The use of sustainable materials is a challenging issue for the construction industry; thus, Fiber Reinforced Polymers (FRP) is of interest to civil and structural engineers for their lightweight and high-strength properties. The paper describes the results of tensile and shear strength testing of Basalt FRP (BFRP) and Hybrid FRP (HFRP) bars. The combination of carbon fibers and basalt fibers leads to a more cost-efficient alternative to Carbon FRP (CFRP) and a more sustainable alternative to BFRP. The bars were subjected to both tensile and shear strength testing in order to investigate their structural behavior and find a correlation between the results. The results of the tests done on BFRP and HFRP bars showed that the mechanical properties of BFRP bars were lower than for HFRP bars. The maximum tensile strength obtained for a BFRP bar with a diameter of 10 mm was equal to approximately 1150 MPa, whereas for HFRP bars with a diameter of 8 mm, it was higher, approximately 1280 MPa. Additionally, better results were obtained for HFRP bars during shear testing; the average maximum shear stress was equal to 214 MPa, which was approximately 22% higher than the average maximum shear stress obtained for BFRP bars. However, HFRP bars exhibited the lowest shear strain of 57% that of BFRP bars. This confirms the effectiveness of using HFRP bars as a replacement for less rigid BFRP bars. It is worth mentioning that after obtaining these results, shear testing can be performed instead of tensile testing for future studies, which is less complicated and takes less time to prepare than tensile testing. Concrete structures built during the previous century will be in need of modernization and renovation, which will lead to an increase in resources and costs associated with these buildings. Hence, it is necessary to develop inexpensive and efficient modernizing techniques in order to prevent structural damage. Throughout the years, researchers have experimented with different materials in various contexts, and Fiber Reinforced Polymers (FRP) have proved to be an applicable material for strengthening and reinforcing concrete structures.For decades, FRP has been widely used in the construction industry as a substitute for steel due to its lightweight, increased corrosion resistance, and improved strength. FRP can be used in a wide range of applications in construction, such as reinforcement of concrete, formwork, modular structures, bridge decks, and external reinforcement for strengthening structures.FRP reinforcement exhibits a linear elastic behavior prior to failure but fails in a brittle manner. The weakness of FRP reinforcement in terms of its mechanical behavior is observed in the transverse direction, which can be seen in shear resistance testing, where the strength characteristics of tested samples are significantly reduced. Moreover, the FRP reinforcement lacks plastic behavior. However, FRP reinforcement has several advantages, such as high tensile strength in the longitudinal direction, long durability, high corrosion resistance, and easy application.Tensile tests have been conducted by several researchers over the past decades on different FRP bars to determine their mechanical and physical properties ,4,5,6,7.In the studies of Walsh, Chang, and Wallenberger et al. ,9,10, exNanni et al.  investigZhishen Wu et al. found that the tensile modulus of the fibers influenced the fatigue failure mode, where the Glass Fiber Reinforced Polymers (GFRP) and Basalt Fiber Reinforced Polymers (BFRP) composites exhibited transverse cracks that indicated a stress transfer to the adhesive layer with a modulus of about 90 GPa, whereas the Carbon Fiber Reinforced Polymers (CFRP) and Poly(p-phenylene benzobisoxazole) (PBO) composites showed longitudinal cracks along the test coupons with a modulus of approximately 250 GPa .Marta Kosior-Kazberuk performed an investigation on the application of Basalt FRP (BFRP) bars and Hybrid FRP (HFRP) bars as reinforcement for full-scale concrete beams. The results showed that the FRP bars used in the tensile zone worked in a similar way to steel reinforcement. Rapid destruction in the tensile zone with crushing of concrete in the compression zone, deformation of compressed bars, stirrups breaking within cracks, and failure of tensile reinforcement were observed .Shi et al.  investigMany researchers have studied the shear strength behavior of different FRP bars. The limited application of BFRP in the past decades led to the limited investigation of its shear behavior. However, Benmokrane et al.  proved tAlam et al.  studied Khalifa et al. , TäljsteBased on the literature, axial tensile testing is most commonly used for defining the mechanical properties of composite bars, and some of the researchers have studied the shear properties of the bars through tensile testing. Current research is concentrated on the defining properties of BFRP and HFRP bars through axial-tensile testing and shear testing, as well as finding a correlation between these two testing methods. The variables in this study were types of FRP bars and the diameter of bars. The outcomes were compared, and a correlation between the results was found. Establishing this correlation allowed the identification of mechanical properties of FRP bars basing on shear testing, which is easier to prepare and takes less time than tensile testing. RC bending elements are the basic structural components that cover the operational load as well as the load from partition walls and transfer them further to vertical elements . Reinforcement of these elements should be characterized by high strength values and appropriate corrosion resistance. In order to determine these properties, strength tests should be carried out. On the one hand, the tensile strength test for FRP bars is complex, relatively labor-intensive, and expensive. On the other hand, testing the shear strength of FRP bars is simple, low-cost, and fast. By carrying out a quick and simple shear test, it is possible to determine the tensile strength and stiffness (modulus of elasticity) of both homogeneous BFRP bars and HFRP bars on the basis of the method presented in the article. Additionally, the method allows optimizing the tensile strength and stiffness based on the determined correlation between shear strength and tensile strength.In spite of the fact that FRP has numerous prevalent material properties, such as high corrosion resistance, high specific stiffness, high specific strength, and durability, the brittle nature and high cost of FRP limits its extensive usage in many industries. To overcome these obstacles, combinations of FRP and ordinary materials are being explored by several researchers . ApproprHFRP bars can be an optimal solution since hybrid bars have a better combination of mechanical properties and are less expensive than FRP bars with homogeneous fibers. On the other hand, they are corrosion resistant and have significantly better strength characteristics than conventional steel reinforcements . HybridiDue to the high cost of carbon, the ideal solution is the usage of HFRP bars, where the part of low-cost basalt fibers was substituted by high-cost carbon fibers. Hence, the hybridization of carbon/basalt fibers is less expensive than CFRP; concurrently, this combination is characterized by better mechanical properties in comparison with BFRP ,35,36.Carbon fibers are chosen due to their high properties in the longitudinal direction as well as their strong anisotropy. However, basalt fibers were chosen for their environmentally friendly producing process and low-cost. In addition, basalt fibers are significantly less brittle when used in composites . An addiComposite materials properties can be calculated according to the Rule of Mixtures (ROM)  based on the literature ,35,36. BThe Voigt model does not consider the location of the fibers; therefore, numerical analysis was made for two configurations: (i) carbon fibers in the core region and basalt fibers in the near-surface region, (ii) carbon fibers in the near-surface region and basalt fibers in the core region of the bar section. The numerical simulation of the tensile strength test for HFRP bars was performed by Finite Element Methods (FEM). The bars consisted of two parts, the core region  and the surface region (tube form), which were perfectly interconnected. The constant pressure was applied on both sides along the longitudinal axis. The obtained results from numerical modeling were compared with analytical considerations and were found to be in a good convergence with each other.Numerical results indicated that the arrangement of the fibers was not of high importance for the final mechanical properties of HFRP bars with different combinations of fibers. More on the analytical and numerical investigation of Hybrid FRP bars can be found in companion papers ,37. Two HFRP bars utilized for this work were composed of epoxy matrix and basalt fibers, with a volume ratio of 1:4 . Due to this substitution, HFRP bars are characterized by a much higher stiffness, which enables more efficient application as reinforcement for concrete elements subjected to bending and compression ,38,39. MIn the tensile and shear strength tests conducted, two types of FRP were used: BFRP and HFRP. Twisting FRP braids were done in order to improve adhesion with concrete  .The tensile strength testing of different BFRP and HFRP bars was carried out according to the ACI 440.3R and ASTM D7205/D7205M methodologies [In the tensile strength test, anisotropic FRP materials of different diameters were tested according to the methodologies. The two different types of FRP bars had different diameters, specifically Ø6, Ø8, Ø10, Ø12, Ø14, and Ø18 mm. Hence, for each type of BFRP and HFRP bar, five samples of each diameter were tested. In total, tensile tests were carried out on sixty bars.Basalt fiber strength in the transverse direction is very low compared to its extremely high longitudinal strength. Hence, the use of appropriate anchorage at both ends of the bar subjected to the tensile test on the testing apparatus is a requirement. To meet this requirement, two steel pipes with a length of 400 mm, an external diameter of 40 mm, and a thickness of 5 mm each were designed. The steel caps at the end of the steel pipes were designed with an opening in the center for the bar.The free space between the bar and the pipe was filled with a special adhesive layer. The