Genome-wide analysis of bZIP transcription factors and their expression patterns in response to methyl jasmonate and low-temperature stresses in Platycodon grandiflorus.

Background: Platycodon grandiflorus belongs to the genus Platycodon and has many pharmacological effects, such as expectorant, antitussive, and anti-tumor properties. Among transcription factor families peculiar to eukaryotes, the basic leucine zipper (bZIP) family is one of the most important, which exists widely in plants and participates in many biological processes, such as plant growth, development, and stress responses. However, genomic analysis of the bZIP gene family and related stress response genes has not yet been reported in P. grandiflorus. Methods: P. grandiflorus bZIP (PgbZIP) genes were first identified here, and the phylogenetic relationships and conserved motifs in the PgbZIPs were also performed. Meanwhile, gene structures, conserved domains, and the possible protein subcellular localizations of these PgbZIPs were characterized. Most importantly, the cis-regulatory elements and expression patterns of selected genes exposed to two different stresses were analyzed to provide further information on PgbZIPs potential biological roles in P. grandiflorus upon exposure to environmental stresses. Conclusions: Forty-six PgbZIPs were identified in P. grandiflorus and divided into nine groups, as displayed in the phylogenetic tree. The results of the chromosomal location and the collinearity analysis showed that forty-six PgbZIP genes were distributed on eight chromosomes, with one tandem duplication event and eleven segmental duplication events identified. Most PgbZIPs in the same phylogenetic group have similar conserved motifs, domains, and gene structures. There are cis-regulatory elements related to the methyl jasmonate (MeJA) response, low-temperature response, abscisic acid response, auxin response, and gibberellin response. Ten PgbZIP genes were selected to study their expression patterns upon exposure to low-temperature and MeJA treatments, and all ten genes responded to these stresses. The real-time quantitative polymerase chain reaction (RT-qPCR) results suggest that the expression levels of most PgbZIPs decreased significantly within 6 h and then gradually increased to normal or above normal levels over the 90 h following MeJA treatment. The expression levels of all PgbZIPs were significantly reduced after 3 h of the low-temperature treatment. These results reveal the characteristics of the PgbZIP family genes and provide valuable information for improving P. grandiflorus's ability to cope with environmental stresses during growth and development.

Platycodon grandiflorus polysaccharide inhibits the inflammatory response of 3D4/21 cells infected with PCV2.

Porcine circovirus type 2 (PCV2) infection cause multi-systemic inflammation in pigs. Platycodon grandiflorus polysaccharide (PGPSt) has been reported to have the effects of immune regulation and disease resistance. Nevertheless, the role and mechanism of PGPSt in the inflammatory response of 3D4/21 cells induced by PCV2 infection remain unclear. The present study aims to investigate effects of PGPSt on inflammatory response and its possible underlying mechanisms in vitro models. Cells were treated with PCV2 for 36 h to construct a cell inflammation model. The 3D4/21 cell lines were pretreated with or without PGPSt, and the changes of inflammation-related markers and the signaling pathway were detected by CCK-8, ELISA, qPCR and Western blot. The results showed that PGPSt was non-toxic to cells and protected PCV2-infected cells from inflammatory damage. PGPSt could significantly inhibit the high acetylation of histone H3 (AcH3) and histone H4 (AcH4), down-regulate HAT and up-regulate HDAC activity, and reduce the expression of pro-inflammatory enzymes iNOS and COX-2 proteins levels. Then the levels of IL-1ß, IL-6 and TNF-α were significantly inhibited, and the level of IL-10 was promoted. We also observed that PGPSt inhibited the phosphorylation of p65, p38 and Erk1/2, which subsequently inhibited nuclear translocation of NF-κB p65 to express pro-inflammatory factors. In conclusion, PGPSt can reduce the inflammatory response by regulating histone acetylation, reducing the release of inflammatory factors, reducing the expression of pro-inflammatory enzymes, and inhibiting the activation of NF-κB and MAPKs signaling pathways. This suggests that PGPSt had an anti-inflammatory effect on the inflammatory response caused by PCV2 infection, which provided theoretical data support for the research.

