Functional characterization of polyphenol oxidase OfPPO2 supports its involvement in parallel biosynthetic pathways of acteoside.

Acteoside is a bioactive phenylethanoid glycoside widely distributed throughout the plant kingdom. Because of its two catechol moieties, acteoside displays a variety of beneficial activities. The biosynthetic pathway of acteoside has been largely elucidated, but the assembly logic of two catechol moieties in acteoside remains unclear. Here, we identified a novel polyphenol oxidase OfPPO2 from Osmanthus fragrans, which could hydroxylate various monophenolic substrates, including tyrosine, tyrosol, tyramine, 4-hydroxyphenylacetaldehyde, salidroside, and osmanthuside A, leading to the formation of corresponding catechol-containing intermediates for acteoside biosynthesis. OfPPO2 could also convert osmanthuside B into acteoside, creating catechol moieties directly via post-modification of the acteoside skeleton. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis and subcellular localization assay further support the involvement of OfPPO2 in acteoside biosynthesis in planta. These findings suggest that the biosynthesis of acteoside in O. fragrans may follow "parallel routes" rather than the conventionally considered linear route. In support of this hypothesis, the glycosyltransferase OfUGT and the acyltransferase OfAT could direct the flux of diphenolic intermediates generated by OfPPO2 into acteoside. Significantly, OfPPO2 and its orthologs constitute a functionally conserved enzyme family that evolved independently from other known biosynthetic enzymes of acteoside, implying that the substrate promiscuity of this PPO family may offer acteoside-producing plants alternative ways to synthesize acteoside. Overall, this work expands our understanding of parallel pathways plants may employ to efficiently synthesize acteoside, a strategy that may contribute to plants' adaptation to environmental challenges.

Multi-omics analysis reveals mechanism of Schisandra chinensis lignans and acteoside on EMT in hepatoma cells via ERK1/2 pathway.

BACKGROUND: Hepatocellular carcinoma (HCC), a globally common cancer, often presents late and shows high resistance to chemotherapy, resulting in suboptimal treatment efficacy. Components from traditional Chinese medicines have been recognized for their anti-cancer properties. OBJECTIVE: Exploring the mechanism of Schisandra chinensis lignans and acteoside in suppressing Epithelial-Mesenchymal Transition (EMT) in hepatoma cells through the Extracellular signal-Regulated Kinases (ERK)1/2 pathway and identifying biomarkers, molecular subtypes, and targets via multi-omics for precision oncology. METHODS: Proliferation was assessed using cell counting kit-8 (CCK-8) assays, with scratch and transwell assays for evaluating invasion and migration. Flow cytometry quantified apoptosis rates. Expression levels of CCL20, p-ERK1/2, c-Myc, Vimentin, and E-cadherin/N-cadherin were analyzed by real-time PCR and Western blot. Tumor volume was calculated with a specific formula, and growth. RESULTS: The Schisandra chinensis lignans and acteoside combination decreased CCL20 expression, inhibited hepatoma proliferation and migration, and enhanced apoptosis in a dose- and time-dependent manner. Molecular analysis revealed increased E-cadherin and decreased N-cadherin, p-ERK1/2, c-Myc, and Vimentin expression, indicating ERK1/2 pathway modulation. In vivo, treated nude mice showed significantly reduced tumor growth and volume. CONCLUSION: Schisandra chinensis lignans and acteoside potentially counteract CCL20-induced EMT, invasion, and migration in hepatocellular carcinoma cells via the ERK1/2 pathway, enhancing apoptosis. Multi-omics analysis further aids in pinpointing novel biomarkers for precision cancer therapy.

Acteoside ameliorates learning and memory impairment in APP/PS1 transgenic mice by increasing Aß degradation and inhibiting tau hyperphosphorylation.

Alzheimer's disease (AD) is a neurodegenerative disease. Senile plaques and intracellular neurofibrillary tangles are pathological hallmarks of AD. Recent studies have described the improved cognitive and neuroprotective functions of acteoside (AS). This study aimed to investigate whether the improved cognition of AS was mediated by Aß degradation and tau phosphorylation in APP/PS1 mice. The open field, Y maze, and novel object recognition tests were used to assess cognitive behavioral changes. We evaluated the levels of Aß40 and Aß42 in serum, cortex, and hippocampus, and Aß-related scavenging enzymes, phosphorylated GSK3ß and hyperphosphorylated tau in the cortex and hippocampus of APP/PS1 mice by western blotting. Our results revealed that AS treatment ameliorated anxious behaviors, spatial learning, and memory impairment in APP/PS1 mice and significantly reduced Aß deposition in their serum, cortex, and hippocampus. AS significantly increased Aß degradation, inhibited the hyperphosphorylation of tau, and significantly decreased the activity of GSK3ß, which is involved in tau phosphorylation. Altogether, these findings indicated that the beneficial effects of AS on AD-associated anxious behaviors and cognitive impairments could be attributed to promoting Aß degradation and inhibiting tau hyperphosphorylation, which might be partly mediated by GSK3ß.

