Transcriptome and metabolome analysis reveals mechanism of light intensity modulating iridoid biosynthesis in Gentiana macrophylla Pall.

Light intensity is a key factor affecting the synthesis of secondary metabolites in plants. However, the response mechanisms of metabolites and genes in Gentiana macrophylla under different light intensities have not been determined. In the present study, G. macrophylla seedlings were treated with LED light intensities of 15 µmol/m2/s (low light, LL), 90 µmol/m2/s (medium light, ML), and 200 µmol/m2/s (high light, HL), and leaves were collected on the 5th day for further investigation. A total of 2162 metabolites were detected, in which, the most abundant metabolites were identified as flavonoids, carbohydrates, terpenoids and amino acids. A total of 3313 and 613 differentially expressed genes (DEGs) were identified in the LL and HL groups compared with the ML group, respectively, mainly enriched in KEGG pathways such as carotenoid biosynthesis, carbon metabolism, glycolysis/gluconeogenesis, amino acids biosynthesis, plant MAPK pathway and plant hormone signaling. Besides, the transcription factors of GmMYB5 and GmbHLH20 were determined to be significantly correlated with loganic acid biosynthesis; the expression of photosystem-related enzyme genes was altered under different light intensities, regulating the expression of enzyme genes involved in the carotenoid, chlorophyll, glycolysis and amino acids pathway, then affecting their metabolic biosynthesis. As a result, low light inhibited photosynthesis, delayed glycolysis, thus, increased certain amino acids and decreased loganic acid production, while high light got an opposite trend. Our research contributed significantly to understand the molecular mechanism of light intensity in controlling metabolic accumulation in G. macrophylla.

A fused hybrid enzyme of 8-hydroxygeraniol oxidoreductase (8HGO) from Gardenia jasminoides and iridoid synthase (ISY) from Catharanthus roseus significantly enhances nepetalactol and iridoid production.

MAIN CONCLUSION: The operation of 8HGO-ISY fusion enzymes can increase nepetalactol flux to iridoid biosynthesis, and the Gj8HGO-CrISY expression in Gardenia jasminoides indicates that seco-iridoids and closed-ring iridoids share a nepetalactol pool. Nepetalactol is a common precursor of (seco)iridoids and their derivatives, which are a group of noncanonical monoterpenes. Functional characterization of an 8HGO (8-hydroxygeraniol oxidoreductase) from Catharanthus roseus, a seco-iridoids producing plant, has been reported; however, the 8HGO from G. jasminoides with plenty of closed-ring iridoids remains uninvestigated. In this work, a Gj8HGO was cloned and biochemically characterized. In addition, the relatively low production of nepetalactol in plants and engineered microbial host is likely to be attributed to the fact that Cr8HGO and CrISY (iridoid synthase) are substrate-promiscuous enzymes catalyzing unexpected substrates to the undesired products. Herein, a bifunctional enzyme consisting of an 8HGO fused to an ISY was designed for the proximity to the substrate and recycling of NADP+ and NADPH cofactor to reduce the undesired intermediate in the synthesis of nepetalactol. Of four fusion enzymes (i.e., Gj8HGO-GjISY, Gj8HGO-GjISY2, Gj8HGO-GjISY4, and Gj8HGO-CrISY), interestingly, only the last one can enable cascade reaction to form cis-trans-nepetalactol. Furthermore, we establish a reliable Agrobacterium-mediated transformation system. The expression of Gj8HGO-CrISY in G. jasminoides led to a significant enhancement of nepetalactol production, about 19-fold higher than that in wild-type plants, which further resulted in the twofold to fivefold increase of total iridoids and representative iridoid such as geniposide, indicating that seco-iridoids in C. roseus and closed-ring iridoids in G. jasminoides share a nepetalactol pool. All results suggest that 8HGO and ISY can be manipulated to maximize metabolic flux for nepetalactol and iridoid production.

Concise Synthesis of 11-Noriridoids via Pauson-Khand Reaction.

