Integrated metabolomic and transcriptomic analysis of triterpenoid accumulation in the roots of Codonopsis pilosula var. modesta (Nannf.) L.T.Shen at different altitudes.

INTRODUCTION: Codonopsis Radix is a beneficial traditional Chinese medicine, and triterpenoid are the major bioactive constituents. Codonopsis pilosula var. modesta (Nannf.) L.T.Shen (CPM) is a precious variety of Codonopsis Radix, which is distributed at high mountain areas. The environment plays an important role in the synthesis and metabolism of active ingredients in medicinal plants, but there is no report elaborating on the effect of altitude on terpenoid metabolites accumulation in CPM. OBJECTIVES: This study aims to analyse the effects of altitude on triterpenoid biosynthetic pathways and secondary metabolite accumulation in CPM. MATERIAL AND METHODS: The untargeted metabolomics based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and 10 triterpenoids based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) method were analysed at the low-altitude (1480 m) and high-altitude (2300 m) CPM fresh roots. The transcriptome based on high-throughput sequencing technology were combined to analyse the different altitude CPM triterpenoid biosynthetic pathways. RESULTS: A total of 17,351 differentially expressed genes (DEGs) and 55 differentially accumulated metabolites (DAMs) were detected from the different altitude CPM, and there are significant differences in the content of the 10 triterpenoids. The results of transcriptome study showed that CPM could significantly up-regulate the gene expression levels of seven key enzymes in the triterpenoid biosynthetic pathway. CONCLUSIONS: The CPM at high altitude is more likely to accumulate triterpenes than those at low altitude, which was related to the up-regulation of the gene expression levels of seven key enzymes. These results expand our understanding of how altitude affects plant metabolite biosynthesis.

Study on the mechanism of Fufang E'jiao Jiang on precancerous lesions of gastric cancer based on network pharmacology and metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang E'jiao Jiang (FEJ) is a prominent traditional Chinese medicine prescription, which consists of Asini Corii Colla (Donkey-hide gelatin prepared by stewing and concentrating from the hide of Equus asinus Linnaeus., ACC), Codonopsis Radix (the dried roots of Codonopsis pilosula (Franch.) Nannf., CR), Ginseng Radix et Rhizoma Rubra (the steamed and dried root of Panax ginseng C.A. Mey., GRR), Crataegi Fructus (the mature fruits of Crataegus pinnatifida Bunge., CF), and Rehmanniae Radix Praeparata (the steamed and sun dried tuber of Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C.A. Mey., RRP). It is a popularly used prescription for "nourishing Qi and nourishing blood". AIM OF THE STUDY: To explore the potential mechanism of FEJ on precancerous lesion of gastric cancer in rats by combining network pharmacology and metabolomics. METHODS: Traditional Chinese Medicine Systems Pharmacology and Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine were used to identify the ingredients and potential targets of FEJ. GeneCards database was used to define PLGC-associated targets. We built a herb-component-disease-target network and analyzed the protein-protein interaction network. Underlying mechanisms were identified using Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. In addition, 40% ethanol, N-methyl-N'-nitro-N-nitroguanidine and irregular eating were used to establish PLGC rats model. We also evaluated the efficacy of FEJ on MNNG-induced PLGC rats by body weight, histopathology, blood routine and cytokine levels, while the predicted pathway was determined by the Western blot. Ultra-performance liquid chromatography-tandem mass spectrometry-based serum non-targeted metabolomics was used to select potential biomarkers and relevant pathways for FEJ in the treatment of PLGC. RESULTS: Network pharmacology showed that FEJ exhibited anti-PLGC effects through regulating ALB, TNF, VEGFA, TP53, AKT1 and other targets, and the potential pathways mainly involved cancer-related, TNF, PI3K-AKT, HIF-1, and other signaling pathways. Animal experiments illustrated that FEJ could suppress inflammation, regulate gastrointestinal hormones, and inhibit the expression of PI3K/AKT/HIF-1α pathway-related proteins. Based on serum non-targeted metabolomics analysis, 12 differential metabolites responding to FEJ treatment were identified, and metabolic pathway analysis showed that the role of FEJ was concentrated in 6 metabolic pathways. CONCLUSION: Based on network pharmacology, animal experiments and metabolomics, we found that FEJ might ameliorate gastric mucosal injury in PLGC rats by regulating gastrointestinal hormones and inhibiting inflammation, and its mechanism of action is related to the inhibition of excessive activation of PI3K/AKT/HIF-1α signaling pathway and regulation of disorders of body energy metabolism. This comprehensive strategy also provided a reasonable way for unveiling the pharmacodynamic mechanisms of multi-components, multi-targets, and multi-pathways in Traditional Chinese Medicine.