In vitro immune-enhancing effects of Platycodon grandiflorum combined with Salvia plebeian via MAPK and NF-κB signaling in RAW264.7 cells.

The immune-enhancing activity of the combination of Platycodon grandiflorum and Salvia plebeian extracts (PGSP) was evaluated through macrophage activation using RAW264.7 cells. PGSP (250-1000 µg/mL) showed a higher release of NO in a dose-dependent manner. The results showed that PGSP could significantly stimulate the production of PGE2, COX-2, TNF-α, IL-1ß, and IL-6 in RAW264.7 cells and promote iNOS, COX-2, TNF-α, IL-1ß, IL-4, and IL-6 mRNA expression. Western blot analysis demonstrated that the protein expression of iNOS and COX-2 and the phosphorylation of ERK, JNK, p38, and NF-κB p65 were greatly increased in PGSP-treated cells. PGSP also promoted the phagocytic activity of RAW264.7 cells. All these results indicated that PGSP might activate macrophages through MAPK and NF-κB signaling pathways. Taken together, PGSP may be considered to have immune-enhancing activity and might be used as a potential immune-enhancing agent.

Heterologous Expression of Platycodon grandiflorus PgF3'5'H Modifies Flower Color Pigmentation in Tobacco.

Flavonoid-3',5'-hydroxylase (F3'5'H) is the key enzyme for the biosynthesis of delphinidin-based anthocyanins, which are generally required for purple or blue flowers. Previously, we isolated a full-length cDNA of PgF3'5'H from Platycodon grandiflorus, which shared the highest homology with Campanula medium F3'5'H. In this study, PgF3'5'H was subcloned into a plant over-expression vector and transformed into tobacco via Agrobacterium tumefaciens to investigate its catalytic function. Positive transgenic tobacco T0 plants were obtained by hygromycin resistance screening and PCR detection. PgF3'5'H showed a higher expression level in all PgF3'5'H transgenic tobacco plants than in control plants. Under the drive of the cauliflower mosaic virus (CaMV) 35S promoter, the over-expressed PgF3'5'H produced dihydromyricetin (DHM) and some new anthocyanin pigments (including delphinidin, petunidin, peonidin, and malvidin derivatives), and increased dihydrokaempferol (DHK), taxifolin, tridactyl, cyanidin derivatives, and pelargonidin derivatives in PgF3'5'H transgenic tobacco plants by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, resulting in a dramatic color alteration from light pink to magenta. These results indicate that PgF3'5'H products have F3'5'H enzyme activity. In addition, PgF3'5'H transfer alters flavonoid pigment synthesis and accumulation in tobacco. Thus, PgF3'5'H may be considered a candidate gene for gene engineering to enhance anthocyanin accumulation and the molecular breeding project for blue flowers.

Histone Deacetylase Inhibitor, Sodium Butyrate-Induced Metabolic Modulation in Platycodon grandiflorus Roots Enhances Anti-Melanogenic Properties.

While the status of histone acetylation is a critical regulator of chromatin's structure with a significant impact on plant physiology, our understanding of epigenetic regulation in the biosynthesis of active compounds in plants is limited. In this study, Platycodon grandiflorus was treated with sodium butyrate (NaB), a histone deacetylase inhibitor, to investigate the influence of histone acetylation on secondary metabolism. Its treatment with NaB increased the acetylation of histone H3 at lysine 9, 14, and 27 and enhanced the anti-melanogenic properties of P. grandiflorus roots. Through transcriptome and differentially expressed gene analyses, we found that NaB influenced the expression of genes that were involved in both primary and secondary metabolic pathways. In addition, NaB treatment caused the accumulation of polyphenolic compounds, including dihydroquercetin, gallic acid, and 2,4-dihydroxybenzoic acid. The NaB-induced transcriptional activation of genes in the phenylpropanoid biosynthetic pathway influenced the anti-melanogenic properties of P. grandiflorus roots. Overall, these findings suggest the potential of an epigenomic approach to enhance the medicinal qualities of medicinal plants.

MALDI Imaging Assisted Discovery of a Di-O-glycosyltransferase from Platycodon grandiflorum Root.

A matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) assisted genome mining strategy was developed for the discovery of glycosyltransferase (GT) from the root of Platycodon grandiflorum. A di-O-glycosyltransferase PgGT1 was discovered and characterized that is capable of catalyzing platycoside E (PE) synthesis through the attachment of two ß-1,6-linked glucosyl residues sequentially to the glucosyl residue at the C3 position of platycodin D (PD). Although UDP-glucose is the preferred sugar donor for PgGT1, it could also utilize UDP-xylose and UDP-N-acetylglucosamine as weak donors. Residues S273, E274, and H350 played important roles in stabilizing the glucose donor and positioning the glucose in the optimal orientation for the glycosylation reaction. This study clarified two key steps involved in the biosynthetic pathway of PE and could greatly contribute to improving its industrial biotransformation.

Improvement of loperamide-hydrochloride-induced intestinal motility disturbance by Platycodon grandiflorum polysaccharides through effects on gut microbes and colonic serotonin.

Constipation is a common gastrointestinal symptom characterized by intestinal motility disorder. The effects of Platycodon grandiflorum polysaccharides (PGP) on intestinal motility have not been confirmed. We established a rat model of constipation induced by loperamide hydrochloride to elucidate the therapeutic effect of PGP on intestinal motility disorder and to explore the possible mechanism. After PGP treatment (400 and 800 mg/kg) for 21 d, PGP clearly relieved gastrointestinal motility, including fecal water content, gastric emptying rate, and intestinal transit rate. Moreover, the secretion of motility-related hormones, gastrin and motilin, were increased. Enzyme-linked immunosorbent assay, western blot, immunohistochemistry, and immunofluorescence results confirmed that PGP significantly increased the secretion of 5-hydroxytryptamine (5-HT) and the expression of related proteins, such as tryptophan hydroxylase 1, 5-HT4 receptor, and transient receptor potential ankyrin 1. 16S rRNA gene sequencing showed that PGP significantly increased the relative abundance of Roseburia, Butyricimonas, and Ruminiclostridium, which were positively correlated with 5-HT levels. However, the relative abundance of Clostridia_UCG-014, Lactobacillus, and Enterococcus were decreased. PGP improved intestinal transport by regulating the levels of 5-HT, which interacts with the gut microbiota and the intestinal neuro-endocrine system, further affecting constipation. Overall, PGP is a potential supplement for the treatment of constipation.

Genome-wide identification and expression profiling analysis of the trihelix gene family and response of PgGT1 under abiotic stresses in Platycodon grandiflorus.

The trihelix gene family plays an important role in plant growth and abiotic stress responses. Through the analysis of genomic and transcriptome data, 35 trihelix family members were identified for the first time in Platycodon grandiflorus; they were classified into five subfamilies: GT-1, GT-2, SH4, GTγ, and SIP1. The gene structure, conserved motifs and evolutionary relationships were analyzed. Prediction of physicochemical properties of the 35 trihelix proteins founded, the number of amino acid molecules is between 93 and 960, theoretical isoelectric point is between 4.24 and 9.94, molecular weight is between 9829.77 and 107435.38, 4 proteins among them were stable, and all GRAVY is negative. The full-length cDNA sequence of the PgGT1 gene of the GT-1 subfamily was cloned by PCR. It is a 1165 bp ORF encoding a 387 amino acid protein, with a molecular weight of 43.54 kDa. The predicted subcellular localization of the protein in the nucleus was experimentally verified. After being treated with NaCl, PEG6000, MeJA, ABA, IAA, SA, and ethephon, the expression of PgGT1 gene showed an up-regulated trend except for the roots treated with NaCl and ABA. This study laid a bioinformatics foundation for the research of trihelix gene family and the cultivation of excellent germplasm of P. grandiflorus.

An evaluation of the qualitative superiority of the Mongolian medicinal herb hurdan-tsagaan (Platycodi Radix) from five different geographic origins based on anti-inflammatory effects.

