Inhibitory Effect Of Chlorogenic Acid Research Articles

Modeling Inhibitory Effects of Chlorogenic Acid on Amyloid Beta Aggregation

Modeling Inhibitory Effects of Chlorogenic Acid on Amyloid Beta Aggregation

Synergistic interaction of Cu(II) with caffeic acid and chlorogenic acid in α-glucosidase inhibition.

Phenolic acids are widespread in foods and are beneficial to human health. However, the role of metal ions in influencing the binding of proteins with phenolic acids that contain the same parent nucleus structure remains unclear. This study investigated the inhibitory effect of caffeic acid (CA) and chlorogenic acid (CHA) on α-glucosidase and the biological effect of copper on this process. It was found that the esterification of CA with quinic acid could increase the fluorescence quenching, conformational change, and inhibitory effect of CHA on α-glucosidase. Copper ions reduced their fluorescence quenching and conformation-changing ability by binding to the neighboring phenolic hydroxyl group but also increased their ability to alter secondary structure and to inhibit α-glucosidase and in vitro anti-glycation. Overall, this study shows that the binding of copper ions to the phenolic hydroxyl group adjacent to CA and CHA synergistically inhibited α-glucosidase. The findings will offer a theoretical basis for investigating the properties of metal ions and phenolic acid in food chemistry and their potential applications in the prevention and treatment of diabetes mellitus. © 2023 Society of Chemical Industry.

Inhibitory Effects of Chlorogenic Acid Containing Green Coffee Bean Extract on Lipopolysaccharide-Induced Inflammatory Responses and Progression of Colon Cancer Cell Line.

An inflammatory response, related to colorectal cancer (CRC) progression, is a major subsequent result of bacterial infection following CRC surgery and should be of serious concern. Lipopolysaccharide (LPS), from the bacterial membrane, is a vital mediator of this event through binding with a Toll-like receptor 4 (TLR4) and activating through NF-κB in CRC. To identify a novel inhibitor of LPS-induced colon cancer cells (SW480), green coffee bean extract (GBE) was investigated. Ethyl acetate insoluble fraction (EIF) was mainly collected from GBE and classified as chlorogenic acid (CGA)-rich fractions. EIF and CGA inhibited TLR4 expression in LPS-induced SW480 cells. However, EIF was more dominant than CGA, via inhibition of expression and secretion of several associated mediators in inflammatory responses and CRC metastasis through NF-κB inactivation, which resulted in the abrogation of CRC migration and invasion. Thus, CGA-rich fraction from GBE can be further developed as an alternative treatment, coupled with CRC surgical treatment, to increase therapeutic efficiency and survival rate.

Chlorogenic Acid Inhibited Epithelial-Mesenchymal Transition to Treat Pulmonary Fibrosis through Modulating Autophagy.

Chlorogenic acid (CGA), derived from dicotyledons and ferns, has been demonstrated with anti-inflammatory, anti-bacterial, and free radical-scavenging effects and can be used to treat pulmonary fibrosis (PF). However, the specific mechanism by which CGA treats PF needs to be further investigated. In this study, in vivo experiment was firstly performed to evaluate the effects of CGA on epithelial-mesenchymal transition (EMT) and autophagy in bleomycin (BLM)-induced PF mice. Then, the effects of CGA on EMT and autophagy was assessed using transforming growth factor beta (TGF-β) 1-induced EMT model in vitro. Furthermore, autophagy inhibitor (3-methyladenine) was used to verify that the inhibitory mechanism of CGA on EMT was associated with activating autophagy. Our results found that 60 mg/kg of CGA treatment significantly ameliorated lung inflammation and fibrosis in mice with BLM-induced PF. Besides, CGA inhibited EMT and promoted autophagy in mice with PF. In vitro studies also demonstrated that 50 µM of CGA treatment inhibited EMT and induced autophagy related factors in TGF-β1-induced EMT cell model. Moreover, the inhibitory effect of CGA on autophagy and EMT in vitro was abolished after using autophagy inhibitor. In conclusion, CGA could inhibit EMT to treat BLM-induced PF in mice through, activating autophagy.