[Influence of processing factors on honey-fried Codonopsis Radix].

The present study investigated the influence of heating and honey addition on the appearance, chemical component content, and pharmacological activity of Codonopsis Radix decoction pieces in the honey-frying process, and explored the processing mechanism of honey-fried Codonopsis Radix. The color, sweetness, and content of macromolecular components(e.g., oligosaccharides and polysaccharides) and small molecular components(e.g., lobetyolin and atractylenolide Ⅲ) of raw Codonopsis Radix, fried Codonopsis Radix, honey-mixed Codonopsis Radix, and honey-fried Codonopsis Radix were determined, and the antioxidant activities in vitro of their water extract, polysaccharide extract, and oligosaccharide extract were compared. The results showed that in terms of color and sweetness, compared with the raw Codonopsis Radix, the fried Codonopsis Radix slightly changed, the honey-mixed Codonopsis Radix changed significantly, and the honey-fried Codonopsis Radix changed with high significance. In terms of the content of lobetyolin, atractylenolide Ⅲ, and polysaccharides, the samples were ranked as raw Codonopsis Radix > fried Codonopsis Radix > honey-mixed Codonopsis Radix > honey-fried Codonopsis Radix, which indicated that heating and honey addition could reduce the content of these three components. In terms of the content of oligosaccharides, the samples were ranked as honey-fried Codonopsis Radix ≈ honey-mixed Codonopsis Radix > fried Codonopsis Radix ≈ raw Codonopsis Radix, indicating that honey addition could increase the content of oligosaccharides. In terms of antioxidant activity in vitro, ABTS radical scavenging ability of water extract, polysaccharides, and oligosaccharides of honey-fried Codonopsis Radix was most potent, while the change of antioxidant activity in vitro of each extract in the other three processed products was different. In short, both heating and honey addition can affect the appearance, chemical component content, and antioxidant activity in vitro of Codonopsis Radix decoction pieces, but the effect of the combination of the two factors is the best. The comprehensive analysis of the effects of heating and honey addition on Codonopsis Radix decoction pieces indicates that honey addition followed by heating at high temperature is the necessary condition for honey-fried Codonopsis Radix to enhance its activity.

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.

Two new aromatic derivatives from Codonopsis pilosula and their α-glucosidase inhibitory activities.

The ethanol extract of the roots of Codonopsis pilosula was subjected to chromatographic fractionation, which result in the isolation and characterization of two new aromatic derivatives 2,3-dihydroxypropyl 2,4-dihydroxy-3,6-dimethylbenzoate (1) and 2-oxopropyl 3-hydroxy-4-methoxybenzoate (2), along with three known compounds pilosulinene A (3), pollenfuran B (4) and (+)-pinoresinol (5). Their structures were demonstrated by HRESIMS and spectroscopic methods including NMR and IR. It is worth noting that compound 4 was isolated for the first time from the genus Codonopsis. The potential hypoglycemic properties of compounds 2-5 were evaluated by measuring their α-glucosidase inhibitory effects. As a result, compounds 2 and 3 showed weak α-glucosidase inhibitory activities with IC50 values of 154.8 ± 11.0 µM and 24.0 ± 2.2 µM, respectively.[Formula: see text].

Network pharmacology unveils spleen-fortifying effect of Codonopsis Radix on different gastric diseases based on theory of "same treatment for different diseases" in traditional Chinese medicine.

