Synergism with Shikimic Acid Restores ß-Lactam Antibiotic Activity against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved into a dangerous pathogen resistant to beta-lactam antibiotics (BLAs) and has become a worrisome superbug. In this study, a strategy in which shikimic acid (SA), which has anti-inflammatory and antibacterial activity, is combined with BLAs to restart BLA activity was proposed for MRSA treatment. The synergistic effects of oxacillin combined with SA against oxacillin resistance in vitro and in vivo were investigated. The excellent synergistic effect of the oxacillin and SA combination was confirmed by performing the checkerboard assay, time-killing assay, live/dead bacterial cell viability assay, and assessing protein leakage. SEM showed that the cells in the control group had a regular, smooth, and intact surface. In contrast, oxacillin and SA or the combination treatment group exhibited different degrees of surface collapse. q-PCR indicated that the combination treatment group significantly inhibited the expression of the mecA gene. In vivo, we showed that the combination treatment increased the survival rate and decreased the bacterial load in mice. These results suggest that the combination of oxacillin with SA is considered an effective treatment option for MRSA, and the combination of SA with oxacillin in the treatment of MRSA is a novel strategy.

Exploiting the synergistic antibacterial activity of shikimic acid and ceftiofur against methicillin-resistant Staphylococcus aureus.

Efforts to curtail the escalating health threat posed by methicillin-resistant Staphylococcus aureus (MRSA), a formidable superbug, necessitate the development of innovative treatment strategies. Leveraging potential compounds from natural sources in tandem with antibiotics has emerged as a promising approach against MRSA. These strategies should enhance the antibiotic efficacy, reduce dosage and toxicity, and bypass MRSA resistance. In this study, we used a checkerboard assay to illustrate the significant synergistic anti-MRSA effect of shikimic acid (SA), a naturally occurring compound, and ceftiofur (CF). Time-kill curves further revealed that a combination of 1/4 of the minimum inhibitory concentration (MIC) of SA and 1/8 MIC of the sodium CF eradicated MRSA within 2 h, with no noticeable toxicity observed with these concentrations. In vivo experiments confirmed that this combination therapy demonstrated robust antimicrobial activity against MRSA-induced bacteremia in mice, significantly reducing bacterial loads in the kidneys, liver, and spleen, attenuating inflammatory cell infiltration, and alleviating pathological damage. This study not only offers a compelling strategy, capitalizing on the synergistic potential of SA and CF, to rapidly address antibiotic resistance but also contributes significantly to the refinement of antimicrobial therapeutic strategies.

Effects of direct-fed microbial supplement on ruminal and plasma metabolome of early-lactation dairy cows: Untargeted metabolomics approach.