ETHNOPHARMACOLOGICAL RELEVANCE: The contents and types of the active compounds in medicinal herbs depend greatly on their extraction methods, sources of origin and the modes of cultivation. Platycodon grandiflorus (Jacq.) A.DC. is an ethnic medicinal herb widely cultivated in China, and its dried root, Platycodi Radix (PR), is an important ingredient in herbal formulae for attenuating lung issues in Mongolian medical practice. However, research evaluating the superiority of PR based on harvesting regions is relatively limited. AIM: This study aimed to evaluate the qualitative superiority of PR from different regions based on anti-inflammatory effect. MATERIALS AND METHODS: A total of three commercial PR samples were obtained from Anguo, Bozhou and Shangluo, and two wild samples were obtained from Chifeng and Hinggan. PR extract (PRE) was prepared by water distillation, and platycodin D content in the extract was examined by HPLC-UVD. An optimal dose of PRE was administered to BALB/c mice with S. pneumoniae pneumonia, and IL-10 and TNF-α levels in lung tissue were examined by ELISA. HepG2 cells were treated with PRE, and an analysis of differentially expressed gene and functional enrichment was performed using an HTS2 assay. RESULTS: The contents of moisture, total ash, crude extract and platycodin D in the raw roots met the quality control requirements outlined in the Chinese Pharmacopoeia (2020 edition). The platycodin D content in the aqueous extract of the roots in descending order was 24.16% in PRE_Shangluo, 22.91% in PRE_Hinggan, 21.41% in PRE_Bozhou, 17.8% in PRE_Chifeng and 15.92% in PRE_Anguo. Furthermore, administration of PREs at an optimal dose of 2.0 g/kg resulted in some anti-inflammatory effect in mice with Streptococcus pneumoniae pneumonia, among which PRE_Shangluo administration exhibited a more obvious anti-inflammatory impact as shown by a significant decrease in the plasma white cell count (p < 0.05) and IL-10 level elevation and TNF-α reduction in lung tissue (p < 0.05) after treatment. In HepG2 cells treated with 100 µg/ml of each PRE, PRE_Hinggan and PRE_Shangluo resulted in significant differential expression of genes such as nuclear factor kappa B subunit 1 (NFKB1) and significant enrichment of pathways involved in the immune system, such as PI3K-Akt, MAPK and NF-kappa B signaling pathways. CONCLUSIONS: In this study, based on the anti-inflammatory effect, the quality of PR of Shangluo origin was superior to that of PR from the other four regions.

Identification and expression analysis of YABBY family genes in Platycodon grandiflorus.

Platycodon grandiflorus set ornamental, edible, and medicinal plant with broad prospects for further application development. However, there are no reports on the YABBY transcription factor in P. grandiflorus. Identification and analysis of the YABBY gene family of P. grandiflorus using bioinformatics means. Six YABBY genes were identified and divided into five subgroups. Transcriptome data and qRT-PCR were used to analyze the expression patterns of YABBY. YABBY genes exhibited organ-specific patterns in expression in P grandiflorus. Upon salt stress and drought induction, P. grandiflorus presented different morphological and physiological changes with some dynamic changes. Under salt treatment, the YABBY gene family was down-regulated; PgYABBY5 was up-regulated in leaves at 24 h. In drought treatment, PgYABBY1, PgYABBY2, and PgYABBY3 were down-regulated to varying degrees, but PgYABBY3 was significantly up-regulated in the roots. PgYABBY5 was up-regulated gradually after being down-regulated. PgYABBY5 was significantly up-regulated in stem and leaf at 48 h. PgYABBY6 was down-regulated at first and then significantly up-regulated. The dynamic changes of salt stress and drought stress can be regarded as the responses of plants to resist damage. During the whole process of salt and drought stress treatment, the protein content of each tissue part of P grandiflorus changed continuously. At the same time, we found that the promoter region of the PgYABBY gene contains stress-resistant elements, and the regulatory role of YABBY transcription factor in the anti-stress mechanism of P grandiflorus remains to be studied. PgYABBY1, PgYABBY2, and PgYABBY5 may be involved in the regulation of saponins in P. grandiflorus. PgYABBY5 may be involved in the drought resistance mechanism in P. grandiflorus stems and leaves. This study may provide a theoretical basis for studying the regulation of terpenoids by the YABBY transcription factor and its resistance to abiotic stress.

Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model.

The occurrence of non-alcoholic fatty liver disease (NAFLD) has been increasing at an alarming rate worldwide. Platycodon grandiflorum is widely used as a traditional ethnomedicine for the treatment of various diseases and is a typical functional food that can be incorporated into the everyday diet. Studies have suggested that platycodin D (PD), one of the main active ingredients in Platycodon grandiflorum, has high bioavailability and significantly mitigates the progress of NAFLD, but the underlying mechanism of this is still unclear. This study aims to investigate the therapeutic effect of PD against NAFLD in vitro. AML-12 cells were pretreated with 300 µM palmitic acid (PA) for 24 h to model NAFLD in vitro. Then, the cells were either treated with PD or received no PD treatment for 24 h. The levels of reactive oxygen species (ROS) were analyzed using 2',7'-dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining, and the mitochondrial membrane potential was determined by the JC-1 staining method. Moreover, the protein expression levels of LC3-II/LC3-I and p62/SQSTM1 in the cell lysates were analyzed by western blotting. PD was found to significantly decrease the ROS and mitochondrial membrane potential levels in the PA-treated group compared to the control group. Meanwhile, PD increased the LC3-II/LC3-I levels and decreased the p62/SQSTM1 levels in the PA-treated group compared to the control group. The results indicated that PD ameliorated NAFLD in vitro by reducing oxidative stress and stimulating autophagy. This in vitro model is a useful tool for studying the role of PD in NAFLD.

Cloning, Expression, and Functional Analysis of the Full-Length cDNA of Acetyl-CoA C-acetyltransferase (AACT) Genes Related to Terpenoid Synthesis in Platycodon grandiflorus.

Platycodon grandiflorus is a well-known and widely distributed traditional herbal medicine and functional food in Asia, with triterpenoids as the main bioactive component in its roots. Acetyl-CoA C-acetyltransferase (AACT) is the initiation enzyme in the mevalonate pathway and plays an important role in the biosynthesis of terpenoids. OBJECTIVE: The objective of this study was to clone and identify the PgAACT function in P. grandiflorus. METHODS: The full-length sequence of PgAACT genes was isolated and cloned from P. grandiflorus by polymerase chain reaction (PCR). The recombinant plasmid was constructed using the pET-32a vector and expressed in E. coli Transetta (DE3) cells. Subcellular localization of AACT was observed in the epidermal cells of N. tabacum. Quantitative reverse transcription-PCR (qRT-PCR) was used to identify the PgAACT gene transcription levels. After MeJA treatment, the changes in AACT gene expression were observed, and UHPLC-Q-Exactive Orbitrap MS/MS was used to detect the changes in P. grandiflorus saponins. RESULTS: In this study, two full-length cDNAs encoding AACT1 (PgAACT1) and AACT2 (PgAACT2) were isolated and cloned from P. grandiflorus. The deduced PgAACT1 and PgAACT2 proteins contain 408 and 416 amino acids, respectively. The recombinant vectors were constructed, and the protein expression was improved by optimizing the reaction conditions. Sodium dodecyl sulphate-polycrylamide gel electrophloresis and western blot analysis showed that the PgAACT genes were successfully expressed, with molecular weights of the recombinant proteins of 61 and 63 kDa, respectively. Subcellular localization showed that the PgAACT genes were localized in the cytoplasm. Tissue specificity analysis of P. grandiflorus from different habitats showed that PgAACT genes were expressed in the roots, stems, and leaves. After MeJA treatment, the expression level of PgAACT genes and the content of total saponins of P. grandiflorus were significantly increased, suggesting that PgAACT genes play an important role in regulating plant defense systems. CONCLUSION: Cloning, expression, and functional analysis of PgAACT1 and PgAACT2 will be helpful in understanding the role of these two genes in terpene biosynthesis.

Transcriptome-wide identification of WRKY transcription factors and their expression profiles in response to methyl jasmonate in Platycodon grandiflorus.