Objective: "Same treatment for different diseases" is a unique treatment strategy under the guidance of traditional Chinese medicine (TCM) theory. Codonopsis Radix (Codonopsis pilosula, Dangshen in Chinese) with spleen-fortifying effect was employed to understand the strategy of "Same treatment for different diseases", based on its common mechanism in the treatment of gastric diseases including gastric ulcer, gastritis and gastric cancer via network pharmacology research. Methods: Network pharmacology research methods were used to analyze the interaction network and potential mechanisms of Dangshen in treating gastric ulcer, gastritis and gastric cancer. The active components and their target proteins of Dangshen were integrated from TCMSP, BATMAN-TCM databases. The targets of gastric ulcer, gastritis and gastric cancer were collected through GeneCards, PubMed, TDD and DisGeNET Database. Through screening, the key components and the key targets of Dangshen in treating gastric ulcer, gastritis and gastric cancer were obtained. After KEGG pathway analysis and GO analysis, the important pathways and biological processes were analyzed. Results: Through data and literature mining, the common and specific pharmaceutical effects and mechanism of Dangshen were summarized in these three gastric lesions. It was shown that Dangshen mainly acted on gastric ulcer, gastritis and gastric cancer through the overall regulation of the PI3K-AKT signaling pathway. With the development of the disease, it will gradually increase the control of inflammation through TNF, NF-κB and other inflammation-related signaling pathways to reduce inflammatory damage. For tumorigenesis, it pays more attention to inhibiting the ErbB signaling pathways to reduce the proliferation and migration of tumor cells. In addition, Dangshen's regulation of HIF-1 signaling pathway may also be beneficial for the treatment of gastric ulcer, gastritis and gastric cancer. Conclusion: Dangshen achieves spleen-fortifying effect on gastric diseases including gastric ulcer, gastritis and gastric cancer through multiple targets in multiple pathways, especially PI3K-AKT pathway and HIF-1 pathway. It could provide a scientific basis for understanding the strategy of "Same treatment for different diseases" in traditional Chinese medicine.

Immune-enhancement effects of oligosaccharides from Codonopsis pilosula on cyclophosphamide induced immunosuppression in mice.

As an important edible traditional Chinese medicine, Codonopsis pilosula has good immunomodulation effects. This study focuses on C. pilosula oligosaccharides (CPO), which are the sweetness components of C. pilosula. CPO were obtained through systematic separation and purification (the yield is 14.3%), and the effect of CPO on the immunological activities of immunocompromised mice induced by cyclophosphamide (CTX) was evaluated. The results showed that CPO could increase immune organ indices, phagocytic index and immunoglobulin contents, stimulate the proliferation of splenic lymphocytes (coordinating with ConA and LPS), enhance the earlap swelling of the DTH reaction, promote the production of NO and cytokines (IL-2 and IFN-γ) and upregulate the expression of the corresponding mRNA. In addition, CPO upregulated the protein expression of phosphorylated p38, phosphorylated ERK1/2 and phosphorylated JNK, which indicated that CPO might exert immunomodulatory effects through the MAPK signaling pathway. These findings indicated that CPO are important immunomodulatory components in C. pilosula and could be developed as immunomodulators in medicine or functional food areas.

The pharmacokinetic study of rutin in rat plasma based on an electrochemically reduced graphene oxide modified sensor.

An electrochemical method based on a directly electrochemically reduced graphene oxide (ERGO) film coated on a glassy carbon electrode (GCE) was developed for the rapid and convenient determination of rutin in plasma. ERGO was modified on the surface of GCE by one-step electro-deposition method. Electrochemical behavior of rutin on ERGO/GCE indicated that rutin underwent a surface-controlled quasi-reversible process and the electrochemical parameters such as charge transfer coefficient (α), electron transfer number (n) and electrode reaction standard rate constant (ks ) were 0.53, 2 and 3.4 s-1, respectively. The electrochemical sensor for rutin in plasma provided a wide linear response range of 4.70×10-7-1.25×10-5 M with the detection limit (s/n=3) of 1.84×10-8 M. The assay was successfully used to the pharmacokinetic study of rutin. The pharmacokinetic parameters such as elimination rate half-life (t1/2), area under curve (AUC), and plasma clearance (CL) were calculated to be 3.345±0.647 min, 5750±656.0 µg min/mL, and 5.891±0.458 mL/min/kg, respectively. The proposed method utilized a small sample volume of 10 µL and had no complicated sample pretreatment (without deproteinization), which was simple, eco-friendly, and time- and cost-efficient for rutin pharmacokinetic studies.

Ultrasensitive Detection of Ferulic Acid Using Poly(diallyldimethylammonium chloride) Functionalized Graphene-Based Electrochemical Sensor.