Iridoids, which are a class of monoterpenoids, are attractive synthetic targets due to their diversely substituted cis-fused cyclopenta[c]pyran skeletons. Additionally, various biological activities of iridoids raise the value of synthetic studies on this class of compounds. Here, our synthetic efforts toward 11-noriridoids; (±)-umbellatolide B (6), (±)-10-O-benzoylglobularigenin (9) and 1-O-pentenylaucubigenin (34) are described. For the efficient synthesis of target compounds, common synthetic intermediates (tricyclic enones 17 and 26) were prepared by the Pauson-Khand reaction. The cleavage of the acetal bond on the tricyclic enones and 1,2-reduction introduced the two hydroxy groups on the cyclopentane ring of the core scaffold. Furthermore, the C3-C4 olefin part was constructed by the syn-elimination of a thiocarbonate moiety to obtain 34. The developed synthetic routes for 6, 9, and 34 will be useful for the preparation of iridoid analogs that have a polyfunctionalized core skeleton.

An Insight of Plant Source, Toxicological Profile, and Pharmacological Activities of Iridoid Loganic Acid: A Comprehensive Review.

This study evaluates the pharmacological effects of iridoid glucoside loganic acid, a plant constituent with diverse properties, based on literature, and explores the underlying cellular mechanisms for treating several ailments. Data were collected from reliable electronic databases, including PubMed, Scopus, Web of Science, and Google Scholar, etc. The results demonstrated the anti-inflammatory, anti-oxidant, and other protective effects of loganic acid on metabolic diseases and disorders such as atherosclerosis, diabetes, and obesity, in addition to its osteoprotective and anticancer properties. The antioxidant activity of loganic acid demonstrates its capacity to protect cells from oxidative damage and mitigates inflammation by reducing the activity of inflammatory cytokines involving TNF-α and IL-6, substantially upregulating the expression of PPAR-γ/α, and decreasing the clinical signs of inflammation-related conditions related to hypertriglyceridemia and atherosclerosis. Meanwhile, loganic acid inhibits bone loss, exhibits osteoprotective properties by increasing mRNA expression levels of bone synthesizing genes such as Alpl, Bglap, and Sp7, and significantly increases osteoblastic proliferation in preosteoblast cells. Loganic acid is an anti-metastatic drug that reduces MnSOD expression, inhibits EMT and metastasis, and prevents cellular migration, proliferation, and invasion in hepatocellular carcinoma cells. However, additional clinical trials are required to assess its safety, efficacy, and human dose.

Four new iridoid glycosides from the roots of Rehmannia glutinosa.

Four new iridoid glycosides (1-4), rehmaglutosides L-O, were isolated from the air-dried roots of Rehmannia glutinosa. Their structures were established from the spectroscopic data obtained and by chemical evidence. The known mellittoside (5) and ajugol (6) were also obtained in the current investigation, and the structure of mellittoside was unequivocally defined using X-ray diffraction data. Compounds 1-6 were tested for their cytotoxicity against five human tumor cell lines and proliferation effects on Lactobacillus Reuteri.

Six new iridoid glycosides from the whole plants of Hedyotis diffusa.

Six new iridoid glycosides were isolated from the ethyl acetate fraction of the whole plants of Hedyotis diffusa Willd. They were identified as E-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (1), Z-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (2), E-6-O-caffeoyl scandoside methyl ester (3), E-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (4), Z-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (5), and E-6-O-p-coumaroyl-4'-O-acetyl scandoside methyl ester (6). The structures of them were elucidated based on unambiguous spectroscopic data (UV, IR, HRESIMS, and NMR). They were screened for anti-inflammatory effect, antioxidant effect, antitumor effect, and neuroprotective effect and did not show potent activities.

Two new iridoid glycosides from the whole plant of Rehmania piasezkii.

Two new iridoid glycosides, piasezkiiosides A (1) and B (2), were isolated from aqueous extract of the whole plant of Rehmannia piasezkii. Their structures were established from the spectroscopic data, chemical transformation, and X-ray diffraction analysis. Compound 1 exhibited weak hepatoprotective activity against APAP-induced HepG2 cell damage.

Antinociceptive iridoid glycosides from the aerial parts of Paederia foetida.

