An inulin-type fructan CP-A from Codonopsis pilosula attenuates experimental colitis in mice by promoting autophagy-mediated inactivation of NLRP3 inflammasome.

Inulin-type fructan CP-A, a predominant polysaccharide in Codonopsis pilosula, demonstrates regulatory effects on immune activity and anti-inflammation. The efficacy of CP-A in treating ulcerative colitis (UC) is, however, not well-established. This study employed an in vitro lipopolysaccharide (LPS)-induced colonic epithelial cell model (NCM460) and an in vivo dextran sulfate sodium (DSS)-induced colitis mouse model to explore CP-A's protective effects against experimental colitis and its underlying mechanisms. We monitored the clinical symptoms in mice using various parameters: body weight, disease activity index (DAI), colon length, spleen weight, and histopathological scores. Additionally, molecular markers were assessed through enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting assays. Results showed that CP-A significantly reduced reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), and interleukins (IL-6, IL-1ß, IL-18) in LPS-induced cells while increasing IL-4 and IL-10 levels and enhancing the expression of Claudin-1, ZO-1, and occludin proteins in NCM460 cells. Correspondingly, in vivo findings revealed that CP-A administration markedly improved DAI, reduced colon shortening, and decreased the production of myeloperoxidase (MPO), malondialdehyde (MDA), ROS, IL-1ß, IL-18, and NOD-like receptor protein 3 (NLRP3) inflammasome-associated genes/proteins in UC mice. CP-A treatment also elevated glutathione (GSH) and superoxide dismutase (SOD) levels, stimulated autophagy (LC3B, P62, Beclin-1, and ATG5), and reinforced Claudin-1 and ZO-1 expression, thereby aiding in intestinal epithelial barrier repair in colitis mice. Notably, the inhibition of autophagy via chloroquine (CQ) diminished CP-A's protective impact against colitis in vivo. These findings elucidate that CP-A's therapeutic effect on experimental colitis possibly involves mitigating intestinal inflammation through autophagy-mediated NLRP3 inflammasome inactivation. Consequently, inulin-type fructan CP-A emerges as a promising drug candidate for UC treatment.

Codonopsis lanceolata polysaccharide ameliorates high-fat diet induced-postpartum hypogalactia via stimulating prolactin receptor-mediated Jak2/Stat5 signaling.

This study aims to investigate the ameliorative effect of Codonopsis lanceolata polysaccharide (PCL) on mice with hypogalatia induced by a high-fat diet (HFD) and the potential underlying mechanism. We found that oral administration of PCL demonstrated significant benefits in countering the negative effects of HFD, including weight gain, hepatic steatosis, mesenteric adipocyte hypertrophy, and abnormal glucose/lipid metabolism. In addition, PCL improved mammary gland development and enhanced lactogenesis performance. Histologically, PCL ameliorated the retardation of ductal growth, reduced mammary fat pad thickness, improved the incomplete linear encapsulation of luminal epithelium and myoepithelium, and increased the proliferation of mammary epithelial cells. Flow cytometry analysis showed that PCL mitigated the detrimental effects of HFD on mammary gland development by promoting the proliferation and differentiation of mammary epithelial cells. Mechanistic studies revealed that PCL upregulated the levels of prolactin (PRL) and its receptor (PRLR) in the mammary gland, activated JAK2/STAT5 signaling pathway, and increased the expression of p63, ERBB4, and NRG1. Overall, PCL can ameliorate HFD-induced hypogalactia by activating PRLR-mediated JAK2/STAT5 signaling. Our findings offer a methodological and theoretical foundation for investigating the functional constituents of traditional Chinese medicine in the treatment of hypogalactia.

Nutritional, biological, and structural properties of bioactive peptides from bellflower, Persian-willow, and bitter-orange pollens.