Seasonal and Circadian Variation of Non-Volatile Metabolites in Lippia alba Leaves.

The present study describes the seasonal and circadian variations of the major compounds from Lippia alba leaves. SPSS was used to identify, quantify, and associate the variations in the secondary metabolites of this species through HPLC/DAD analysis of the leaves hydroethanolic extracts of six selected L. alba specimens. For the circadian study, the samples were collected at four different daily hours in each year's season. For the seasonal study, the samples were collected monthly from the same individuals for two consecutive years (2018 and 2019). These samples were analyzed and quantified using a validated HPLC method for flavonoids, iridoids, and phenyl ethanoid glycoside. Mussaenoside, acteoside, and tricin-7-O-diglucuronide showed a moderate positive correlation between their biosynthesis and the precipitation index, while epi-loganin had a moderate negative correlation. Acteoside showed a moderate positive correlation between the minimum registered temperature and its production. Compared with previous studies, a drastic reduction (about 95 %) in the production of tricin-7-O-diglucuronide compared with previous study and this difference could be attributed to the plant's aging. Thus, the data demonstrated that lower temperatures and high rainfall could favor the production of the major L. alba active compounds (acteoside and tricin-7-O-diglucuronide) and that older plants harm their production.

Clerodendrum trichotomum Thunberg-An Ornamental Shrub with Medical Properties.

Harlequin glory bower (Clerodendrum trichotomum) is a shrub or small tree belonging to the Lamiaceae family, native to Japan, Korea, and eastern China. It has esthetic value and in Europe, it is cultivated as an ornamental plant. Its sweet-smelling flowers have a white or pink crown. The calyx turns from green to pink-purple over time, providing an especially decorative touch around surrounding the ripe deep-blue fruits that persist until winter. In the areas of its natural occurrence, the leaves and young shoots of C. trichotomum, and sometimes the roots, flowers and fruits, are used in folk medicine due to its anti-inflammatory, analgesic, anticancer, sedative, and hypotensive effects. Products based on Harlequin glory are also used in the treatment of rheumatoid arthritis, joint pain, skin inflammation, or asthma. Preliminary research on the composition of raw material suggests that its health-promoting effect is associated with the presence of numerous secondary metabolites, including phenylpropanoids, flavonoids, lignans, terpenoids, steroids, alkaloids, and anthraquinones. This work reviews the current state of knowledge about the phytochemistry and in vitro and in vivo pharmacological properties of the extracts and main active components isolated from C. trichotomum. It also indicates that before it can be used in modern medicine, further research is necessary regarding the safety and efficacy of the raw material, its mechanisms of action, and dosage.

[Role of mitochondrial autophagy and the curative effect of rehmannia glutinosa leaves total glycoside capsules on nucleos(t)ide drug-induced renal injury].

