The Tao Hong Si Wu Decoction ameliorates diabetes-associated cognitive dysfunction by inhibiting the formation of amyloid plaques.

OBJECTIVES: The herbs in Tao Hong Si Wu Decoction (THSWD) are beneficial in the treatment of cognitive impairment. However, the underlying mechanisms of THSWD in treating diabetes-associated cognitive dysfunction (DACD) are not entirely explored. This study is aimed to investigate the therapeutic effects of THSWD in DACD model rats and the underlying mechanism. METHODS: Ultra-high-phase liquid chromatography was employed to identify the main compounds contained in the THSWD extract. DACD rat model was induced by feeding with a high-sugar and high-fat diet and injecting streptozotocin (35 mg/kg). DACD rats were gavaged with THSWD for 1 week. The learning and memory abilities of the rats were measured by using the Morris water maze. Western blotting was used to detect the changes in DACD rat targets. Statistical methods were used to evaluate the correlation between proteins. RESULTS: The results show that THSWD effectively reduced the escape latency, hippocampal neuron damage, glycosylated hemoglobin, type A1C, and blood lipid levels in DACD rats. Furthermore, DACD rats showed significantly increased amyloid precursor protein, ß-secretase, Aß1-40 , Aß1-42 , Tau phosphorylation, and advanced glycation end products (AGEs) expression. However, THSWD treatment can reverse this phenomenon. CONCLUSIONS: THSWD can improve the learning and memory abilities of DACD rats by inhibiting the expression of AEGs-AGE receptors pathway, which provides an experimental basis for the clinical application of THSWD. In addition, the experiment combines pharmacological and statistical methods, which offers a new perspective for the study of Chinese herbal medicine.

Ultra-performance liquid chromatography-quadrupole time-of-flight mass combined with UNIFI to study the mechanism of Tao Hong Si Wu Decoction in the treatment of postpartum blood stasis.

Postpartum hemorrhage can lead to a variety of maternal complications. Tao Hong Si Wu Decoction (THSWD) is a traditional Chinese medicine used for treating gynecological diseases. However, the active ingredients of THSWD and its pharmacological mechanism of treatment for postpartum blood stasis still remained unclear. In this study, 201 components were identified in THSWD ethanol extract using ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry, including 59 terpenoids and volatile oil, 61 Phenylpropanoids, 41 flavonoids, 22 alkaloids, and other 18 components. A total of 45 active compounds were identified in the blood and 33 active compounds were identified in the uterine. Taking the common components into the blood and into the uterus combined with network pharmacology. It was demonstrated that the active compounds can bind to the core target with good affinity through molecular docking. The results of this study will provide a reference for the quality control and pharmacodynamic material base research of THSWD.

Poria cocos Attenuated DSS-Induced Ulcerative Colitis via NF-κB Signaling Pathway and Regulating Gut Microbiota.

Ulcerative colitis (UC), as a chronic inflammatory disease, presents a global public health threat. However, the mechanism of Poria cocos (PC) in treating UC remains unclear. Here, LC-MS/MS was carried out to identify the components of PC. The protective effect of PC against UC was evaluated by disease activity index (DAI), colon length and histological analysis in dextran sulfate sodium (DSS)-induced UC mice. ELISA, qPCR, and Western blot tests were conducted to assess the inflammatory state. Western blotting and immunohistochemistry techniques were employed to evaluate the expression of tight junction proteins. The sequencing of 16S rRNA was utilized for the analysis of gut microbiota regulation. The results showed that a total of fifty-two nutrients and active components were identified in PC. After treatment, PC significantly alleviated UC-associated symptoms including body weight loss, shortened colon, an increase in DAI score, histopathologic lesions. PC also reduced the levels of inflammatory cytokines TNF-α, IL-6, and IL-1ß, as evidenced by the suppressed NF-κB pathway, restored the tight junction proteins ZO-1 and Claudin-1 in the colon, and promoted the diversity and abundance of beneficial gut microbiota. Collectively, these findings suggest that PC ameliorates colitis symptoms through the reduction in NF-κB signaling activation to mitigate inflammatory damage, thus repairing the intestinal barrier, and regulating the gut microbiota.