We examined the effects of 2 multispecies direct-fed microbial (DFM) supplements on ruminal and plasma metabolome of early-lactation dairy cows using a high-coverage untargeted metabolomics approach. A total of 45 multiparous Holstein cows (41 ± 7 DIM) were enrolled for the 14-d pre-experimental and 91-d experimental period and were a subset from a lactation performance study, which used 114 cows. Cows were blocked using pre-experimental energy-corrected milk yield and randomly assigned within each block to 1 of 3 treatments: (1) corn silage-based diet with no DFM supplement (control; CON), (2) basal diet top-dressed with a mixture of Lactobacillus animalis and Propionibacterium freudenreichii at 3 × 109 cfu/d (PRO-A), or (3) basal diet top-dressed with a mixture of L. animalis, P. freudenreichii, Bacillus subtilis, and Bacillus licheniformis at 11.8 × 109 cfu/d (PRO-B). The basal diet was fed ad libitum daily as a TMR at 0600 and 1200 h for a duration of 91 d. Rumen fluid and blood samples were taken on d -3, 28, 49, 70, and 91 and immediately stored at -80°C. Before analysis, ruminal and plasma samples from d 28, 49, 70, and 91 were composited. An in-depth, untargeted metabolome profile of the composite rumen and plasma samples and the d -3 samples was developed by using a chemical isotope labeling/liquid chromatography-mass spectrometry (LC-MS)-based technique. Differentially abundant metabolites (taking into account fold change [FC] values and false discovery rates [FDR]) were identified with a volcano plot. In the rumen, compared with the CON diet, supplemental PRO-A increased (FC ≥1.2; FDR ≤0.05) the relative concentrations of 9 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, quinolinic acid, and shikimic acid, and PRO-B increased relative concentrations of 16 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, 16-hydroxypalmitic acid, and 2 propionate precursors (succinic and methylsuccinic acids). Relative to PRO-A, supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative rumen concentrations of 3 metabolites, 16-hydroxypalmitic acid, indole-3-carboxylic acid, and 5-aminopentanoic acid, but reduced relative rumen concentrations of 13 metabolites, including carnitine, threonic acid, and shikimic acid. Compared with the CON diet, relative concentrations of 13 plasma metabolites, including myxochelin A and glyceraldehyde, were increased (FC ≥1.2; FDR ≤0.05) by PRO-A supplementation, whereas those of 9 plasma metabolites, including 4-(2-aminophenyl)-2,4-dioxobutanoic acid, N-acetylornithine, and S-norlaudanosolin, were reduced (FC ≤0.83; FDR ≤0.05). Supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative concentrations of 9 plasma metabolites, including trans-o-hydroxybenzylidenepyruvic acid and 3-methylsalicylaldehyde, and reduced relative concentrations of 4 plasma metabolites, including ß-ethynylserine and kynurenine. Pathway analysis of the differentially abundant metabolites in both rumen and plasma revealed that these metabolites are involved in AA and fatty acid metabolism and have antimicrobial and immune-stimulating properties. The results of this study demonstrated that dietary supplementation with either PRO-A or PRO-B altered the plasma and ruminal metabolome. Notably, ruminal and plasma metabolites involved in the metabolism of AA and fatty acids and those with immunomodulatory properties were altered by either or both of the 2 microbial additives.

An overview of mechanism and chemical inhibitors of shikimate kinase.

Tuberculosis is a highly infectious disease other than HIV/AIDS and it is one of the top ten causes of death worldwide. Resistance development in the bacteria occurs because of genetic alterations, and the molecular insights suggest that the accumulation of mutation in the individual drug target genes is the primary mechanism of multi-drug resistant tuberculosis. Chorismate is an essential structural fragment for the synthesis of aromatic amino acids and synthesized biochemically by a number of bacteria, including Mycobacterium tuberculosis, utilizing the shikimate pathway. This shikimate kinase is the newer possible target for the generation of novel antitubercular drug because this pathway is expressed only in mycobacterium and not in Mammals. The discovery and development of shikimate kinase inhibitors provide an opportunity for the development of novel selective medications. Multiple shikimate kinase inhibitors have been identified via insilico virtual screening and related protein-ligand interactions along with their in-vitro studies. These inhibitors bind to the active site in a similar fashion to shikimate. In the current review, we present an overview of the biology and chemistry of the shikimate kinase protein and its inhibitors, with special emphasis on the various active scaffold against the enzyme. A variety of chemically diversified synthetic scaffolds including Benzothiazoles, Oxadiazoles, Thiobarbiturates, Naphthoquinones, Thiazoleacetonitriles, Hybridized Pyrazolone derivatives, Orthologous biological macromolecule derivatives, Manzamine Alkaloids derivatives, Dipeptide inhibitor, and Chalcones are discussed in detail. These derivatives bind to the specific target appropriately proving their potential ability through different binding interactions and effectively explored as an effective and selective Sk inhibitor.Communicated by Ramaswamy H. Sarma.

Characterization and antibacterial activity of ruthenium-based shikimate cross-linked chitosan composites.