Objective: To study the curative effect of rehmannia glutinosa leaves total glycoside capsules and the role of mitochondrial autophagy on nucleos(t)ide drug-induced renal injury. Methods: Adefovir dipivoxil (ADV) was used to construct a hepatitis B virus (HBV) transgenic mouse model for renal injury. Renal function was measured in each group at one and two weeks of modeling. Mitochondrial autophagy indicators were measured at two weeks of modeling in renal tissue. Transmission electron microscopy was used to detect mitochondrial autophagy phenomena in renal tissue. The model was established for two weeks. Mouse with renal injury were treated with rehmannia glutinosa leaves total glycoside capsules or isotonic saline for eight weeks by intragastric administration. Renal function was measured. Renal tissue morphology was observed. Mitochondrial autophagy indicators were detected in renal tissue. The protective effect of different concentrations of verbascoside (the main active ingredient of rehmannia glutinosa capsule) was observed on HK-2 cell damage induced by ADV. HK-2 cells were divided into control, ADV, and ADV plus verbascoside groups. The effects of verbascoside at different times and concentrations were observed on the HK-2 mitochondrial autophagy indicators. Fifty patients with chronic hepatitis B were collected who presented with renal injury after treatment with nucleos(t)ide analogs. The random number method was used to divide 29 cases into a control group that received conventional treatment. The treatment group of 21 cases was treated with rehmannia glutinosa leaves total glycoside capsules on the basis of the control group. Serum creatinine (Scr) and urinary protein were detected at eight weeks.The χ(2) test or t-test was used for statistical analysis. Results: Compared with the control group, two weeks of modeling in the ADV group induced renal function injury in HBV mice. The expression of autophagy indicators was higher in the renal tissue of the ADV group than that of the control group. Transmission electron microscopy had revealed mitochondrial autophagy in the renal tissue of the ADV group. Compared with the control group, the renal function of HBV mice treated with rehmannia glutinosa leaves total glycoside capsules improved for two months, and the expressions of autophagy indicators were down-regulated.Verbascoside promoted proliferation in ADV-damaged HK-2 cells, and the expression of autophagy indicators was down-regulated compared with the ADV alone group. In 50 patients with renal function injury, the urinary protein improvement was significantly superior in the treatment group than that in the control group, with eighteen and three cases being effective and ineffective in the treatment group and 12 and 17 cases being effective and ineffective in the control group, with a statistically significant difference (χ(2) = 9.975 0, P = 0.001 6). Serum creatinine was decreased in the treatment group compared with the control group, with 11 and 10 cases being effective and ineffective in the treatment group and 12 and 17 cases being effective and ineffective in the control group, with no statistically significant difference (χ(2) = 0.593 5, P = 0.441 1). Conclusion: Rehmannia glutinosa leaves total glycoside capsule can improve the nucleos(t)ide drug-induced renal function injury in chronic hepatitis B, possibly playing a role via inhibiting PINK1/Parkin-mediated mitochondrial autophagy.

[Mechanism of acteoside in prevention and treatment of gouty arthritis based on liver metabolomics].

This study aims to reveal the effect of acteoside on gouty arthritis(GA) in rats based on liver metabolomics. The ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was employed to search for the potential biomarkers and metabolic pathways. SD rats were randomly assigned into blank, model, colchicine(0.3 mg·kg~(-1)), and high-, medium-, low-dose(200, 100, and 50 mg·kg~(-1), respectively) acteoside groups(n=7). The rats were administrated once a day for 7 continuous days. Monosodium urate(MSU) was used to induce GA model in rats during administration. The degree of joint swelling and pathological changes of synovial tissue in rats were observed, and the levels of interleukin(IL)-1ß, IL-18 and tumor necrosis factor(TNF)-α in the synovial tissue of rats were measured. UPLC-Q-TOF-MS was employed to collect rat liver data, and Progenesis QI and EZ info were used for data analysis. Human Metabolomics Database(HMDB) and Kyoto Encyclopedia of Genes and Genomes(KEGG) were employed to predict the potential biomarkers and metabolic pathways. The results showed that acteoside alleviated joint swelling, reduced synovial tissue damage, and lowered the levels of inflammatory cytokines in GA rats. A total of 19 common biomarkers were identified, 17 of which can be regulated by acteoside. Seven metabolic pathways were enriched, such as glycerophospholipid metabolism, linoleic acid metabolism, and taurine and hypotaurine metabolism, among which glycerophospholipid metabolism was strongly disturbed. The metabolomics analysis suggested that acteoside may down-regulate the expression of inflammatory cytokines and alleviate the symptoms of GA rats by regulating glycerophospholipid metabolism, linoleic acid metabolism, and taurine and hypotaurine metabolism. The findings provide a reference for future research and development of acteoside.

Molecular Identification of UDP-Sugar-Dependent Glycosyltransferase and Acyltransferase Involved in the Phenylethanoid Glycoside Biosynthesis Induced by Methyl Jasmonate in Sesamum indicum L.