An anti-inflammatory active ingredients in Poriae cutis: Screening based on network pharmacology.

Hyperuricemia (HUA) is a disorder of uric acid metabolism, which can lead to the formation of gouty arthritis, kidney inflammation and other damages. Previous studies have found that the alcohol extract of Poria cutis can reduce the level of uric acid and protect against kidney injury. Based on network pharmacology, the core targets and main active components of P. cutis intervention in HUA were determined. Most of the potential active ingredients are triterpenoid acids such as tumulosic acid (TA) and eburicoic acid (EA), and the potential targets are TNF and IL-6, which are associated with inflammation. In vitro experiments have shown that TA can significantly inhibit the release of NO, TNF-α and IL-6 in inflammatory RAW264.7 cell culture medium and the expression of TNF-α and IL-6 in RAW264.7 cells. This study suggests that TA based on network pharmacological screening has obvious anti-inflammatory effect on inflammatory RAW264.7 cells and is a promising anti-inflammatory compound.

CDCT-induced nephrotoxicity in rat by apoptosis via metabolic disturbance.

Compound diclofenac sodium chlorphenamine maleate tablets (CDCT) are widely used for the cold in Asia. However, CDCT can cause hematuria symptoms in clinical, and the underlying mechanism is unknown. This study aims to investigate the CDCT-induced changes of morphology in kidney and metabolites and further explore the possible mechanisms of CDCT-induced nephrotoxicity. Sprague-Dawley rats were exposed to the CDCT at a clinical equivalent dose for 6 days. CDCT exposure can induce kidney injury and death. Pathological changes, including creatinine, urea nitrogen, and histopathology, were observed in rats. Furthermore, metabolomic-driven energy and glycerophospholipid metabolism pathway disorders, accompanied by remarkably changed key metabolites, such as succinate, leukotriene B4 (LTB4 ), and cardiolipin (CL), are observed in the CDCT-induced nephrotoxicity. Functionally, succinate accumulation leads to mitochondrial damage, as evidence by the imbalance of complex I and complex II and an increase in mitochondrial reactive oxygen species (mito SOX). Meanwhile, LTB4 activated the NF-κB signaling, as shown by increased protein of p65, phosphor-p65, and decreased protein of IκBα and phosphor-IκBα. Eventually, the apoptosis pathway was triggered in response to reduced CL, inflammation, and mito SOX, as demonstrated by the expression of cyt c, Bax, Bcl-2, caspase-3, and caspase-9. This study indicated that CDCT-induced metabolic disorders triggered nephrotoxicity and provided a comprehensive information to elucidate the mechanism of CDCT induced nephrotoxicity.

Anti-tumor Effect of Gambogenic Acid and Its Effect on CYP2C and CYP3A after Oral Administration.

Gambogenic acid (GNA), which has a broad spectrum of anti-tumor activity, is considered as a potential anticancer ingredient. In this study, we examined the anti-tumor effect and the effect of GNA on CYP and pregnane X receptor (PXR). In anti-tumor experiments, an A549 cells tumor-bearing nude mice model was established. Tumor weights and volumes were measured. Inhibition ratio (IR) was calculated. In a pharmacokinetic study, after intragastrical administration of GNA in rats, a cocktail method was adopted to evaluate the activities of CYP2C6, 2C11 and 3A1; RT-quantitative PCR (RT-qPCR) and Western blot (WB) assays were applied to evaluate the mRNA and protein expression levels, respectively. Compared with injection, oral administration also can inhibit tumor growth. Moreover, GNA increased the activities of CYP2C11 and CYP3A1 in the high-dose group as well as the mRNA and protein expression levels. The mRNA and protein expression levels of PXR were also slightly induced. Our study suggested that, oral administration of GNA was effective in inhibiting tumor growth in mice and could induced the activities of CYP2C and CYP3A in rats.

The Effect of Poria cocos Polysaccharide PCP-1C on M1 Macrophage Polarization via the Notch Signaling Pathway.