Two previously undescribed iridoid glycosides, 6'-O-trans-feruloyl-(4S,6R)-3,4-dihydro-3ß-ethoxypaederoside (1) and 6'-O-trans-caffeoyl-(4S,6R)-3,4-dihydro-2'-O-3α-paederoside (2), were isolated from the 90% EtOH extract of the air dried aerial parts of Paederia Foetida. Structural elucidation of all the compounds was performed by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy. The two isolated iridoid glycosides were tested in vivo for their antinociceptive properties. As a result, 2 showed potent antinociceptive effect and its ID50 value (53.4 µmol/kg) was 2-fold less than those of the positive control drugs aspirin and acetaminophen.

Cytotoxic iridoid glycosides from the leaves of Paederia scandens.

A phytochemical investigation on the 80% EtOH extract of the leaves of Paederia scandens (Lour.) Merr. resulted into the isolation of three undescribed iridoid glycosides, 10-O-trans-p-coumaroyl-(4R,6R)-3,4-dihydro-3α-methylthiopaederoside (1), 10-O-trans-feruloyl-(4S,6R)-3,4-dihydro-2'-O-3α-paederoside (2), and 10-O-trans-caffeoyl-paederosidic acid ethyl ester (3). The structures of the new compounds were elucidated by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, as well as high resolution mass spectrometry. The isolated compounds were tested in vitro for cytotoxic activity against five endocrine tumor cell lines. As a result, compound 1 exhibited some cytotoxicities against all the tested tumor cell lines with IC50 value less than 20.0 µM.

Bioactive neolignan, iridoid and flavonoid glycosides from the leaves of Vaccinium bracteatum.

Two neolignan glycosides including a new one (1), along with seven iridoid glycosides (3 - 9) and nine flavonoid glycosides (10 - 18), were isolated from the leaves of Vaccinium bracteatum. Their structures were established mainly on the basis of 1D/2D NMR and ESIMS analyses, as well as comparison to known compounds in the literature. The structure of 1 with absolute stereochemistry was also confirmed by chemical degradation and ECD calculation. Selective compounds showed antiradical activity against ABTS and/or DPPH. Moreover, several isolates also suppressed the production of ROS in RAW264.7 cells and exerted neuroprotective effect toward PC12 cells.

Neuroprotective Effects of Aucubin against Cerebral Ischemia and Ischemia Injury through the Inhibition of the TLR4/NF-κB Inflammatory Signaling Pathway in Gerbils.

Aucubin, an iridoid glycoside, possesses beneficial bioactivities in many diseases, but little is known about its neuroprotective effects and mechanisms in brain ischemia and reperfusion (IR) injury. This study evaluated whether aucubin exhibited neuroprotective effects against IR injury in the hippocampal CA1 region through anti-inflammatory activity in gerbils. Aucubin (10 mg/kg) was administered intraperitoneally once a day for one week prior to IR. Neuroprotective effects of aucubin were assessed by neuronal nuclei (NeuN) immunofluorescence and Floro-Jade C (FJC) histofluorescence. Microgliosis and astrogliosis were evaluated using immunohistochemistry with anti-ionized calcium binding adapter protein 1 (Iba1) and glial fibrillary acidic protein (GFAP). Protein levels of proinflammatory cytokines interleukin1 beta (IL1ß) and tumor necrosis factor alpha (TNFα) were assayed using enzyme-linked immunosorbent assay and Western blot. Changes in toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway were assessed by measuring levels of TLR4, inhibitor of NF-κB alpha (IκBα), and NF-κB p65 using Western blot. Aucubin treatment protected pyramidal neurons from IR injury. IR-induced microgliosis and astrogliosis were suppressed by aucubin treatment. IR-induced increases in IL1ß and TNFα levels were significantly alleviated by the treatment. IR-induced upregulation of TLR4 and downregulation of IκBα were significantly prevented by aucubin treatment, and IR-induced nuclear translocation of NF-κB was reversed by aucubin treatment. Briefly, aucubin exhibited neuroprotective effects against brain IR injury, which might be related to the attenuation of neuroinflammation through inhibiting the TLR-4/NF-κB signaling pathway. These results suggest that aucubin pretreatment may be a potential approach for the protection of brain IR injury.

Cornelian Cherry (Cornus mas L.) Fruit Extract Lowers SREBP-1c and C/EBPα in Liver and Alters Various PPAR-α, PPAR-γ, LXR-α Target Genes in Cholesterol-Rich Diet Rabbit Model.