In this study, the composition of amino acids, nutritional characteristics, degree of hydrolysis (DH), antioxidant properties, and antibacterial activity of proteins and hydrolysates of bellflower (Campanula latifolia), Persian willow (Salix aegyptiaca), and bitter orange (Citrus aurantium L.) were investigated under the influence of different proteases (Alcalase: Al, trypsin: Tr, pancreatin: Pa, and pepsin: Pe). Evaluation of the structural features of the proteins showed amide regions (amide A, B, I-III) and secondary structures. Hydrophobic amino acids (∼38%), antioxidants (∼21%), and essential types (∼46%) form a significant part of the structure of flower pollen. The digestibility and nutritional quality (PER) of the hydrolyzed samples (CP: 1.67; CA: 1.89, and PW: 1.93) were more than the original protein. Among proteins and peptides, the highest degree of hydrolysis (34.6%: Al-PWH), inhibition of free radicals DPPH (84.2%: Al-CPH), ABTS (95.2%: Pa-CPH), OH (86.7%: Tr-CAH), NO (57.8%: Al-CPH), reducing power (1.31: Pa-CPH), total antioxidant activity (1.46: Pa-CPH), and chelation of iron ions (80%: Al-CPH and Al-CAH) and copper (50.3%: Pa-CAH) were affected by protein type, enzyme type, and amino acid composition. Also, the highest inhibition of the growth of Escherichia coli (25 mm) and Bacillus cereus (24 mm) were related to CP and PW hydrolysates, respectively. The results of this research showed that hydrolyzed flower pollens can be used as a rich source of essential amino acids as well as natural antioxidants and antibacterial in food and dietary products. PRACTICAL APPLICATION: Enzymatic hydrolysis of Campanula latifolia, Persian willow, and Citrus aurantium pollen proteins was performed. The hydrolyzed ones had high nutritional quality and digestibility (essential amino acids and PER index). Antioxidant properties and chelation of metal ions of peptides were affected by the type of protein and enzyme. The hydrolysates showed inhibitory activity against the growth of Esherichia coli and Bacillus cereus.

Antimicrobial activity and enzymatic analysis of endophytes isolated from Codonopsis pilosula.

The roots of the medicinal plant Codonopsis pilosula (Franch.) Nannf (C. pilosula) possess most medicinal supplements. In current research on C. pilosula root endophytes were isolated, identified, and evaluated for their antimicrobial activity against human pathogens such as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Salmonella typhi, and Pseudomonas aeruginosa and the fungi Candida albicans and Aspergillus niger. Endophytes C.P-8 and C.P-20 exhibited very significant antimicrobial activity, the secondary metabolite of C.P-8 registered at retention time 24.075 by HPLC analysis. Significant minimum inhibitory concentration (MIC) of C.P-8 was exhibited at 250 µg/ml against S. aureus and 500 µg/ml against B. subtilis. Qualitative, quantitative analyses, and partial purification of enzymes and purity was analysed by molecular weight determined by SDS‒PAGE of enzymes produced by C.P-20, amylase-64 kDa, protease-64 kDa, chitinase-30 kDa, and cellulase-54 kDa. Optimum pH and temperature of the partially purified enzymes, was carried out. The partially purified enzymes from C.P-20 displayed maximum activity at pH 6-7 and temperatures of 40°C-45°C. Moreover, the above endophytes will be useful tools for producing active enzymes and active bioantimicrobial agents against human pathogens.

A polysaccharide from Codonopsis pilosula roots attenuates carbon tetrachloride-induced liver fibrosis via modulation of TLR4/NF-κB and TGF-ß1/Smad3 signaling pathway.

The present work reported the extraction, purification, characterization of a polysaccharide from roots of Codonopsis pilosula (CPP-A-1) and its effect on liver fibrosis. The findings exhibited that the molecular weight of CPP-A-1 was 9424 Da, and monosaccharide composition were glucose and fructose and minor contents of arabinose. Structural characterization of CPP-A-1 has a backbone consisting of→(2-ß-D-Fruf-1)n→ (n ≈ 46-47). Treatment with CPP-A-1 inhibited the proliferation of transforming growth factor-beta 1 (TGF-ß)-activated human hepatic stellate cell line (LX-2), and induced cell apoptosis. We used carbon tetrachloride (CCl4) to construct mice model of liver fibrosis and subsequently administered CPP-A-1 treatment. The results showed that CPP-A-1 alleviated CCl4-induced liver fibrosis as demonstrated by reversing liver histological changes, decreased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) contents, collagen deposition, and downregulated fibrosis-related collagen I and α-smooth muscle actin (α-SMA), and inhibited the generation of excessive extracellular matrix (ECM) components by restoring the balance between matrix metalloproteinases (MMPs) and its inhibitor (TIMPs). Moreover, CPP-A-1 improved anti-oxidation effects detected by promoting liver superoxide dismutase (SOD), glutathione (GSH) and Mn-SOD levels, and inhibition of liver malondialdehyde (MDA) and iNOS levels. CPP-A-1 also ameliorated the inflammatory factor (tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6), and expression of inflammatory factor genes (TNF-α, IL-11 mRNA). In addition, our results showed that CPP-A-1 inhibited Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) and transforming growth factor-ß1 (TGF-ß1)/drosophila mothers against decapentaplegic 3 (Smad3) signaling pathways. Furthermore, In vitro tests of LX-2 cells demonstrated that CPP-A-1 not only inhibited α-SMA expression with lipopolysaccharide (LPS) or TGF-ß1 stimulation, but also inhibited TLR4/NF-κB and TGF-ß1/Smad3 signaling, similar to corresponding small-molecule inhibitors. Therefore, CPP-A-1 might exert suppressive effects against liver fibrosis by regulating TLR4/NF-κB and TGF-ß1/Smad3 signaling, our findings support a possible application of CPP-A-1 for the treatment of liver fibrosis.