The homogeneous galactoglucan PCP-1C extracted from Poria cocos sclerotium has multiple biological activities. The present study demonstrated the effect of PCP-1C on the polarization of RAW 264.7 macrophages and the underlying molecular mechanism. Scanning electron microscopy showed that PCP-1C is a detrital-shaped polysaccharide with fish-scale patterns on the surface, with a high sugar content. The ELISA assay, qRT-PCR assay, and flow cytometry assay showed that the presence of PCP-1C could induce higher expression of M1 markers, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-12 (IL-12), when compared with the control and the LPS group, and it caused a decrease in the level of interleukin-10 (IL-10), which is the marker for M2 macrophages. At the same time, PCP-1C induces an increase in the CD86 (an M1 marker)/CD206 (an M2 marker) ratio. The results of the Western blot assay showed that PCP-1C induced activation of the Notch signaling pathway in macrophages. Notch1, ligand Jagged1, and Hes1 were all up-regulated with the incubation of PCP-1C. These results indicate that the homogeneous Poria cocos polysaccharide PCP-1C improves M1 macrophage polarization through the Notch signaling pathway.

Transcriptomic responses to cold stress in Dendrobium huoshanense C.Z. Tang et S.J. Cheng.

Dendrobium huoshanense C.Z. Tang et S.J. Cheng is a perennial epiphytic herb of the family Orchidaceae. The main metabolites of D. huoshanense include polysaccharides and flavonoids. Low temperature is the main environmental factor that limits the growth and development of plants. However, changes that occur at the molecular level in response to low temperatures in D. huoshanense are poorly understood. We performed a transcriptome analysis at two time points of 0 d (control group) and 7 d (cold stress group) under culture of D. huoshanense at 4 °C. A total of 37.63 Gb transcriptomic data were generated using the MGI 2000 platform. These reads were assembled into 170,754 transcripts and 23,724 differentially expressed genes (DEGs) were obtained. Pathway analysis indicated that "flavonoid biosynthesis," "anthocyanin biosynthesis," "flavone and flavonol biosynthesis," and "plant hormone signal transduction" might play a vital role in the response of D. huoshanense to cold stress. Several important pathway genes were identified to be altered under cold stress, such as genes encoding polysaccharides, flavonoids, and plant hormone-signaling transduction kinase. In addition, the content of mannose and total flavonoids increased under cold stress. Twelve DEGs related to polysaccharides, flavonoid, and hormone pathways were selected from the transcriptome data for validation with real-time quantitative PCR (RT-qPCR). Our results provide a transcriptome database and candidate genes for further study of the response of D. huoshanense to cold stress. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01385-7.

[Active components and potential mechanism of Taohong Siwu Decoction in regulating ischemic stroke based on target cell trapping combined with network pharmacology, molecular docking, and experimental validation].

The potential anti-stroke active components in Taohong Siwu Decoction(THSWD) were identified by target cell trapping coupled with ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS). The underlying mechanism of active components in THSWD in the treatment of ischemic stroke(IS) was explored by network pharmacology, molecular docking, and experimental validation. The UPLC-Q-TOF-MS technology combined with the UNIFI data analysis platform was used to analyze the composition of the cellular fragmentation fluid after co-incubation of THSWD with target cells. The targets of potential active components and IS were collected by network pharmacology, and the common targets underwent protein-protein interaction(PPI), Gene Ontology(GO), and Kyoto Encyclopedia of Genes and Genomes(KEGG) signaling pathway enrichment analyses. The target cell trapping component-core target-signaling pathway network was constructed, and the active components were molecularly docked to the top targets in the PPI network, followed by pharmacodynamic validation in vitro. Fifteen active components were identified in the target cellular fragmentation fluid, including bicyclic monoterpenes, cyanoglycosides, flavonols, quinoid chalcones, phenylpropanoids, and tannins. As revealed by the analysis of network pharmacology, THSWD presumably regulated PI3K-AKT, FoxO, MAPK, Jak-STAT, VEGF, HIF-1, and other signaling pathways to affect inflammatory cascade reaction, angiogenesis, oxidative stress, pyroptosis, apoptosis, and other pathological processes via paeoniflorin, butylphthalide, dehydrated safflower yellow B, 3,4-dicaffeoylquinic acid, amygdalin, paeoniflorin, and ligusticolactone. Molecular docking and in vitro pharmacodynamic validation revealed that the target cell trapping active components could promote neovascularization in rat brain microvascular endothelial cells(rBMECs) in the oxygen-glucose deprivation/reoxygenation(OGD/R) model. The application of target cell trapping coupled with UPLC-Q-TOF-MS technology can rapidly screen out the potential active components in THSWD. The active components of THSWD can be predicted to intervene in the pathogenesis of IS through network pharmacology, and molecular docking combined with experimental validation can further clarify the efficacy, thus providing a theoretical basis for research ideas on the pharmacodynamic substance basis of traditional Chinese medicine compounds.