Sesame (Sesamum indicum L.) plants contain large amounts of acteoside, a typical phenylethanoid glycoside (PhG) that exhibits various pharmacological activities. Although there is increasing interest in the biosynthesis of PhGs for improved production, the pathway remains to be clarified. In this study, we established sesame-cultured cells and performed transcriptome analysis of methyl jasmonate (MeJA)-treated cultured cells to identify enzyme genes responsible for glucosylation and acylation in acteoside biosynthesis. Among the genes annotated as UDP-sugar-dependent glycosyltransferase (UGT) and acyltransferase (AT), 34 genes and one gene, respectively, were upregulated by MeJA in accordance with acteoside accumulation. Based on a phylogenetic analysis, five UGT genes (SiUGT1-5) and one AT gene (SiAT1) were selected as candidate genes involved in acteoside biosynthesis. Additionally, two AT genes (SiAT2-3) were selected based on sequence identity. Enzyme assays using recombinant SiUGT proteins revealed that SiUGT1, namely, UGT85AF10, had the highest glucosyltransferase activity among the five candidates against hydroxytyrosol to produce hydroxytyrosol 1-O-glucoside. SiUGT1 also exhibited glucosyltransferase activity against tyrosol to produce salidroside (tyrosol 1-O-glucoside). SiUGT2, namely, UGT85AF11, had similar activity against hydroxytyrosol and tyrosol. Enzyme assay using the recombinant SiATs indicated that SiAT1 and SiAT2 had activity transferring the caffeoyl group to hydroxytyrosol 1-O-glucoside and salidroside (tyrosol 1-O-glucoside) but not to decaffeoyl-acteoside. The caffeoyl group was attached mainly at the 4-position of glucose of hydroxytyrosol 1-O-glucoside, followed by attachment at the 6-position and the 3-position of glucose. Based on our results, we propose an acteoside biosynthetic pathway induced by MeJA treatment in sesame.

Acteoside inhibits high glucose-induced oxidative stress injury in RPE cells and the outer retina through the Keap1/Nrf2/ARE pathway.

Diabetes retinopathy (DR) is one of the most common microvascular complications of diabetes. Retinal pigment epithelial (RPE) cells exposed to a high glucose environment experience a series of functional damages, which is an important factor in promoting the progression of DR. Acteoside (ACT) has strong antioxidant and anti-apoptotic properties, but the mechanism of ACT in DR is not completely clear. Therefore, the purpose of the present study was to explore whether ACT inhibits the damage to RPE cells in a high glucose environment through antioxidative effects to alleviate the DR process. The DR in vitro cell model was constructed by treating RPE cells with high glucose, and the DR in vivo animal model was constructed by injecting streptozotocin (STZ) into the peritoneal cavity of mice to induce diabetes. The proliferation and apoptosis of RPE cells were detected by CCK-8 and flow cytometry assays, respectively. The expression changes in Nrf2, Keap1, NQO1 and HO-1 were evaluated by qRT‒PCR, Western blot and immunohistochemistry analyses. The MDA, SOD, GSH-Px and T-AOC contents were detected by kits. The changes in ROS and nuclear translocation of Nrf2 were observed by immunofluorescence assays. HE staining was used to measure the thickness of the outer nuclear layer (ONL) of the retina, and TUNEL staining was used to detect the number of apoptotic cells in the retinas of mice. In the present study, ACT effectively ameliorated outer retina damage in diabetic mice. In high glucose (HG)-induced RPE cells, ACT treatment had the following effects: improved proliferation, decreased apoptosis, inhibited Keap1 expression, promoted the nuclear translocation and expression of Nrf2, upregulated NQO1 and HO-1 (the target genes of Nrf2) expression, decreased ROS concentration, and increased the levels of the SOD, GSH-Px and T-AOC antioxidant indicators. However, knockdown of Nrf2 reversed the above phenomena, which indicated that the protective function of ACT in HG-induced RPE cells are closely related to Nrf2. In summary, the present study demonstrated that HG-induced oxidative stress injury is inhibited by ACT in RPE cells and the outer retina through the Keap1/Nrf2/ARE pathway.

Acteoside protects podocyte against apoptosis through regulating AKT/GSK-3ß signaling pathway in db/db mice.

BACKGROUND: Podocyte apoptosis is one of the important pathological mechanisms of diabetic kidney disease (DKD). Acteoside (Act), a major active component of Rehmannia glutinosa leaves total glycoside, has a strong renoprotective action. Our study aims to demonstrate Act's renoprotective actions in db/db mice. METHODS: We adopted C57BLKS/J db/db mice as DKD animal models. After 8 weeks of Act administration, the 24-hour urine albumin, renal function index, and blood lipid levels were quantified using matching kits. Renal pathology was evaluated by HE and PAS staining. The podocyte damage and apoptosis-related signaling pathway were observed by using immunohistochemistry, western blot, and TUNEL staining. RESULTS: The albuminuria of db/db mice was reduced from 391 ug/24 h to 152 ug/24 h, and renal pathology changes were alleviated after Act administration. The western blot and immunohistochemistry showed that Act treatment upregulated the synaptopodin and podocin expression compared with db/db mice, while the TUNEL staining indicated podocyte apoptosis was inhibited. The B-cell lymphoma-2 (Bcl-2) level was upregulated in the Act group, but cleaved caspase-3 and Bcl-2 associated X protein (Bax) expression declined, while the protein kinase B/glycogen synthase kinase-3ß (AKT/GSK-3ß) signaling pathway was repressed. CONCLUSIONS: By inhibiting the AKT/GSK-3ß signaling pathway, Act protected podocytes from apoptosis, decreasing the urine albumin of db/db mice and delaying the course of DKD.