De Novo Transcriptome Sequencing of Codonopsis lanceolata for Identification of Triterpene Synthase and Triterpene Acetyltransferase.

Codonopsis lanceolata (Campanulaceae) is a perennial plant commonly known as the bonnet bellflower. This species is widely used in traditional medicine and is considered to have multiple medicinal properties. In this study, we found that shoots and roots of C. lanceolata contained various types of free triterpenes (taraxerol, ß-amyrin, α-amyrin, and friedelin) and triterpene acetates (taraxerol acetate, ß-amyrin acetate, and α-amyrin acetate). The content of triterpenes and triterpene acetates by GC analysis was higher in the shoot than in the roots. To investigate the transcriptional activity of genes involved in triterpenes and triterpene acetate biosynthesis, we performed de novo transcriptome analysis of shoots and roots of C. lanceolata by sequencing using the Illumina platform. A total of 39,523 representative transcripts were obtained. After functional annotation of the transcripts, the differential expression of genes involved in triterpene biosynthetic pathways was investigated. Generally, the transcriptional activity of unigenes in the upstream region (MVA and MEP pathway) of triterpene biosynthetic pathways was higher in shoots than in roots. Various triterpene synthases (2,3-oxidosqualene cyclase, OSC) participate to produce triterpene skeletons by the cyclization of 2,3-oxidosqualene. A total of fifteen contigs were obtained in annotated OSCs in the representative transcripts. Functional characterization of four OSC sequences by heterologous expression in yeast revealed that ClOSC1 was determined as taraxerol synthase, and ClOSC2 was a mixed-amyrin synthase producing α-amyrin and ß-amyrin. Five putative contigs of triterpene acetyltransferases showed high homology to the lettuce triterpene acetyltransferases. Conclusively, this study provides the basis of molecular information, particularly for the biosynthesis of triterpenes and triterpene acetates in C. lanceolata.

Chemical and metabolic profiling of Codonopsis Radix extract with an integrated strategy using ultra-high-performance liquid chromatography coupled with mass spectrometry.

Codonopsis radix was commonly used as food materials or herbal medicines in many countries. However, the comprehensive analysis of chemical constituents, and in vivo xenobiotics of Codonopsis radix remain unclear. In the present study, an integrated strategy with feature-based molecular networking using ultra-high-performance liquid chromatography coupled with mass spectrometry was established to systematically screen the chemical constituents and the in vivo xenobiotics of Codonopsis radix. A step-by-step manner based on a composition database, visual structure classification, discriminant ions, and metabolite software prediction was proposed to overcome the complexities due to the similar structure of chemical constituents and metabolites of Codonopsis radix. As a result, 103 compounds were tentatively characterized, 20 of which were identified by reference standards. Besides, a total of 50 xenobiotics were detected in vivo, including 26 prototypes and 24 metabolites, while the metabolic features of the pyrrolidine alkaloids were elucidated for the first time. The metabolism reactions of pyrrolidine alkaloids and sesquiterpene lactones included oxidation, methylation, hydration, hydrogenation, demethylation, glucuronidation, and sulfation. This study provided a generally applicable approach to the comprehensive investigation of the chemical and metabolic profile of traditional Chinese medicine and offered reasonable guidelines for further screening of quality control indicators and pharmacodynamics mechanism of Codonopsis radix.