Platycodon grandiflorus, a perennial flowering plant widely distributed in China and South Korea, is an excellent resource for both food and medicine. The main active compounds of P. grandiflorus are triterpenoid saponins. WRKY transcription factors (TFs) are among the largest gene families in plants and play an important role in regulating plant terpenoid accumulation, physiological metabolism, and stress response. Numerous studies have been reported on other medicinal plants; however, little is known about WRKY genes in P. grandiflorus. In this study, 27 PgWRKYs were identified in the P. grandiflorus transcriptome. Phylogenetic analysis showed that PgWRKY genes were clustered into three main groups and five subgroups. Transcriptome analysis showed that the PgWRKY gene expression patterns in different tissues differed between those in Tongcheng City (Southern Anhui) and Taihe County (Northern Anhui). Gene expression analysis based on RNA sequencing and qRT-PCR analysis showed that most PgWRKY genes were expressed after induction with methyl jasmonate (MeJA). Co-expressing PgWRKY genes with triterpenoid biosynthesis pathway genes revealed four PgWRKY genes that may have functions in triterpenoid biosynthesis. Additionally, functional annotation and protein-protein interaction analysis of PgWRKY proteins were performed to predict their roles in potential regulatory networks. Thus, we systematically analyzed the structure, evolution, and expression patterns of PgWRKY genes to provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in triterpenoid biosynthesis.

Integrated metabolomic and transcriptomic analysis reveals variation in the metabolites and genes of Platycodon grandiflorus roots from different regions.

INTRODUCTION: Platycodon grandiflorum root (PG), a popular traditional Chinese medicine, contains considerable chemical components with broad pharmacological activities. The complexity and diversity of the chemical components of PG from different origins contribute to its broad biological activities. The quality of southern PG is superior to that of northern PG, but the mechanisms underlying these differences remain unclear. OBJECTIVES: In order to study variation in the differentially accumulated metabolites (DAMs), differentially expressed genes (DEGs), as well as their interactions and signalling pathways among PG from Anhui and Liaoning. METHODS: The metabolomes based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the transcriptome based on high-throughput sequencing technology were combined to comprehensively analyse PGn and PGb. RESULTS: A total of 6515 DEGs and 83 DAMs from the comparison of PG from Anhui and Liaoning were detected. Integrated analysis of metabolomic and transcriptomic data revealed that 215 DEGs and 57 DAMs were significantly enriched in 48 pathways according to KEGG pathway enrichment analysis, and 15 DEGs and 10 DAMs significantly enriched in the main pathway sesquiterpenoid and triterpenoid and phenylpropanoid biosynthesis might play a key role in complex response or regulatory processes. CONCLUSION: Differences in PG from southern and northern China might thus stem from differences in environmental factors, such as precipitation, light duration, and humidity. The results of our study provide new insight into geographic variation in gene expression and metabolite accumulation and will enhance the utilisation of PG resources.

Platycodon grandiflorum root fermentation broth reduces inflammation in a mouse IBD model through the AMPK/NF-κB/NLRP3 pathway.

The effect of Platycodon grandiflorum (PG) on colitis and its underlying mechanism were rarely studied. In this study, Lactobacillus rhamnosus 217-1 was used to ferment PG roots, and the concentrations of platycodin-D, flavonoids, and polyphenols and the DPPH free radical scavenging rate were significantly increased. Treatment with a PG root fermentation broth (PGRFB) could reduce dextran sulfate sodium (DSS) induced ulcerative colitis (UC) in mice. Meanwhile, the PGRFB significantly reduced the content of inflammatory factors in mouse serum and the expression of inflammatory factor mRNA in the intestinal tract, regulated the polarization of M1/M2 macrophages, and increased the expression of tight junction protein mRNA in intestinal epithelial cells. In summary, it was proved that the PGRFB could inhibit the nuclear factor kappa B (NF-κB) signaling pathway and the expression of Nod-like receptor protein 3 (NLRP3) inflammasomes by activating AMP-activated protein kinase (AMPK) and lowering the release of pro-inflammatory cytokines.

Exploring the effects of different processing techniques on the composition and biological activity of Platycodon grandiflorus (Jacq.) A.DC. by metabonomics and pharmacologic design.

