Integrating network pharmacology and experimental verification to reveal the anti-inflammatory ingredients and molecular mechanism of pycnogenol

Introduction: Pycnogenol (PYC), a standardized extract from French maritime pine, has traditionally been used to treat inflammation. However, its primary active components and their mechanisms of action have not yet been determined. Methods: This study employed UPLC-MS/MS (Ultra-high performance liquid chromatography-tandem mass spectrometry) and network pharmacology to identify the potential active components of PYC and elucidate their anti-inflammatory mechanisms by cell experiments. Results: 768 PYC compounds were identified and 19 anti-inflammatory compounds were screened with 85 target proteins directly involved in the inflammation. PPI (protein-protein interaction) analysis identified IL6, TNF, MMP9, IL1B, AKT1, IFNG, CXCL8, NFKB1, CCL2, IL10, and PTGS2 as core targets. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis suggested that the compound in PYC might exert anti-inflammatory effects through the IL17 and TNF signal pathways. Cell experiments determined that PYC treatment can reduce the expression of IL6 and IL1β to relieve inflammation in LPS (lipopolysaccharide)-induced BV2 cells. Conclusion: PYC could affect inflammation via multi-components, -targets, and -mechanisms.


Introduction
Inflammation is a critical physiological and pathological process that prevents diseases, removes necrotic and damaged tissues, and targets infectious pathogens (Rafiyan and Sadeghmousavi, 2023;D'Arcy, 2019).Normally, inflammation resolves quickly and benefits the host; however, when acute inflammation becomes chronic, it can lead to a harmful cycle of aberrant inflammatory responses and prolonged tissue damage, contributing to the development of conditions such as diabetes, neurological disorders, heart disease, and cancer (Alderton and Scanlon, 2021;Langworth-Green et al., 2023;Nigam et al., 2023).Therefore, it is crucial to control inflammation through effective therapeutic strategies.
Existing literature suggests that PYC significantly enhances antioxidant mediators, indicating that procyanidins may be its primary anti-inflammatory component (Shin et al., 2013).However, PYC also contains 25%-35% of other compounds, and the association of these ingredients with PYC's anti-inflammatory effects remains unclear.Moreover, identifying and understanding the mechanisms of PYC's anti-inflammatory compounds pose significant challenges.UPLC-MS/MS (Ultra-high performance liquid chromatography-tandem mass spectrometry) has high sensitivity, resolution, and selectivity; therefore, it has been utilized to characterize unknown trace components in herbal medicines rapidly (Wang et al., 2021).Network pharmacology, a novel interdisciplinary tool, merges aspects of pharmacology, computational and systems biology, network analysis, and bioinformatics to explore the relationships between traditional herbs and various diseases, facilitating the identification of signaling pathways and targets to enhance drug discovery efficiency holistically (Xian X et al., 2023).Therefore, integrating UHPLC-MS and network pharmacology offers a robust approach to assessing the anti-inflammatory components of PYC and their mechanisms.
This investigation employed the UPLC-MS/MS to assess the active PYC compounds.Then, network pharmacology was used to identify key active targets, components, and potential signaling pathways related to the anti-inflammatory effects, enabling a detailed analysis of PYC's anti-inflammatory mechanism.This research aims to provide evidence supporting the comprehensive evaluation and clinical application of PYC for treating inflammation.The experimental procedures of this study are depicted in Figure 1.

Materials
PYC was acquired from the Horphag Research Ltd. (Geneva, Switzerland).UNIQ-10 column total RNA purification kit and CCK8 (Cell Counting Kit-8) were bought from Sangon Biotech Co., Ltd.(Shanghai, China).MagicSYBR Mixture and HiFiScript cDNA Synthesis Kit were sourced from Jiangsu Cowin Biotech Co., Ltd (Nanjing, China).The BV 2 cell line and its specified media were brought from Procell Life Science &Technology Co., Ltd.(Wuhan, China).Lipopolysaccharides (LPS), Micro NO (nitric oxide) content and Malondialdehyde (MDA) content kits were obtained from Solarbio Science and Technology Co., Ltd.(Beijing, China).

