Mechanism of Gentisic Acid on Rheumatoid Arthritis Based on miR-19b-3p/RAF1 Axis.

OBJECTIVE: To investigate the therapeutic effect of gentisic acid (GA) on rheumatoid arthritis (RA) based on the miR-19b-3p/RAF1 axis. METHODS: The cell counting kit-8 method was used to detect the growth inhibitory effect of different concentrations of GA on MH7A cells, and the drug concentration of GA was determined in the experiment. The quantificational real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-19b-3p and RAF1. RAF1, extracellular regulated protein kinases1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2) were examined by Western blotting. Three methods (dual-luciferase assay, qRT-PCR and Western blot analysis) were used to verify miR-19b-3p targeting RAF1. Flow cytometry was performed to detect MH7A cell apoptosis. Transwell and wound healing assays were used to determine the invasion and migration capacities of MH7A cells. RESULTS: The growth of MH7A cells was gradually inhibited with increasing GA concentration. When the GA concentration exceeded 80 mmol/L, GA was significantly cytotoxic to MH7A cells, so the half maximal inhibitory concentration of GA for MH7A cells was calculated as 67.019 mmol/L. GA upregulated miR-19b-3p expression, downregulated RAF1 expression, inhibited ERK1/2 phosphorylation, induced MH7A cell apoptosis and suppressed MH7A cell invasion and migration (P<0.05 or P<0.01). RAF1 was identified as the target of miR-19b-3p and reversed inhibitory effects on miR-19b-3p expression (P<0.05 or P<0.01). The miR-19b-3p inhibitor upregulated RAF1 expression and ERK1/2 phosphorylation, suppressed MH7A cell apoptosis and induced MH7A cell invasion and migration (P<0.01). CONCLUSION: GA regulated miR-19b-3p/RAF1 axis to mediate ERK pathway and inhibit the development of RA.

Influence of calcium channel modulators on the production of serotonin, gentisic acid, and a few other biosynthetically related phenolic metabolites in seedling leaves of salt tolerant rice variety Nonabokra.

Rice, a most salt-sensitive cereal plant, adopts diverse pathways to withstand sodium chloride-induced salinity-related adversities. During the present study, attempt was made to understand the role of calcium on metabolite profile of the leaves of salt tolerant rice seedlings of variety of Nonabokra under sodium chloride induced salinity, by Gas Chromatography-Mass Spectrometry-based metabolomics approach. Calcium availability in the seedlings was reduced or enhanced applying inhibitors (vanadyl sulfate, lanthanum chloride, and verapamil) or promoters of calcium influx (calcimycin also known as calcium ionophore A23187) in the sodium chloride (100 mM) supplemented growth medium. Growth medium of ten-day-old seedlings was replaced by sodium chloride supplemented hydroponic solution with promotor or inhibitors of calcium channel. Fifteen days old seedlings were harvested. It was observed that depletion of calcium availability increased the level of serotonin and gentisic acid whereas increased calcium level decreased these metabolites. It was concluded from the results that production of the signaling molecules serotonin and gentisic acids was elevated in calcium-deficient seedlings under salt stress the condition that was considered as control during the experiment. The two signaling molecules probably help this tolerant rice variety Nonabokra to withstand the salt-induced adversities.

Screening of antimicrobial synergism between phenolic acids derivatives and UV-A light radiation.