material filling the anchoring pipes was determined on the basis of previous tests done at the Warsaw University of Technology ,40. Figui,FRPA. To experimentally determine the modulus of elasticity of FRP bars, Equation (1) can be used.i,FRPA—cross-sectional area for BFRP and HFRP bars, respectively.The tensile strength test was carried out in accordance with  standardThe deformation measurement was performed up to 70% of the breaking load of the bars due to the possibility of strain gauge damage.The average values obtained for BFRP and HFRP bars of tensile strength, tensile strain, and tensile modulus for six different diameters of each type are presented in In the tensile property values obtained for BFRP and HFRP, the deformation values obtained for HFRP were lower than the analogous deformation values for BFRP at rupture.Additionally, comparing the  values of the two types of FRPs used in the test, much higher (COV) values were obtained for HFRP bars compared to the tested BFRP bars. However, this phenomenon might occur due to technical issues indicated in the previous chapter, which might occur during the manufacturing of bars composed of several FRP roving. Hence, the (COV) value depends on the diameter of the bars. The average (COV) values for BFRP bars were twice as small as the corresponding COV values for HFRP. The changes in mechanical properties for the different diameters and FRP types are represented in Comparing the tensile strength of BFRP and HFRP bars with diameters of Ø8 and Ø14 mm, the values for BFRP bars of diameter Ø8 mm were smaller than HFRP bars of the same diameter by 15% and slightly smaller for bars of diameter Ø14 mm by approximately 5%. However, the tensile strength values for BFRP bars with diameters Ø6, Ø10, Ø12, and Ø18 mm were slightly higher than HFRP bars with the same diameters. Values for BFRP bars with diameters Ø6, Ø12, and Ø18 mm were slightly higher than HFRP bars by approximately 5%, and for diameter Ø10 mm by approximately 2%.The rupture strain for all HFRP bar diameters is about 1/3 lower than the rupture strain of the BFRP bars. This is due to the smaller ultimate tensile strains of the carbon fiber  contained in the HFRP bars.Observations made during the study confirm theoretical forecasts. The ultimate strain of hybrid bars corresponds to the strain of its fibers, which are characterized by the lowest ultimate strain among the fibers contained in the HFRP bar. The values obtained for the tensile modulus of elasticity for HFRP bars for all diameters were greater than the values obtained for the tensile modulus of elasticity for BFRP bars for all diameters. The results obtained during this test confirm the viability of using HFRP bars as a replacement for less rigid BFRP bars.The shear strength test of FRP bars was carried out in accordance with the methodology described in ACI 440.3R-04 and ASTM D7617/D7617M–11 [The shear strength testing was carried out on BFRP and HFRP bars with diameters Ø6, Ø8, Ø10, Ø12, Ø14, and Ø18 mm. For the shear strength test, in order to ensure that the bar sample shears in two planes simultaneously, a steel device was constructed with blades converging along a surface perpendicular to the sample axis. The device consists of one upper blade, two lower blades, and a sample holder. The sample holder has a V-shaped groove for placing FRP samples and a rectangular notch for holding the lower and upper blades in the center of the upper part of the device. The sample holder has 100 mm width, 110 mm height, and 230 mm length. It is attached to the instrument stand with screws that stabilize it in order to eliminate horizontal movement of the bar. All FRP bars that were tested for shear strength were composed of a spiral braid of basalt fiber. All the FRP bars were tested at speeds up to 50 MPa/min for a range of time between 3.5 min and 4.5 min, with the test ending when a sharp decrease in shear stress was observed.2.According to ACI 440.6-08 and ASTM A615/A615M [Failure of the tested bars due to shearing could occur in two ways; two failure modes are shown in the It was impossible to clearly determine the values of the shear modulus due to the non-uniform slope of the shear curves, in contrast to the stress–strain relationship. Comparing BFRP bars to HFRP bars, it was noticed that BFRP bars had greater shear deformation and less shear strength compared to HFRP bars. The type of FRP bar affects the COV variation for shear strength. The quite significant spread of FRP bar test results confirms that they are heterogeneous materials consisting of a matrix and fibers. Studies show that the average lateral shear strength of BFRP bars ranges from 170 to 210 MPa. While for HFRP bars, it is up to 20% higher due to the participation of carbon fibers.The transverse shear strength displays a slight downward trend as the bar diameter increases, most evidently in HFRP bars. However, minor variation is observed in the shear strength data, with the COV greater than in the rebar tension tests. Comparing the average shear strength of BFRP bars to HFRP bars, the values for BFRP bars were lower than the average shear strength values of HFRP bars except for diameter Ø6 mm, where it was slightly higher by approximately 1%. However, regarding the other diameters, the average shear strength of BFRP bars was lower for diameters Ø8, Ø10, and Ø14 mm by approximately 19% and for diameters Ø12 and Ø18 mm by approximately 1%. This might suggest that the shear strength of BFRP bars was lower than for HFRP.From the results obtained from the tensile and shear strength testing of BFRP and HFRP with different diameters, a correlation was made between the obtained values in order to find a method for calculating tensile strength from the data obtained from shear strength experimental testing.In dk) according to bar diameter can be calculated from Equation (3):From Based on Equation (3), the coefficient of accuracy corresponding to a given diameter can be identified. The values are presented in Accuracy represented in dk) varied between −7% and +7% in comparison with the tensile strength experimental values, using the Equation (5) for BFRP and HFRP:dk) can be identified for any specific type of FRP, which will allow us to determine tensile strength based on shear testing.Thus, it is possible to obtain the tensile strength of FRP bars by applying shear test strength only using Equation (5), taking into consideration the additional number of samples to be tested in order to eliminate errors and obtaining better accuracies. The coefficient (The modulus of elasticity for the tested bar diameters depends mostly on the volume fraction of carbon and basalt fibers. The differences in volume fraction can be caused due to a various number of rovings used in the pultrusion process, which do not correspond to the bar diameter. In the case of BFRP bars with diameters of 10, 12, and 14 mm, the volume fraction of basalt fibers was greater than the average value by about 10%, while for the diameters of 18 mm, it was lower by over 10%. On the other hand, for the HFRP bars with diameters of 14 mm, the volume fraction of fibers was higher by more than 10% than the average value, while for the diameters of 18 mm, it was 20% lower.The tensile strength, in turn, is influenced by the shear lag effect, which causes that as the diameter of the bar increases, there is an increasing disproportion of the stress of the fibers in the cross-section of the bar.Summarizing, the values of modulus of elasticity and tensile strength, as well as shear strength, depend on a combination of two factors, specifically the volume fraction of fibers and the shear lag effect.The tests conducted in this study provided insight into the mechanical behavior of the types of FRP bars analyzed. Considering the results observed during tests done on BFRP bars for all diameters, it can be seen that the modulus of elasticity was quite low compared to HFRP bars, lower, on average, by approximately 18%. However, the main objective of this research was to check the maximum strength of HFRP and how it is compared to BFRP, as well as to find the stress–strain correlation. The influence of material hybridization was explored in order to examine the possibility of using less complicated shear testing as an alternative to tensile testing.1.The tensile stress–strain correlation of BFRP bars was tested for given diameters. The average maximum stress obtained equaled to approximately 700 MPa, with an average strain approximately equal to 17%. However, the HFRP bars tested exhibited average maximum stress approximately equal to 725 MPa with the average strain approximately equal to 11%. This confirmed the viability of using HFRP bars as a replacement for less rigid BFRP bars.2.Based on the tests performed, HFRP bars exhibited better results in terms of their tensile stress–strain relationship. However, comparing the obtained results for HFRP to BFRP bars, the tensile strength and modulus of elasticity of HFRP bars were higher by approximately 68% and 16%, respectively. The 6% lower elongation of HFRP bars could be explained by the superior extensibility properties of carbon fiber.3.Additionally, according to the shear stress–strain relationship, better results were obtained for HFRP bars with average maximum shear stress equal to 214 MPa, which was approximately 22% higher than the average maximum shear stress obtained for BFRP bars. However, HFRP bars obtained a lower shear strain, 57% compared to BFRP bars.4.Comparing experimental to analytical/numerical calculations done by the authors, the predicted analytical/numerical results were much better than those obtained experimentally. The difference in mechanical properties of FRP bars could be explained by the exact chemistry of the polymer matrix and the strength of polymer/BFRP or polymer/HFRP interactions.5.Comparing the tensile stress–strain relationship to shear stress–strain for all bars and diameters, it was observed that BFRP and HFRP bars could withstand more tensile force and had a lower accompanying strain in tensile tests than shear tests.Based on experimental tests and numerical and analytical considerations for BFRP and HFRP bars, the following conclusive remarks can be drawn:The preparation for shear testing is less complicated than for tensile testing; therefore, on the basis of the results and observations, shear testing can substitute tensile testing for this class of composite materials.