Verbascoside Elicits Its Beneficial Effects by Enhancing Mitochondrial Spare Respiratory Capacity and the Nrf2/HO-1 Mediated Antioxidant System in a Murine Skeletal Muscle Cell Line.

Muscle weakness and muscle loss characterize many physio-pathological conditions, including sarcopenia and many forms of muscular dystrophy, which are often also associated with mitochondrial dysfunction. Verbascoside, a phenylethanoid glycoside of plant origin, also named acteoside, has shown strong antioxidant and anti-fatigue activity in different animal models, but the molecular mechanisms underlying these effects are not completely understood. This study aimed to investigate the influence of verbascoside on mitochondrial function and its protective role against H2O2-induced oxidative damage in murine C2C12 myoblasts and myotubes pre-treated with verbascoside for 24 h and exposed to H2O2. We examined the effects of verbascoside on cell viability, intracellular reactive oxygen species (ROS) production and mitochondrial function through high-resolution respirometry. Moreover, we verified whether verbascoside was able to stimulate nuclear factor erythroid 2-related factor (Nrf2) activity through Western blotting and confocal fluorescence microscopy, and to modulate the transcription of its target genes, such as heme oxygenase-1 (HO-1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), by Real Time PCR. We found that verbascoside (1) improved mitochondrial function by increasing mitochondrial spare respiratory capacity; (2) mitigated the decrease in cell viability induced by H2O2 and reduced ROS levels; (3) promoted the phosphorylation of Nrf2 and its nuclear translocation; (4) increased the transcription levels of HO-1 and, in myoblasts but not in myotubes, those of PGC-1α. These findings contribute to explaining verbascoside's ability to relieve muscular fatigue and could have positive repercussions for the development of therapies aimed at counteracting muscle weakness and mitochondrial dysfunction.

[Acteoside promotes autophagy and apoptosis of hepatoma cells by regulating JNK signaling pathway].

This study explored the molecular mechanism of acteoside against hepatoma 22(H22) tumor in mice through c-Jun N-terminal kinase(JNK) signaling pathway. H22 cells were subcutaneously inoculated in 50 male BALB/c mice, and then the model mice were classified into model group, low-dose, medium-dose, and high-dose acteoside groups, and cisplatin group. The administration lasted 2 weeks for each group(5 consecutive days/week). The general conditions of mice in each group, such as mental status, diet intake, water intake, activity, and fur were observed. The body weight, tumor volume, tumor weight, and tumor-inhibiting rate were compared before and after administration. Morphological changes of liver cancer tissues were observed based on hematoxylin and eosin(HE) staining, and the expression of phosphorylated(p)-JNK, JNK, B-cell lymphoma-2(Bcl-2), Beclin-1, and light chain 3(LC3) in each tissue was detected by immunohistochemistry and Western blot. qRT-PCR was performed to detect the mRNA expression of JNK, Bcl-2, Beclin-1, and LC3. The general conditions of mice in model and low-dose acteoside groups were poor, while the general conditions of mice in the remaining three groups were improved. The body weight of mice in medium-dose acteoside group, high-dose acteoside group, and cisplatin group was smaller than that in model group(P<0.01). The tumor volume in model group was insignificantly different from that in low-dose acteoside group, and the volume in cisplatin group showed no significant difference from that in high-dose acteoside group. Tumor volume and weight in medium-dose and high-dose acteoside groups and cisplatin group were lower than those in the model group(P<0.001). The tumor-inhibiting rates were 10.72%, 40.32%, 53.79%, and 56.44% in the low-dose, medium-dose, and high-dose acteoside groups and cisplatin group, respectively. HE staining showed gradual decrease in the count of hepatoma cells and increasing sign of cell necrosis in the acteoside and cisplatin groups, and the necrosis was particularly obvious in the high-dose acteoside group and cisplatin group. Immunohistochemical results suggested that the expression of Beclin-1, LC3, p-JNK, and JNK was up-regulated in acteoside and cisplatin groups(P<0.05). The results of immunohistochemistry, Western blot, and qRT-PCR indicated that the expression of Bcl-2 was down-regulated in the medium-dose and high-dose acteoside groups and cisplatin group(P<0.01). Western blot showed that the expression of Beclin-1, LC3, and p-JNK was up-regulated in acteoside and cisplatin groups(P<0.01), and there was no difference in the expression of JNK among groups. qRT-PCR results showed that the levels of Beclin-1 and LC3 mRNA were up-regulated in the acteoside and cisplatin groups(P<0.05), and the level of JNK mRNA was up-regulated in medium-dose and high-dose acteoside groups and cisplatin group(P<0.001). Acteoside promotes apoptosis and autophagy of H22 cells in mice hepatoma cells by up-regulating the JNK signaling pathway, thus inhibiting tumor growth.