Cornelian cherry (Cornus mas L.) fruits, abundant in iridoids and anthocyanins, are natural products with proven beneficial impacts on the functions of the cardiovascular system and the liver. This study aims to assess and compare whether and to what extent two different doses of resin-purified cornelian cherry extract (10 mg/kg b.w. or 50 mg/kg b.w.) applied in a cholesterol-rich diet rabbit model affect the levels of sterol regulatory element-binding protein 1c (SREBP-1c) and CCAAT/enhancer binding protein α (C/EBPα), and various liver X receptor-α (LXR-α), peroxisome proliferator-activated receptor-α (PPAR-α), and peroxisome proliferator-activated receptor-γ (PPAR-γ) target genes. Moreover, the aim is to evaluate the resistive index (RI) of common carotid arteries (CCAs) and aortas, and histopathological changes in CCAs. For this purpose, the levels of SREBP-1c, C/EBPα, ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), fatty acid synthase (FAS), endothelial lipase (LIPG), carnitine palmitoyltransferase 1A (CPT1A), and adiponectin receptor 2 (AdipoR2) in liver tissue were measured. Also, the levels of lipoprotein lipase (LPL), visceral adipose tissue-derived serine protease inhibitor (Vaspin), and retinol-binding protein 4 (RBP4) in visceral adipose tissue were measured. The RI of CCAs and aortas, and histopathological changes in CCAs, were indicated. The oral administration of the cornelian cherry extract decreased the SREBP-1c and C/EBPα in both doses. The dose of 10 mg/kg b.w. increased ABCA1 and decreased FAS, CPT1A, and RBP4, and the dose of 50 mg/kg b.w. enhanced ABCG1 and AdipoR2. Mitigations in atheromatous changes in rabbits' CCAs were also observed. The obtained outcomes were compared to the results of our previous works. The beneficial results confirm that cornelian cherry fruit extract may constitute a potentially effective product in the prevention and treatment of obesity-related disorders.

Implications of the Propagation Method for the Phytochemistry of Nepeta cataria L. throughout a Growing Season.

Catnip (Nepeta cataria L.) plants produce a wide array of specialized metabolites with multiple applications for human health. The productivity of such metabolites, including nepetalactones, and natural insect repellents is influenced by the conditions under which the plants are cultivated. In this study, we assessed how field-grown catnip plants, transplanted after being propagated via either single-node stem cuttings or seeds, varied regarding their phytochemical composition throughout a growing season in two distinct environmental conditions (Pittstown and Upper Deerfield) in the state of New Jersey, United States. Iridoid terpenes were quantified in plant tissues via ultra-high-performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-QqQ-MS), and phenolic compounds (phenolic acids and flavonoids) were analyzed via UHPLC with diode-array detection (UHPLC-DAD). The highest contents of total nepetalactones in Pittstown were found at 6 weeks after transplanting (WAT) for both seedlings and cuttings (1305.4 and 1223.3 mg/100 g, respectively), while in Upper Deerfield, the highest contents for both propagules were at 11 WAT (1247.7 and 997.1 mg/100 g, respectively) for seed-propagated and stem cuttings). The highest concentration of nepetalactones was associated with floral-bud to partial-flowering stages. Because plants in Pittstown accumulated considerably more biomass than plants grown in Upper Deerfield, the difference in nepetalactone production per plant was striking, with peak productivity reaching only 598.9 mg per plant in Upper Deerfield and 1833.1 mg per plant in Pittstown. Phenolic acids accumulated in higher contents towards the end of the season in both locations, after a period of low precipitation, and flavone glycosides had similar accumulation patterns to nepetalactones. In both locations, rooted stem cuttings reached their maximum nepetalactone productivity, on average, four weeks later than seed-propagated plants, suggesting that seedlings have, overall, better agronomic performance.

Study on the Neuroprotective Effects of Eight Iridoid Components Using Cell Metabolomics.