Lipid mediators are major factors in multiple biological functions and are strongly associated with disease. Recent lipidomics approaches have made it possible to analyze multiple metabolites and the associations of individual lipid mediators. Such systematic approaches have enabled us to identify key changes of biological relevance. Against this background, a knowledge-based pathway map of lipid mediators would be useful to visualize and understand the overall interactions of these factors. Here, we have built a precise map of lipid mediator metabolic pathways (LimeMap) to visualize the comprehensive profiles of lipid mediators that change dynamically in various disorders. We constructed the map by focusing on ω-3 and ω-6 fatty acid metabolites and their respective metabolic pathways, with manual curation of referenced information from public databases and relevant studies. Ultimately, LimeMap comprises 282 factors  and 279 reactions derived from 102 related studies. Users will be able to modify the map and visualize measured data specific to their purposes using CellDesigner and VANTED software. We expect that LimeMap will contribute to elucidating the comprehensive functional relationships and pathways of lipid mediators. They are produced by lipid-metabolizing enzymes in vivo and function through specific G protein-coupled receptors (GPCRs). Prostaglandins (PGs), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs), which are metabolites of arachidonic acid (AA) and ω-6 polyunsaturated FA (PUFA), are classic lipid mediators and form a family of bioactive lipids called “eicosanoids”. Enzymes involved in eicosanoid metabolism have been extensively studied, and the metabolic pathway is referred to as the AA cascade. In addition, GPCRs for eicosanoid lipid mediators have been mostly identified and characterized8. As a result, the AA cascade is recognized as an effective drug target. For example, nonsteroidal anti-inflammatory drugs including aspirin that targets cyclooxygenases (COXs), key enzymes of PG biosynthesis, are some of the most widely used drugs; drugs that target LT receptors (e.g. Pranlukast) are used as antiallergy therapies; and PGE1-derivatives and PGF2α are used as anti-platelet/vasodilators and labor-inducing drugs, respectively12.Accumulating evidence has revealed the roles of eicosanoids in various pathological and physiological responses, including inflammatory response, vasodilation, platelet aggregation, asthma, fever, pain, and cancer17.In addition to the AA cascade, recent studies have extended the repertoire of lipid mediators to ω-3 PUFA, for example, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are oxidized in a similar manner to AA, producing various anti-inflammatory and pro-resolving mediators such as resolvins and protectinsResearch on the biological roles of lipid mediators has advanced through the identification and functional analysis of protein factors, such as lipid mediator-producing enzymes and lipid mediator receptors. Studies using genetically modified animals have revealed various pathophysiological roles of individual lipid mediators. However, because lipid mediators share precursors and biosynthetic pathways and often have functional interactions, it is clear that a comprehensive analysis covering a large number of lipid mediators is necessary to uncover their diverse roles in vivo.20. Currently, more than a hundred lipid mediator-related metabolites can be simultaneously quantified in a single sample, and the resulting data can be assessed by multivariate analyses such as principal component analysis (PCA), partial least squares regression (PLS) or PLS-discriminant analysis (PLS-DA), and clustering analysis22. Analyses based on the quantitative profiles of lipid mediators can identify those that are significant for specific biological functions, and can also reveal functional similarities or reciprocity among different lipid mediators. Without a pathway-based interpretation, however, the analysis sometimes fails to demonstrate biological meaning. Integrating pathway information with metabolite quantification data is an effective metabolomics strategy: as lipid mediators and enzymes  are linked on the metabolic pathway map, so a comprehensive overview of the biological processes becomes apparent. To this end, there are growing demands for resources and tools that can disentangle a complex omics dataset as well as visualize it. For general metabolomics and lipidomics purposes, public databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.kegg.jp)23, PANTHER (http://www.pantherdb.org)24, LIPID MAPS (http://www.lipidmaps.org)25, Human Metabolome Database (HMDB) (http://www.hmdb.ca)26, and PubChem (https://pubchem.ncbi.nlm.nih.gov) have become essential research resources. There also are tools that are more domain- or interest-specific, aimed for the visualization of specific types of omics data on biological pathway networks35. At present, however, there is no pathway map with sufficient accuracy and coverage for visualizing lipid mediator datasets. The aim of the present study was, therefore, to fill this gap. Lipid Mediator Metabolic Pathway Map (LimeMap), which maps lipid mediators and related metabolites of ω3-FA and ω6-FA origin onto the metabolic pathway with relevant enzymes and receptors, has been built to simultaneously visualize and annotate experimental data obtained from widely-targeted mediator lipidomics analysis. It integrates information from manually curated literature, public knowledge bases, and databases. To demonstrate its application, we measured lipid mediators in a mouse model of acute inflammation and mapped them onto LimeMap, implemented in CellDesigner- and VANTED-compatible formats. We demonstrate that LimeMap can be used to effectively visualize datasets derived from comprehensive lipid mediator analysis.Recent improvements to mass spectrometers in terms of speed, sensitivity, and selectivity have accelerated the development of strategies for the comprehensive analysis of lipid mediators, which were previously assessed via single-target analyses such as an enzyme-linked immunosorbent assay33. The files generated by the software in XML format (LimeMap.xml)33 can be flexibly edited and expanded with user information and data. By integration of current knowledge, LimeMap can support researchers in a comprehensive analysis of the interactions of lipid mediators  and 279 reactions derived from 102 related studies, each of which is annotated in the “notes” section for each node, link, enzyme and receptor. The map has also been ported to VANTED software in Geography Markup Language (GML) format (LimeMap.gml)34. LimeMap in both files are available as LimeMap.xml/ LimeMap.gml. The initial version of LimeMap reported in this study mainly focuses on ω6- and ω3- FA metabolites even though there are several other classes of bioactive lipids, such as lysophospholipids which also have important roles. Both of the ω6- and ω3 lipid mediator pathways are critically important and interrelate to control biological functions such as initiation and resolution of the inflammatory response16. The metabolites derived from AA: PGs, LTs, HETEs, and EETs, which form a family of bioactive lipids called eicosanoids, are included in the map. The map also includes ω3-FA metabolites, resolvins and maresins, which are essential in anti-inflammatory processes and resolving inflammation. Furthermore, we used mouse gene symbols to describe enzymes and receptors which are related to lipid mediators in the map. Although human and mouse lipid mediator pathways are thought to be generally conserved, they are known to differ for some factors. For instance, part of the role of lipoxygenases differs between human and mouse36: expression of human ALOX15 mainly produces a 15-lipoxygenating activity while mouse Alox15 expression mainly produces 12-HpETE. Human ALOX15B produces 15-HpETE from AA and, in contrast, mouse Alox15b has arachidonic acid 8-lipoxygenating activity. Therefore, species differences should be noted.We built LimeMap by integrating current knowledge of the metabolic pathways of lipid mediators via the manual curation of related studies and databases, such as the Alliance of Genome Resources, HMDB, KEGG, LIPID MAPS, and PubChem. The relationships among metabolic processes were constructed by using CellDesigner software in a standard Systems Biology Markup Language (SBML) format, and visualized as a Systems Biology Graphical Notation (SBGN) diagramors Fig. 33. Ultimhttp://www.garuda-alliance.org/).LimeMap can be used with CellDesigner software and VANTED software on the GARUDA platform 19. The profile data will provide important information on the inflammatory responses of lipid mediators; however, it is important to consider the sampling conditions, such as anesthesia, when comparing the profile data between different experiments. We measured a total of 158 metabolites, In this manner, LimeMap is useful as a means for comprehensive comparison of lipid mediator profiles.In summary, we have developed LimeMap as a tool to facilitate comprehensive analysis of lipid mediator data. Users can modify the map and visualize measured data for their purposes via CellDesigner and VANTED software. At present, coverage of the map is limited to what we curated and what we knew. A continuous effort to curate and extend the pathways based on feedback, correction and the most recent data from the research community will be necessary to develop the map and keep it updated, and to further enhance its usability.44.As a future project for using LimeMap, kinetic laws regarding the metabolic reactions can be assigned to the map in SBML format; in addition, we are planning to make a simulation model. Lastly, we are planning to accumulate lipid mediator profiles in specific tissues and under various physiological conditions such as aging, and are trying to reveal the functional networks of lipid mediators using the LimeMap pathways19 who developed a method for the simultaneous analysis of major lipid mediators. In addition, to describe the metabolic pathways, we manually curated information on lipid mediators, their metabolic interactions, and factors related to metabolism and function such as enzymes and receptors/ion channels, as described in both public databases  and published manuscripts, as noted in the “notes” section of the map. The basic LimeMap was built mainly through the use of CellDesigner version 4.4 software. The map conforms to SBML, a data exchange format based on XML, and is represented in the graphical notation of CellDesigner, which adheres to the standards of SBGN. The map content, factors, and reactions were produced using an SBGN process description language, which facilitates the visualization of state transitions.The metabolites in LimeMap mainly include those described by Yamada et al,http://www.alliancegenome.org), while the gene name, synonyms, and human and rat orthologous genes were included the notes section. To enable visualization of the measured value by several graphical methods, we also built a map in GML format using VANTED software based on the XML-formatted LimeMap.For map construction, each lipid mediator was described as a simple molecule and constituted the edge of the pathway. Information, such as molecular formula, molecular weight, chemical name, and InChI Key, was mainly collected from the PubChem database and included in the notes section for each lipid mediator. The molecules were connected in a stepwise manner in accordance with the metabolic process. Metabolic enzymes, described by their mouse gene symbol, were connected to the nodes as catalytic factors. Furthermore, receptors associated with specific lipid mediators were placed in the map as related lipid mediators. Both enzymes and receptors were described mainly by their mouse gene symbol, referenced from the Alliance of Genome Resources  and used for lipid profiling at 8 weeks of age after habituation. Mice were housed individually in a cage with paper chips, and permitted free access to food and water. The rearing conditions were room temperature 23 °C, relative humidity 60%, and 12 h light-dark cycle (7:00-19:00). Mice were maintained and used for the experiments in accordance with the Guidelines for the Care and Use of Laboratory Animals of Tsumura & Co. Before the experiment, the average body weight was matched between groups and used. All experimental procedures were carried out upon approval from the Laboratory Animal Committee of Tsumura & Co., and performed in accordance with guidelines for the conduct of animal experiments in ministry of health, labour and welfare, Japan.Six milligrams of polyI:C supplied by InvivoGene  was dissolved in 10 ml of saline  by heating the mixture for 10 min at 70 °C. The solution was cooled for 1 h at room temperature to ensure proper annealing before administration to mice by intraperitoneal injection at a dose of 6 mg/kg. polyI:C was administered to nine mice, and saline was administered to ten mice as the control group. We did not consider the blinding. Distilled water was given to all mice at the same time as the injection.Two hours after treatment with polyI:C, mice were anesthetized with isoflurane , and blood was collected from the inferior vena cava with EDTA.2 K . Blood samples were centrifuged at 4 °C to prepare plasma samples, which were stored at −80 °C until use.g for 3 min. The supernatant was diluted with 4 ml of 0.1% formic acid in water and gently mixed. The mixture was loaded onto a preconditioned solid-phase extraction cartridge , which was washed with 1 ml of 0.1% formic acid, followed by 1 ml of 15% ethanol. The lipids were eluted with 250 μl of 0.1% formic acid in methanol; the eluent was then reduced by vacuum evaporator and reconstituted in 20 μl of methanol. Five microliters of sample were used for LC/MS analysis. The LC/MS system consisted of two LC-30AD pumps, a SIL-30AC auto-sampler, a CTO-20A column oven, a CBM-20A system controller, and a triple-quadrupole mass spectrometer LCMS-8050 (Shimadzu). A reversed-phase column  was used for chromatographic separation. Chromatogram acquisition, detection of mass spectral peaks, and waveform processing were performed using LCMS solution software and the LC-MS/MS Method Package for Lipid Mediators version 2 (Shimadzu), which contains method files specifying the analytical conditions, and data analysis parameters for 158 lipid mediators derived from AA, EPA or DHA, among others. The peak area of each quantified ion was calculated and normalized to those of the internal standards. For normalization, the intensity of detected peaks was divided by the peak intensity of the standard in the same category. Processing of metabolomics data was performed with Excel software . Missing values in the raw data were replaced by half of the minimum positive value, and these data were used for subsequent statistical analysis.Lipid mediators were measured in mouse plasma sampled 2 h after treatment with polyI:C. To extract low molecular weight metabolites for LC/MS analysis, 1 ml of methanol containing a mixture of internal standards  was mixed with 100 μl of plasma sample for 5 min at room temperature and then centrifuged at 15,000 t-test, and differences with P < 0.05 were considered significant.Statistical analysis was conducted using Excel software. Statistical significance was tested by two-sided Welch’s http://www.garuda-alliance.org)45.The measured data were visualized in LimeMap using CellDesigner and VANTED software on the GARUDA platform (Supplementary informationSupplementary DataSupplementary Data