ETHNOPHARMACOLOGICAL RELEVANCE: Platycodon grandiflorus (Jacq.) A.DC. (PG) is a common natural medicine with a history of thousands of years. The processing products were mainly recorded as raw, honey-processed, wine-fried, yellow-fried, and bran-fried PG, which were respectively used for different clinical purposes. Therefore, it is necessary to study the chemical composition and pharmacological activity of PG after processing. AIM OF THE STUDY: To explore the effects of different processing methods on the composition and biological activity of PG using metabonomics and pharmacologic design. MATERIALS AND METHODS: UPLC-QTOF-MS combined with multivariate statistical analysis was used to identify different metabolites before and after the processing of PG. Network pharmacology was used to construct the metabolite-target-disease network. CCK-8 assay, flow cytometry, and western blotting were used to detect cell viability, apoptosis, and the expression of related proteins, respectively. RESULT: A total of 43 differentially expressed metabolites (VIP >10) were detected and identified in the analyzed groups. Based on their chemical nature, these metabolites were divided into five categories, namely, saccharolipids, flavonoid glycosides, alkynes, saponins, and lipids (including fatty acids, phospholipids, fatty aldehydes, and sterols). The content of lipids in the five processed groups (CH, FC, JZ, MZI, and MZG) was found to be higher than that in raw PG. In particular, the processing approaches explored herein increased the contents of many phospholipids, such as, glycerophosphoinositols, phosphatidic acids, and lysophosphatidyle·thanolamines. The 8 metabolites were found by venn diagram to distinguish different processed products (metabolites 2, 6, 19, 20, 21, 26, 28, and 38). The results of network pharmacology analysis showed that the primary anti-cancer targets of 43 metabolites of PG processing products are PIK3CA, Akt, and STAT3, and based on CCK-8 assay, MZI has a significant killing effect on A549 cells, compared to other processing techniques. Moreover, flow cytometry analysis showed that the cells treated with MZI exhibit significantly increased cell apoptosis, and that the effect is dose-dependent. Finally, the western blots performed herein demonstrated that the MZI effectively inhibits the expression of p-Akt and p-STAT3, which is consistent with the network pharmacology results. CONCLUSION: Depending on the processing technique, the contents of 43 different metabolites in PG were varied significantly. Specifically, the contents of phospholipids and fatty acids increase, whereas the contents of large Mw saponins decrease. Compared to the other investigated processing methods, MZI increases the potential of PG in inducing cell apoptosis and inhibiting cell proliferation by affecting the Akt and STAT3 signaling pathways. The increased levels of 3-O-ß-glucopyranosyl polygalacic acid and platycoside F after honey-frying confirm these results.

A candidate gene identified in converting platycoside E to platycodin D from Platycodon grandiflorus by transcriptome and main metabolites analysis.

Platycodin D and platycoside E are two triterpenoid saponins in Platycodon grandiflorus, differing only by two glycosyl groups structurally. Studies have shown ß-Glucosidase from bacteria can convert platycoside E to platycodin D, indicating the potential existence of similar enzymes in P. grandiflorus. An L9(34) orthogonal experiment was performed to establish a protocol for calli induction as follows: the optimal explant is stems with nodes and the optimum medium formula is MS + NAA 1.0 mg/L + 6-BA 0.5 mg/L to obtain callus for experimental use. The platycodin D, platycoside E and total polysaccharides content between callus and plant organs varied wildly. Platycodin D and total polysaccharide content of calli was found higher than that of leaves. While, platycoside E and total polysaccharide content of calli was found lower than that of leaves. Associating platycodin D and platycoside E content with the expression level of genes involved in triterpenoid saponin biosynthesis between calli and leaves, three contigs were screened as putative sequences of ß-Glucosidase gene converting platycoside E to platycodin D. Besides, we inferred that some transcription factors can regulate the expression of key enzymes involved in triterpernoid saponins and polysaccharides biosynthesis pathway of P. grandiflorus. Totally, a candidate gene encoding enzyme involved in converting platycoside E to platycodin D, and putative genes involved in polysaccharide synthesis in P. grandiflorus had been identified. This study will help uncover the molecular mechanism of triterpenoid saponins biosynthesis in P. grandiflorus.