The unsustainable antibacterial activity of ruthenium antibacterial agents is an important factor limiting their applications. This present work attempts to prepare ruthenium (Ru) coordination polymer composites with chitosan quaternary ammonium polymers (CQ) and shikimic acid (SA) through the interaction of ionic bonds and covalent bonds by microwave-assisted high-pressure homogenization methods. The prepared CQ@Ru-SA was characterized by size distribution, zeta potential, TEM, UV-vis, FTIR, XPS and XRD analyses. The coordination structure and morphology of Bridge-CQ-NH-Ru-SA were verified. The CQ@Ru-SA was well-dispersed in both the aqueous or anhydrous states. MIC and MBC, time-killing curves, biofilm formation inhibition assay, mature biofilm disruption assay, SEM, Ca2+ mobilization assay and Ca2+-Mg2+-ATPase activity studies revealed that CQ@Ru-SA had a stronger inhibitory effect against S. aureus than CQ and showed sustained antibacterial properties in the dynamic time-killing curves. Meanwhile, CQ@Ru-SA had good antibacterial effects against S. aureus and inhibited their biofilm forming ability in a dose-dependent manner. Further studies on antibacterial mechanisms revealed that CQ@Ru-SA influenced cell membrane integrity, Ca2+-Mg2+-ATPase activity on the cell membrane and intracellular Ca2+ levels of S. aureus. This study will provide the necessary data for the further design and development of ruthenium-based photosensitive antibacterial agents.

Inhibition of Biofilm Formation and Virulence Factors of Cariogenic Oral Pathogen Streptococcus mutans by Shikimic Acid.

Streptococcus mutans is known as an important oral pathogen causing dental caries, a widespread oral infectious disease. S. mutans synthesize exopolysaccharide (EPS) using glucosyltransferases (Gtfs), resulting in biofilm formation on the tooth surface. Bacterial cells in the biofilms become strongly resistant to a harsh environment, such as antibiotics and host defense mechanisms, making biofilm-based infections difficult to eliminate. Discovering novel antibiofilm agents, especially from natural products, helps to develop effective strategies against this kind of diseases. The present study investigated the inhibitory effect of shikimic acid (SA), one abundant compound derived from Illicium verum extract, on the biofilm formation of S. mutans. We found SA can reduce the EPS synthesized by this oral pathogen and modulate the transcription of biofilm formation related genes, leading to fewer bacterial cells in its biofilm. SA also interacted with cell membrane and membrane proteins, causing damage to bacterial cells. Ex vivo testing of biofilm formation on bovine teeth showed SA strongly decreased the number of S. mutans cells and the number of EPS accumulated on dental enamel surfaces. Moreover, SA exhibits almost no toxicity to human oral cells evaluated by in vitro biocompatibility assay. In conclusion, shikimic acid exhibits remarkable antibiofilm activity against S. mutans and has the potential to be further developed as a novel anticaries agent. IMPORTANCE Natural products are an important and cost-effective source for screening antimicrobial agents. Here, we identified one compound, shikimic acid, from Illicium verum extract, exhibiting antimicrobial activity against S. mutans proliferation. It also inhibits biofilm formation of this bacteria through decreasing Gtf expression and EPS synthesis. Furthermore, this compound exhibits no significant cytotoxicity at its MIC against S. mutans, providing evidence for its clinical application.

Natural Xanthine Oxidase Inhibitor 5-O-Caffeoylshikimic Acid Ameliorates Kidney Injury Caused by Hyperuricemia in Mice.

Xanthine oxidase (XOD) inhibition has long been considered an effective anti-hyperuricemia strategy. To identify effective natural XOD inhibitors with little side effects, we performed a XOD inhibitory assay-coupled isolation of compounds from Smilacis Glabrae Rhizoma (SGR), a traditional Chinese medicine frequently prescribed as anti-hyperuricemia agent for centuries. Through the in vitro XOD inhibitory assay, we obtained a novel XOD inhibitor, 5-O-caffeoylshikimic acid (#1, 5OCSA) with IC50 of 13.96 µM, as well as two known XOD inhibitors, quercetin (#3) and astilbin (#6). Meanwhile, we performed in silico molecular docking and found 5OCSA could interact with the active sites of XOD (PDB ID: 3NVY) with a binding energy of -8.6 kcal/mol, suggesting 5OCSA inhibits XOD by binding with its active site. To evaluate the in vivo effects on XOD, we generated a hyperuricemia mice model by intraperitoneal injection of potassium oxonate (300 mg/kg) and oral gavage of hypoxanthine (500 mg/kg) for 7 days. 5OCSA could inhibit both hepatic and serum XOD in vivo, together with an improvement of histological and multiple serological parameters in kidney injury and HUA. Collectively, our results suggested that 5OCSA may be developed into a safe and effective XOD inhibitor based on in vitro, in silico and in vivo evidence.