Sample preparation
The PYC powder (2 g) was macerated with 50 mL double distilled H 2 O (dd H 2 O) in a 250 mL sample bottle and stored at 4 °C for 12 h.This mixture was sonicated in an ice-cold water bath for 1 h and centrifuged at 4 °C for 10 min at 10,000 × g to separate the buoyant fraction as sample (A), which was subsequently stored at 4 °C.The remaining PYC residue was mixed with 30 mL of dd H 2 O, then sonicated, centrifuged, and collected as sample (B) using the abovementioned parameters.The extracted supernatant was then mixed and diluted to 100 mL with dd H 2 O as the final PYC sample for subsequent chemical analyses.

UPLC-MS/MS analysis
For chemical analysis, the UHPLC system (Vanquish, Thermo Fisher Scientific) fitted with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to an Orbitrap Exploris 120 mass spectrometer (Orbitrap MS, Thermo) was employed.The mobile phases included 5 mM/L acetic acid in acetonitrile and 5 mM/L ammonium acetate in water.The analysis was conducted at 40 °C with a 0.4 mL/min flow rate.
With the help of the mass spectrometer, MS/MS spectra were obtained using the information-dependent acquisition (IDA) mode.The ESI source parameters were: 50 Arb flow rate of sheath gas, full MS resolution was 60000, 15 Arb flow rate of Aux gas, MS/MS resolution was 15000 collision energy as 10/30/60 in NCE mode, 320 °C capillary temperature, spray Voltage was −3.4 kV (negative) or 3.8 kV (positive).
For 30 s, the extract supernatant (0.3 mL) and methanol extract solution (1 mL) were vortexed, then for 10 min was sonicated on icecold temperature before incubation for 1 h at −40 °C and centrifugation at −40 °C for 15 min at 12,000 rpm.The acquired supernatant was utilized for product assessment.The raw data extraction and chemical identification were based on the secondary and primary spectral data interpreted against the MS2 database.The UPLC-MS/MS analysis were commissioned by a professional biological company (Biotree DB, Shanghai).

Network pharmacology
The chemical components of PYC were characterized using UHPLC-MS.The compounds with anti-inflammatory bioactivity in PYC were identified using the Inflammatory Natural Products Database and Predictor (http://www.inflamnat.com/#/main/home).Compounds were further filtered based on ADME (absorption, distribution, metabolism, and excretion) properties from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, https://tcmsp-e.com/index.php) with the criteria: drug-likeness (DL) ≥ 0.18, oral bioavailability (OB) ≥ 25%, and rotatable bond number (RBN) ≤ 10 (Li et al., 2022).Target predictions for these anti-inflammatory compounds were obtained from the TCMSP, High-throughput Experiment-and Reference-guided database of TCM (HERB, http://herb.ac.cn), and the Comparative Toxicogenomics Database (CTD, http://ctdbase.org).The human genes associated with the inflammation were screened from the Genecards database (https://www.genecards.org/)using the keyword "inflammation," and to ensure results credibility, the relevance score was set to ≥5.The potential targets of the final anti-inflammatory compounds were assessed and compared with disease targets to acquire potential target genes for treating inflammation.Using the CTD, the common overlapping genes were depicted through the Venn diagram.Furthermore, these genes were uploaded to the STRING database (https://www.string-db.org/)to establish a protein-protein interaction (PPI) network and were graphically visualized using the Cytoscape (version 3.9.1).Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment assessments were conducted using the Database for Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov/).The target organism was Homo sapiens, the false discovery rate (FDR)-adjusted p-value The flowchart of the experimental procedures in this study.
Frontiers in Pharmacology frontiersin.orgwas employed for elucidating the enrichment analysis, and a p-value < 0.05 was termed significant.

Cell culture
Under the influence of various inflammatory factors, the BV2 microglial cell line can be over-activation and subsequently release inflammatory mediators such as TNFα, IL1β, and IL6, so here the BV2 microglial cell line were chosen for further study and was cultured at 37 °C in the BV2 cell specified media containing DMEM with 10% FBS (Fetal Bovine Serum) and 1% Penicillin-Streptomycin Solution, which is also the culture media for BV2 cells.Meanwhile, Both LPS and PYC were dissolved in BV2 cell specific media to obtain stocking solutions with concentrations of 10 μg/mL and 200 μg/mL, respectively.