Chlorogenic acid induces endoplasmic reticulum stress in Botrytis cinerea and inhibits gray mold on strawberry

Chlorogenic acid (CGA) is a plant-derived compound with antifungal activity. However, the antifungal mechanism of CGA remains not fully revealed. The antifungal effect of CGA against Botrytis cinerea was assessed in the present study, and its mode of action was studied. Results demonstrated that CGA suppressed the growth of B. cinerea, as well as the development of gray mold (B. cinerea) on strawberry. Further analysis indicated that CGA induced endoplasmic reticulum (ER) stress in hyphae of B. cinerea resulting in an efflux of calcium (Ca2+) from the ER to the cytoplasm and then into mitochondria. The Ca2+ overload in mitochondria elicited the Ca2+-calmodulin-calcineurin cascade, resulting in a superoxide burst in the mitochondria. Finally, a decrease in ATP content and cell viability was observed in B. cinerea. This is the first report showing that the inhibitory effect of CGA towards B. cinerea involves an ER stress, followed by a Ca2+ release from the ER to the mitochondria, resulting in a superoxide burst and subsequent oxidative injury that reduces cell viability. Thus, CGA can be considered as an effectively substance to control gray mold on strawberry.

Inactivation of Salmonella typhimurium SL1344 by Chlorogenic Acid and the Impairment of Cellular Integrity.

Chlorogenic acid (CGA) is an antibacterial agent that can be isolated from Eucommia ulmoides Oliver, a Chinese medicinal and edible plant food. The inhibitory effect of CGA on bacterial growth and stiffness of the outer membrane (OM) had been reported, while more evidence were required to elucidate its impairment of cell wall. In this study, the morphological and physiochemical changes of Salmonella cells under CGA treatment were investigated. Firstly, the minimum inhibitory concentration (MIC) of CGA against Salmonella was assayed. Later, the permeability of OM and activity of the proteins released were measured and observed to reveal the alteration of OM characteristic and cellular morphology. Finally, reactive oxygen species and cell membrane fluidity were analyzed, respectively, to elucidate how CGA damaged cell surface. The results showed that MIC of CGA against Salmonella was 6.25 mg/L. Under sub-lethal doses of CGA, the OM permeability and the release of soluble proteins were enhanced evidently, and Salmonella cells showed more deformed and shrunken, confirming the impairment of cellular integrity under CGA. Finally, the possible cause of cell surface damage was investigated. the fluidity of the membrane was increased upon CGA treatment, which may the possible cause of OM by CGA.

Gold nanoparticles enhance microRNA 31 detection in colon cancer cells after inhibition with chlorogenic acid.

In the present study, the inhibitory effect of chlorogenic acid (CGA), a phenolic compound with potential antitumor effects, on circulating microRNA 31 (miR-31), was evaluated in RKO colon cancer cells. The capacity of gold nanoparticles (AuNPs) to enhance miR-31 quantification after treatment with CGA was assessed. RKO cells were treated with different concentrations of CGA for 24, 48 and 72 h, after which AuNPs coupled to CD81 were added to the supernatants. Total RNA was extracted, and miR-31 was quantified by reverse transcription-quantitative PCR. The results revealed an 85% decrease in miR-31 level following treatment with 1,000 µM CGA for 72 h, and the highest capacity to detect miR-31 (after treatment and isolation with AuNPs + CD81) was observed at 24 h. Furthermore, CGA decreased the expression of the miR-31 oncogene in an in vitro colon cancer model, and the use of AuNPs enhanced the levels of miRNA detection. The results suggest that miR-31 inhibition is one mechanism by which CGA decreases colon cancer cell proliferation. Moreover, AuNPs can increase the capacity of miR-31 quantification, representing a new strategy to develop non-invasive tools for the molecular diagnosis and monitoring of colon cancer.

The mechanism of chlorogenic acid inhibits lipid oxidation: An investigation using multi-spectroscopic methods and molecular docking

Endogenous lipase and lipoxygenase play important roles in accelerating lipid oxidation. Polyphenols are a series of commonly used chemicals for preserving fish and seafood products, due to their positive inhibitory effects on lipid oxidation. However, the mechanism involved is still unknown. The inhibitory effects of chlorogenic acid (CGA) on lipase and lipoxygenase were investigated and explored with multi- spectroscopic and molecular docking approaches. Results showed that CGA could inhibit the activities of lipase and lipoxygenase with concentration increased in a highly dose-dependent manner. CGA quenched intrinsic fluorescence intensities of enzymes by static quenching and binding with CGA which led to changes in 3D structures of enzymes. Results of the molecular docking confirmed binding modes, binding sites and major interaction forces between CGA and enzymes, which reduced the corresponding activity. Thus, this study could provide basic mechanisms of the inhibitory effects of polyphenols on lipid oxidation during food preservation.