The electrochemical redox of ferulic acid (FA) was investigated systematically by cyclic voltammetry (CV) with a poly(diallyldimethylammonium chloride) functionalized graphene-modified glassy carbon electrode (PDDA-G/GCE) as a working electrode. A simple and sensitive differential pulse voltammetry (DPV) technique was proposed for the direct quantitative determination of FA in Angelica sinensis and spiked human urine samples for the first time. The dependence of the intensities of currents and potentials on nature of the supporting electrolyte, pH, scan rate, and concentration was investigated. Under optimal conditions, the proposed sensor exhibited excellent electrochemical sensitivity to FA, and the oxidation peak current was proportional to FA concentration in the range of 8.95 × 10(-8) M ~5.29 × 10(-5) M, with a relatively low detection limit of 4.42 × 10(-8) M. This fabricated sensor also displayed acceptable reproducibility, long-term stability, and high selectivity with negligible interferences from common interfering species. Besides, it was applied to detect FA in Angelica sinensis and biological samples with satisfactory results, making it a potential alternative tool for the quantitative detection of FA in pharmaceutical analysis.

Electrochemical study and application on rutin at chitosan/graphene films modified glassy carbon electrode.

Graphene (G) was dispersed into 0.5% chitosan (Chit) solution, then the composite films were coated on glassy carbon electrode (GCE), the electrochemical behavior of rutin on a Chit/G modified GCE was investigated and the electrochemical parameters of rutin were calculated. Rutin effectively accumulated on the Chit/G/GCE and caused a pair of redox peaks at around 408 mV and 482 mV (vs. SCE) in 0.1 M phosphate buffer solution (pH 4.0). Under optimized conditions, the anodic peak current was linear to the rutin concentration in the range of 5×10-7-1.04×10-5 M. The regression equation was: y=9.9219x-0.0025, r= 0.9958. The proposed method was successfully used for the determination of rutin content in tablet samples with satisfactory results.

Quantification of six bioactive compounds in Zhenqi Fuzheng preparation by high-performance liquid chromatography coupled with diode array detector and evaporative light scattering detector.

A simple and accurate high-performance liquid chromatography (HPLC) coupled with diode array detector (DAD) and evaporative light scattering detector (ELSD) was established for the determination of six bioactive compounds in Zhenqi Fuzheng preparation (ZFP). The monitoring wavelengths were 254, 275 and 328 nm. Under the optimum conditions, good separation was achieved, and the assay was fully validated in respect of precision, repeatability and accuracy. The proposed method was successfully applied to quantify the six ingredients in 31 batches of ZFP samples and evaluate the variation by hierarchical cluster analysis (HCA), which demonstrated significant variations on the content of these compounds in the samples from different manufacturers with different preparation procedures. The developed HPLC method can be used as a valid analytical method to evaluate the intrinsic quality of this preparation.

Evaluation preparation technology of Xiaochaihu granules using fingerprint-peak pattern matching.

An approach was proposed to evaluate preparation technology by means of fingerprint-peak matching technology of high Performance liquid chromatography with diode array detector (HPLC-DAD). Similarity and hierarchical clustering analysis (HCA) were applied to identify the 15 batches of Xiaochaihu granules from different manufacturers and our laboratory, and peak pattern matching between the composite formulae and Radix Bupleuri Chinensis, which was one of the main ingredients of Xiaochaihu granules, was utilized to evaluate the preparation technology of Xiaochaihu granules via the indexes of the relative deviation of retention time (RT) and UV spectrum feature similarity of their corresponding peaks. Eleven matching peaks were found between Xiaochaihu granules and Radix Bupleuri Chinensis. However, the saikosaponin A and saikosaponin D, which are the important active components in Radix Bupleuri Chinensis, were not found in Xiaochaihu granules from any manufacturers. The peak areas of 11 characteristic peaks of Xiaochaihu granules samples formed a matrix of 11 × 15. The result of HCA showed that Xiaochaihu granules samples were divided into four kinds of category. Xiaochaihu granules samples from the same manufacturer were basically clustered of the same category. The results suggested that the saikosaponin A and saikosaponin D are prone to structural transformation under the condition of decoction and in the presence of the organic acidic components. These active components, existing in raw herb, might transform to a series of non-active secondary saikosaponin due to unfavourable preparation technology. So the conventional decoction-based preparation technology of Xiaochaihu granules might greatly affect its quality and therapeutic effectiveness. This study demonstrates that fingerprint-peak matching technology can not only be used for quality control of this composite formulae, but also provide some guidance for preparation technology of Xiaochaihu granules.