The objective of this study was to investigate synergistic antibacterial activity based on a combination of UV-A light and three classes of food grade compounds: benzoic acid derivatives, cinnamic acid derivatives, and gallates. By using Escherichia coli O157:H7 as the model strain, it was observed that three cinnamic acid derivatives (ferulic acid, coumaric acid, and caffeic acid) and one benzoic acid derivative (2,5-dihydroxybenzoic acid) presented strong synergistic antibacterial activity with UV-A light radiation, where 1 mM levels of these compounds plus with 15 min of UV-A light (total light dose of 6.1 cm-2) led to more than 7-log CFU mL-1 of bacterial inactivation. In contrast, synergistic antibacterial activity between UV-A light and most benzoic acid derivatives (benzoic acid, gallic acid, vanillic acid, and 2,5-dimethoxybenzoic acid) were only observed after higher concentrations of these compounds were applied (10 mM). Lastly, from the three gallates tested (methyl gallate, ethyl gallate, and propyl gallate), only propyl gallate showed strong antibacterial synergism with UV-A light, where 10 mM of propyl gallate plus 15 min of UV-A light led to approximately 6.5-log of bacterial reduction. Presence of antioxidant compounds mitigated the light-mediated antibacterial activity of gallic acid, 2,5-dihydroxybenzoic acid, and propyl gallate. Similarly, the light-mediated antibacterial activity of these compounds was significantly (P < 0.05) reduced against metabolic-inhibited bacterial cells (sodium azide pretreatment). On the other hand, the antibacterial synergism between ferulic acid and UV-A light was not affected by the presence of antioxidants or the metabolic state of the bacterial cells. Due to the increasing concerns of antimicrobial resistant (AMR) pathogens, the study also investigated the proposed synergistic treatment on AMR Salmonella. Combinations of 1 mM of ferulic acid or 1 mM of 2,5-dihydroxybenzoic acid with UV-A light radiation was able to inactivate more than 6-log of a multi-drug resistant Salmonella Typhimurium strain.

Gentisic Acid Stimulates Keratinocyte Proliferation through ERK1/2 Phosphorylation.

Keratinocyte proliferation is important for skin wound healing. The wound healing process includes blood clotting around the wound, removal of dead cells and pathogens through inflammation, and then re-epithelialization through proliferation and maturation. Proliferation assay was performed on acid natural compounds to identify candidates for natural-derived components of skin injury treatment. We found that gentisic acid promoted high cell proliferation activity compared with other compounds. Gentisic acid improved HaCaT cell proliferation by over 20% in MTT assay. Gentisic acid also had higher healing activity in an in vitro wound healing assay than allantoin as a positive control. Furthermore, we have identified how the treatment of gentisic acid can increase proliferation in the cell. Western blot analysis of proteins in the mitogen-activated protein (MAP) kinase signaling pathway showed that ERK1/2 phosphorylation was increased by gentisic acid treatment. Thus, our study indicates that gentisic acid promotes the proliferation of keratinocyte by phosphorylation of ERK1/2.

Rapid quality control of medicine and food dual purpose plant polysaccharides by matrix assisted laser desorption/ionization mass spectrometry.

With their multiple biological activities and health benefit effects, polysaccharides from medicine and food dual purpose plants (MFDPPPs) have been extensively applied in many fields, including in medical treatments, stock farming, and cosmetics. However, to date, quality issues of MFDPPPs and technologies for the analysis of polysaccharides have posed challenges to chemists. Reported herein is a rapid and high-throughput quality control method for analyzing MFDPPPs, based on matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). For the analysis of illegally added and doped substances, ferroferric oxide nanoparticles were employed as the MALDI matrix to avoid small molecule interference. Qualitatively, high sensitivity was obtained for both illegal drugs and glucose. Quantitatively, the best linear response (R2 > 0.99) was attained in the concentration range from 0.005 to 1 mg mL-1 for glucose. For the analysis of polysaccharides, 2,5-dihydroxybenzoic acid/N-methylaniline was employed as the MALDI matrix to increase the detection sensitivity and mass range coverage. Furthermore, the established method was successfully applied to the analysis of supplements from Astragalus polysaccharides and Lentinan real samples, showing its potential in quality control for MFDPPPs.

A review on gentisic acid as a plant derived phenolic acid and metabolite of aspirin: Comprehensive pharmacology, toxicology, and some pharmaceutical aspects.