Inhibitory effect of chlorogenic acid on polyphenol oxidase and browning of fresh-cut potatoes

Abstract In our study, the inhibition mechanism of chlorogenic acid (CGA) on PPO was investigated by enzyme kinetic analysis, fluorescence quenching, thermodynamic parameters analysis, circular dichroism and molecular docking. Our results showed that CGA inhibited PPO activity in a reversible mixed-type manner and Ki was estimated to be 3.577 × 10−4 mol L-1. PPO activity was significantly inactivated by CGA in a dose-dependent manner and IC50 was calculated as 1.55 × 10−4 mol L-1. CGA interacts with PPO and quench its intrinsic fluorescence. Furthermore, results indicated that CGA bound with PPO by hydrophobic interaction. In addition, CGA changed the hydrogen bonding network of PPO and resulted in rearragement of secondary structure. The molecular docking results suggested that CGA bound to active site of PPO. Importantly, the efficacy of CGA inhibiting the enzymatic browning of fresh-cut potato slices was confirmed by the inhibition of PPO. Therefore, based on the inhibition of PPO, CGA might represent a new type of inhibitor for fresh-cut potatoes.

Correction to: the Inhibitory Effect of Chlorogenic Acid on Lipid Oxidation of Grass Carp (Ctenopharyngodon idellus) during Chilled Storage

Correction to: the Inhibitory Effect of Chlorogenic Acid on Lipid Oxidation of Grass Carp (Ctenopharyngodon idellus) during Chilled Storage

The inhibitory effect of chlorogenic acid on lipid oxidation of grass carp (Ctenopharyngodon idellus) during chilled storage

Grass carp (Ctenopharyngodon idellus) is a kind of freshwater fish which is rich in polyunsaturated fatty acids and easily exposed to lipid oxidation during refrigeration. The effect of chlorogenic acid (CGA) on lipid oxidation, protein oxidation, enzymatic activities, and color stability of grass carp muscle during chilled storage was investigated. The lipid oxidation was inhibited by CGA, as evidenced by lower thiobarbituric acid values, peroxide values, carbonyl valence, less free fatty acid content, and higher amount of unsaturated fatty acid compared to the control group. CGA also had a positive effect on the whiteness value and the stability of protein oxidation of fish samples. In addition, the inhibitory study of CGA on endogenous lipase and lipoxygenase activities of fish muscle can help to partly illustrate the mechanism that retains its freshness effect. The results indicate that CGA is a novel natural additive which can be used to inhibit lipid and protein oxidation and be applied in the storage of aquatic products or some similar fields.

Potential Oral Health Care Agent from Coffee Against Virulence Factor of Periodontitis.

Background: Coffee is a major dietary source of polyphenols. Previous research found that coffee had a protective effect on periodontal disease. In this study, we aimed to investigate whether coffee extract and its primary phenolic acid, chlorogenic acid, affect the growth and protease activity of a periodontopathogen Porphyromonas gingivalis (P. gingivalis). Methods: Coffee extract and chlorogenic acid were prepared by a two-fold serial dilution. The turbid metric test and plate count method were used to examine the inhibitory effects of chlorogenic acid on P. gingivalis. The time-kill assay was used to measure changes in the viability of P. gingivalis after exposure to chlorogenic acid for 0–24 h. The protease activity of P. gingivalis was analyzed using the optical density of a chromogenic substrate. Results: As a result, the minimum inhibitory concentration (MIC) of chlorogenic acid was 4 mg/mL, and the minimum bactericidal concentration was 16 mg/mL. Chlorogenic acid at concentrations above MIC resulted in a longer-lasting inhibitory effect on P. gingivalis viability and significantly reduced associated protease activity. The coffee extract showed antibacterial activity as observed by the disk diffusion test, whereas these inhibitory effects were not affected by different roast degrees of coffee. Conclusions: Collectively, our novel findings indicate that chlorogenic acid not only has antimicrobial activity but also reduced the protease activity of P. gingivalis. In addition, coffee extract inhibits the proliferation of P. gingivalis, which may partly be attributed to the effect of chlorogenic acid.

Attenuation of Palmitic Acid-Induced Lipotoxicity by Chlorogenic Acid through Activation of SIRT1 in Hepatocytes.

Saturated free fatty acids (FFAs) induce hepatocyte lipotoxicity, wherein oxidative stress-associated mitochondrial dysfunction is mechanistically involved. Chlorogenic acid (CGA), a potent antioxidant and anti-inflammatory compound, protects against high-fat-diet-induced oxidative stress and mitochondrial dysfunction in liver. This study investigates whether CGA protects against FFA-induced hepatocyte lipotoxicity via the regulation of mitochondrial fission/fusion and elucidates its underlying mechanisms. AML12 cell, a non-transformed hepatocyte cell line, is treated with palmitate. Here, it is shown that CGA prevents palmitate-induced lipotoxicity by activation of SIRT1 regulated mitochondrial morphology. CGA treatment mitigates oxidative stress and mitochondrial dysfunction, as evidenced by a decrease in reactive oxygen species (ROS) production, and an increase in mitochondrial mass and mitochondrial membrane potential. CGA also significantly decreases Bax expression and thereby reduces mitochondria-mediated caspase-dependent apoptosis. Mechanistically, CGA attenuates ROS-induced mitochondrial fragmentation by inhibiting dynamin-related protein 1 (Drp1) and enhancing Mfn2 expression. In contrast, the inhibitory effects of CGA on the generation of mitochondrial ROS and Drp1 are blocked by siRNA knockdown of SIRT1. Collectively, these findings show that supplementation with CGA protects hepatocytes from FFA-induced lipotoxicity through activation of SIRT1, which reverses the oxidative stress and dysfunction of mitochondrial biogenesis directly.