Shikimic acid protects skin cells from UV-induced senescence through activation of the NAD+-dependent deacetylase SIRT1.

UV radiation is one of the main contributors to skin photoaging by promoting the accumulation of cellular senescence, which in turn induces a proinflammatory and tissue-degrading state that favors skin aging. The members of the sirtuin family of NAD+-dependent enzymes play an anti-senescence role and their activation suggests a promising approach for preventing UV-induced senescence in the treatment of skin aging. A two-step screening designed to identify compounds able to protect cells from UV-induced senescence through sirtuin activation identified shikimic acid (SA), a metabolic intermediate in many organisms, as a bona-fide candidate. The protective effects of SA against senescence were dependent on specific activation of SIRT1 as the effect was abrogated by the SIRT1 inhibitor EX-527. Upon UV irradiation SA induced S-phase accumulation and a decrease in p16INK4A expression but did not protect against DNA damage or increased polyploidies. In contrast, SA reverted misfolded protein accumulation upon senescence, an effect that was abrogated by EX-527. Consistently, SA induced an increase in the levels of the chaperone BiP, resulting in a downregulation of unfolded protein response (UPR) signaling and UPR-dependent autophagy, avoiding their abnormal hyperactivation during senescence. SA did not directly activate SIRT1 in vitro, suggesting that SIRT1 is a downstream effector of SA signaling specifically in the response to cellular senescence. Our study not only uncovers a shikimic acid/SIRT1 signaling pathway that prevents cellular senescence, but also reinforces the role of sirtuins as key regulators of cell proteostasis.

A simple and effective method for the preparation of high-purity shikimic acid from chromatography wash effluent of Ginkgo biloba leaf extract by macroporous resin considering the effect of varying feed solution compositions.

OBJECTIVES: The present study investigated the feasibility of preparing high-purity shikimic acid (SA) from the chromatography wash effluent of Ginkgo biloba leaf extract by macroporous resin. METHODS: First, static/dynamic adsorption and desorption were conducted to screen out the optimal resin. Second, the key parameters of the chromatographic process were optimised with face-centred central composite design (CCD). Third, wash effluent indices were measured, different batches of wash effluent were used to prepare SA under the optimised parameters, and the effect of varying feed solution compositions on final products was investigated. KEY FINDINGS: It was found that the final purity and recovery rate of SA prepared with ADS-21 resin were not lower than 70 and 60%, respectively, when the purity of SA in the wash effluent was higher than 21.4%. The quality of the final product can be predicted based on the properties of wash effluent. CONCLUSIONS: The proposed method could not only provide a simple, green and promising approach for the large-scale purification of SA from wash effluent but also be used to develop process intermediate quality standards for other natural products.

Structural investigation on damaged hair keratin treated with α,ß-unsaturated Michael acceptors used as repairing agents.

Many restoring formulations for damaged hair keratin have been developed. Some patents claim that the hair repair occurs through the reconstruction of disulfide bridges of keratin, through α,ß-unsaturated Michael acceptors, such as shikimic acid and bis-aminopropyl diglycol dimaleate. To gain more insights into the possible repairing mechanism, this study is aimed at assessing, by IR and Raman spectroscopies coupled to scanning electron microscopy (SEM), the structural changes induced in keratin from bleached hair by the treatment with commercial reconstructive agents as well as shikimic acid and dimethyl maleate, chosen as model compounds. Vibrational spectroscopy revealed that shikimic acid- and maleate-based restoring agents interacted with hair fibers modifying both their cortex and cuticle regions. None of the investigated treatments induced an increase in the SS disulfide bridges content of the hair cortex, although it cannot be excluded that this phenomenon could have occurred in the cuticle. SS rearrangements were found to occur. None of our results can be interpreted as direct evidence of the sulfa-Michael reaction/cross-linking. From a morphological point of view, beneficial effects of the restoring agents were observed by SEM analyses, in terms of a more regular hair surface and more imbricated scales.