Cells treatments
BV2 cells were cultured at 37 °C in a 6-well plate at a density of 2 × 10 5 per well overnight in 5% CO 2 using.Those cells were categorized into three groups: the blank (Control; received no treatment, the LPS (received BV2 special medium augmented with 1 μg/mL LPS), and the treatment (received BV2 special medium augmented with 80 μg/mL of PYC).The treatment time was 24 h.Each group contained four replicates.

Determination of cell physiological and biochemical indexes
The NO and MDA levels were quantified using commercial assay kits from Solarbio.The concentration of NO was determined using the Griess reagent.In short, 100 μL of cell lysis supernatant was combined with an equal volume of Griess reagent, containing a final concentration of 0.1% N-[1-naphthyl] ethylenediamine dihydrochloride in distilled water and 1% sulfanilamide in 5% phosphoric acid, in a 96-well flat-bottom plate.The absorbance was then measured at 550 nm after a 10min incubation period.Finally, the content of NO was calculated from a standard curve plotted using sodium nitrite.The MDA concentration was assessed by TBA (Thiobarbituric Acid) method, and the reaction mixture included 100 μL of a cell lysis supernatant, 300 μL of MDA test fluid (containing TBA) and 100 μL of acetic acid.The mixed solution was incubated in a water bath at 100 °C for 60 min, then tightly covered to prevent moisture loss.It was then cooled in an ice bath, centrifuged at 10,000 g for 10 min at room temperature.200μL of supernatant was transferred to 96-well plate, and the absorbance of each sample at 532 nm was measured and the content of MDA can be estimated.

Measurement of proinflammatory cytokine production
The total cellular RNA was obtained with the help of the UNIQ-10 column total RNA purification kit, per the manufacturer's guide.cDNA was prepared using the HiFiScript cDNA synthesis kit, followed by qPCR assessment via a CFX Connect Real-Time PCR Detection System (Bio-Rad, United States) using using a total 20 μL reaction volume containing 10 μL of Magic SYBR Mixture (2×), 4 μL of each primer (2 μM), and 2 μL of cDNA.The -ΔΔCq formula was applied to identify the target genes (IL6 and IL1β) expression.β-actin was set as an endogenous control.All qPCR primers are as follows, β-actin (Sense: GGCTGTATTCCCCTCCATCG, Antisense: CCAGTTGGTAACAATGCCATGT), IL1β (Sense: GCAACTGTTCCTGAACTCAACT, Antisense: ATCTTTTGG GGTCCGTCAACT), IL6 (Sense: CTGCAAGAGACTTCCATC CAG, Antisense: AGTGGTATAGACAGGTCTGTTGG).

Analysis of proinflammatory cytokine production
All assays were performed in quadruplicate, and the data are presented as mean ± standard error of the mean (SEM).The data assessment and statistical measurements were performed using the GraphPad Prism (9.0 version) software.The inter-group statistically significant variations were assessed using the one-way ANOVA followed by Duncan's multiple range test.The significance threshold was set as p < 0.05.

Screening of anti-inflammatory compounds in PYC
The chemical components of PYC were characterized using UHPLC-MS/MS.The mixed quality control sample's total ions current was the sum of all ion's intensities in the mass spectrum at each time point versus time (Figure 2).A total of 768 compounds were analyzed (Supplementary Table SA1).The InflamNat database indicated >1,351 (up to 2021) natural compounds with anti-inflammatory activity (Zhang et al., 2022).Using the InflamNat database, 53 chemical components in PYC were identified with anti-inflammatory bioactivity.Components were screened using the TCMSP database based on the following criteria: DL ≥ 0.18, RBN ≤10, and OB ≥ 25%.After eliminating repetitions, 19 anti-inflammatory compounds in PYC were screened (Table 1; Figure 3).

Targets prediction and establishment of compound-target-disease network
HERB database identified 630 targets associated with the 19 anti-inflammatory compounds (Supplementary Table SA2).After duplicate removal, 251 unique targets remained for further analysis.From the GeneCards database, 607 inflammationrelated targets were identified among 13,622 entries with a relevance score >5.0 (Supplementary Table SA3).Venn analysis identified 85 genes associated with PYC's antiinflammatory compounds and inflammation (Figure 4A), highlighting potential treatment targets.A network of 375 edges and 106 nodes was constructed to depict PYCtarget-inflammation relationships (Figure 4B).