Beneficial therapeutic effects of phenolic acids have been proven in various research projects including in vivo and in vitro studies. Gentisic acid (GA) is a phenolic acid that has been associated with useful effects on human health, such as antiinflammatory, antigenotoxic, hepatoprotective, neuroprotective, antimicrobial, and especially antioxidant activities. It is an important metabolite of aspirin and also widely distributed in plants as a secondary plant product such as Gentiana spp., Citrus spp., Vitis vinifera, Pterocarpus santalinus, Helianthus tuberosus, Hibiscus rosa-sinensis, Olea europaea, and Sesamum indicum and in fruits such as avocados, batoko plum, kiwi fruits, apple, bitter melon, black berries, pears, and some mushrooms. This study was undertaken to review the pharmacological effects, pharmacokinetic properties as well as toxicity and pharmaceutical applications of GA.

Signaling pathway played by salicylic acid, gentisic acid, nitric oxide, polyamines and non-enzymatic antioxidants in compatible and incompatible Solanum-tomato mottle mosaic virus interactions.

Plants are exposed to a vast array of pathogens. The interaction between them may be classified in compatible and incompatible. Polyamines (PAs) are involved in defense responses, as well as salicylic acid (SA), gentisic acid (GA) and nitric oxide (NO), which can increase the content of reactive oxygen species (ROS), creating a harsh environment to the pathogen. ROS can also damage the host cell and they can be controlled by ascorbate and glutathione. Among phytopathogens, one of the major threats to tomato crops is tomato mottle mosaic virus (ToMMV). Resistance against this virus probably involves the Tm-22 gene. This work aimed to analyze signaling and antioxidant molecules in the defense response against ToMMV in Solanum pimpinellifolium and in S. lycopersicum 'VFNT'. In S. pimpinellifolium plants inoculated with ToMMV, an increase in NO, SA, GA, ascorbate and oxidized glutathione and a decrease in the content of PAs were observed. Characteristic symptoms of diseased plants and high absorbance values in PTA-ELISA indicated a compatible interaction. In VFNT-inoculated plants, less significant differences were noticed. Symptoms and viral concentration were not detected, indicating an incompatible interaction, possibly associated with the effector-triggered immunity (ETI) response.

Assessing Anticancer Potential of Blueberry Flavonoids, Quercetin, Kaempferol, and Gentisic Acid, Through Oxidative Stress and Apoptosis Parameters on HCT-116 Cells.

In recent years, natural products gained popularity with their anti-inflammatory and antioxidant effects mediated by chemical compounds within their composition. Study results offering them as palliative therapy options in cancer or as anticancer agents with high levels of cytotoxicity brought a new approach to combine cancer treatment protocols with these products. From a different perspective, edible types of these products are suggested in daily diets due to their potential cancer preventive effects. Our preliminary work was on blueberry extracts (Vaccinium myrtillus) as a main representative of these natural products, and the contents of the extracts were analyzed with liquid chromatography tandem mass spectrometry (LC MS/MS) to reveal the composition and distribution of polyphenolic compounds within. The most abundant polyphenols detected in V. myrtillus extracts were quercetin, kaempferol, and a phenolic acid, gentisic acid (GA). The compounds were further evaluated on treated HCT-116 cells for their potential anticancer effects by measuring total antioxidant status, total oxidant status, and 8-hydroxydeoxyguanosine levels for evaluation of oxidative stress and through protein array analysis and flow cytometric analysis for evaluation of apoptosis. In analysis of oxidative stress parameters, reduced total oxidant levels and reduced oxidative stress index levels were found in cells treated with the compounds in comparison with untreated cells. In apoptosis-related protein profiles, at least twofold reduction in various apoptotic proteins was observed after quercetin and kaempferol treatment, whereas a different profile was observed for GA. Overall, results of this study showed that quercetin and kaempferol have strong cytotoxic, antioxidant, and apoptotic effects, although GA is mostly effective as an antioxidant polyphenol on HCT-116 cells.

Protective effect of Tremella fuciformis Berk extract on LPS-induced acute inflammation via inhibition of the NF-κB and MAPK pathways.