Xianlinggubao formula (XLGB), a Chinese State Food and Drug Administration-permitted traditional Chinese herbal medicine, has been extensively used to treat osteoporosis. Although XLGB was shown to improve bone mass in ovariectomized rats and clinically alleviate osteoporosis symptoms, its pharmacological mechanisms remain unclear.In this study, we used a network pharmacological approach to explore the potential mechanism of XLGB in treating osteoporosis. We obtained XLGB compounds from the TCMSP and TCMID databases and identified potential targets of these compounds through target fishing based on the TCMSP and Swiss Target Prediction databases. Next, we identified the osteoporosis targets by using the CTD, TTD, GeneCards, OMIM and PharmGKB databases. Then, the overlapping genes between the XLGB potential targets and the osteoporosis targets were used to establish a protein-protein interaction (PPI) network and to analyze their interactions and identify the major hub genes in this network. Subsequently, the Metascape database was utilized to conduct the enrichment of Gene Ontology biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.There were 104 active compounds and 295 related targets identified overall. After the Metascape enrichment analysis, we identified the top 25 cellular biological processes and top 15 pathways based on the logP value and found that the XLGB-mediated anti-osteoporosis effect was mainly associated with reactive oxygen species, organonitrogen compound response and cell migration. Furthermore, 36 hub genes of XLGB, such as EGF, EGFR, MTOR, MAPK14 and NFKB1, were considered potential therapeutic targets, suggesting the underlying mechanisms of XLGB acting on osteoporosis.We investigated the possible therapeutic mechanisms of XLGB from a systemic perspective. These key targets and pathways provide promising directions for future research to reveal the exact regulatory mechanisms of XLGB. Osteoporosis (OP) is a systemic bone disorder characterized by low bone mass and an accompanying increased incidences of fractures. And OP is usually considered to be the result of an imbalance between osteoclasts promoting bone absorption and osteoblasts inducing bone remodeling . A recenTraditional Chinese medicine (TCM) has attracted worldwide attention and served as a main alternative treatment in East Asia, North America, and Europe due to its satisfactory curative effect, relatively low toxicity and low cost –8. TCM hEpimedium sagittatum Maxim (70%), Dipsacus inermis Wall. (10%), Salvia miltiorrhiza Bunge (5%), Anemarrhena asphodeloides Bunge (5%), Psoralea corylifolia L. (5%), and Rehmannia glutinosa Steud. (5%) [Dipsacus inermis Wall. on osteoporosis [As an important type of TCM, Xianlinggubao formula (XLGB) has been proven to effectively improve bone mass in ovariectomized rats  and clinud. (5%) . Due to ud. (5%) . Althougud. (5%) . Recentlud. (5%) . Similaroporosis .In this network pharmacology study, we aimed to comprehensively dissect the mechanisms of XLGB in treating osteoporosis. We identified compounds related to the XLGB capsules based on multiple databases and obtained the compounds’ potential targets via target fishing. Then, we matched them with osteoporosis-related targets collected through a multisource database. Next, using overlapping targets from the previous process, we built a protein-protein network to analyze their internal interactions and then identified the hub genes. Furthermore, we used the Metascape database to conduct the biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The protocol of our experimental procedures is shown in Fig. http://lsp.nwu.edu.cn/tcmsp.php) [http://119.3.41.228:8000/) [To establish a compound ingredient database, we utilized the Traditional Chinese Medicine System Pharmacology Database  , which iOral bioavailability (OB) refers to the percent of an orally administered drug that reaches systemic circulation and is one of the most important pharmacokinetic indexes for drug screening. The TCMSP platform has adopted the OBioavail1.1 system, which integrates P450, 3A4 and P-glycoprotein information to obtain the OB value . To detehttp://www.swissadme.ch/) [Drug-likeness (DL) is a qualitative index that represents the “drug-like” degree of the target compound and is used to remove chemically unsuitable compounds. TCMSP used the Tanimoto similarity method to calculate the DL index by comparing the target compound to all 6511 molecules in the DrugBank database . In thisdme.ch/)  to condudme.ch/) . Thus, tThe permeability of the target drug is another critical part of drug development. The participation of villi and microvilli strongly increases the small intestinal absorption of orally administered drugs. Thus, the TCMSP platform used the Caco-2 permeability model based on 100 drug molecules with satisfactory statistical results (R2 > 0.8) to find compounds with good permeability. Therefore, we set the Caco-2 threshold at 0, and the compounds that met these criteria were used for further experiments.http://www.swisstargetprediction.ch/index.php). After obtaining canonical descriptors of ingredients, we assessed their potential targets via this webserver based on chemical similarity.The compound ingredients have effects on the targets to exert their biological function. Thus, we used the TCMSP platform to predict the targets of active ingredients. For herbs that were not included in TCMSP, we used the Swiss Target Prediction database instead (http://ctdbase.org/) [https://www.genecards.org/) [https://db.idrblab.org/ttd/) [https://www.omim.org/) [https://www.pharmgkb.org/) [We used multiple databases to collect OP-related targets, and the term ‘Osteoporosis’ was searched as the key word. The databases in this step included the Comparative Toxicogenomics Database  , GeneCards.org/) , the Therg/ttd/) , OMIM (him.org/)  and Pharkb.org/) . In totahttps://string-db.org/) [Then, we constructed a Venn diagram to determine the overlapping targets between the OP and active ingredient targets because these overlapping targets play a critical role when XLGB treats OP. These targets were analyzed by String (db.org/) , and thehttp://www.cytoscape.org/) was used in all network visualizations and is one of most powerful open-source software programs for constructing different networks visually [Three main networks were built in this process, including 1. The compound-target network, 2. The XLGB target-OP target interaction network and 3. The target-pathway network. Target information was obtained from the KEGG pathway enrichment results. Cytoscape 3.6.2  [http://www.kegg.jp/) data obtained from Metascape.We used the Metascape database (ex.html)  to conduex.html)  , 202 (36.5%) in Dan Shen , 76 (13.7%) in Di Huang , 31 (5.6%) in , 81 (14.6%) in Zhi Mu , and 33 (6.0%) in Bu Gu Zhi . After screening by the ADME thresholds  and five different rule-based filters of the Swiss ADME database, we obtained 104 active compounds. Then, the herb-compound network was constructed as shown in Fig. From the TCMSP and TCMID databases, we identified 553 related compounds in the formula, among which there were 130 ingredients 23.5%) in Yin Yang Huo  terms in ascending order of logP value, we found that the top 25 biological processes Fig.  were maiWe further conducted KEGG pathway enrichment of 146 overlapping genes to determine the potential therapeutic mechanism of the XLGB capsules for OP. Then, we sorted 15 pathways based on the logP value; these pathways included pathways in cancer hsa05200), fluid shear stress and atherosclerosis hsa05418), AGE-RAGE signaling pathway in diabetic complications (hsa04933), Th17 cell differentiation (hsa04659), PI3K-Akt signaling pathway (hsa04151), IL-17 signaling pathway (hsa04657), prostate cancer (hsa05215), endocrine resistance (hsa01522), HIF-1 signaling pathway (hsa04066), EGFR tyrosine kinase inhibitor resistance (hsa01521), TNF signaling pathway (hsa04668), sphingolipid signaling pathway (hsa04071), estrogen signaling pathway (hsa04915), neurotrophin signaling pathway (hsa04722) and pancreatic cancer (hsa05212)  mice, which may result from the interaction between its active compounds and related osteoporosis targets. Thus, we investigated this potential mechanism through a network pharmacology method.Based on the major hub gene-compound network Fig. , we founOn the basis of the analysis of the target-pathway network Fig.  as well Osteoporosis is a skeletal disease that primarily results from excessive osteoclast activity or deficient osteogenesis. Reactive oxygen species (ROS), produced during aerobic respiration, have been found to play a critical role in regulating cell proliferation, apoptosis, migration and differentiation , as wellMAPK14 is a common gene in reactive oxygen species pathways, the PI3K-Akt signaling pathway and the TNF signaling pathway. MAPKs have important roles in cell proliferation, differentiation and apoptosis, as well as in regulating inflammation . ROS canEpimedii herba and Radix Salviae, may promote the expression of EGFR and then activate the EGFR-Akt-mTOR pathway to improve osteoblast functions. Similarly, diosgenin may also negatively regulate MTOR and subsequently activate the PI3K-Akt-mTOR signaling pathway to improve the osteoblastic differentiation of bone marrow mesenchymal stem cells. Moreover, anhydroicaritin may have an adverse effect on MAPK14, which further inhibits the PI3K-Akt signaling pathway and ultimately alleviates osteoporosis symptoms.Therefore, XLGB capsule’s regulatory effect on ROS is possibly due to the regulatory function of luteolin, diosgenin and anhydroicaritin. For example, luteolin, originally originating from Bone mass maintenance depends upon bone formation, which is mediated by mesenchyma stem cell (MSC)-derived osteoblasts. However, the imbalance of bone formation and resorption induced by osteoblasts and osteoclasts, respectively, will cause bone diseases such as osteoporosis. MSC migration is the early stage of bone formation, as MSCs differentiate into osteoblasts after they transmigrate to the bone surface . TherefoEGF and EGFR were the overlapping targets between the cell migration biological process and PI3K-Akt signaling pathway. EGF and EGF-like ligands are effective chemoattractants for mesenchymal progenitors as well as for some epithelial cells, which are based on the interaction with the EGF receptor (EGFR) to promote the Akt pathway and ultimately increase MSC migration . FurtherIn XLGB capsules, ingredients that act on these genes include luteolin, bavacoumestan B, psoralenol and bavachalcone. Based on these important components, XLGB may play a key role in regulating the migration of MSCs and then alleviate the symptoms of osteoporosis.This cellular response to organonitrogen compounds is related to the cell state or activity change after stimulation with a compound containing at least one carbon-nitrogen bond. Bisphosphonates (BPs) are the most frequently used treatments for osteoporosis to inhibit excessive osteoclast resorption activity. There are two different kinds of BPs that restrain osteoclast resorption. Non-nitrogen-containing BPs induce osteoclast apoptosis by inhibiting adenosine triphosphate-dependent enzymes. However, nitrogen-containing bisphosphonates (N-BPs) suppress the activation of farnesyl diphosphate synthase, which is necessary for osteoclast differentiation . NotablyIn the case of this organonitrogen compound response process, NF-kB1 is enriched in the PI3K-Akt, TNF, TH17, IL17, HIF and fluid shear signaling pathways. NF-κB is a critical transcription factor composed of Rel proteins, NF-κB1 and NF-κB2. NF-κB signaling activation is one of the downstream events after the stimulation of RANKL, which induces the differentiation and bone resorption of osteoclasts . The XLGBy utilizing network pharmacology, we explored the potential targets of XLGB compounds and investigated the underlying mechanism of their anti-osteoporosis effects, which may be based on three key biological processes: the inhibition of reactive oxygen species, promotion of the organonitrogen compound response and cell migration. According to the KEGG pathway enrichment results, we found that the PI3K-Akt signaling pathway is the main pathway when XLGB capsules are used to treat osteoporosis. Thus, we believe that the anti-osteoporotic effect of XLGB is mainly based on its direct or indirect regulation of the above potential targets and pathways, and our results provide promising directions for future research, which is essential to reveal the exact regulatory mechanisms.