Lancemaside A from Codonopsis lanceolata: Studies on Antiviral Activity and Mechanism of Action against SARS-CoV-2 and Its Variants of Concern.

Several plant-derived natural products with anti-SARS-CoV-2 activity have been evaluated for the potential to serve as chemotherapeutic agents for the treatment of COVID-19. Codonopsis lanceolata (CL) has long been used as a medicinal herb in East Asian countries to treat inflammatory diseases of the respiratory system but its antiviral activity has not been investigated so far. Here, we showed that CL extract and its active compound lancemaside A (LA) displayed potent inhibitory activity against SARS-CoV-2 infection using a pseudotyped SARS-CoV-2 entry assay system. We demonstrated that this inhibitory effect of LA was due to the alteration of membrane cholesterol and blockade of the membrane fusion between SARS-CoV-2 and host cells by filipin staining and cell-based membrane fusion assays. Our findings also showed that LA, as a membrane fusion blocker, could impede the endosomal entry pathway of SARS-CoV-2 and its variants of concern (VOCs), including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), in Vero cells with similar of IC50 values ranging from 2.23 to 3.37 µM as well as the TMPRSS2-mediated viral entry pathway in A549 cells overexpressing ACE2 and TMPRSS2 with IC50 value of 3.92 µM. We further demonstrated that LA could prevent the formation of multinucleated syncytia arising from SARS-CoV-2 spike protein-mediated membrane fusion. Altogether, the findings reported here suggested that LA could be a broad-spectrum anti-SARS-CoV-2 therapeutic agent by targeting the fusion of viral envelope with the host cell membrane.

Astragalus polysaccharides combined with Codonopsis pilosula polysaccharides modulates the physiological characteristics of trophoblasts via miR-92a-1-5p/CCR7 axis.

BACKGROUND: When the abnormality occurs in proliferation, differentiation and apoptosis of trophoblasts, the invasion ability of placental trophoblast is weakened, which is prone to trigger the occurrence of various pregnancy diseases such as repeated spontaneous abortion (RSA). Clinically, Astragalus and Codonopsis pilosula polysaccharides (APS and CPPS) are used for the treatment of unexplained recurrent spontaneous abortion (URSA). Therefore, we aimed to probe into the roles of APS and CPPS in biological behaviors of placental trophoblasts. METHODS: The trophoblasts were treated with APS and CPPS, and transfected with miR-92a-1-5p mimic and CCR7 plasmid to explore the roles of APS and CPPS. Cell viability and apoptosis were determined by CCK-8 and flow cytometry, respectively. The levels of miRNA/mRNA and protein were measured by qRT-PCR and western blot, respectively. The interaction between miR-92a-1-5p and CCR7 was analyzed by TargetScan and dual-luciferase reporter assay. Invasion and migration rates were assessed by Transwell and wound healing assays, respectively. RESULTS: APS combined with CPPS enhanced viability, Bcl-2 expression, and migration and invasion capabilities, while suppressing apoptosis, and expressions of Bax, Bim and miR-92a-1-5p in trophoblasts. Nevertheless, miR-92a-1-5p mimic produced the inverse modulations in trophoblasts, and partially reversed the effects of APS and CPPS. Furthermore, overexpression of CCR7, the target of miR-92a-1-5p, partially offset the effect of miR-92a-1-5p mimic in trophoblasts. CONCLUSION: Astragalus combined with Codonopsis pilosula polysaccharides modulates the biological behaviors of trophoblasts via miR-92a-1-5p/CCR7 axis. The regulatory axis we studied will be helpful for the treatment of URSA in the future.

Improved growth and metabolite accumulation in Codonopsis pilosula (Franch.) Nannf. by inoculation with the endophytic Geobacillu sp. RHBA19 and Pseudomonas fluorescens RHBA17.