[Origin identification of Poria cocos based on hyperspectral imaging technology].

To realize the non-destructive and rapid origin discrimination of Poria cocos in batches, this study established the P. cocos origin recognition model based on hyperspectral imaging combined with machine learning. P. cocos samples from Anhui, Fujian, Guangxi, Hubei, Hunan, Henan and Yunnan were used as the research objects. Hyperspectral data were collected in the visible and near infrared band(V-band, 410-990 nm) and shortwave infrared band(S-band, 950-2 500 nm). The original spectral data were divided into S-band, V-band and full-band. With the original data(RD) of different bands, multiplicative scatter correction(MSC), standard normal variation(SNV), S-G smoothing(SGS), first derivative(FD), second derivative(SD) and other pretreatments were carried out. Then the data were classified according to three different types of producing areas: province, county and batch. The origin identification model was established by partial least squares discriminant analysis(PLS-DA) and linear support vector machine(LinearSVC). Finally, confusion matrix was employed to evaluate the optimal model, with F1 score as the evaluation standard. The results revealed that the origin identification model established by FD combined with LinearSVC had the highest prediction accuracy in full-band range classified by province, V-band range by county and full-band range by batch, which were 99.28%, 98.55% and 97.45%, respectively, and the overall F1 scores of these three models were 99.16%, 98.59% and 97.58%, respectively, indicating excellent performance of these models. Therefore, hyperspectral imaging combined with LinearSVC can realize the non-destructive, accurate and rapid identification of P. cocos from different producing areas in batches, which is conducive to the directional research and production of P. cocos.

[Origin identification of Polygonatum cyrtonema based on hyperspectral data].

In this study, visual-near infrared(VNIR), short-wave infrared(SWIR), and VNIR + SWIR fusion hyperspectral data of Polygonatum cyrtonema from different geographical origins were collected and preprocessed by first derivative(FD), second derivative(SD), Savitzky-Golay smoothing(S-G), standard normalized variate(SNV), multiplicative scatter correction(MSC), FD+S-G, and SD+S-G. Three algorithms, namely random forest(RF), linear support vector classification(LinearSVC), and partial least squares discriminant analysis(PLS-DA), were used to establish the identification models of P. cyrtonema origin from three spatial scales, i.e., province, county, and township, respectively. Successive projection algorithm(SPA) and competitive adaptive reweighted sampling(CARS) were used to screen the characteristic bands, and the P. cyrtonema origin identification models were established according to the selected characteristic bands. The results showed that(1)after FD preprocessing of VNIR+SWIR fusion hyperspectral data, the accuracy of recognition models established using LinearSVC was the highest, reaching 99.97% and 99.82% in the province origin identification model, 100.00% and 99.46% in the county origin identification model, and 99.62% and 98.39% in the township origin identification model. The accuracy of province, county, and township origin identification models reached more than 98.00%.(2)Among the 26 characteristic bands selected by CARS, after FD pretreatment, the accuracy of origin identification models of different spatial scales was the highest using LinearSVC, reaching 98.59% and 97.05% in the province origin identification model, 97.79% and 94.75% in the county origin identification model, and 90.13% and 87.95% in the township origin identification model. The accuracy of identification models of different spatial scales established by 26 characteristic bands reached more than 87.00%. The results show that hyperspectral imaging technology can realize accurate identification of P. cyrtonema origin from different spatial scales.