Analysis of the PPI network
The PPI data for the specified targets were retrieved from the STRING database and imported into Cytoscape 3.9.1 to construct a function-related PPI network.Figure 5A shows the PPI network, which consists of 2,193 edges and 85 nodes.The average node degree was 51.6, and the edge referred to the interaction of nodes.After assessing each node's degree value (Supplementary Table SA4), the gene with a degree value ≥54 (median) was identified as the key gene target, and a total of 43 genes were screened out.The top 18 targets with ≥70-degree value included IL6, TNF, MMP9, IL1B, AKT1, IFNG, CXCL8, NFKB1, CCL2, IL10, PTGS2, ICAM1, IL1A, TGFB1, CASP3, HIF1A, JUN, STAT3 (Figure 5B; the horizontal coordinate is the degree value of each target point).

Enrichment analysis
Enrichment analysis using the DAVID database identified 85 PYC targets involved in treating inflammation.GO   SA5).Each category's top 10 GO entries were selected according to the FDR <0.05 (Figure 6).The BPs enrichment items included positive transcription regulation, inflammatory response, positive regulation of transcription from RNA polymerase II promoter, positive gene expression regulation, negative apoptosis regulation, positive DNA-template modulation of pri-miRNA transcription from RNA polymerase II promoter, etc.The CCs enrichment items comprised extracellular region, macromolecular complex, cytoplasm, extracellular space, membrane raft, etc.The MFs enrichment items contained enzyme binding, cytokine activity, identical protein binding, protein homodimerization activity, protease binding, etc. KEGG pathway enrichment analysis identified 105 significant pathways with Bonferroni-corrected p-value < 0.05 (Supplementary Table SA6).These pathways were further classified into four major groups: six pathways of cellular processes, 21 of organismal systems, 13 of environmental information processing, and 65 were related to human disease (Figure 7).The pathways from the environmental information processing, organismal systems, and cellular processes groups were selected for the Sankey and dot plot chart: the IL17, TNF, C-type lectin receptor, Toll-like receptor, and NF-kappa B were the main signaling pathways associated with inflammation (Figure 8).
To further elucidate and understand the PYC pharmacological mechanism against inflammation, a network of 19 anti-inflammatory compounds, 85 anti-inflammatory targets, and 40 signaling pathways was constructed (Figure 9).This network indicated that the PYC's components could regulate various targets, which regulates different signaling pathways.

The influence of PYC on cell viability
The cell viability of BV2 cells is depicted in Figure 10.When treating BV2 cells with different concentrations of PYC, compared to the blank, there was no significant difference in cell viability, with the highest viability occurring at 80 μg/mL.Compared to the blank group (100% viability), the viability of the model cells significantly decreased to 30.96% (p < 0.001) but improved following PYC treatment, as shown in the PYC group (Figure 10B).