[Study on biomarkers of acteoside in treating puromycin aminonucleoside nephropathy in young rats based on non-targeted urine metabolomics technology].

This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted urine metabolomics and decipher the potential mechanism of action. Biochemical indicators in the urine of rats from each group were determined by an automatic biochemical analyzer. The potential biomarkers and related core metabolic pathways were identified by ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). MetaboAnalyst 5.0 was used to establish the receiver operating characteristic(ROC) curve for evaluating the clinical diagnostic performance of core metabolites. The results showed that acteoside significantly decreased urinary protein-to-creatinine ratio in PAN young rats. A total of 17 differential metabolites were screened out by non-targeted urine metabolomics in PAN young rats and they were involved in phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. Thirtten differential metabolites were screened by acteoside intervention in PAN young rats, and they were involved in phenylalanine metabolism and arginine and proline metabolism. Among them, leucylproline and acetophenone were the differential metabolites that were significantly recovered after acteoside treatment. These pathways suggest that acteoside treats PAN in young rats by regulating amino acid metabolism. The area under the curve of two core biomarkers, leucylproline and acetophenone, were both greater than 0.9. In summary, acteoside may restore amino acid metabolism by regulating endogenous differential metabolites in PAN young rats, which will help to clarify the mechanism of acteoside in treating chronic glomerulonephritis in children. The characteristic biomarkers screened out have a high diagnostic value for evaluating the treatment of chronic glomerulonephritis in children with acteoside.

[Quality evaluation of Compound Cheqian Tablets based on UPLC-Q-TOF-MS/MS, network pharmacology and "double external standards" QAMS].

The Compound Cheqian Tablets are derived from Cheqian Power in Comprehensive Recording of Divine Assistance, and they are made by modern technology with the combination of Plantago asiatica and Coptis chinensis. To investigate the material basis of Compound Cheqian Tablets in the treatment of diabetic nephropathy, in this study, the chemical components of Compound Cheqian Tablets were characterized and analyzed by UPLC-Q-TOF-MS/MS, and a total of 48 chemical components were identified. The identified chemical compounds were analyzed by network pharmacology. By validating with previous literature, six bioactive compounds including acteoside, isoacteoside, coptisine, magnoflorine, palmatine, and berberine were confirmed as the index components for qua-lity evaluation. Furthermore, the content of the six components in the Compound Cheqian Tablets was determined by the "double external standards" quantitative analysis of multi-components by single marker(QAMS), and the relative correction factor of isoacteoside was calculated as 1.118 by using acteoside as the control; the relative correction factors of magnoflorine, palmatine, and berberine were calculated as 0.729, 1.065, and 1.126, respectively, by using coptisine as the control, indicating that the established method had excellent stability under different conditions. The results obtained by the "double external standards" QAMS approximated those obtained by the external standard method. This study qualitatively characterized the chemical components in the Compound Cheqian Tablets by applying UPLC-Q-TOF-MS/MS and screened the pharmacodynamic substance basis for the treatment of diabetic nephropathy via network pharmacology, and primary pharmacodynamic substance groups were quantitatively analyzed by the "double external stan-dards" QAMS method, which provided a scientific basis for clarifying the pharmacodynamic substance basis and quality control of Compound Cheqian Tablets.

Functional characterization of tyrosine decarboxylase genes that contribute to acteoside biosynthesis in Rehmannia glutinosa.