[Shikimic acid extracted from Chaenomeles speciosa reduces chondrocyte differentiation by inhibiting RBL-2H3 cell degranulation].

Objective To investigate the reducing effects of shikimic acid from the total extract of Chaenomeles speciose on the differentiation of chondrocytes into hypertrophic chondrocytes by inhibiting RBL-2H3 cell degranulation. Methods The chondrocytes were identified by toluidine blue staining and tryptase immunohistochemical staining. The chondrocytes were divided into normal chondrocytes control group, C48/80 activated RBL-2H3 cell culture supernatant treatment group, 3, 10 and 30 µg/mL SA activated RBL-2H3 cell culture supernatant treatment groups. The toxicity of SA and RBL-2H3 cell supernatant were detected by MTT assay. Western blotting was used to detect the expression of collagen type II (Col2) and collagen type X (Col10) in chondrocytes. The levels of matrix metalloproteinase 13 (MMP13), soluble nuclear factor B receptor activated protein ligand (sRANKL) and bone protective factor (OPG) were determined by ELISA, and glycosaminoglycan polysaccharide (GAG) were tested by dimethylmethylene blue (DMB) colorimetry. Results (0~30) µg/mL SA had no significant effects on the growth of chondrocytes. Compared with the C48/80 activated RBI-2H3 cell supernatant treatment group, the expression of Col2 and GAG proteins increased significantly, while the expression of Col10 and MMP13 and the ratio of sRANKL/OPG decreased significantly in the SA treatment groups in a dose-dependent manner. Conclusion SA can effectively reduce the differentiation of chondrocytes into hypertrophic chondrocytes by inhibiting RBL-2H3 cell degranulation.

Virucidal Action Against Avian Influenza H5N1 Virus and Immunomodulatory Effects of Nanoformulations Consisting of Mesoporous Silica Nanoparticles Loaded with Natural Prodrugs.

BACKGROUND: Combating infectious diseases caused by influenza virus is a major challenge due to its resistance to available drugs and vaccines, side effects, and cost of treatment. Nanomedicines are being developed to allow targeted delivery of drugs to attack specific cells or viruses. MATERIALS AND METHODS: In this study, mesoporous silica nanoparticles (MSNs) functionalized with amino groups and loaded with natural prodrugs of shikimic acid (SH), quercetin (QR) or both were explored as a novel antiviral nanoformulations targeting the highly pathogenic avian influenza H5N1 virus. Also, the immunomodulatory effects were investigated in vitro tests and anti-inflammatory activity was determined in vivo using the acute carrageenan-induced paw edema rat model. RESULTS: Prodrugs alone or the MSNs displayed weaker antiviral effects as evidenced by virus titers and plaque formation compared to nanoformulations. The MSNs-NH2-SH and MSNs-NH2-SH-QR2 nanoformulations displayed a strong virucidal by inactivating the H5N1 virus. They induced also strong immunomodulatory effects: they inhibited cytokines (TNF-α, IL-1ß) and nitric oxide production by approximately 50% for MSNs-NH2-SH-QR2 (containing both SH and QR). Remarkable anti-inflammatory effects were observed during in vivo tests in an acute carrageenan-induced rat model. CONCLUSION: Our preliminary findings show the potential of nanotechnology for the application of natural prodrug substances to produce a novel safe, effective, and affordable antiviral drug.

Shikimic acid, a mannose bioisostere, promotes hair growth with the induction of anagen hair cycle.