Despite the wealth of information gained over the years on Taxol research, there still remains supply issues to meet increasing clinical demand. Although alternative Taxol production methods have been developed, they still face several drawbacks that cause supply shortages and high production costs. It is highly desired to develop biotechnological production platforms for Taxol, however, there are still gaps in our understanding of the biosynthetic pathway, catalytic enzymes, regulatory and control mechanisms that hamper production of this critical drug by synthetic biology approaches. Over the past 5 years, significant advances were made in metabolic engineering and optimization of the Taxol pathway in different hosts, leading to accumulation of taxane intermediates. Computational and experimental approaches were leveraged to gain mechanistic insights into the catalytic cycle of pathway enzymes and guide rational protein engineering efforts to improve catalytic fitness and substrate/product specificity, especially of the cytochrome P450s (CYP450s). Notable breakthroughs were also realized in engineering the pathway in plant hosts that are more promising in addressing the challenging CYP450 chemistry. Here, we review these recent advances and in addition, we summarize recent transcriptomic data sets of Taxus species and elicited culture cells, and give a bird’s-eye view of the information that can be gleaned from these publicly available resources. Recent mining of transcriptome data sets led to discovery of two putative pathway enzymes, provided many lead candidates for the missing steps and provided new insights on the regulatory mechanisms governing Taxol biosynthesis. All these inferences are relevant to future biotechnological production of Taxol.The diterpenoid paclitaxel (Taxol Though it went through a long developmental phase, it later became the most successful plant natural product in use as an effective chemotherapy drug since its initial Food and Drug Administration (FDA) approval in 1992  plant cell fermentation (PCF) involving use of Taxus cell suspension cultures  to produce 1 g pure Taxol or ca. 3,000 yew trees to produce only 1 kg of the drug . To add solvents . Total csolvents , but arecultures .Taxus twigs, but it suffers from its own shortcomings; (i) instability of cell lines in the long fermentation periods (ii) use of endogenous biosynthetic pathways, thus no genetic engineering targets to improve flux toward Taxol and (iii) poor Taxol yields in the fermenters, even with elicitors  production from twigs is still dependent on yew trees, thus is susceptible to weather and environmental factors, (ii) long maturity time of the trees and (iii) extraction process still involves use of organic solvents. The PCF route presents obvious advantages over direct extraction from field-grown licitors .For these reasons, it is highly desirable to establish biotechnological production systems for Taxol production that will address most of these issues. Production in more amenable, fast-growing heterologous hosts offers more advantages in terms of control and manipulation of metabolic flux by improving enzyme expression, pathway regulation, availability of cofactors and engineering competing pathways. However, there are challenges that prevent development of desired sustainable Taxol biotechnological platforms: the biosynthetic pathway is long and complicated, involving 19 expected steps from geranylgeranyl diphosphate (GGPP) the universal precursor of diterpenoids  and therIn this review, we discuss various strategies conducted in the last 5 years to develop heterologous systems for Taxol biosynthesis. Building from the breakthrough strategy 10 years ago , recent Metabolic engineering of the Taxol pathway in heterologous hosts is highly desirable as it will establish a versatile, direct route to the critical anticancer drug. Biosynthetic production systems are expected to improve supply of Taxol, as well as lower the production costs through improved efficiency and use of inexpensive sugars as starting material in microbial factories. Another potential avenue under exploration is production in cheaper, high biomass and fast-growing plants like tobacco that can be engineered for high-level production of non-native metabolites using carbon resources from photosynthesis. Before realizing this dream, there are several challenges with Taxol biosynthesis that should be addressed.5 terpenoid universal precursors, dimethylallyl diphosphate (DMAPP) and isopentenyl pyrophosphate (IPP) through geranylgeranyl diphosphate (GGDP), the C20 universal precursor of all diterpenoids. The structure of the Taxol molecule is complex and likewise, the biosynthetic pathway is equally complex; five decades after its discovery, the pathway is not yet fully understood, with several steps still undefined, and several enzymes still missing. These gaps in our understanding of the biosynthetic pathway deprive us of the critical framework to manipulate Taxol biosynthesis and develop heterologous production systems. The pathway is hypothesized to involve 19 steps from GGPP (N-benzoyl transferase (DBTNBT)] and phenylalanine aminomutase (PAM).Taxol is derived from the Crom GGPP , involvirom GGPP , five CYrom GGPP , five acT. baccata cell cultures . T5αH accepts both taxa-4(5),11(12) diene  and its close alternative isomer taxa-4(20),11(12) diene , that are both products of TXS to form taxadiene-5α-ol (T-5α-ol) . Most research efforts in the past 5 years were focused on T5αH, and more synthetic biology tools have been dedicated to T5αH than any other pathway enzyme to overcome this bottleneck on the second step of the pathway, thus, this review also has more comprehensive discussions on this enzyme.Oxygenation chemistry of Taxol presents a formidable barrier to both pathway elucidation and metabolic engineering efforts, starting with the first hydroxylation of the C5 position of taxadiene by T5αH . All TaxT-5α-ol) . This CYSaccharomyces cerevisiae). Expression and enzyme kinetics of T5αH in this construct was the lowest compared to other enzymes, leading to the first detection of a bottleneck at this first oxygenation step that restricted flux toward downstream steps. Two years later, an attempt to introduce TXS and T5αH in wild tobacco (Nicotiana sylvestris), targeting trichomes after knocking down production of cembratrien-diols similarly failed to produce the desired T-5α-ol, but instead led to production of a cyclic ether, 5(12)-oxa-3(11)-cyclotaxane (OCT) (Escherichia coli strain that was engineered via a multivariate modular metabolic engineering (MMME) approach that achieved a 15,000-fold increase in taxadiene production (titers of ∼1 g/L) but lost optimality and titers on introduction of T5αH   (summaryne (OCT) . Intrigu of T5αH . Recent ompounds . A numbeT and T1, as discussed below. The T5αH- catalyzed second step splits T into many other products, dominated by OCT and its isomer, iso-OCT, branching the pathway into many directions. Several other downstream enzymes also accept different substrates and have broad product profiles, which present an emerging picture of a highly branched pathway. This lack of linearity is at the core of the problems with the pathway that has not only slowed metabolic engineering advances, but has even prevented pathway elucidation and identification of key downstream intermediates. Not surprisingly, recent work has focused on improving the catalytic efficiency and selectivity of pathway enzymes through experimental methods supported by computational tools to improve our mechanistic understanding of catalysis, as covered in the next sections.Despite that much of the Taxol pathway enzymes and genes have been identified, success in engineering at least the known segments of the pathway in heterologous systems have been mainly hampered by inherent poor enzyme catalysis, protein interdependency issues and product promiscuity of key enzymes that lead to a highly branched pathway. Carbon flux is channeled toward off-target products by the promiscuous enzymes, presenting a formidable challenge to synthetic biologists that cannot be solved by simple redirection of IPP and DMAPP precursors. The first enzyme, TXS is now known to have a broad substrate profile including verticillenes and reports of cembrene A, in addition to Taxus cell suspension cultures, but the mechanisms through which MeJA activates Taxol biosynthesis have not been elucidated in detail. Knowledge of the regulatory mechanisms, identification of key transcription factors and any feedback loops in the pathway is critical in informing metabolic engineering efforts. Likewise, there are also many gaps in our understanding of the transport mechanisms of taxane intermediates in plant cells. This wealth of information will be very vital in designing a biosynthetic route to Taxol especially in plant cells.Not much is known on the regulation of the Taxol pathway at transcription, translation and post-translational levels. Elicitors like methyl jasmonate (MeJA) have been used for a relatively long time in improving secondary metabolism in Synthetic biology tools have been widely applied in advancing Taxol biosynthesis research from gene discovery to pathway designs and construction in heterologous hosts, resulting in successes in detection and accumulation of taxane intermediates. Recent metabolic engineering strategies and achievements in different hosts are summarized in E,E,E)-geranylgeranyl diphosphate (GGPP) and cyclizes it to the endocyclic diterpene olefin taxa-4(5),11(12)-diene  and four other minor products: taxa-4(20), 11(12)-diene , verticillia-3(4),7(8),12(13)-triene (V), verticillia- 4(20),7(8),11(12)-triene (V1) and verticillia-3(4),7(8),11(12)-triene (V2) (The class 1 terpene cyclase taxadiene synthase (TXS) is the first and rate-limiting enzyme of the Taxol biosynthesis pathway. It accepts the acyclic C20 diterpenoid precursor, (ene (V2)  and alsoene (V2) . Though ene (V2) , it lackene (V2) .Consequently, labeling and computational tools have been used to get insights on the energetics of proton transfer and carbocation formation in the TXS active site. The generally agreed mechanism follows TXS-catalyzed cleavage of the pyrophosphate moiety (PPi) from GGPP to form charged carbocations followed by subsequent cyclizations and proton transfer, leading to the mixed product profile stated above . Using QUsing the TXS model constructed by E. coli as a proof of concept and achieved a fivefold increase in oxygenated taxanes, reaching the highest oxygenated diterpene titer to date (∼570 mg/L). The work built from the MMME breakthrough  is introduced in a second plasmid to bind and activate the rSFP, creating a gated ON/OFF switch for the downstream gene . Protein engineering is an effective tool that has already been harnessed