MAIN CONCLUSION: The RgTyDCs possess typical decarboxylase functional activity in vitro and in vivo and participate in acteoside biosynthesis in R. glutinosa, positively controlling its production via activated acteoside/tyrosine-derived pathways. Acteoside is an important ingredient in Rehmannia glutinosa and an active natural component that contributes to human health. Tyrosine decarboxylase (TyDC) is thought to play an important role in acteoside biosynthesis. Several plant TyDC family genes have been functionally characterized and shown to play roles in some bioactive metabolites' biosynthesis by mediating the decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-DOPA); however, one TyDC (named RgTyDC1) in R. glutinosa has been identified to date, but the family genes that contribute to acteoside biosynthesis remain largely characterized. Here, by in silico and experimental analyses, we isolated and identified three RgTyDCs (RgTyDC2 to RgTyDC4) in this species; these genes' sequences showed 50.92-82.55% identity, included highly conserved domains with homologues in other plants, classified into two subsets, and encoded proteins that localized to the cytosol. Enzyme kinetic analyses of RgTyDC2 and RgTyDC4 indicated that they both efficiently catalysed L-tyrosine and L-dopa. The overexpression of RgTyDC2 and RgTyDC4 in R. glutinosa, which was associated with enhanced TyDC activity, significantly increased tyramine and dopamine contents, which was positively correlated with improved acteoside production; moreover, the overexpression of RgTyDCs led to upregulated expression of some other genes-related to acteoside biosynthesis. This result suggested that the overexpression of RgTyDCs can positively activate the molecular networks of acteoside pathways, enhancing the accumulation of tyramine and dopamine, and promoting end-product acteoside biosynthesis. Our findings provide an evidence that RgTyDCs play vital molecular roles in acteoside biosynthesis pathways, contributing to the increase in acteoside yield in R. glutinosa.

Xuanfei Baidu Decoction reduces acute lung injury by regulating infiltration of neutrophils and macrophages via PD-1/IL17A pathway.

The pathogenic hyper-inflammatory response has been revealed as the major cause of the severity and death of the Corona Virus Disease 2019 (COVID-19). Xuanfei Baidu Decoction (XFBD) as one of the "three medicines and three prescriptions" for the clinically effective treatment of COVID-19 in China, shows unique advantages in the control of symptomatic transition from moderate to severe disease states. However, the roles of XFBD to against hyper-inflammatory response and its mechanism remain unclear. Here, we established acute lung injury (ALI) model induced by lipopolysaccharide (LPS), presenting a hyperinflammatory process to explore the pharmacodynamic effect and molecular mechanism of XFBD on ALI. The in vitro experiments demonstrated that XFBD inhibited the secretion of IL-6 and TNF-α and iNOS activity in LPS-stimulated RAW264.7 macrophages. In vivo, we confirmed that XFBD improved pulmonary injury via down-regulating the expression of proinflammatory cytokines such as IL-6, TNF-α and IL1-ß as well as macrophages and neutrophils infiltration in LPS-induced ALI mice. Mechanically, we revealed that XFBD treated LPS-induced acute lung injury through PD-1/IL17A pathway which regulates the infiltration of neutrophils and macrophages. Additionally, one major compound from XFBD, i.e. glycyrrhizic acid, shows a high binding affinity with IL17A. In conclusion, we demonstrated the therapeutic effects of XFBD, which provides the immune foundations of XFBD and fatherly support its clinical applications.

Exploration of a ternary deep eutectic solvent for the efficient extraction of plantamajoside, acteoside, quercetin and kaempferol from Plantago asiatica L.

INTRODUCTION: In the present study, ternary deep eutectic solvent-based ultrasound-assisted extraction was developed for the efficient extraction of plantamajoside, acteoside, quercetin and kaempferol from Plantago asiatica L. METHODOLOGY: Six kinds of choline chloride-based ternary deep eutectic solvents (TDESs) were prepared as potential extraction solutions. In order to obtain optimal extraction efficiency, a series of extraction conditions were investigated by single-factor test and orthogonal test. RESULTS: The extraction efficiency of choline chloride/lactic acid/ethylene glycol (ChCl-LA-EG) was much higher than that of other TDESs. ChCl-LA-EG-11 synthesised with choline chloride, lactic acid and ethylene glycol (1:4:2) was considered to have a higher extraction efficiency. The optimal ultrasound-assisted extraction conditions were as follows: water content in ChCl-LA-EG-11, 50%; extraction temperature, 70°C; ratio of solid/liquid, 20 mg/mL; ultrasonic power, 60 W; extraction time, 35 min; pH of the solution, 8. Under the optimal extraction conditions, the extraction efficiencies of plantamajoside, acteoside, quercetin and kaempferol were 3.83 ± 0.41, 4.23 ± 0.45, 0.56 ± 0.15 and 0.19 ± 0.08 mg/g, respectively. The extraction efficiency of the total target components was 9.21 ± 0.63 mg/g, which was much higher than that of conventional solvents (water, methanol, ethanol, 50% methanol, 50% ethanol). The target components were isolated efficiently from the TDES solution by an AB-8 macroporous resin column with a recovery rate of 95.6%. CONCLUSION: This study demonstrated that TDESs possessed excellent physical and chemical properties and had enormous potential for active component extraction of traditional Chinese medicinal materials.