Use of Steaming Process to Improve Biochemical Activity of Polygonatum sibiricum Polysaccharides against D-Galactose-Induced Memory Impairment in Mice.

Polysaccharide from Polygonatum sibiricum (PSP) possesses antioxidant, antiaging, and neuroprotective activities. However, whether and how the steaming process influences the biological activities of PSP, especially against aging-related memory impairment, is not yet known. In this study, Polygonatum sibiricum rhizome was subjected to a "nine steaming and nine drying" process, then PSPs with different steaming times were abstracted. Thereafter, the physicochemical properties were qualified; the antioxidant activities of PSPs were evaluated in a D-gal-induced HT-22 cell model, and the effects of PSPs (PSP0, PSP5 and PSP9) on memory was evaluated using D-gal-injured mice. Our results showed that while the steamed PSPs had a low pH value and a large negative charge, they shared similar main chains and substituents. Cellular experiments showed that the antioxidant activity of steamed PSPs increased. PSP0, PSP5, and PSP9 could significantly ameliorate the memory impairment of D-gal-injured mice, with PSP5 showing the optimal effect. Meanwhile, PSP5 demonstrated the best effect in terms of preventing cell death and synaptic injury in D-gal-injured mice. Additionally, the steamed PSPs increased anti-oxidative stress-related protein expression and decreased inflammation-related protein expression in D-gal-injured mice. Collectively, the steaming process improves the effects of PSPs against D-gal-induced memory impairment in mice, likely by increasing the antioxidant activity of PSPs.

Isolation and Characterization of Natural Nanoparticles in Naoluo Xintong Decoction and Their Brain Protection Research.

Currently, researchers use modern analytical techniques in a unique perspective of physical pharmacy to analyze the phase composition of traditional Chinese medicine (TCM) and have discovered that natural nanoparticles commonly exist in decoctions. This study aims to isolate and characterize the structure and composition of nanoparticles in Naoluo Xintong (NLXT) and investigate whether the brain protection effect of NLXT is closely related to NLXT-Nanoparticles (NLXT-NPs). Firstly, the dialysis-centrifugation method was used to separate the nanoparticles and then their size distribution, potential, and morphology were characterized. In addition, infrared spectroscopy and ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometer (UPLC-Q-TOF-MS) technology were used to analyze the composition of nanoparticles. As for the pharmacodynamic experiment, Sprague Dawley (SD) rats were randomly divided into sham, Middle cerebral artery occlusion (MCAO) model, NLXT, NLXT with nanoparticles removing (NLXT-RN), NLXT-RN+Nanoparticles (NLXT-RN+NPs), and NLXT-NPs groups. After administration, the neurological function, histopathological changes, oxidative stress, and apoptosis level were measured. Our research showed that NLXT-NPs are mainly composed of polysaccharides, proteins, and saponins, with typical characteristics of two hundred-nanometer size and negatively loaded. NLXT can improve nerve function, reduce oxidative stress, and inhibit cell apoptosis. However, removing nanoparticles can significantly reduce the brain-protective effect of NLXT, which indicates that NLXT-NPs play an essential role in the efficacy of NLXT.

[Anti-inflammatory and hemostatic effects of total extract, saponins, and flavonoids of Clinopodium chinense in female rats with abnormal uterine bleeding and mechanism].

