet. In addition, the content of polysac- charides of Rehmanniae Radix Praeparata is increased obviously after “steamed for nine times and dried for nine times”. Scientists be- lieved the glycosides degraded during Z “stea- med for nine times and dried for nine times”. and monosaccharides such as glucose and rhamnose were polymerized. Lots of scholars believed that the Maillard reaction played an important role in the processing of Rehmanniae Radix Praeparata . However, the molecular mechanism of the changes in pharmacological action before and after “steamed for nine times and dried for nine times” has not been clear yet. In this study, 101 potential targets were predicted for 12 different components and a PPI network was constructed. It is suggested that the change in carbohydrate composition may be the main reason for the change to en- hancement of the tonic function (such as medi- ating Notch signaling pathway, neuroactive ligand-receptor interaction) after “steamed for nine times and dried for nine times”.. Effect of Rehmanniae Radix Praeparata changing to “enhancement of the tonifying ef- fect” after “steamed for nine times and dried for nine times”. Rehmanniae Radix Praeparata has the cha- racteristics of promoting development, bone growth, and myelopoiesis. It plays an important role in the differentiation of stem cells. Notch. Figure 2. Component-target network of Rehmanniae Radix Praeparata before and after “steamed for nine times and dried for nine times”.. Processing technology and Rehmanniae Radix Praeparata. 13509 Int J Clin Exp Med 2019;12(12):13504-13513. Figure 3. Protein-protein net- work of discrepant components and top 30 targets.. Processing technology and Rehmanniae Radix Praeparata. 13510 Int J Clin Exp Med 2019;12(12):13504-13513. signaling pathway plays an important role in regulating cell differentiation, proliferation, and. apoptosis. In recent years, scholars concen- trate on Notch signaling pathway because it is. Table 2. Main enriched GO terms related to Rehmanniae Radix Praeparata before and after “steamed for nine times and dried for nine times” Catalogy GO ID Description Genes P Value CC GO: 0005887 integral component of plasma membrane 30 1.71E-16 BP GO: 0007220 Notch receptor processing 5 7.62E-08 BP GO: 0071880 adenylate cyclase-activating adrenergic receptor signaling pathway 6 1.59E-07 BP GO: 0001975 response to amphetamine 5 1.51E-06 CC GO: 0070765 gamma-secretase complex 4 2.26E-06 BP GO: 0043085 positive regulation of catalytic activity 5 2.85E-06 MF GO: 0001609 G-protein coupled adenosine receptor activity 4 4.15E-06 MF GO: 0004935 adrenergic receptor activity 4 4.15E-06 BP GO: 0007200 phospholipase C-activating G-protein coupled receptor signaling pathway 6 6.17E-06 BP GO: 0001973 adenosine receptor signaling pathway 4 6.47E-06 MF GO: 0030246 carbohydrate binding 7 7.64E-06 BP GO: 0042493 response to drug 7 9.66E-06 MF GO: 0004175 endopeptidase activity 5 1.09E-05 MF GO: 0030594 neurotransmitter receptor activity 5 1.36E-05 BP GO: 0051209 release of sequestered calcium ion into cytosol 5 1.47E-05 BP GO: 0006730 one-carbon metabolic process 5 2.50E-05 BP GO: 0005980 glycogen catabolic process 4 2.68E-05 CC GO: 0031012 extracellular matrix 7 3.12E-05 MF GO: 0004993 G-protein coupled serotonin receptor activity 5 4.72E-05 MF GO: 0051378 serotonin binding 4 4.85E-05. Figure 4. Main enriched KEGG pathways related to Rehmanniae glotinosa before and after “steamed for nine times and dried for nine times”.. Processing technology and Rehmanniae Radix Praeparata. 13511 Int J Clin Exp Med 2019;12(12):13504-13513. related to stem cell differentiation. Fu et al. fo- und that Rehmanniae Radix Praeparata poly- saccharide significantly inhibited the expres- sion of Notch 1 protein and induced BMSCs to neurolike cells . Du et al. believed that some glycosyl components or ligand-receptor patterns in the ground yellow polysaccharide affected the extracellular subunit of the Notch protein, reducing the expression of the Note receptor in the cell, thereby inhibiting the acti- vation of the signaling pathway and the expres- sion of Hesl, hence promoting stem cells differ- entiating to nerve cells . This study found that the carbohydrate difference components changed (mainly salicose, cottonose, and man- nose triose, etc.) before and after “steamed for nine times and dried for nine times”, These can mediate neuroactive ligand-receptor inter- actions by regulating DRD1, DRD2, DRD3, HTR- 2A, HTR1B, HTR6, HTR2B, HTR2C, ADRA2A, ADRA1A, ADRA2C, ADRA2B, ADRA1D, and TRP- V1. This type of carbohydrate component can participate in the Notch signal pathway through multiple targets such as PSEN1, PSEN2, PSE- NEN, APH1A, and APH1N1 B. In addition, the potential targets of different components of sugar before and after “steamed for nine times and dried for nine times” (NCSTN, APP, TNF, PSEN1, APH1A, APH1N1 B, PSEN2, PSENEN) are also enriched into pathways involved in ner- vous system diseases, such as Alzheimer’s dis- ease. Those results show that these carbohy- drate components may play an important role in anti-aging and inducing BMSCs into neuro- like cells through these pathways. This may pro- vide a basis for further exploration of the mod- ern pharmacological mechanisms for the treat- ment of Alzheimer’s disease, promoting neural stem cell differentiation, paracrine, and the efficacy (“tonifying”) about Rehmanniae Radix Praeparata.. Differences of hypoglycemic effect of Rehmanniae Radix Praeparata after “steamed for nine times and dried for nine times”. Rehmanniae Radix undergoing different pro- cessing methods has a great influence on the hyperglycemic and hyperlipemia. Wu et al. found that Rehmanniae Radix was better for hypoglycemic effect and improving blood lipid than Rehmanniae Radix Praeparata in diabetic mice induced by streptozotocin, possibly due to the loss of valid components after steamed for. nine times and dried for nine times [23, 24]. We found that the potential targets of the different components before and after “steamed for nine times and dried for nine times” were en- riched into glucose metabolism pathways, such as starch and sucrose metabolism, galactose metabolism, fructose and mannose metabo- lism. Catalpol can regulate these glycometa- bolic pathways by regulating PYGM, MGAM, SI, HK1, HK2, and TREH. Other components, like ajugol and Rehmannioside_A, also have hypo- glycemic effect by regulating MGAM, SI, TREH and other targets. The potential targets of sac- charides are also enriched into the sugar me- tabolism pathway, such as sucrose [three gly- cogen phosphatases (PYGB, PYGL, PYGM), MG- AM, SI], cotton sugar (MGAM, GAA), glucose (PYGB, PYGM) and so on. Changes in the com- position of iridoids and sugars may be related to the weakening of the hypoglycemic effect of Rehmanniae Radix Praeparata after “steamed for nine times and dried for nine times” .. 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