Shikimic acid (SA) has recently been found to be a major component of plant stem cells. The exact effects of SA on human hair follicles (HFs) is unknown. The purpose of this study was to examine the effects of SA on hair growth. We investigated the effect of SA on an in vivo C57BL/6 mouse model. We examined the expression of mannose receptor (MR), which is a known receptor of SA, in human HFs and the effect of SA on human dermal papilla cells (hDPCs), outer root sheath cells (hORSCs), and on ex vivo human hair organ culture. SA significantly prolonged anagen hair growth in the in vivo mouse model. We confirmed expression of the MR in human HFs, and that SA increased the proliferation of hDPCs and hORSCs. It was found that SA enhanced hair shaft elongation in an ex vivo human hair organ culture. SA treatment of hDPCs led to increased c-myc, hepatocyte growth factor, keratinocyte growth factor and vascular endothelial growth factor levels and upregulation of p38 MAPK and cAMP response element-binding protein levels. Our results show that SA promotes hair growth and may serve as a new therapeutic agent in the treatment of alopecia.

Mangiterpenes A-C and 2',3'-seco-manginoid C, four sesquiterpene/monoterpene-shikimate-conjugated spirocyclic meroterpenoids from Guignardia mangiferae.

Mangiterpenes A-C and 2',3'-seco-manginoid C, four undescribed sesquiterpene/monoterpene-shikimate-conjugated meroterpenoids with spiro ring systems, were isolated from Guignardia mangiferae. The structures and absolute configurations of these compounds were established by comprehensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. Mangiterpenes A-C represent the first examples of sesquiterpene-shikimate-conjugated spirocyclic meroterpenoids, and 2',3'-seco-manginoid C features an unexpected 2',3'-seco-manginoids skeleton. Mangiterpene C strongly inhibited the production of NO inducted by LPS, with an IC50 value of 5.97 µM. It showed an anti-inflammatory effect by means of blocking in the NF-κB signaling pathway and decreasing the expression of inflammatory mediators.

Potential Mechanisms of 3, 4-Oxo-Isopropylidene-Shikimic Acid in Ameliorating 2, 4, 6-Trinitrobenzenesulfonic Acid-Induced Colitis in Rats.

Previously, we reported that 3, 4-oxo-isopropylidene-shikimic acid (ISA) has therapeutic potential in experimental colitis in rats. This study aimed to elucidate the potential mechanisms of ISA on the inflammatory response in rats with 2, 4, 6-trinitrobenzenesulfonic acid-induced colitis. After the induction of colitis, rats were orally administered ISA for 12 days. Then, the expression levels of inflammatory cytokines, cell adhesion molecules, and matrix metalloproteinase (MMP) in the blood and colon tissues, and the protein level of nuclear factor kappa B (NF-κB) p65 in cytoplasm and nucleus of colon tissues were evaluated. As a result, an enhanced inflammatory response was observed in rats with experimental colitis. However, the treatment with ISA significantly ameliorated the inflammatory response, which was manifested as a significant decrease in the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, interferon (IFN)-γ, IL-8, TNF-α mRNA, P-selectin, E-selectin, intercellular cell adhesion molecule-1, MMP9 and MMP9 mRNA in rat blood and colon tissues, respectively, and a significant decrease in the levels of IFN-γ/IL-4, and the NF-κBp65 activity coefficient. Therefore, the therapeutic effect of ISA on experimental colitis may be related to its inhibitory effect on the expression of cytokines, adhesion molecules, and MMP9, which may be involved in the inhibition of the activation and nuclear translocation of NF-κBp65.

Shikimic acid promotes estrogen receptor(ER)-positive breast cancer cells proliferation via activation of NF-κB signaling.