The PYC's anti-inflammatory impact on LPS-induced BV2 cells
MDA levels were significantly higher in the model cells than the controls but were markedly reduced after PYC and aspirin treatment (Figure 11A).Estimation of NO content and cytokines (IL1β and IL6) mRNA levels revealed significantly higher values in the model group compared to the control group.Furthermore, these levels were substantially decreased in the treatment group than in the model group (Figures 11B-D).[a] Chromatographic peak area presents the relative abundance of the anti-inflammatory compounds in PYC.
Frontiers in Pharmacology frontiersin.org08 Liu et al. 10.3389/fphar.2024.1408304Inflammation is a physiological response of the immune system to irritation, injury, or infection by pathogens, autoimmune processes, or radiation exposure.Controlled inflammation is beneficial as it helps remove irritants, damaged cells, and pathogens (Nakkala et al., 2021).However, dysregulated uncontrolled inflammation can cause several chronic diseases, even death (Ma and Willey, 2022).Glucocorticoids, broad-spectrum anti-inflammatory agents, are used to treat inflammatory diseases but are associated with several adverse effects, including high blood pressure, hyperglycemia, and adrenal insufficiency (Sun et al., 2022).Aspirin is a nonsteroidal anti-inflammatory drug that is known to act by blocking cyclooxygenase activity (Nyambuya et al., 2023), but a variety of side effects can be associated with long-term aspirin use, which include varying degrees of damage to the gastrointestinal tract, liver, kidney, and cardiovascular system (Shi et al., 2023).Thus, finding agents with substantial anti-inflammatory effects and few side effects is important.As an herb, pine bark extract is traditionally used as an antiinflammatory in Europe with few side effects (D'Andrea, 2010).
Like macrophages, microglia are vital immune system components, stimulating proinflammatory cytokines involved in inflammatory responses (Xiong et al., 2016;Zhang et al., 2021).In order to better demonstrate the anti-inflammatory activity of PYC, based on our previous cytotoxicity experiments, we chose the concentration of PYC as 80 μg/mL, which is above any concentration that can be expected in vivo (except for concentrations in the intestine).This study identified Chemical structure of anti-inflammatory compounds in PYC.
Frontiers in Pharmacology frontiersin.org11 Liu et al. 10.3389/fphar.2024.1408304acid, a phenolic acid, significantly inhibited NO in RAW 264.7 cells (Yang et al., 2011).Furthermore, Rhein can effectively suppress NO release and substantially reduce the expression of iNOS and COX-2 proteins after 18 h of LPS treatment (Choi et al., 2013).In RAW264.7 cells, cryptotanshinone significantly suppressed LPS-mediated NO generation and cytokines release like IL6 and TNFα (Gao et al., 2018).Auraptene and beta-sitosterol also showed anti- Classified enriched KEGG pathways of PYC candidate targets against inflammation.The pathways were classified into four major groups: human diseases, organismal systems, cellular processes, and environmental information processing.
These studies further confirm the potential of the 19 antiinflammatory compounds identified in PYC.
Frontiers in Pharmacology frontiersin.org13 Liu et al. 10.3389/fphar.2024.1408304have shown that levels of IL1β, IL6, and TNF are significantly associated with the intensity of systemic inflammation (Del Valle et al., 2020).MMP9, usually expressed in inflammatory cells, is a known activator of inflammatory cytokines like TNF and IL1β (Cheeran and Munuswamy-Ramanujam, 2020).AKT1, a significant subtype of serine/threonine kinase (AKT), regulates macrophage innate immunity by producing mitochondrial H 2 O 2, and its levels are crucial for stimulating inflammatory reactions (Tang et al., 2017).NFKB1 is a pleiotropic transcription factor in almost all cell types and has been indicated as a crucial inflammatory regulator (Tu et al., 2020).CCL2 exerts its effect as an inflammatory chemokine, essential for recruiting leukocytes and memory T cells during inflammation (Ranjbar et al., 2022).PTGS2 expression is promoted during inflammation and regulates inflammatory responses by generating prostaglandins (Li et al., 2022).Usually, PYC can reduce inflammation by acting as a blocker of inflammatory transcription factors NF-kB and lowering the levels of MMP-9 (Grimm et al., 2006), which is consistent with our analysis results for the PYC's target.When pycnogenol is administered, are lowered, displaying the inhibitory effect of pycnogenol on NF-kB All identified core targets were closely associated with the inflammation.The KEGG and GO enrichment tests predicted the potential functions of the 85 targets and signaling pathways associated with the anti-inflammatory response of PYC.The GO analysis revealed that the BP of the targets was mainly involved in the inflammatory response, transcription regulation, and cell Network of compound-target-pathway. Green triangles, blue circles, and purple boxes represent compounds, targets, and pathways, respectively.
Frontiers in Pharmacology frontiersin.org14 response; the CC affected by the targets included extracellular structures, and the MF of the targets was binding and cytokine activity (Figure 5A).Furthermore, it was indicated that PYC's anti-inflammatory compounds are involved in various biological processes, cellular components, and molecular functions as defined in the GO categories.KEGG enrich analysis suggested that the anti-inflammatory compounds in PYC might exert their anti-inflammatory effect through the IL17 and TNF signal pathways.Interestingly, among the 11 core targets (IL6, TNF, MMP9, IL1B, AKT1, IFNG, CXCL8, NFKB1, CCL2, IL10 and PTGS2) analyzed by PPI, 10 genes except IL10 participated in IL17 signaling pathway.This pathway is initiated after the interaction of cytokine (IL17A/F, IL17F/F, or IL17A/A) with their receptors IL17RC and IL17RA, which activates TRAF proteins, which in turn stimulate C/EBPβ, NF-κB, C/EBPδ, and MAPK pathways, aggravating inflammation (Amatya et al., 2017).Contrasting with the IL17 signaling pathway, the TNF signaling pathway, a traditional proinflammatory pathway, involves six core targets: IL6, TNF, MMP9, IL1B, AKT1, and CCL2.Furthermore, it was found that PYC treatment reduced the expression of IL6 and IL1B, thereby alleviating inflammation.
Traditional anti-inflammatory agents usually act on a single target, which can lead to drug resistance during long-term use.For example, dexamethasone binds to the cytoplasmic GC receptor to form a heterodimer that translocated from the cytosol to the nucleus, where it transactivates or transrepresses target genes, leading to cell-cycle arrest and apoptosis (Zhang et al., 2023); aspirin is known to block cyclooxygenase activity (Nyambuya et al., 2023).Therefore, treatment of the chronic inflammation, dexamethasone and aspirin showed no effect (Thuo et al., 2022).Different from traditional anti-inflammatory agents, PYC could affect inflammation via multi-components, -targets and -mechnism, which avoid drug resistance and is suitable for longterm use.Moreover, The results shown in this in vitro study demonstrate that PYC was observed to be non-toxic shown by the cytotoxicity analyses in BV2 cells at the conctration below 100 μg/mL.Other studies also shown that no acute toxicity was identified with intraplantar injection of P. brutia bark extract into rat at a dose of 2000 mg/kg body wt (Ince et al., 2009).Therefore, PYC can be utilized as an anti-inflammatory agent.
Nevertheless, the study still presents certain limitations.Our research primarily centers on investigating the anti-inflammatory properties of the compounds found in PYC.We have solely examined the direct anti-inflammatory effects of these compounds in PYC, neglecting to explore the potential antiinflammatory effects of their metabolites post-assimilation in the body.Consequently, a comprehensive understanding of the overall anti-inflammatory potential of PYC remains elusive, and and further in vivo and in vitro research need to be conducted.