This study aims to explore the anti-inflammatory and hemostatic effects of the total extract of Clinopodium chinense(TEC), total saponins of C. chinense(TSC), and total flavonoids of C. chinense(TFC) in female rats with abnormal uterine bleeding(AUB), and the possible mechanism. Mifepristone(i.g., 12.4 mg·kg~(-1)) and misoprostol(i.g., 130 µg·kg~(-1)) were used to induce AUB in SD female rats conceiving on the same day. Then the AUB rats were randomized into model group, TEC group, TSC group, TFC group, Yimucao Granules(LG) group, and estradiol valerate(EV) group, with 8 rats in each group. Another 8 non-pregnant female rats were selected as normal group. During the experiment, each group was given the corresponding drug by gavage once a day for 7 days. After the administration, blood and uterine tissue were collected. The uterine bleeding volume was measured by ultraviolet spectrophotometry and the pathological changes of endometrium were observed based on hematoxylin-eosin(HE) staining. In addition, the microvessel density of endometrium was determined by immunohistochemistry, and the content of thromboxane B2(TXB2), 6-keto-PGF_(1α), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in plasma and levels of lutenizing hormone(LH), follicle stimulating hormone(FSH), estradiol(E_2), and progesterone in serum were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA and protein expression of estrogenreceptor α(ERα), progesterone receptor(PR), matrix metalloproteinase(MMP)-2, MMP-9, and vascular endothelial growth factor(VEGF) in uterine tissue was determined by Western blot. Compared with the model group, TEC, TSC, and TFC can reduce uterine bleeding volume, alleviate the pathological damage of endometrium, and increase the microvessel density in endometrium. Moreover, TEC and TSC can significantly raise plasma TXB2 level and ratio of TXB2 to 6-keto-PGF_(1α), and TEC and TFC can significantly reduce the levels of IL-6 and TNF-α. In addition, TEC significantly elevated serum progesterone level and TFC significantly increased serum levels of E_2, FSH, and LH. TSC can significantly raise serum progesterone and FSH levels. In addition, TEC can significantly down-regulate the protein expression of PR, MMP-2, and VEGF and TSC significantly reduced the expression of MMP-9. TFC significantly decreased the expression of PR, MMP-9, and VEGF, and up-regulated the expression of ERα. In conclusion, TEC, TSC, and TFC all show therapeutic effects on AUB, particularly TEC. TSC exerts the effects by enhancing the coagulation function and promoting endometrial repair, and TFC by regulating estrogen levels and reducing inflammatory response. This study reveals the mechanism of C. chinense against AUB and also explains the holistic characteristics of Chinese medicine.

[Simultaneous determination and pharmacokinetic study of five compounds from total extract of Clinopodium chinense in abnormal uterine bleeding rat plasma by UPLC-MS/MS].

Clinopodium chinense, a traditional folk medicinal herb, has been used to treat abnormal uterine bleeding(AUB) for many years. Saponins and flavonoids are the main active components in C. chinense. To study the pharmacokine-tics of multiple components from the total extract of C. chinense(TEC), we established a sensitive and rapid method of ultra-perfor-mance liquid chromatography coupled with tandem mass spectrometry(UPLC-MS/MS) for simultaneous determination of five compounds in the plasma of AUB rats. After validation, the AUB model was established with SD female rats which got pregnant on the same day by gavage with mifepristone(12.4 mg·kg~(-1)) and misoprostol(130 µg·kg~(-1)). The established method was applied to the detection of hesperidin, naringenin, apigenin, saikosaponin a, and buddlejasaponin Ⅳb in AUB rats after the administration of TEC. The pharmacokinetic parameters were calculated by DAS 2.0. The five compounds showed good linear relationship within the detection range. The specificity, accuracy, precision, recovery, matrix effect, and stability of the method all matched the requirements of biolo-gical sample detection. The above 5 compounds were detected in the plasma of AUB rats after the administration of TEC. The C_(max) va-lues of hesperidin, naringenin, apigenin, saikosaponin a, and clinoposide A were 701.6, 429.5, 860.7, 75.1, and 304.1 ng·mL~(-1), respectively. All the compounds owned short half-life and quick elimination rate in vivo, and the large apparent volume of distribution indicated that they were widely distributed in tissues. Being rapid, accurate, and sensitive, this method is suitable for the pharmacokinetic study of extracts of Chinese herbal medicines and provides a reference for the study of pharmacodynamic material basis of C. chinense in treating AUB.

[Extracts of Poria cocos polysaccharides improves alcoholic liver disease in mice via CYP2E1 and NF-κB inflammatory pathways].