Tremella fuciformis Berk (TFB) has long been used as a traditional medicine in Asia. Although TFB exhibits antioxidant and anti-inflammatory effects, the mechanisms of action responsible have remained unknown. We confirmed the anti-inflammatory effects of Tremella fuciformis Berk extract (TFE) in RAW 264.7 cells and observed significantly suppressed LPS-induced iNOS/NO and COX-2/PGE2 production. TFE also suppressed LPS-induced IKK, IkB, and p65 phosphorylation, as well as LPS-induced translocation of p65 from the cytosol. Additionally, TFE inhibited LPS-induced phosphorylation of MAPKs. In an acute inflammation study, oral administration of TFE significantly inhibited LPS-induced IL-1ß, IL-6 and TNF-α production and iNOS and COX-2 expression. The major bioactive compounds from TFB extract were identified as gentisic acid, protocatechuic acid, 4-hydroxybenzoic acid, and coumaric acid. Among these compounds, protocatechuic acid showed the strongest inhibitory effects on LPS-induced NO production in RAW 264.7 cells. Overall, these results suggest that TFE is a promising anti-inflammatory agent that suppresses iNOS/NO and COX-2/PGE2 expression, as well as the NF-κB and MAPK signaling pathways.

In vitro antioxidant and inhibitory activity of water decoctions of carob tree (Ceratonia siliqua L.) on cholinesterases, α-amylase and α-glucosidase.

This work reports the in vitro inhibitory activity of water decoctions of leaves, germ flour, pulp, locust bean gum and stem bark of carob tree on α-amylase, α-glucosidase, acetylcholinesterase and butyrylcholinesterase. The antioxidant activity and the chemical characterisation of the extracts made by spectrophotometric assays and by high-performance liquid chromatography are also reported. Leaves and stem bark decoctions strongly inhibited all the enzymes tested, had significant antioxidant activity and the highest total phenolics content. The major compounds were identified as gallic acid in the leaves and gentisic acid in the stem bark.

Qualitative and quantitative analysis of Potentilla fulgens roots by NMR, matrix-assisted laser desorption/ionisation with time-of-flight MS, electrospray ionisation MS/MS and HPLC/UV.

INTRODUCTION: Potentilla fulgens is a commonly used folk medicine by natives of northeast India, Nepal and Bhutan and is rich in polyphenolic and triterpene constituents. OBJECTIVE: To identify chemomarkers in the roots of P. fulgens by an interplay of (13)C-NMR, matrix-assisted laser desorption/ionisation with time-of-flight (MALDI/TOF) MS, electrospray ionisation (ESI) MS/MS and HPLC/UV. MATERIAL AND METHODS: The (13)C-NMR spectrum of crude methanolic extract was recorded in deuterated dimethyl sulphoxide. For MALDI/TOF/MS analysis, 2,5-dihydroxybenzoic acid was used as the matrix. For determination of chemical constituents, two independent simple isocratic HPLC/UV methods for monomeric/oligomeric flavanols and triterpene acids were developed and validated. RESULTS: The (13)C-NMR spectrum of the methanolic extract indicated the presence of B-type oligomeric polyphenolics containing mainly epicatechin/catechin (epicat/cat) and epiafzelechin/afzelechin (epiafz/afz) as the monomeric units. Several isobaric monomeric and oligomeric flavanols and triterpenoids were tentatively identified by MALDI/TOF/MS and ESI/MS/MS. Fourteen compounds (four monomeric and five dimeric flavanols and five triterpene acids) were isolated using repeated column chromatography and semi-preparative HPLC, and were quantitated using HPLC/UV. CONCLUSION: It is evident from these analyses that roots of P. fulgens contain flavans, including oligomeric flavanols, as major constituents followed by triterpene acids. The methods described can be applied to other Potentilla species to identify their constituents.

Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection.

Competition for iron influences host-pathogen interactions. Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron. In response, the host secretes siderophore-binding proteins, such as lipocalin 24p3, which limit siderophore-mediated iron import into bacteria. Mammals produce 2,5-dihydroxy benzoic acid, a compound that resembles a bacterial siderophore. Our data suggest that bacteria use both mammalian and bacterial siderophores. In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro. In addition, mice lacking the mammalian siderophore resist E. coli infection. Finally, we show that the host responds to infection by suppressing siderophore synthesis while up-regulating lipocalin 24p3 expression via TLR signaling. Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

Endogenous siderophore 2,5-dihydroxybenzoic acid deficiency promotes anemia and splenic iron overload in mice.