In this study, we focused on the plant-growth-promoting properties of two strains isolated from Codonopsis pilosula, and the effect of inoculation with different strain treatments on physiological and metabolite accumulation of C. pilosula. The strains RHBA19 and RHBA17 were isolated and identified as Geobacillu sp. and Pseudomonas fluorescens, respectively. The two strains produced indole acetic acid (IAA), siderophore, biofilm, and various exoenzymes. Based on the pot experiments, inoculation of RHBA19 (G group) and the two mixed bacteria (M group) significantly improved the growth, root development, and photosynthesis of C. pilosula. Compared with the no-inoculation group (CK), the total polysaccharide content of root in the G and M groups was dramatically enhanced by 59.27% and 96.07%, and the lobetyolin (root) improved by 58.4% and 66.0%, respectively. After inoculation with bacteria agents, the activities of antioxidant enzymes (CAT, POD, SOD) of C. pilosula increased differentially. Inoculation with two types of bacterial agents significantly increased the activities of sucrose synthase (SS) and sucrose phosphate synthase (SPS) in root, and phenylalanine ammonia lyase (PAL) in leaf of C. pilosula. In addition, the content of signaling molecules (NO and H2O2) in three types of tissue increased significantly. The magnitude of these results was higher with mixtures than with individual strains. These results imply that the two types of bacterial agents induce physiological metabolism changes to accumulate polysaccharides and lobetyolin by regulating stress resistance enzymes and signal molecules, especially NO and H2O2.

Characterization of chemical composition variations in raw and processed Codonopsis Radix by integrating metabolomics and glycomics based on multiple chromatography-mass spectrometry technology.

Codonopsis Radix, a popular food homology medicine, is widely used in clinical traditional Chinese medicine and food supplement, raw products and three types of processed products are the main forms of decoction pieces in China. However, there is no scientific basis for comprehensive chemical characterization of raw and three types of processed products. Herein, we investigated qualitatively and quantificationally secondary and primary metabolites in raw Codonopsis Radix and three types of processed products by metabolomics and glycomics employing multiple chromatography-mass spectrometry technology combined with chemometric analysis further to look for differential compounds and propose the processing-induced chemical mechanisms. The results indicated that Codonopsis Radix became dark-colored and the smell of burnt incense odor was observed after processing. The principal component analysis demonstrated that secondary metabolome and glycome were significantly altered between raw and processed products, and 36 differential secondary metabolites and 11 differential primary metabolites were finally screened through orthogonal partial least-squares-discriminant analysis. The main types of compounds are alkaloids, terpenoids, glycosides, amino acids, monosaccharides, oligosaccharides, and furfural derivatives. Meanwhile, Chemical mechanisms could be involved, including oxidation, glycosidic hydrolysis, esterification, dehydration, and Maillard reaction. This work supplies a chemical basis for the application of various types of Codonopsis Radix decoction pieces.

Investigating the Mechanism of Shengmaiyin (Codonopsis pilosula) in the Treatment of Heart Failure Based on Network Pharmacology.

BACKGROUND AND OBJECTIVE: To explore the molecular mechanism by which Shengmaiyin (Codonopsis pilosula) (SMY) improves isoproterenol (ISO)-induced heart failure (HF) in rats via a traditional Chinese medicine (TCM) integrated pharmacology research platform, The Chinese Medicine Integrated Pharmacology Platform (TCMIP V2.0). METHOD: The chemical constituents and drug targets of SMY medicines were identified through TCMIP, and HF disease target information was collected. A prescription Chinese medicinecomponent- core target network was constructed through the TCM network mining module, and biological process and pathway enrichment analyses of core targets were conducted. In vivo experiments in rats were performed to verify the pathway targets. Hematoxylin and eosin staining was used to observe myocardial tissue morphology. ELISA kits were used to detect cAMP content, and Western blotting was used to detect the expression levels of signaling pathway-related proteins. RESULTS: The TCMIP analysis indicated that SMY treatment of HF activates the GS-ß-adrenergic receptor (ßAR)-cAMP-protein kinase A (PKA) signaling pathway. The in vivo experimental results confirmed this finding. High-dose SMY significantly improved the morphology of ISO-injured myocardium. The levels of G-protein-coupled receptor (GPCR), adenylate cyclase (AC), ßAR, and PKA proteins in myocardial tissue were significantly increased in the SMY group. In addition, the content of cAMP in myocardial tissue was increased, and the content of cAMP in serum was decreased. CONCLUSION: Based on the analysis of TCMIP, SMY treatment of HF may activate the GS-ßARcAMP- PKA signaling pathway. The findings provide a theoretical basis for further research on the anti-HF mechanism of SMY.