The Beneficial Effects of Triterpenic Acid and Acteoside in an In Vitro Model of Nonalcoholic Steatohepatitis (NASH).

Triterpenic acid (TA) and acteoside (ACT), the major components of APPLIVER and ACTEOS, respectively, have been reported to exert hepatoprotective effects, but the molecular mechanisms remain elusive, particularly in the NAFLD/NASH context. We assessed their effects in our well-established in vitro model resembling the pathophysiological mechanisms involved in NASH. Human hepatocytes and hepatic stellate cells were exposed to free fatty acids (FFA) alone or in combination with APPLIVER and ACTEOS as a mono- or co-culture. Steatosis, inflammation, generation of reactive oxygen species (ROS), and collagen deposition were determined. ACTEOS reduced both the TNF-α and ROS production, and, most importantly, attenuated collagen deposition elicited by the excess of FFA in the co-culture model. APPLIVER also showed inhibition of both TNF-α production and collagen deposition caused by FFA accumulation. The compounds alone did not induce any cellular effects. The present study showed the efficacy of APPLIVER and ACTEOS on pathophysiological mechanisms related to NASH. These in vitro data suggest that these compounds deserve further investigation for possible use in NASH treatment.

Identification and Functional Characterization of Tyrosine Decarboxylase from Rehmannia glutinosa.

Rehmannia glutinosa is an important medicinal plant that has long been used in Chinese traditional medicine. Acteoside, one of the bioactive components from R. glutinosa, possessed various pharmacological activities for human health; however, the molecular mechanism of acteoside formation is not fully understood. In the current study, a novel tyrosine decarboxylase (designated as RgTyDC2) was identified from the R. glutinosa transcriptome. Biochemical analysis of RgTyDC2 showed RgTyDC2 uses tyrosine and dopa as the substrate to produce tyramine and dopamine, respectively, and it displays higher catalytic efficiency toward tyrosine than dopa. Moreover, the transcript level of RgTyDC2 was consistent with the accumulation pattern of acteoside in R. glutinosa, supporting its possible role in the biosynthesis of acteoside in vivo.

Pedicularis resupinata Extract Prevents Depressive-like Behavior in Repeated Corticosterone-Induced Depression in Mice: A Preliminary Study.

Excessive corticosterone (CORT), resulting from a dysregulated hypothalamic-pituitary-adrenal (HPA) axis, is associated with cognitive impairment and behavioral changes, including depression. In Korean oriental medicine, Pedicularis resupinata is used for the treatment of inflammatory diseases such as rheumatoid arthritis. However, the antidepressant properties of P. resupinata have not been well characterized. Here, the antidepressant-like effects of P. resupinata extract (PRE) were evaluated in terms of CORT-induced depression using in vivo models. HPLC confirmed that acteoside, a phenylethanoid glycoside, was the main compound from PRE. Male ICR mice (8 weeks old) were injected with CORT (40 mg/kg, i.p.) and orally administered PRE daily (30, 100, and 300 mg/kg) for 21 consecutive days. Depressive-like behaviors were evaluated using the open-field test, sucrose preference test, passive avoidance test, tail suspension test, and forced swim test. Treatment with a high dose of PRE significantly alleviated CORT-induced, depressive-like behaviors in mice. Additionally, repeated CORT injection markedly reduced brain-derived neurotrophic factor levels, whereas total glucocorticoid receptor (GR) and GR phosphorylation at serine 211 were significantly increased in the mice hippocampus but improved by PRE treatment. Thus, our findings suggest that PRE has potential antidepressant-like effects in CORT-induced, depressive-like behavior in mice.