The present study investigated the effect of extract of Poria cocos polysaccharides(PCP) on cytochrome P450 2 E1(CYP2 E1) and nuclear factor κB(NF-κB) inflammatory signaling pathways in alcoholic liver disease(ALD) mice and explored its protective effect and mechanism. Sixty male C57 BL/6 N mice of SPF grade were randomly divided into a control group, a model group, a positive drug group(bifendate, 200 mg·kg~(-1)), and high-(200 mg·kg~(-1)) and low-dose(50 mg·kg~(-1)) PCP groups. Gao-binge mo-del was induced and the mice in each group were treated correspondingly. Liver morphological and pathological changes were observed and organ index was calculated. Serum levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were detected. Malondialdehyde(MDA) and superoxide dismutase(SOD) in liver tissues were detected by assay kits. The levels of interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were detected by ELISA. The activation of macrophages was observed by immunofluorescence staining and protein expression of CYP2 E1, Toll-like receptor 4(TLR4), NF-κB p65, and phosphorylated NF-κB p65(p-NF-κB p65) were analyzed by Western blot. The ALD model was properly induced. Compared with the model group, the PCP groups significantly improved the pathological injury of liver tissues. Immunofluorescence staining revealed that compared with the model group, the groups with drug intervention showed decreased macrophages in liver tissues. Additionally, the PCP groups showed reduced ALT, AST, MDA, IL-6, and TNF-α(P<0.05), and potentiated activity of SOD(P<0.01). PCP extract has the protective effect against alcoholic liver injury in mice, and the underlying mechanism may be related to the regulation of the expression of CYP2 E1 and inhibition of TLR4/NF-κB inflammatory signaling pathway to reduce oxidative stress and inflammatory injury, thereby inhibiting the development of ALD.

Putative genes in alkaloid biosynthesis identified in Dendrobium officinale by correlating the contents of major bioactive metabolites with genes expression between Protocorm-like bodies and leaves.

BACKGROUND: Dendrobium officinale, an endangered Chinese herb, possesses extensive therapeutic effects and contains bioactive ingredients such as major polysaccharides, alkaloids, and minimal flavonoids. We first obtained the protocorm-like bodies (PLBs) of this plant through tissue culture in order to determine the distribution of the main secondary metabolites in each organelle and the PLBs. We then analyzed the correlation between gene expression level from comparative transcriptome sequencing and metabolite content in different organs to identify putative genes encoding enzymes involved in the biosynthesis of polysaccharides, alkaloids, and flavonoids. RESULTS: We used seeds as explants for protocorm induction and PLB propagation of D. officinale. The optimal medium formula for PLB propagation was 1/2 MS + α-NAA 0.5 mg·L- 1 + 6-BA 1.0 mg·L- 1 + 2, 4-D 1.5-2.0 mg·L- 1 + potato juice 100 g·L- 1. Stems, PLBs and leaves of D. officinale had the highest content of polysaccharides, alkaloids and flavonoids, respectively. Naringenin was only produced in stem; however, PLBs with high alkaloid content can replace other organs producing alkaloids. The hot water extraction method outperformed the ultrasound-assisted extraction method for extracting polysaccharides from D. officinale. A comparative transcriptome analysis of PLBs and leaves of D. officinale revealed differential expression of genes encoding enzymes involved in polysaccharide, alkaloid and flavonoid biosynthetic pathways. Putative genes encoding enzymes involved in these biosynthetic pathways were identified. Notably, we identified genes encoding the alkaloid biosynthesis enzymes strictosidine ß-D-Glucosidase, geissoschizine synthase and vinorine synthase in D. officinale. CONCLUSIONS: The identification of candidate genes encoding enzymes involved in metabolite biosynthesis will help to explore and protect this endangered species and facilitate further analysis of the molecular mechanism of secondary metabolite biosynthesis in D. officinale.

Transcriptome analysis identifies putative genes involved in triterpenoid biosynthesis in Platycodon grandiflorus.