Shikimic acid (SA), a widely-known hydroaromatic compound enriched in Bracken fern and Illicium verum (also known as Chinese star anise), increases the risk of gastric and esophageal carcinoma, nevertheless, the influence of SA on breast cancer remains indistinct. Herein we found that, with models in vitro, SA significantly promoted estrogen receptor(ER) positive cells proliferation and NF-κB activation was involved in it. Moreover, our data showed that IκBα, a critically endogenous inhibitor of NF-κB, was repressed. Subsequently, we found increase of miR-300 by SA treatment sand miR-300 could target IκBα mRNA. Additionally, inhibition of miR-300 abrogated the repression of IκBα by SA. As a result, miR-300 was also involved in NF-κB activation and breast cancer cells proliferation promotion due to SA exposure. Taken together, with ER-positive breast cancer cell models in vitro, MCF-7 and T47D, our results implied that SA promoted breast cancer cells proliferation via a miR-300-induced NF-κB dependent pathway controlling cell cycle proteins.

Hypolipogenic Effect of Shikimic Acid Via Inhibition of MID1IP1 and Phosphorylation of AMPK/ACC.

Although shikimic acid from Illicium verum has antioxidant, antibacterial, anti-inflammatory, and analgesic effects, the effect of shikimic acid on lipogenesis has not yet been explored. Thus, in the present study, hypolipogenic mechanism of shikimic acid was examined in HepG2, Huh7 and 3T3-L1 adipocyte cells. Shikimic acid showed weak cytotoxicity in HepG2, Huh7 and 3T3-L1 cells, but suppressed lipid accumulation in HepG2, Huh7 and 3T3-L1 cells by Oil Red O staining. Also, shikimic acid attenuated the mRNA expression of de novo lipogenesis related genes such as FAS, SREBP-1c, and LXR-α in HepG2 cells by RT-PCR analysis and suppressed the protein expression of SREBP-1c and LXR-α in HepG2 and 3T3-L1 cells. It should be noted that shikimic acid activated phosphorylation of AMP-activated protein kinase (AMPK)/Aacetyl-coenzyme A carboxylase (ACC) and reduced the expression of MID1 Interacting Protein 1 (MID1IP1) in HepG2, Huh7 and 3T3-L1 cells. Conversely, depletion of MID1IP1 activated phosphorylation of AMPK, while overexpression of MID1IP1 suppressed phosphorylation of AMPK in HepG2 cells. However, AMPK inhibitor compound c did not affect the expression of MID1IP1, indicating MID1IP1 as an upstream of AMPK. Taken together, our findings suggest that shikimic acid has hypolipogenic effect in HepG2 and 3T3-L1 cells via phosphorylation of AMPK/ACC and inhibition of MID1IP1 as a potent candidate for prevention or treatment of fatty liver and hyperlipidemia.

The regulatory role of shikimate in plant phenylalanine metabolism.

In higher plants, the amino acid phenylalanine is a substrate of both primary and secondary metabolic pathways. The primary pathway that consumes phenylalanine, protein biosynthesis, is essential for the viability of all cells. Meanwhile, the secondary pathways are not necessary for the survival of individual cells, but benefit of the plant as a whole. Here we focus on the monolignol pathway, a secondary metabolic pathway in the cytosol that rapidly consumes phenylalanine to produce the precursors of lignin during wood formation. In planta monolignol biosynthesis involves a series of seemingly redundant steps wherein shikimate, a precursor of phenylalanine synthesized in the plastid, is transiently ligated to the main substrate of the pathway. However, shikimate is not catalytically involved in the reactions of the monolignol pathway, and is only needed for pathway enzymes to recognize their main substrates. After some steps the shikimate moiety is removed unaltered, and the main substrate continues along the pathway. It has been suggested that this portion of the monolignol pathway fulfills a regulatory role in the following way. Low phenylalanine concentrations (viz. availability) correlate with low shikimate concentrations. When shikimate concentratios are low, flux into the monolignol pathway will be limited by means of the steps requiring shikimate. Thus, when the concentration of phenylalanine is low it will be reserved for protein biosynthesis. Here we employ a theoretical approach to test this hypothesis. Simplified versions of plant phenylalanine metabolism are modelled as systems of ordinary differential equations. Our analysis shows that the seemingly redundant steps can be sufficient for the prioritization of protein biosynthesis over the monolignol pathway when the availability of phenylalanine is low, depending on system parameters. Thus, the phenylalanine precursor shikimate may signal low phenylalanine availability to secondary pathways. Because our models have been abstracted from plant phenylalanine metabolism, this mechanism of metabolic signalling, which we call the Precursor Shutoff Valve (PSV), may also be present in other biochemical networks comprised of two pathways that share a common substrate.