to address the T5αH bottleneck, and to improve other enzymes of the Taxol pathway . NotwithT and T1) undergo catalysis through different transition states en-route to T-5α-ol; T1 follows the radical-rebound mechanism (abstraction at the C20- position), while T follows epoxidation route to an unstable epoxide intermediate that decomposes non-selectively to several products like OCT, iso-OCT, and T-5α-ol (T can be epoxidized in a regio- and diastereoselective manner to yield the intermediate taxadiene-4(5)-epoxide that can be further rearranged into T-5α-ol, OCT, and iso-OCT  engineering T5αH to improve turnover and (iii) searching for an epoxidase enzyme to improve selectivity of the hypothesized epoxidase intermediate , or other unidentified compounds, most likely cembrene A  that not only improved catalytic efficiency but could more efficiently utilize vinyl acetate, a cheaper alternative acyl donor compared to acetyl CoA . CompareComputational tools are increasingly becoming useful in gaining mechanistic insights on the catalysis cycles of Taxol pathway enzymes. The above examples highlight how versatile and powerful the tool was leveraged for several proteins. Cytochrome P450s are very sensitive to mutations, thus computational and phylogenetically guided mutagenesis studies of T5αH have so far not yet identified significantly improved mutants for solving this bottleneck. Nevertheless, given the lack of a X-ray crystal structure of T5αH or any CYP450 of the Taxol pathway, homology modeling and molecular docking have thus far provided three models  that canN. benthamiana leaves  but the nature of the exchange of intermediates among these different locations is not known. We considered inter-organellar transport of taxane intermediates to be the major barrier blocking access of endoplasmic reticulum-localized CYP450s to their diterpenoid substrate produced in the plastid in plant cells and used a compartmentalized engineering strategy that led to production of T-5α-ol in a heterologous plant host for the first time . A very a leaves . Given tTaxus cell cultures, differential display of mRNA-reverse transcription-PCR, screening of EST libraries and use of available substrates and surrogate substrates  that provided a total of 15 candidate transcripts identified as potential lead candidate genes encoding the six remaining enzymes  of the Taxol pathway. Functional characterization of these candidates led to the isolation of PCL  and six novel CYP725A subfamily genes . The same three sets of publicly available transcriptome datasets from T. chinensis cultured cells were mined for WRKY transcription factors leading to identification of 61 transcripts of TcWRKY of which six selected genes were all upregulated by MeJA  and screened for activation of different organic acids for N-substitution of 3-phenylisoserine side chain of taxoids  was mined from three publicly available RNA-Seq data sets  transcription factors  were identified as negative regulators of MeJA-induced Taxol biosynthesis through their interaction with E-boxes in the promoters of Taxol pathway genes . Computational and experimental approaches were used to improve our understanding of the catalytic mechanism of TXS and T5αH, shedding more light on the transition states governing the observed product profile. Models of the closed TXS and those of T5αH and DBAT were also built that provided intricate details of the active site architecture to guide semi-rational protein engineering strategies to improve catalytic activity and alter product profile.S. cerevisiae. Nicotiana benthamiana, a high biomass plant with available technologies for DNA manipulation and agrobacterium-mediated transient expression systems is very promising as a chassis for heterologous expression of the Taxol pathway and is increasingly being favored for production of many other terpenoids. The Taxol pathway that comprises nine CYP450s that trigger membrane envelope stress on their expression in host cells as demonstrated with T5αH, is a very good candidate for expression in N. benthamiana.As highlighted throughout this review, most strategies in the past 5 years were focused on T5αH. This is because this enzyme catalyzes the most important bottleneck of the pathway. With a taxadiene conversion rate of less than 10%, and a product profile comprising several monooxygenated and few dioxygenated taxanes, it’s not surprising that strategies that aimed to increase supply of precursors, or those aiming to improve catalytic activity did not achieve much improvements since this is a major branching point of the pathway that splits flux into several off-target taxanes. The successes recorded through optimized expression , a clever approach to couple expression of the protein to cell envelope stress through rSFPs, compartmentalized engineering in chloroplasts of plant cells and engineering of TXS to favor iso-taxadiene product that proved to exclusively lead to T-5α-ol are approaches that we expect to be further developed in the near future, utilizing such enabling tools as the CRISPR/Cas9 toolkit developed for Taxus species to be assembled that will complement transcriptome data sets and accelerate gene discovery of the remaining CYP450s, PCL, and an epoxidase. Synthetic biology tools are expected to play an increasingly important role in enzyme discovery, construction and optimization of the pathway in different chassis and silencing of competing pathways.Advances is high throughput sequencing technologies have enabled generation of several Taxol-related transcriptomes, and recent mining of these publicly available resources have led to isolation of two missing genes, PCL and T2′αH, though wide testing of PCL in different heterologous hosts is yet to be conclusive. Dozens of lead candidate genes for the missing steps were identified through mining transcriptomes, and with more screening platforms being established, we anticipate full elucidation of the pathway in the near future. Furthermore, we anticipate gas phase QM/MM calculations and experimental methods to reveal the mechanism of catalysis of T5αH that will guide protein engineering to overcome the bottleneck, as was done for TXS and DBAT. As sequencing technology advances in the coming years, we also expect a high-resolution genome of IM and JL drafted the manuscript with support from FX. IM, JL, FX, and YW revised the manuscript. YW supervised this work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gouty arthritis is a common metabolic disease caused by long-term purine metabolic disorder and elevated serum uric acid. Jiang-Suan-Chu-Bi recipe (JSCBR), a traditional Chinese herbal formula prescribed according to utilization frequency and cluster analysis, has been clinically validated remedy for gouty arthritis. However, its therapeutic composition and mechanism remains unclear.In the present study, a simple, rapid, and sensitive ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS)-based chemical profiling was firstly established for comprehensively identifying the major constituents in JSCBR. A phytochemistry-based network pharmacology analysis was further performed to explore the potential therapeutic targets and pathways involved in JSCBR bioactivity. Finally, THP-1 cell model was used to verify the prediction results of network pharmacology by western blot analysis.A total of 139 compounds containing phenolic acids, flavonoids, triterpenoid saponins, alkaloids, amino acids, fatty acids, anthraquinones, terpenes, coumarins, and other miscellaneous compounds were identified, respectively. 175 disease genes, 51 potential target nodes, 80 compounds, and 11 related pathways based on network pharmacology analysis were achieved. Among these pathways and genes, NOD-like receptor signaling pathway may play an important role in the curative effect of JSCBR on gouty arthritis by regulation of NRLP3/ASC/CASP1/IL1B. The results of cellular and molecular experiments showed that JSCBR can effectively reduce the protein expression of ASC, caspase-1, IL-1β, and NRLP3 in monosodium urate-induced THP-1 cells, which indicated that JSCBR mediated inflammation in gouty arthritis by inhibiting the activation of NOD-like receptor signaling pathway.Thus, the integrated approaches adopted in the present study could contribute to simplifying the complex system and providing directions for further research of JSCBR. Gouty arthritis is a metabolic disease caused by the deposition of monosodium urate crystals in joints and soft tissues  and closAt present, western medicines including colchicine, allopurinol, benzbromarone, and febuxostat have been used as the conventional therapy method for gouty arthritis. Although their short-term effect on inhibiting uric acid production, promoting uric acid excretion, or analgesic effect is optimistic, the side effects such as skin mucosal injury, kidney and liver injury, digestive tract injury, as well as some potential neurotoxicity and muscular toxicity caused by long-term use could not be ignored. Meanwhile, as the main form of traditional Chinese medicine (TCM) that based on the compatibility theory, traditional Chinese herbal formula has been applied for the treatment of gouty arthritis for thousands of years in view of remarkable therapeutic effect and little adverse reactions .Jiang-Suan-Chu-Bi recipe (JSCBR) is a formula prescribed according to utilization frequency and cluster analysis by retrieving gout-related data obtained from multiple databases , which hIn the present study, a systematic dissection of JSCBR was employed by integrating chemical profile, network pharmacology, and experimental support using molecular cell biology. The schematic diagram of the present study was depicted in UHPLC-MS grade acetonitrile and methanol were supplied by Merck Company Inc., . MS grade formic acid was purchased from Fisher Scientific Company . Ultrapure water (18.2 MΩ) was prepared with a Milli-Q water purification system . All other reagents were of analytical grade and purchased from Tianjin Concord Technology Co. Ltd. .56), polydatin (57), paeoniflorin (79), atractylodin (81), liquiritin (84), aloe-emodin (94), rhein (115), glycyrrhizic acid (119), osthole (126), columbianadin (127), and oleanolic acid (134) were purchased from Chengdu Pufei De Biotech . The reference compounds astilbin (60), chysophanol (93), physcion (100), and emodin (122) were purchased from Baoji Herbest Biotech . The purity of each reference standard was determined to be over 98% by UHPLC analysis. All the 12 herbs of JSCBR were purchased from Beijing Tong-Ren-Tang Technologies Co., Ltd. , and authenticated by Professor Yunpeng Diao . Voucher specimens were deposited at the authors’ laboratory.The reference compounds ferulic acid . RPMI 1640 cell culture mediums and pencillin-streptomycin were purchased from Gibco