MAIN CONCLUSION: Comprehensive transcriptome analysis of different Platycodon grandiflorus tissues discovered genes related to triterpenoid saponin biosynthesis. Platycodon grandiflorus (Jacq.) A. DC. (P. grandiflorus), a traditional Chinese medicine, contains considerable triterpenoid saponins with broad pharmacological activities. Triterpenoid saponins are the major components of P. grandiflorus. Here, single-molecule real-time and next-generation sequencing technologies were combined to comprehensively analyse the transcriptome and identify genes involved in triterpenoid saponin biosynthesis in P. grandiflorus. We quantified four saponins in P. grandiflorus and found that their total content was highest in the roots and lowest in the stems and leaves. A total of 173,354 non-redundant transcripts were generated from the PacBio platform, and three full-length transcripts of ß-amyrin synthase, the key synthase of ß-amyrin, were identified. A total of 132,610 clean reads obtained from the DNBSEQ platform were utilised to explore key genes related to the triterpenoid saponin biosynthetic pathway in P. grandiflorus, and 96 differentially expressed genes were selected as candidates. The expression levels of these genes were verified by quantitative real-time PCR. Our reliable transcriptome data provide valuable information on the related biosynthesis pathway and may provide insights into the molecular mechanisms of triterpenoid saponin biosynthesis in P. grandiflorus.

Determination and analysis of monosaccharides in Polygonatum cyrtonema Hua polysaccharides from different areas by ultra-high-performance liquid chromatography quadrupole trap tandem mass spectrometry.

We wanted to explore a new method for the determination of monosaccharides in Polygonatum cyrtonema Hua polysaccharide using the ultra-high-performance liquid chromatography quadrupole trap tandem mass spectrometry. In this study, hydrochloric acid was used instead of trifluoroacetic acid to hydrolyze polysaccharides, and hydrolysis time was greatly reduced from 5-9 h to 1 h. The 1-phenyl-3-methyl-5-pyrazolone was used for pre-column derivatization of monosaccharides. The parameters of linearity (R2  > 0.999), stability (1.63-2.52%), intra-day and inter-day precision (0.69-0.95%, 1.81-2.77%), repeatability (1.89-2.65%), and recovery (97.63-102.24%) of the method were verified. Satisfactory validation results showed this method could be used to determine the target components. The results indicated the polysaccharide contained glucose, mannose, rhamnose, galactose, ribose, and arabinose. Technique for order preference by similarity to an ideal solution and principal component analysis were used to build an evaluation model based on the monosaccharide composition. The evaluation results showed that the samples from the Qingyang County of Anhui Province were the best when the monosaccharides were used as the evaluation index. Therefore, a new method was established to detect the monosaccharide content of polysaccharides from Polygonatum cyrtonema Hua and comprehensively evaluate the quality of Chinese medicines with polysaccharides as the main active ingredient.

Determination of monosaccharides in Lycium barbarum fruit polysaccharide by an efficient UHPLC-QTRAP-MS/MS method.

INTRODUCTION: Pre-column derivatisation using 1-phenyl-3-methyl-5-pyrazolone (PMP) is a common method for monosaccharide determination. Herein, a specific ultra-high-performance liquid chromatography quadrupole trap tandem mass spectrometry (UHPLC-QTRAP-MS/MS) method was developed and used for the determination of monosaccharide composition of Lycium barbarum polysaccharide (LBP). OBJECTIVE: Exploration of a new efficient method for monosaccharide determination. METHODS: In our study, hydrochloric acid was used to hydrolyse the polysaccharide obtained from L. barbarum fruits. Principal component analysis (PCA) and technique for order of preference by similarity to ideal solution (TOPSIS) analysis were used for comprehensive evaluation of the samples. The results showed that LBP was composed of seven monosaccharides: galactose, arabinose, mannose, rhamnose, xylose, ribose, and glucose. The linear relationship of the seven monosaccharides was optimum within a certain concentration range. Quantitative recoveries of the seven monosaccharides from the samples ranged from 94.76% to 102.11%. RESULTS: A rapid quantitative detection method was established, in which the hydrolysis time was reduced from 12 h to 2 h. By using LBP as one of the indexes to evaluate the quality of L. barbarum, L. barbarum from Zhongning County, Ningxia Province, was identified as having the best quality among the varieties tested. CONCLUSION: The UHPLC-QTRAP-MS/MS pre-column derivatisation method used in this study was simple, accurate, and sensitive, with good repeatability, and can be used for quality evaluation and origin distinction of L. barbarum and other medicinal plants.