Iridoid components have been reported to have significant neuroprotective effects. However, it is not yet clear whether the efficacy and mechanisms of iridoid components with similar structures are also similar. This study aimed to compare the neuroprotective effects and mechanisms of eight iridoid components (catalpol (CAT), genipin (GE), geniposide (GEN), geniposidic acid (GPA), aucubin (AU), ajugol (AJU), rehmannioside C (RC), and rehmannioside D (RD)) based on corticosterone (CORT)-induced injury in PC12 cells. PC12 cells were randomly divided into a normal control group (NC), model group (M), positive drug group (FLX), and eight iridoid administration groups. Firstly, PC12 cells were induced with CORT to simulate neuronal injury. Then, the MTT method and flow cytometry were applied to evaluate the protective effects of eight iridoid components on PC12 cell damage. Thirdly, a cell metabolomics study based on ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was performed to explore changes in relevant biomarkers and metabolic pathways following the intervention of administration. The MTT assay and flow cytometry analysis showed that the eight iridoid components can improve cell viability, inhibit cell apoptosis, reduce intracellular ROS levels, and elevate MMP levels. In the PCA score plots, the sample points of the treatment groups showed a trend towards approaching the NC group. Among them, AU, AJU, and RC had a weaker effect. There were 38 metabolites (19 metabolites each in positive and negative ion modes, respectively) identified as potential biomarkers during the experiment, among which 23 metabolites were common biomarkers of the eight iridoid groups. Pathway enrichment analysis revealed that the eight iridoid components regulated the metabolism mainly in relation to D-glutamine and D-glutamate metabolism, arginine biosynthesis, the TCA cycle, purine metabolism, and glutathione metabolism. In conclusion, the eight iridoid components could reverse an imbalanced metabolic state by regulating amino acid neurotransmitters, interfering with amino acid metabolism and energy metabolism, and harmonizing the level of oxidized substances to exhibit neuroprotective effects.

Bioactivity evaluation and active compounds identification of Symphoricarpos orbiculatus as potential botanical herbicide.

BACKGROUND: Evaluation of herbicidal activity and identification of active compounds are important bases for the development of new botanical herbicides. RESULTS: This study confirmed that Symphoricarpos orbiculatus has high herbicidal activities against mono-dicotyledonous weeds, including Echinochloa crusgalli, Digitaria sanguinalis, Amaranthus retroflexus and Portulaca oleracea. By bioassay-guided isolation, 12 compounds were isolated and identified from S. orbiculatus for the first time, including iridoids: naucledal (K1), loganin (K2), loganigenin (K3), loganin acid (K4), glucologanin (K5) and vogeloside (K6), as well as flavonoids: quercetine (K7), luteolin (K8), nobiletin (K9), astragalin (K10), isorhamnetin 3-d-glucoside (K11) and rutin (K12). Biological assays showed that iridoids are the main active ingredients of S. orbiculatus. The compounds of K5 and K6 could inhibit both the root (IC50 = 37.54 and 38.91 µg mL-1, respectively) and shoot (IC50 = 42.78 and 45.72 µg mL-1, respectively) of Portulaca oleracea, which have a weeding toxicity similar to that of the commercialized plant-based herbicide pelargonic acid. In addition, the results of pot culture assay showed that S. orbiculatus ethanol extracts had high fresh weight control effect against Digitaria sanguinalis and P. oleracea at the concentration of 40 g L-1. After 7 days, both the soil treatment and the stem and leaf spray method resulted in severe leaf necrosis and significant leaf etiolation. CONCLUSION: Symphoricarpos orbiculatus and its herbicidal active compounds have the potential to develop into botanical herbicides, and are first reported in the present study. © 2024 Society of Chemical Industry.

Therapeutic potentials of iridoids derived from Rubiaceae against in vitro and in vivo inflammation: A scoping review.