Shikimic acid from Artemisia absinthium inhibits protein glycation in diabetic rats.

This study investigated the impact of Shikimic Acid (SA) obtained from leaves of Artemisia absinthium on protein glycation in the retina of diabetic rats. The GC/MS analysis of A. absinthium showed that the most abundant bioactive compound was SA (C7H10O5) with a measured retention Index (RI) of 1960 compared to that of the reference sample (1712). Male albino rats were divided into two main groups, Group I (control) and Group II (diabetic); Group II was further divided into four subgroups: Group IIa (diabetic control), Group IIb (diabetic rats were given SA orally [50 mg/kg, body weight (bw)/day], Group IIc diabetic rats were given SA orally [100 mg/kg, bw/day], and Group IId (diabetic rats were given metformin orally [100 mg/kg, bw/day] as positive control). The data obtained suggested that SA reduced glucose and glycated hemoglobin levels. In addition, SA also decreased the formation of glucose-derived advanced glycation end products. Interestingly, SA showed interference with the release of inflammatory mediators in retina and possess antioxidant potential. In conclusion, SA protected the tissues from detrimental effects of hyperglycemia and enhanced antioxidant activity. SA could be a potential lead in the process of drug development in the future to prevent retinopathy in diabetic subjects.

Shikimic Acid Inhibits Osteoclastogenesis in Vivo and in Vitro by Blocking RANK/TRAF6 Association and Suppressing NF-κB and MAPK Signaling Pathways.

BACKGROUND/AIMS: Bone homeostasis is associated with the balance between bone-resorbing osteoclasts and bone-forming osteoblasts. Unbalanced bone homeostasis as a result of reduced osteogenesis or excessive osteoclastogenesis can lead to disorders such as osteoporosis, Paget's disease, and rheumatoid arthritis. Shikimic acid is a cyclohexanecarboxylic acid, reported to exhibit pharmacological properties including anti-inflammatory and antioxidant activities. However, its effects on bone homeostasis remain unknown. METHODS: First, the in vitro MTT cell viability assay was performed. Tartrate-resistant acid phosphatase (TRAP) and actin ring formation assays, as well as immunofluorescence staining were then performed to evaluate osteoclastogenesis. Potential signaling pathways were characterized by western blotting and verified in overexpression experiments. Related factors were examined by western blotting, reverse transcription polymerase chain reaction, electrophoretic mobility shift assay, and co-immunoprecipitation. Ovariectomized mice were used for the in vivo study. RESULTS: TRAP staining showed that shikimic acid significantly inhibited osteoclastogenesis and pit resorption in bone marrow monocytes and RAW264.7 cells, and actin ring formation assays showed that shikimic acid suppressed the bone resorption function of osteoclasts. Furthermore, shikimic acid inhibited the receptor activator of nuclear factor-κB RANK/tumor necrosis factor receptor-associated factor 6 (TRAF6) association, suppressed nuclear factor-κB and mitogen-activated protein kinase signaling pathways, and downregulated nuclear factor of activated T-cell cytoplasmic 1. The expression of osteoclastogenesis biomarkers, including TRAF6, calcitonin receptor, TRAP, cathepsin K, and matrix metalloproteinase-9, was inhibited. In vivo, shikimic acid also significantly ameliorated bone loss and prevented osteoclastogenesis in ovariectomized mice. CONCLUSION: Shikimic acid inhibited osteoclastogenesis and osteoclast function by blocking RANK ligand-induced recruitment of TRAF6, as well as downstream signaling pathways in vitro. Shikimic acid also reduced ovariectomy-induced osteoclastogenesis and bone loss in vivo.