Effect of Codonopsis Radix and Polygonati Rhizoma on the regulation of the IRS1/PI3K/AKT signaling pathway in type 2 diabetic mice.

Objective: Codonopsis Radix and Polygonati Rhizoma (CRPR) has a good hypoglycemic effect. The aims of the present study were to investigate the effect of CRPR on high-fat/high-sugar diet (HFHSD)- and streptozotocin (STZ)-induced type 2 diabetes mellitus (T2DM) mice as well as to investigate the involved mechanism. Methods: A T2DM mouse model was generated by combining HFHSD and STZ. After the model was established, normal and model groups received the same volume of normal saline intragastrically, and the negative control group was treated with metformin (200 mg/kg·BW). The low, medium, and high CRPR groups received four consecutive weeks of oral gavage with CRPR doses of 2.5, 5, and 10 g/kg·BW, respectively, during the course of the study. Body weight and fasting blood glucose (FBG) were measured on a weekly basis. Enzyme-linked immunosorbent assay (ELISAs) were used to evaluate the serum and liver samples. Hematoxylin and eosin (H&E) staining was utilized to observe the pathological status of the liver and pancreas. Western blot (WB) analysis was performed to evaluate the protein expression levels of PI3K, p-PI3K, AKT, and p-AKT. Results: Compared to model mice, each treatment group had significantly elevated levels of FBG, total cholesterol (TC), and triacylglycerol (TG) (P<0.01 and P<0.05, respectively). The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly reduced in the treatment groups compared to the model group (P<0.01). Compared to the model group, fasting insulin (FINS) levels were elevated in all groups of CRPR (P<0.05), and there were significantly higher levels of high-density lipoprotein cholesterol (HDL-C) in both the low-dose and high-dose CRPR groups (P<0.05). H&E staining indicated that CRPR treatment reduced organ enlargement, improved liver lipid accumulation, and repaired islet injury in T2DM mice. Moreover, WB analysis demonstrated that all CRPR groups significantly upregulated the protein expression of IRS1, p-GSK3ß, PI3K, p-Akt and p-FOXO1(P<0.05) as well as significantly downregulated p-IRS1 and FOXO1 protein expression (P<0.05). Conclusion: The present study demonstrated that CRPR effectively improves the metabolic disturbance of lipids, repairs damaged liver tissues, repairs damaged pancreatic tissues, and reduces insulin resistance (IR) in T2DM mice. The mechanism of action may be associated with upregulation of the IRS1/PI3K/AKT signaling pathway and inhibition of IRS1 phosphorylation.

Comparative analysis of carbon and nitrogen metabolism, antioxidant indexes, polysaccharides and lobetyolin changes of different tissues from Codonopsis pilosula co-inoculated with Trichoderma.

Codonopsis pilosula is a traditional Chinese herbal medicinal plant and contains various bioactive components, such as C. pilosula polysaccharides (CPPs) and lobetyolin (Lob). Hydrogen peroxide (H2O2) and nitric oxide (NO) are gaseous molecule and have been well known for their ability to relieve some adverse influences on plant from abiotic stress. Endophytic fungus is non-pathogenic plant-associated fungus that could play a significant role in improving plant tolerance by signal molecule. In this work, we determined how inoculation of Trichoderma strain RHTA01 with C. pilosula changed the plant's growth, metabolite accumulation, and related enzyme activity. Results demonstrated that application of Trichoderma strain RHTA01 significantly improved the growth of C. pilosula. Moreover, it noticeably decreased antioxidant enzyme superoxide dismutase (SOD) and catalase (CAT) activity in C. pilosula leaves, reduced the content of H2O2 and malondialdehyde (MDA), and weakened the peroxidation of cell membrane lipids, which reduced the damage of abiotic stress to C. pilosula. Research has shown that it had obvious effects on levels of nitrogen and carbon metabolic enzymes. For example, sucrose synthase (SS) and acid invertase (AI) levels in C. pilosula roots were nearly 1.43 and 1.7 times higher, respectively, than those in the control (CK) group. In addition, it was notable that the production of CPPs and Lob, the most significant secondary metabolites in C. pilosula, were influenced by Trichoderma strain RHTA01. The obtained results indicate that inoculating C. pilosula with Trichoderma stimulates the carbon and nitrogen metabolism of the plant, and helps to increase the content of CPPs and Lob in the root of the plant.