Acute inflammation may develop into chronic, life-threatening inflammation-related diseases if left untreated or if there are persistent triggering factors. Cancer, diabetes mellitus, stroke, cardiovascular diseases, and neurodegenerative disorders are some of the inflammation-related diseases affecting millions of people worldwide. Despite that, conventional medical therapy such as non-steroidal anti-inflammatory drugs (NSAIDs) is associated with serious adverse effects; hence, there is an urgent need for a newer and safer therapeutic alternative from natural sources. Iridoids are naturally occurring heterocyclic monoterpenoids commonly found in Rubiaceae plants. Plant extracts from the Rubiaceae family were demonstrated to have medicinal benefits against neurodegeneration, inflammation, oxidative stress, hyperglycaemia, and cancer. However, the therapeutic effects of natural iridoids derived from Rubiaceae as well as their prospective impacts on inflammation in vitro and in vivo have not been thoroughly explored. The databases of PubMed, Scopus, and Web of Science were searched for pertinent articles in accordance with PRISMA-ScR guidelines. A total of 31 pertinent articles from in vitro and in vivo studies on the anti-inflammatory potentials of iridoids from Rubiaceae were identified. According to current research, genipin, geniposide, and monotropein are the most researched iridoids from Rubiaceae that reduce inflammation. These iridoids primarily act by attenuating inflammatory cytokines and mediators via inhibition of the NF-κB signalling pathway in various disease models. A comprehensive overview of the current research on the anti-inflammatory properties of iridoids from the Rubiaceae family is presented in this review, highlighting the characteristics of the experimental models used as well as the mechanisms of action of these iridoids. To develop an alternative therapeutic agent from iridoids, more studies are needed to elucidate the effects and mechanism of action of iridoids in a wide variety of experimental models as well as in clinical studies pertaining to inflammation-related diseases.

[Gene cloning and functional analysis of an iridoid oxidase gene in Rehmannia glutinosa].

Rehmannia glutinosa is one of the commonly used Chinese herbal medicines, which has activities of heat-clearing,blood-cooling, Yin-nourishing, and body fluid-promoting. Iridoid glycosides are the main bioactive in R. glutinosa. Iridoid oxidase is a key rate-limiting enzyme in the biosynthetic pathway of iridoid glycosides. In this study, an iridoid oxidase gene Rg IO was screened based on the transcriptome data, followed by bioinformatics analysis, expression characteristic detection, and subcellular localization analysis. The results show that the coding region of Rg IO is 1 536 bp, with 511 amino acids encoded, and the molecular weight is about 58 258. 01. The protein sequence of Rg IO contains the conserved domains and motifs of cytochrome P450 oxidases. Rg IO has the highest sequence identities with its ortholog proteins in Striga asiatica, Striga hermonthica, and Centranthera grandiflora and has good sequence identities(77. 28%) with Catharanthus roseus Cr IO. Rg IO shows specific expression in the leaf of R. glutinosa. In response to MeJA induction, the expression of MeJA in leaves and roots after treatment increases by 3. 15 and 1. 3 times at 3 h and 6 h,respectively. The result of subcellular localization shows that Rg IO is distributed in the endoplasmic reticulum. Agrobacterium-mediated transient expression of Rg IO gene in leaves of R. glutinosa makes the content of catalpol increase by 0. 82 times compared with the transient expression of the empty vector. This study provides a key target gene for the molecular regulation and biosynthesis of catalpol in R. glutinosa and lays a foundation for revealing the complete biosynthetic pathway of catalpol.

[UPLC-Q-TOF-MS-based metabolomics reveals mechanism of Morinda officinalis iridoid glycosides in treating rheumatoid arthritis and bone loss].

This study aimed to investigate the therapeutic effects of Morinda officinalis iridoid glycosides(MOIG) on paw edema and bone loss of rheumatoid arthritis(RA) rats, and analyze its potential mechanism based on ultra-high performance liguid chromatography-guadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS) serum metabolomics. RA rats were established by injecting bovin type Ⅱ collagen. The collagen-induced arthritis(CIA) rats were administered drug by gavage for 8 weeks, the arthritic score were used to evaluate the severity of paw edem, serum bone metabolism biochemical parameters were measured by ELISA kits, Masson staining was used to observe the bone microstructure of the femur in CIA rats. UPLC-Q-TOF-MS was used to analyze the alteration of serum metabolite of CIA rats, principal component analysis(PCA) and partial least squares-discriminant analysis(PLS-DA) were used to screen the potential biomarkers, KEGG database analysis were used to construct related metabolic pathways. The results demonstrated that the arthritic score, serum levels of IL-6 and parameters related with bone metabolism including OCN, CTX-Ⅰ, DPD and TRAP were significantly increased, and the ratio of OPG and RANKL was significantly decreased, the microstructure of bone tissue and cartilage were destructed in CIA rats, while MOIG treatments could significantly reduce arthritis score, mitigate the paw edema, reverse the changes of serum biochemical indicators related with bone metabolism, and improve the microstructure of bone tissue and cartilage of CIA rats. The non-targeted metabolomics results showed that 24 altered metabolites were identified in serum of CIA rats; compared with normal group, 13 significantly altered metabolites related to RA were identified in serum of CIA rats, mainly involving alanine, aspartate and glutamate metabolism; compared with CIA model group, MOIG treatment reversed the alteration of 15 differential metabolites, mainly involving into alanine, aspartate and glutamate metabolism, D-glutamine and D-glutamate metabolism, taurine and hypotaurine metabolism, valine, leucine and isoleucine biosynthesis. Therefore, MOIG significantly alleviated paw edema, improved the destruction of microstructure of bone and cartilage in CIA rats maybe through involving into the regulation of amino acid metabolism.