Conclusion
This study systematically analyzed 768 chemical components in PYC using UPLC-MS/MS.After that, 19 anti-inflammatory compounds targeting 85 proteins directly involved in inflammation were identified using the InflamNat and TCMSP databases.Furthermore, PPI analysis indicated the core targets, including IL6, TNF, MMP9, IL1B, AKT1, IFNG, CXCL8, NFKB1, CCL2, IL10, and PTGS2.GO analysis indicated that these targets were involved in the BP, such as inflammatory response, transcription regulation, and cell response; they affected the CC, mainly the extracellular structures, and their MF included cytokine activity and binding.KEGG pathway analysis suggested that the anti-inflammatory compounds in PYC may exert their effects through the IL17 and TNF signaling pathways.Furthermore, it was found that PYC treatment reduced the expression of IL6 and IL1B, thereby alleviating inflammation.The influence of PYC on viability of BV2 cell (A) and the LPS-induced BV2 cell (B).
However, further research is required to elucidate the underlying mechanisms fully.Shandong Provincial University Youth Innovation Team (2022KJ102).

FIGURE 2
FIGURE 2Mass spectrum chromatograms of PYC (A) Positive mode (B) Negative mode.

FIGURE 4
FIGURE 4 Targets of PYC's anti-inflammatory compounds and inflammation.(A) Venn diagram of overlapping target genes between PYC's anti-inflammatory compounds and inflammation.(B) PYC-compound-target-inflammation network.The yellow round rectangle nodes indicate the PYC's antiinflammatory compounds, the red hexagon node depicts inflammation, the green diamond node displays PYC, and the blue round rectangle nodes represent predicted PYC targets for treating inflammation.

FIGURE 5 PPI
FIGURE 5 PPI network analysis.(A) PPI network reconstructed by Cytoscape.The node color and size are proportional to the target degree in the PPI network.(B) The degree values (≥70) of targets in the PPI network.

TABLE 1
Chemical information of anti-inflammatory compounds in PYC.

TABLE 1 (
Continued) Chemical information of anti-inflammatory compounds in PYC.

TABLE 1 (
Continued) Chemical information of anti-inflammatory compounds in PYC.