Antioxidant activities of sulfated Codonopsis polysaccharides in acute oxidative stress.

This study aimed to explore the protective effect of sulfated Codonopsis polysaccharides (SCP) on acute oxidative stress. SCP was modified by chlorosulfonic acid-pyridine method from Codonopsis polysaccharides (CP), which had 34.48% of sulfate content determined by ultrasonic-acidic barium chromate spectrophotometry. The analysis of Fourier transform-infrared spectroscopy (FT-IR) appeared an absorption peak of SCP at 811.91 cm-1 , which related to C-O-SO3 . In vitro test, the antioxidant activities of CP and SCP was induced by H2 O2 in RAW264.7 cells, results indicated that SCP and CP could significantly enhance the activity of superoxide dismutase (SOD), glutathione peroxidase (GDH-Px) and catalase (CAT), and nitric oxide (NO) and decrease the level of malondialdehyde (MDA), reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) secreted by RAW264.7 cells compared with modeling group (p < .05). The flow cytometry results also revealed that SCP and CP could markedly inhibit the apoptosis of macrophage induced by acute oxidative stress. In vivo test, 50% ethanol was used to induce mice acute oxidative stress, results indicated that the blood biochemical parameters in mice were restored to normal levels following administration of SCP and CP, and alanine aminotransferase (ALT), aspartate transaminase (AST), total protein (TP), albumin (ALB), glucose (GLU), and creatinine (UREA) had significant differences compared with modeling group (p < .05). Quantitative real-time PCR analysis revealed that SCP and CP could promote the expression of Keap1 and Nrf2. In summary, both SCP and CP had protective effects against acute oxidative stress. PRACTICAL APPLICATIONS: Oxidative stress is a kind of stress injury, which can cause a variety of diseases and accelerate physical aging. Codonopsis has many active components, among which Codonopsis polysaccharide has antioxidant effect. Recent studies have found that Codonopsis polysaccharides could be modified by sulfate molecules to obtain higher antioxidant activity. The modified Codonopsis polysaccharides could significantly promote the production of antioxidant enzymes (SOD, CAT, GDH-Px) and reduce the content of oxidative stress marks (ROS, MDA). Moreover, its antioxidant mechanism may be related to the Keap1 /Nrf2 signaling pathway. Therefore, SCP was an effective antioxidant, and could be used as a potential health food with antioxidant and anti-aging effects.

Genome-Wide Characterization of SPL Gene Family in Codonopsis pilosula Reveals the Functions of CpSPL2 and CpSPL10 in Promoting the Accumulation of Secondary Metabolites and Growth of C. pilosula Hairy Root.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors play critical roles in regulating diverse aspects of plant growth and development, including vegetative phase change, plant architecture, anthocyanin accumulation, lateral root growth, etc. In the present study, 15 SPL genes were identified based on the genome data of Codonopsis pilosula, a well-known medicinal plant. Phylogenetic analysis clustered CpSPLs into eight groups (G1-G8) along with SPLs from Arabidopsis thaliana, Solanum lycopersicum, Oryza sativa and Physcomitrella patens. CpSPLs in the same group share similar gene structure and conserved motif composition. Cis-acting elements responding to light, stress and phytohormone widely exist in their promoter regions. Our qRT-PCR results indicated that 15 CpSPLs were differentially expressed in different tissues (root, stem, leaf, flower and calyx), different developmental periods (1, 2 and 3 months after germination) and various conditions (NaCl, MeJA and ABA treatment). Compared with the control, overexpression of CpSPL2 or CpSPL10 significantly promoted not only the growth of hairy roots, but also the accumulation of total saponins and lobetyolin. Our results established a foundation for further investigation of CpSPLs and provided novel insights into their biological functions. As far as we know, this is the first experimental research on gene function in C. pilosula.

Codonopsis pilosula Polysaccharides Alleviate Aß 1-40-Induced PC12 Cells Energy Dysmetabolism via CD38/NAD+ Signaling Pathway.