Eukaryotes produce a siderophore-like molecule via a remarkably conserved biosynthetic pathway. 3-OH butyrate dehydrogenase (BDH2), a member of the short-chain dehydrogenase (SDR) family of reductases, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA). Depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of intracellular iron and mitochondrial iron deficiency in cultured mammalian cells, as well as in yeast cells and zebrafish embryos We disrupted murine bdh2 by homologous recombination to analyze the effect of bdh2 deletion on erythropoiesis and iron metabolism. bdh2 null mice developed microcytic anemia and tissue iron overload, especially in the spleen. Exogenous supplementation with 2,5-DHBA alleviates splenic iron overload in bdh2 null mice. Additionally, bdh2 null mice exhibit reduced serum iron. Although BDH2 has been proposed to oxidize ketone bodies, we found that BDH2 deficiency did not alter ketone body metabolism in vivo. In sum, our findings demonstrate a key role for BDH2 in erythropoiesis.

[Chemical constituents from flower buds of Lonicera japonica].

Eighteen compounds were isolated by a combination of various chromatographic techniques including column chromatography over macroporous resin, MCI gel, silica gel, and sephadex LH-20 and reversed-phase HPLC. Their structures were elucidated by spectroscopic data analysis as adinoside A (1), stryspinoside (2), benzyl alcohol beta-glucopyranoside (3), benzyl 2-o-beta-D-glucopyranosyl-2,6-dihydroxybenzoate (4) , gentisic acid 2-O-beta-D-glucopyranoside (5), eugenyl beta-D-glucopyranoside (6) , eugenyl-P-xylopyranosyl-(1-->6)-beta-glucopyranoside (7), (-)-lyoniresinol 9-O-fP-D-glucopyranoside (8) , (+)-lyoniresinol 9-O-beta-D-glucopyranoside (9) , apigenin-7-O-L-rhamnopyranoside (10), luteolin-3 '-O-L-rhamnoside (11) , ursolic acid (12) , beta-sitosteryl-3beta-glucopyranoside-6'-O-palmitate (13), abscisic acid (14), guanosine (15), 5-methyluracil (16), trans-cinnamic acid (17), and 4-hydroxybenzaldehyde(18). These compounds were obtained from this plant for the first time.

Gentisides C-K: nine new neuritogenic compounds from the traditional Chinese medicine Gentiana rigescens Franch.

Nine new alkyl 2,3-dihydroxybenzoates, gentisides C-K, were isolated from the traditional Chinese medicine Gentiana rigescens Franch. Their structures and stereochemistry were elucidated by spectroscopic methods, and comparison of the specific rotation with that of the gentiside B. These metabolites are additional members of the gentisides which belong to a novel class of neuritogenic compounds. They are structurally different from one another because they possess varying alkyl chain lengths, with or without an isobutyl or isopropyl group at the end of the alkyl chain. These compounds are potent inducers of neurite outgrowth on PC12 cells. The gentiside C possessing the shortest alkyl chain length exhibited the highest neuritogenic activity among all of the gentisides. Gentiside C showed a significant neuritogenic activity at 1 µM against PC12 cells comparable to that seen for the best nerve growth factor (NGF) concentration of 40 ng/mL. In addition, evident neuritogenic activity was observed in the cells when treated with gentiside C at a concentration as low as 0.03 µM. The structure-activity relationships within the gentisides A-K revealed that alkyl chain length is important for the activity, but structure diversity at the end of the alkyl chain is not.

Gentisides A and B, two new neuritogenic compounds from the traditional Chinese medicine Gentiana rigescens Franch.

Two new alkyl 2,3-dihydroxybenzoates, gentisides A and B, were isolated from the traditional Chinese medicine Gentiana rigescens Franch. Their structures and stereochemistry were elucidated by spectroscopic methods and chemical derivatization. These compounds showed a significant neuritogenic activity at 30 microM against PC12 cells that was comparable to that seen for the best nerve growth factor (NGF) concentration of 40 ng/mL. Gentisides A and B showed parallel activity, indicating that the observed structural difference at the end of their alkyl chain did not affect neuritogenic activity.