Regioselective O-acetylation of various glucosides catalyzed by Escherichia coli maltose O-acetyltransferase.

Escherichia coli, a well-known prokaryotic organism, has been widely employed as a versatile host for heterologous overexpression of proteins/biocatalysts and the production of pharmaceutically important intermediates/small molecules. However, some E. coli endogenous enzymes showing substrate promiscuity may disturb the heterologous metabolic flux, which will result in the reduction of substrates, intermediates, and target products. Here we reported an unexpected E. coli-catalyzed regioselective O-acetylation of various glucosides. The regioselectively O-acetylated products, 6'-O-acetyl-loganin and 6'-O-acetyl-loganic acid, were obtained and characterized from the enzymatic reaction in which the supernatants of E. coli expressing either CaCYP72A565 and CaCPR, the key enzymes involved in camptothecin biosynthesis, or empty vector were used as catalyst and loganin and loganic acid as independent substrate. An alkaloidal glucoside strictosamide was converted into the regioselectively O-acetylated product 6'-O-acetyl-strictosamide, implying substrate promiscuity of the E. coli-catalyzed O-acetylation reaction. Furthermore, 8 glucosides, including 5 iridoid glucosides and 3 flavonoid glucosides, were successfully converted into the regioselectively O-acetylated products by E. coli, indicating the wide substrate range for the unexpected E. coli-catalyzed O-acetylation. E. coli maltose O-acetyltransferase was demonstrated to be responsible for the mentioned regioselective O-acetylation at the 6-OH of the glucopyranosyl group of multiple classes of natural product glucosides through candidate acetyltransferase-encoding gene analysis, gene knock-out, gene complementation, and the relevant enzymatic reaction activity assays. The present study not only provides an efficient biocatalyst for regioselective O-acetylation but also notifies cautions for metabolic engineering and synthetic biology applications in E. coli. KEY POINTS: • 6-OH of glucosyl of multiple glucosides was regioselectively O-acetylated by E. coli. • Endogenous EcMAT is responsible for the regioselective O-acetylation reaction.

Hepatoprotective Principles from the Rhizomes of Picrorhiza kurroa.

A methanol extract of rhizomes of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae) showed hepatoprotective effects against D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. We had previously isolated 46 compounds, including several types of iridoid glycosides, phenylethanoid glycosides, and aromatics, etc., from the extract. Among them, picroside II, androsin, and 4-hydroxy-3-methoxyacetophenone exhibited active hepatoprotective effects at doses of 50-100 mg/kg, per os (p.o.) To characterize the mechanisms of action of these isolates and to clarify the structural requirements of phenylethanoid glycosides for their hepatoprotective effects, their effects were assessed in in vitro studies on (i) D-GalN-induced cytotoxicity in mouse primary hepatocytes, (ii) LPS-induced nitric oxide (NO) production in mouse peritoneal macrophages, and (iii) tumor necrosis factor-α (TNF-α)-induced cytotoxicity in L929 cells. These isolates decreased the cytotoxicity caused by D-GalN without inhibiting LPS-induced macrophage activation and also reduced the sensitivity of hepatocytes to TNF-α. In addition, the structural requirements of phenylethanoids for the protective effects of D-GalN-induced cytotoxicity in mouse primary hepatocytes were evaluated.