BACKGROUND: Alzheimer's disease (AD) is the most common type of dementia and has a complex pathogenesis with no effective treatment. Energy metabolism disorders, as an early pathological event of AD,have attracted attention as a promising area of AD research. Codonopsis pilosula Polysaccharides are the main effective components of Codonopsis pilosula, which have been demonstrated to regulate energy metabolism. METHODS: In order to further study the roles and mechanisms of Codonopsis pilosula polysaccharides in AD, this study used an Aß1-40-induced PC12 cells model to study the protective effects of Codonopsis pilosula polysaccharides and their potential mechanisms in improving energy metabolism dysfunction. RESULTS: The results showed that Aß1-40 induced a decrease in PC12 cells viability, energy metabolism molecules (ATP, NAD+, and NAD+/NADH) and Mitochondrial Membrane Potential (MMP) and an increase in ROS. Additionally, it was found that Aß1-40 increased CD38 expression related to NAD+ homeostasis, whereas Silent Information Regulation 2 homolog1 (SIRT1, SIRT3), Peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and SIRT3 activity were decreased. Codonopsis pilosula polysaccharides increased NAD+, NAD+/NADH, SIRT3, SIRT1, and PGC-1α related to NAD+, thus partially recovering ATP. CONCLUSION: Our findings reveal that Codonopsis pilosula polysaccharides protected PC12 cells from Aß1-40-induced damage, suggesting that these components of the Codonopsis pilosula herb may represent an early treatment option for AD patients.

Extraction, characterization and anti-inflammatory activities of an inulin-type fructan from Codonopsis pilosula.

A homogenous polysaccharide from Codonopsis pilosula (CPP) was isolated and identified to be an inulin-type fructan, coded as CPP1-2-1. The polysaccharide exhibited anti-inflammatory effect against lipopolysaccharide (LPS) induced RAW264.7 cells in vitro and dextran sodium sulfate (DSS)-induced colitis mice in vivo. Moreover, the expression of cytokines was further detected by qPCR, and the results showed that the polysaccharides can reduce the expression of inflammatory factors such as TLR4, NF-κB, TNF-α and IL-6 in the cells, indicating the anti-inflammatory activities may be achieved by inhibiting the activation of TLR4/NF-κB pathway.

The structural characterization and immune modulation activitives comparison of Codonopsis pilosula polysaccharide (CPPS) and selenizing CPPS (sCPPS) on mouse in vitro and vivo.

Codonopsis pilosula polysaccharide (CPPS) and selenizing CPPS (sCPPS) were prepared and identified by a combination of chemical and instrumental analysis. Their immune modulation activities were compared by lymphocyte proliferation and flowcytometry tests in vitro or serum antibody responses and cytokines with immunization against OVA mice in vivo. The results showed that the sCPPS was successfully modified in selenylation. In vitro, the sCPPS were more effective compared with CPPS in promoting lymphocyte proliferation synergistically with PHA or LPS and increasing the ratio of CD4+ to CD8 + T cells. In vivo, sCPPS could significantly raised IgG, IgM, IFN-γ, IL-2 and IL-4 contents in the serum of mouse against OVA in comparison with CPPS. These results indicate that selenylation modification can enhance the immune modulation activitives of CPPS. sCPPS would be as a component drug of new-type immunoenhancer.

Selection and Validation of Reference Genes for Gene Expression Studies in Codonopsis pilosula Based on Transcriptome Sequence Data.

Relative gene expression analyses by RT-qPCR (reverse transcription-quantitative PCR) are highly dependent on the reference genes in normalizing the expression data of target genes. Therefore, inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, and the selection and validation of suitable internal reference genes becomes essential. In this study, we retrieved the commonly used reference genes in transcriptome datasets of Codonopsis pilosula by RNA-Seq (unpublished data), and selected 15 candidate reference genes according to the coefficient of variation (CV) and fold change (FC) value of gene expression. The expression levels of candidate reference genes, which is at different growth stages, undergoing cold stress and drought stress, was determined by RT-qPCR. The expression stability of these genes was evaluated using software packages and algorithms including ΔCt, geNorm, NormFinder and Bestkeeper. Then appropriate reference genes were screened and validated by target gene-UDGPase (UDP glucose pyrophosphorylase). The optimal RGs combinations of C. pilosula, including PP2A59γ, CPY20-1, UBCE32, RPL5B and UBC18 for developmental stage, RPL5B, RPL13 and PP2A59γ for cold treatment, RPL13 and PP2A59γ for drought treatment, were found and proposed as reference genes for future work. This paper laid foundations for both the selection of reference genes and exploration in metabolic mechanism of C. pilosula.