In vitro and in vivo evaluation of a water-in-oil microemulsion of platycodin D.

Platycodin D (PD) is the active metabolite of Platycodon grandiflorum. The main purpose of this study was to develop and evaluate a water-in-oil (W/O) microemulsion formulation of PD (PD-ME). The PD-ME was successfully prepared by the water titration method at K m = 2, to draw the pseudoternary phase diagrams. Physical characterization including the particle size, pH, refractive index, average viscosity, and polydispersity index (PDI) was performed. The in vivo characteristics were evaluated by intestinal permeability and pharmacokinetic studies. The optimized microemulsion formulation consisted of 100 mg/ml PD aqueous solution, soybean phospholipids, ethanol, and oleic acid (27:39:19:15, w/w). The average viscosity, pH, droplet size, PDI, and zeta potential of the PD-ME were 78.65 ± 0.13 cPa•s, 5.70 ± 0.05, 30.46 ± 0.20 nm, 0.33 ± 0.00, and -3.13 mV, respectively. The drug concentration of the PD-ME was 26.3 ± 0.6 mg/ml. The PD-ME showed significantly higher apparent permeability coefficients than PD (p < .01). The pharmacokinetic studies showed that the PD-ME had significantly higher values of T 1/2 (2.26-fold), AUC0-24h (area under the curve; 1.65-fold), and MRT0-24h (1.58-fold) than PD (p < .01). It can be seen that W/O ME presents a strategy with great promise for enhancing the intestinal permeability and better oral absorption of drugs with high polarity and poor permeability.

Platycodon grandifloras polysaccharides inhibit mitophagy injury induced by Cr (VI) in DF-1 cells.

This study aimed to investigate the role of Platycodon grandiflorus polysaccharide (PGPS) in chromium (VI)-induced autophagy in a chicken embryo fibroblast cell lines (DF-1 cells). DF-1 cells were exposed to Cr (VI), PGPSt, and Cr (VI) + PGPSt, and their effects on cell viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and autophagy-related proteins were examined. The results showed that the cell viability was reduced after Cr (VI) treatment, and 3-MA, CsA or PGPSt suppressed this decrease. Cr (VI) treatment increased the ROS levels and decreased the MMP, thereby enhancing the expression of mitochondrial autophagy marker proteins (PINK1, Parkin, and LC3-II), inhibiting mitophagy autophagy protein TOMM20 expression, and promoting the degradation of autophagy-related marker p62. These changes led to exceeding mitochondrial autophagy and cell trauma and could be mitigated by PGPSt. Overall, our research showed that Cr (VI) can induce exceeding mitochondrial autophagy in DF-1 cells, whereas PGPSt can improve Cr (VI)-induced mitochondrial autophagy by inhibiting ROS and restoring MMP.

Conversion of Glycosylated Platycoside E to Deapiose-Xylosylated Platycodin D by Cytolase PCL5.

Platycosides, the saponins abundant in Platycodi radix (the root of Platycodon grandiflorum), have diverse pharmacological activities and have been used as food supplements. Since deglycosylated saponins exhibit higher biological activity than glycosylated saponins, efforts are on to enzymatically convert glycosylated platycosides to deglycosylated platycosides; however, the lack of diversity and specificities of these enzymes has limited the kinds of platycosides that can be deglycosylated. In the present study, we examined the enzymatic conversion of platycosides and showed that Cytolase PCL5 completely converted platycoside E and polygalacin D3 into deapiose-xylosylated platycodin D and deapiose-xylosylated polygalacin D, respectively, which were identified by LC-MS analysis. The platycoside substrates were hydrolyzed through the following novel hydrolytic pathways: platycoside E → platycodin D3 → platycodin D → deapiosylated platycodin D → deapiose-xylosylated platycodin D; and polygalacin D3 → polygalacin D → deapiosylated polygalacin D → deapiose-xylosylated polygalacin D. Our results show that cytolast PCL5 may have a potential role in the development of biologically active platycosides that may be used for their diverse pharmacological activities.