BRL, Invitrogen Corporation . Fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. . Cell Counting Kit -8 (CCK-8) was purchased from Yiyuan Biotechnology Co., Ltd. . Phorbol-12-myristate-13-acetate (PMA) was purchased from Abcam Ltd., . Monosodium urate (MSU) was purchased from Sigma-Aldrich . Colchicine was supplied by Shengshi Kangpu Chemical Technology Research Institute . Antibodies against caspase-1, β-actin, apoptosis-associated speck-like protein (ASC), IL-1β, and antirabbit IgG-HRP were purchased from Bioss . The BCA Protein Quantification Kit was purchased from KeyGen Biotech Co., Ltd. . RIPA lysis buffer and PMSF protease inhibitor were purchased from Beyotime Institute of Technology . ECL-plus chemiluminescence reagent was purchased from Bio-rad .The usage of each crude herb were accurately weighed as follows: Smilacis Glabrae Rhizome (30 g), Paeoniae Radix Rubra (10 g), Polygoni Cuspidati Rhizoma et Radix (15 g), Cremastrae Pseudobulbus (8 g), Rhei Radix et Rhizoma (10 g), Clematidis Radix et Rhizome (15 g), Angelicae Pubescentis Radix (10 g), Phellodendri Chinensis Cortex (10 g), Atractylodis Rhizome (10 g), Glycyrrhizae Radix et Rhizome (10 g), Codonopsis Radix (15 g), and Angelicae Sinensis Radix (10 g). They were mixed and prepared by the decocting method as described below: a total of 153 g mixture was immersed in sevenfold mass of water for 2 h, heated and refluxed for 2 h and then filtered with six-layer absorbent gauze. A fivefold mass of water was subsequently added to the residues and boiled for 2 h. After being filtered with six-layer absorbent gauze, the two filters were combined and concentrated to 150 ml . The liquid was finally transformed into powder by vacuum freeze drying technology.A 1.0 g of the freeze-dried powder was accurately weighted and extracted with 50 ml of methanol/water  for 30 min under ultrasound. The extract solution was centrifuged at 13,000 rpm for 10 min at 4°C and the supernatant was filtered through a 0.22 μm membrane filters. 1.0 μl of filtrate was injected to UHPLC-QTOF-MS for analysis.The chromatography and MS conditions were almost the same as those reported in literature . The onlThe in-house library that covers all previous reported compounds from the 12 formulated herbs was established in a Microsoft office excel table, which includes compound name, molecular formula, molecular weight, chemical structures, natural source, and related references . The emphttp://bidd.nus.edu.sg/BIDD-Databases/TTD/TTD.asp); 2. OMIM database (http://omim.org/); 3. PharmGKB database (https://www.pharmgkb.org/); 4. DrugBank database ; 5. GAD database (https://geneticassociationdb.nih.gov/); 6. DisGeNET database (http://www.disgenet.org/web/DisGeNET/menu); 7. GeneCards database (https://www.genecards.org/). We searched these databases and acquired 175 genes totally after removing duplicates. The detailed information is provided in Gouty arthritis related targets were acquired from seven existing resources: 1. TTD database (https://www.ncbi.nlm.nih.gov/pccompound/). In order to get as many targets as possible, two databases were employed to this study, including Swiss Target Prediction (http://www.swisstargetprediction.ch/) and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform . Then, the gene information including name, gene ID, and organism was confirmed using UniProt (http://www.uniprot.org/). The final selected genes are supplied in After identifying the compounds contained in JSCBR by UHPLC-QTOF-MS, the CAS number and Canonical SMILES were collected by PubChem Compound (The protein-protein interactions (PPIs) network of gouty arthritis was constructed and analyzed by STRING database. Those PPIs with high confidence score (>0.95) should be selected for network construction and analysis to ensure the accuracy of the results.All the networks can be performed by employing the network visualization software Cytoscape 3.2.1. Three networks were constructed as follows: (1) PPIs of gouty arthritis targets; (2) Compound-potential targets-disease network analysis; (3) Pathways-targets network analysis. In these network plots, nodes represent herbs and chemicals, and edges indicate interactions between different chemicals, targets, and pathways. The “degree” of a node was defined as the number of its connected edges.https://david.ncifcrf.gov/home.jsp, ver. 6.8) and STRING.To clarify the signaling pathways and functions of potential target genes, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments analysis was performed based on Database for Annotation, Visualization and Integrated Discovery  and streptomycin (100 μg/ml). Cells were maintained in an incubator at 37˚C with 5% CO6 cells/well with a final volume of 100 μl. The cells were treated with PMA (100 nM) for 3 h to induce their differentiation into resting M0 macrophage. Fresh complete culture medium was added and cultured for 24 h after washing the cells with PBS solution. Differentiated THP-1 cells were then stimulated with MSU  with optimal concentration and treated with JSCBR extracts at final concentrations of 1, 2, 3, 4, and 5 mg/ml as well as colchicine  simultaneously at 37˚C for 24 h  (PVDF) membrane. After blocking with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 at room temperature for 2 h, the membrane was incubated with primary antibodies at 4˚C overnight and horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature, respectively. Protein bands were probed with an ECL Plus chemiluminescence reagent and exposed to a Tanon-5200 Imaging System .Data are presented as means ± SD for three independent experiments. Statistical differences between two groups were analyzed using a Student’s t test by GraphPad Prism 5.0 . A significant difference was considered as P < 0.05.In the present study, a specific UHPLC-QTOF-MS protocol was performed to rapidly identify the compounds of JSCBR as many as possible after optimizing the LC and MS conditions systemically. As a result, a total of 139 compounds, including 39 phenolic acids, 19 flavonoids, 13 triterpenoid saponins, 11 alkaloids, 10 amino acids, 10 fatty acids, 8 anthraquinones, 8 terpenes, 6 coumarins, and 15 miscellaneous compounds were identified or tentatively characterized Table 1.56, 57, 60, 79, 81, 84, 93, 94, 100, 115, 119, 122, 126, 127, and 134) were identified as ferulic acid, polydatin, astilbin, paeoniflorin, atractylodin, liquiritin, chysophanol, aloe-emodin, physcion, rhein, glycyrrhizic acid, emodin, osthole, columbianadin, and oleanolic acid by comparing the retention time, quasi-molecular ions with authentic standards, respectively. While the others were tentatively deduced based on their high-accurate quasi-molecular ion such as [M−H]−, [M+HCOO]−, [M+CH3COO]−, [M]+, [M+H]+, and [M+Na]+ with those of the known published compounds recorded in the in-house library. Information regarding the 139 constituents, such as tR (min), identification, formula, negative ion (m/z), positive ion (m/z), and botanical source, is offered in Among them, 15 compounds  were screened for network construction and analysis Figure 3The target genes related to gouty arthritis and compounds were got from associated databases, and 51 overlapping genes were identified by matching the aforementioned genes. Then compound-potential targets-disease network constructed by Cytoscape was shown in To better understand the signaling pathways and functions of these potential target genes, we performed functional enrichment analysis using KEGG pathways. The identified pathways and genes in which they are involved are shown in in vitro with a CCK-8 assay and shown in The induction effect of MSU was evaluated In order to validate the action mechanism of JSCBR screened out by phytochemistry-based network pharmacology, protein expression of ASC, caspase-1, IL-1β, and NLRP3 was examined by Western Blot Analysis. Compared with a control group, the expression of these three proteins in the model group was significantly increased  from Glycyrrhizae Radix et Rhizome and macedonoside C (Compound 113) from Rhei Radix et Rhizoma, as well as a phenolic acid amurenlaetone B (Compound 47) from Phellodendri Chinensis Cortex may be the effective components of JSCBR in treating gouty arthritis due to higher degree. The two triterpenoid saponins could act at least in part, as a glucocorticoid-like drug due to the similar chemical structure related to glucocorticoids. Additionally, reports have provided evidence that treatments with these two types of components can alleviate the symptoms of gouty arthritis by exerting antiinflammatory and antioxidant pharmacological effects  and toll-like receptor-4 (TLR4) plays an important role during gouty arthritis Figure 8Our experiments in molecular cell biology had shown that the expressions of ASC, caspase-1, and IL-1β were significantly increased in the MSU-induced THP-1 cells, while they were significantly downregulated after treatment with JSCBR, which is consistent with network prediction. Therefore, it is speculated that NOD-like receptor signaling pathway is involved in the pathogenesis of gouty arthritis, and JSCBR could treat gouty arthritis by negative regulation of the inflammatory response. Besides, other potential mechanisms of JSCBR explored by above integrated strategy need for further investigation.In the current study, an integrated approach based on chemical profile, network pharmacology and experimental support using molecular cell biology were carried out to reveal the therapeutic composition and mechanisms of JSCBR against gouty arthritis. Following identification of 139 chemical constituents in JSCBR by UHPLC-QTOF MS, 175 disease genes, 51 potential target nodes, 80 compounds, and 11 related pathways were achieved by network pharmacology analysis. Potential key targets NRLP3/ASC/CASP1/IL1B that associated with the negative regulation of in NOD-like receptor signaling pathway were further verified in monosodium urate-induced THP-1 cells, and a plausible mechanism for the multitarget effects of JSCBR on gouty arthritis was consequently proposed Figure 8All datasets generated for this study are included in the article/NX and JQ wrote the manuscript and analyze data. NX, SH, PH, YQ conducted the research and developed the network pharmacology experiment. GL and TP revised the manuscript and edited the graphs. HS did the English proofreading of the manuscript. LZ designed the experiments and edited the manuscript. All the authors read and approved the final version of the manuscript.This work was financially supported by the National Natural Science Foundation of China (No. 81873195 and 81703675), Liaoning Natural Science Foundation (20180550980 and 20180550593), Liaoning Revitalization Talents Program (XLYC1907113), 2018 program for Liaoning Excellent Talents in University, the project of Dalian Young Star on Science and Technology in 2016 (2017RQ122) and Dalian Municipal Medical Research Foundation (no. 17Z2001).