Hyperbaric oxygenation alleviates MCAO-induced brain injury and reduces hydroxyl radical formation and glutamate release.

The present study examined the effect of hyperbaric oxygen (HBO) on the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), the products of salicylate trapping of hydroxyl free radicals, and glutamate release in the striatum during acute ischemia and reperfusion. Non-HBO rats (n = 8) were subjected to 1-h ischemia. Study rats (n = 8) were treated with HBO at 2.8 ATA for 1 h during ischemia. Artificial CSF solution containing 5 mM sodium salicylate was perfused at 1 microl/min. Samples were continuously collected at 15 min intervals and the levels of 2,3-DHBA, 2,5-DHBA, and glutamate were analyzed. The lesion volume was determined by TTC stain. Occlusion of the middle cerebral artery induced a significant increase in the levels of 2,3-DHBA and 2,5-DHBA. A peak of approximately two and fourfold of baseline levels was reached at 45 min and was maintained at elevated levels during reperfusion. The level of glutamate increased approximately two times at 30 min during ischemia, continued to increase, and reached approximately three times baseline level during reperfusion. HBO significantly alleviated brain injury associated with decreased levels of 2,3-DHBA, 2,5-DHBA and glutamate. This study suggests that the decreased glutamate release and the reduced formation of hydroxyl free radicals might contribute to the neuroprotective effect of HBO.

[Studies on the chemical constituents of Sarcopyramis nepalensis].

OBJECTIVE: To study the chemical constituents of Sarcopyramis nepalensis. METHODS: The constituents were isolated and purified with chromatography and the structures were elucidated by spectral analysis. RESULTS: Ten compounds were isolated and their structures were identified as: (E)-1-(3,4-dihydroxy-phenyl) ethyl acrylate (I), stearic acid (II), palmitic acid (III), 4-hydroxybenzonic acid (IV), 3,4-dihydroxy benzoic acid (V), gentisic acid ( VI), gallic acid (VII), beta-sitosterol (VIII), daucosterol (IX), stigmasterolstearate (X). CONCLUSION: Compounds I , IV, V, VI, VII are isolated from this plant for the first time.

Acarbose reduces myocardial infarct size by preventing postprandial hyperglycemia and hydroxyl radical production and opening mitochondrial KATP channels in rabbits.

BACKGROUND: Acarbose, an antidiabetic drug, is an alpha-glucosidase inhibitor that can inhibit glucose absorption in the intestine. A recent large-scale clinical trial, STOP-NIDDM, showed that acarbose reduces the risk of myocardial infarction. We examined whether acarbose reduces myocardial infarct size and investigated its mechanisms. METHODS AND RESULTS: Rabbits were fed with 1 of 2 diets in this study: normal chow, 30 mg acarbose per 100 g chow for 7 days. Rabbits were assigned randomly to 1 of 4 groups: control (n = 10), acarbose (n = 10), acarbose + 5HD (n = 10, intravenous 5 mg/kg of 5-hydroxydecanoate), and 5HD (n = 10, intravenous 5 mg/kg of 5HD). Rabbits then underwent 30 minutes of coronary occlusion followed by 48-hour reperfusion. Postprandial blood glucose levels were higher in the control group than in the acarbose group. The infarct size as a percentage of the left ventricular area at risk was reduced significantly in the acarbose (19.4% +/- 2.3%) compared with the control groups (42.8% +/- 5.4%). The infarct size-reducing effect of acarbose was abolished by 5HD (43.4% +/- 4.7%). Myocardial interstitial 2,5-dihydroxybenzoic acid levels, an indicator of hydroxyl radicals, increased during reperfusion after 30 minutes of ischemia, but this increase was inhibited in the acarbose group. This was reversed by 5HD. CONCLUSION: Acarbose reduces myocardial infarct size by opening mitochondrial KATP channels, which may be related to the prevention of postprandial hyperglycemia and hydroxyl radical production.