The application of digital image recognition to the analysis of two-dimensional fingerprints.

High-performance liquid chromatography (HPLC) fingerprint has been commonly used in the quality control and assessment of herbal medicines, and two-dimensional (2D) fingerprint obtained by means of HPLC-diode array detector (HPLC/DAD) can provide higher reliability. In this paper, an approach to the analysis of the 2D HPLC/DAD fingerprints, which was based on digital image recognition techniques, was developed for the first time. First, wavelet transform was employed to eliminate noise signal in the 2D fingerprint, and then the 2D fingerprint was converted into grayscale image. Second, several features of the image were calculated, and hierarchical clustering. This approach was applied to the qualitative analysis of the different samples of coptis chinensis, and the clustering result of samples was all highly consistent with the real situation. Based on the densities in grayscale image, three components in standard samples were quantitative analyzed, and the obtained correlation coefficients between concentration and grayscale density were more than 0.999. Our study indicated that the analysis of the 2D HPLC/DAD fingerprint was successful based on the idea and techniques of digital image recognition techniques, and this proposed approach provided a new pathway for the analysis of two-dimensional spectrums.

Determination of eleutheroside E and eleutheroside B in rat plasma and tissue by high-performance liquid chromatography using solid-phase extraction and photodiode array detection.

A HPLC method with photodiode array detection (PDA) was developed for the determination and a pharmacokinetic study of eleutheroside E (ELU E) and eleutheroside B (ELU B) in rat plasma and tissue following an eleutherococcus injection. The analysis was performed on a Kromasil C18 column, using water-acetonitrile as the gradient mobile phase and 0.8 mL/min flow rate. Detection wavelengths of ELU E and ELU B were 220 and 206 nm, respectively. Protein from the biological sample was deposited using acetonitrile. ELU E and ELU B were extracted from the biological samples using acetonitrile, separated by solid-phase extraction, and eluted from the cartridge using 60% methanol. The extraction recovery of ELU E and ELU B was 91.2 and 88.8%, respectively. The limit of detection was 37.6 ng/mL for ELU E and 37.0 ng/mL for ELU B (S/N = 3) in plasma. Blood drug level-time cuvers of ELU E and ELU B in Wister rats following administration of an eleutherococcus injection into femoral vein were shown to fit a three-compartment model. The half-life (t1/2) was 4.662 h for ELU E and 2.494 h for ELU B. Following administration of a single eleutherococcus injection, the concentration of ELU E and ELU B in the tissue was Cliver > Ckidney > Cspleen > Cheart and Ckidney > Cliver > Cheart. We believe the method described in the present paper is accurate and reliable and can be used for pharmacokinetic studies of ELU E and ELU B in rats. In addition, the method for sample preparation, using solid phase extraction, is precise, simple and rapid.

Determination of seven compounds in Tang Maikang granule by high-performance liquid chromatography.

An accurate and sensitive high-performance liquid chromatography method is developed and applied to the determination of seven compounds in a kind of traditional Chinese medicinal preparation of Tang Maikang Granule. The method is performed on Hypersil C(18) column (250- x 4.6-mm i.d., 5 microm), and different mobile phases and detectors are selected according to the various compounds. For astragaloside IV, an evaporative light scattering detector (ELSD) is used with a gradient of methanol-water at an eluent gas rate of 2.0 mL/min, under a drift tube temperature of 80 degrees C. Formononetin and calycosin are also eluted by a gradient of methanol-water, but a photodiode array (PDA) detector is used at a wavelength of 254 nm for formononetin and calycosin. A PDA detector at a wavelength of 230 nm is used for paeoniflorin, with methanol-water (30:70, v/v) as the mobile phase. For danshensu and protocate chualdehyde, an eluent of methanol-0.5% acetic acid (12:88, v/v) is used, with PDA detection at 280 nm. For berberine, methanol and water containing 0.1% sodium dodecanesulphonate (SDS) and 0.1% phosphorous acid (70:30, v/v) is employed as the mobile phase, also using a PDA detector, but the detection wavelength is 265 nm. The intra- and interrun precision (relative standard deviation) of this method is less than 5% for seven analytes.