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Red ginseng has been widely used in health-promoting supplements in Asia and is becoming increasingly popular in Western countries. However, its therapeutic mechanisms against most diseases have not been clearly elucidated. The aim of the present study was to provide the biological mechanisms of red ginseng against various metabolic diseases. We used a systems biological approach to comprehensively identify the component-target and target-pathway networks in order to explore the mechanisms underlying the therapeutic potential of red ginseng against metabolic diseases. Of the 23 components of red ginseng with target, 5 components were linked with 37 target molecules. Systematic analysis of the constructed networks revealed that these 37 targets were mainly involved in 9 signaling pathways relating to immune cell differentiation and vascular health. These results successfully explained the mechanisms underlying the efficiency of red ginseng for metabolic diseases, such as menopausal symptoms in women, blood circulation, diabetes mellitus, and hyperlipidemia. Metabolic diseases such as diabetes mellitus and cardiovascular disease manifest through a combination of various risk factors, such as low-grade inflammation, metabolic disorders, excess lipid accumulation, and loss of insulin sensitivity . Most ofBecause metabolic diseases can present serious health problems, early prevention is becoming more important, and botanical products may provide health benefits .Panax ginseng (P. ginseng) is commonly known as Asian or Korean ginseng. P. ginseng has been used in Chinese medicine as a tonic to improve stamina and vitality [P. ginseng has effects on cardiovascular risk factors, hypertension, type 2 diabetes mellitus, cognition, Alzheimer’s disease, and performance enhancement [P. ginseng is classified into three types, depending on how it is processed: fresh ginseng, white ginseng, and red ginseng [2, -20(S)-Rg3, -Rs4, -Rg5, and -Rh4 [vitality . Previouancement ,6,7,8,9. ginseng . When gi ginseng . The act ginseng ,13. The and -Rh4 .Clinical studies have reported that the major active components of red ginseng, such as the ginsenosides having anti-diabetes mellitus effects and anti-hypertensive effects, can improve blood flow and prevent symptoms of menopausal syndrome ,17,18,19However, numerous disease states can only be explained by complex, multimolecular interactions rather than by the alteration of a single gene, gene product, or metabolite . Thus, iIn this study, we have developed a comprehensive systematic approach for understanding the mechanisms by which red ginseng acts on metabolic disease and compared the pathways involved with those affected by white ginseng using a systems biology approach . This inP. ginseng was searched, 669 kinds of compounds were identified. Even with the same generic name, the retrieved components have a unique PubChem ID, so deduplication based on the generic name resulted in 458 components. Besides the COCONUT database analysis, there are 80 kinds of red ginseng compounds provided by the Korea Ginseng Corporation. Of these, 23 were excluded because they were not present on the COCONUT DB, and 26 were added passively. In this process, ginsenoside Rg2 of P. ginseng was excluded as a redundant component, and 36 kinds of red ginseng and P. ginseng were identified as superimposed components  database has been constructed to retrieve the name of herbs by scientific name. When mponents .P. ginseng, 168 components were identified by the COCONUT analysis to be linked with 16,569 target genes. Those compounds were regarded as the components contained in white ginseng. On the contrary, as shown in P. ginseng), 23 components were identified that are linked with 187 target genes. Therefore, throughout this study, the networks of red ginseng and white ginseng were compared.Of the 483 components in There were 127,852, 98,385, 98,075, and 152,403 pathways related with menopausal symptoms in women, blood circulation, hyperlipidemia, and diabetes mellitus, respectively, that were affected by white ginseng. On the other hand, red ginseng has 3767, 2590, 210, and 1439 pathways affecting the health of menopausal women, blood circulation, hyperlipidemia, and diabetes mellitus, respectively. Among them, only the pathways related to biological processes were found because there was no complete phenotype in the phenotypes structure of context-oriented directed associations (CODA) related to menopausal symptoms and blood circulation.To identify the most affected pathways for the same biological process, duplicate data were removed, and the biological processes were sorted by score in descending order. Many routes of analysis of the metabolic diseases affected by white ginseng identified 18 pathways associated with menopausal symptoms in women, 19 related to blood circulation, 62 related to hyperlipidemia, and 375 associated with diabetes mellitus . On the p-value in descending order, and then duplication based on the pathway name.For functional enrichment analysis of white ginseng, all compound-associated genes and genes of CODA network analysis routes were used as raw data. Only those that contained at least one target for a statistically significant pathway for each phenotype were selected. The gene overlaps between Pathway Studio and the CODA network were sorted in descending order, followed by p-value, and then deduplicated based on name.For functional enrichment analysis of red ginseng, compound associated genes and genes from the CODA network analysis routes for each of the 23 components were used as raw data. A single data sheet was generated by selecting only pathways with one or more targets that were statistically significant for each phenotype. Overlapping genes between Pathway Studio and the CODA network were sorted first in descending order, then in descending order of P. ginseng or red ginseng . Although this analysis showed the overall genes and pathways related to ginsenosides, direct target protein or receptors to each ginsenoside could not be identified. Nevertheless, the network demonstrated that ginsenoside Rg1 (G-Rg1) was directly connected with AKT1, APP, BAD, BCL2, MAPK1, MAPK3, MAPK8, MAPK9, NFKBIA, NGF, PLCG1, and TNF, related to anti-apoptosis and blood glucose regulation metabolism [Then, we focused and analyzed the pathways that involved only red ginseng. As a result, we identified nine pathways affected only by red ginseng with give components . Among these five compounds, four compounds including ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re and notoginsenoside R1 were ginsenosides. Thirty-seven targets were assigned to these nine pathways, which showed a higher degree b. The Vatabolism ,25. Ginstabolism . Ginsenotabolism ,25. Nototabolism ,25,26. Stabolism . Among ttabolism ,29,30,31tabolism , sexual tabolism , menopautabolism  are imprtabolism  revealedtabolism . Jovanovtabolism  reportedtabolism ,36, the tabolism ,39,40,41tabolism ,43. AlthTo identify the components of red ginseng and their corresponding target genes in humans, we used the COCONUT database v. 2.0  . For ideTo identify the phenotypes affected by all the red ginseng target genes retrieved from the COCONUT database, we used the random walk (RW) simulator, a utility of CODA v. 2.0  . The deg0 means the vector containing the signal amount of the start nodes. A is a matrix representing the degree of connection between each node and edge on the network. α is the rate control factor through which the signal amount is transmitted.Ft+1, Ft, and the sum of the absolute values of the numbers of each vector becomes smaller than a certain threshold at every step (t). The numbers of the Ft+1 vectors are the final RW score of the node corresponding to each.The starting node uses the gene associated with the red ginseng component found in the COCONUT database. The step stops when the difference between FWe searched the Path Scaffold Finder (PSF), which is a utility of CODA, to determine the route of red ginseng ingredients for efficacy against menopausal symptoms in women, blood circulation, diabetes mellitus, and hyperlipidemia. The format of each path is herb compound-compound associated gene (target)-gene target entity .In each path, a frequency value indicating a specific node corresponding to a gene or a biological process is assigned to each node, and a frequency value of all the nodes in the path is averaged to be scored. Compared to the total number of paths, the higher the score, the higher the frequency of the nodes containing the nodes. For each path with n nodes, the path score is calculated as follows:P. ginseng and red ginseng in the routes.Using the results of the pathway scaffold analysis on the CODA network, we inferred the pathway that is statistically related to the four phenotypes of interest. All gene lists correspond to the compound-associated genes and genes of P. ginseng was regarded as an herb without distinction of components, and 23 kinds of red ginseng components, except sucrose, were deduced by each component. When uploading the gene list, the item type was set to “protein” and the type of identifiers to “name”. For each phenotype, “enriched pathway top 100” was set so that the p-values were sorted in descending order.Functional enrichment analysis is an analytical method that identifies cell mechanisms, functions, and signaling pathways that are statistically significantly associated with a particular gene list. In this method, Statistical significance was analyzed by Fisher’s exact test for biological processes among the eight hit types with Pathway Studio v. 12.0.1.9 .Three networks were constructed using the CODA : (1) tarThe visualized T-P/C-T-P network graphs were constructed by Cytoscape v. 3.6.1 .To the best of our knowledge, this work is the first study to apply a systematic biology approach to investigate the molecular mechanisms of red ginseng against metabolic diseases compared with white ginseng using the CODA network. Although this study did not provide detailed mechanisms and direct target protein or receptor for each ginsenosides, this result generated an example for understanding the multiple targets of red ginseng components and their interactions. This study suggests that red ginseng extract may contribute to lowering cholesterol, controlling blood glucose, increasing blood flow, and improving the health of menopausal women through mediation of inflammation and vascular health pathways. Research is needed to verify whether the mechanisms identified in the virtual human system are exactly matched in the human body and to identify the target proteins and action pathways of red ginseng components.