Chlorogenic acid inhibits proliferation and induces apoptosis in A498 human kidney cancer cells via inactivating PI3K/Akt/mTOR signalling pathway.

Kidney cancer is a highly lethal cancer, of which the most common type is renal cell carcinoma (RCC). The targeted drugs used in treating RCC clinically have a lot of side effects. Therefore, it is urgent to find out effective agents with little toxic effects. The antiproliferation effect of chlorogenic acid (CA) was performed using the CCK-8 assay. Then, we adopted colony formation assay, Annexin V/PI staining assay and JC-1 mitochondrial membrane potential assay to explore the mechanism of anticancer effect of CA. We also conducted qPCR and Western blot to determine the pathway involved. We identified that CA selectively suppressed proliferation of human RCC cell line A498 but not the human embryonic kidney cell HEK293. Mechanistic studies showed that CA significantly induced apoptosis, as indicated by activation of caspase protein and increased ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 (P<0.05). Furthermore, we found that PI3K/Akt/mTOR signalling pathway is involved in the inhibitory effect of CA on A498 cells. Activation of this pathway increased proliferation and decreased apoptosis of A498 cells, exhibiting antagonism function against CA. Our research firstly reports the efficacy of CA against RCC cells and elucidates the underlying molecular mechanisms. These findings indicate that CA is a potential agent for treating RCC.

Amelioration of bleomycin-induced pulmonary fibrosis by chlorogenic acid through endoplasmic reticulum stress inhibition

To investigate the inhibitory effects of chlorogenic acid on pulmonary fibrosis and the internal mechanisms in vivo and in vitro. 30 male BALB/C mice were randomized into 5 groups: control group, pulmonary fibrosis model group, low, middle and high dose of chlorogenic acid groups. Mice in pulmonary fibrosis model group were administered 5.0mg/kg bleomycin with intracheal instillation and mice in 3 chlorogenic acid groups were treated with chlorogenic acid every day for 28 days after bleomycin administration. Lung tissue histology was observed using HE staining. Primary pulmonary fibroblasts were isolated and cultured. The expressions of fibrosis related factors (α-SMA and collagen I), as well as ER stress markers (CHOP and GRP78) were determined by both real-time PCR assay and Western blotting, while the expressions of other ER stress signaling pathway factors PERK, IRE-1, ATF-6 and protein levels of caspase-12, caspase-9, caspase-3, PARP were determined by Western blotting. RLE-6TN cell line induced by TGF-β1 was also used to verify the amelioration effects in vitro study. In both in vivo and in vitro studies, TUNEL staining was used to evaluate cell apoptosis. Expressions of collagen I, α-SMA, GRP78, and CHOP were significantly inhibited by chlorogenic acid in dose-dependent manner. Similarly, decreasing levels of cleaved caspase-12, caspase-9, caspase-3 and increasing level of uncleaved PARP were observed in chlorogenic acid groups compared with those in the fibrosis group both in vivo and in vitro. Chlorogenic acid could also significantly down-regulate the level of phosphorylation of PERK and cleaved ATF-6 in vivo study. Moreover, MTT assay demonstrated chlorogenic acid could enhance proliferation of RLE-6TN cells induced by TGFβ1 in vitro. And the apoptosis assays indicated that chlorogenic acid could significantly inhibit cell apoptosis both in vivo and in vitro studies. Chlorogenic acid could inhibit the pulmonary fibrosis through endoplasmic reticulum stress inhibition in vivo and in vitro.

Inhibitory effect of chlorogenic acid on digestion of potato starch

The effect of the chlorogenic acid isomer 5-O-caffeoylquinic acid (5-CQA) on digestion of potato starch by porcine pancreatic alpha amylase (PPAA) was investigated using isolated starch and cooked potato tuber as substrates. In vitro digestion was performed on five varieties of potato with varying phenolic content. Co- and pre-incubation of PPAA with 5-CQA significantly reduced PPAA activity in a dose dependent manner with an IC50 value of about 2mgmL−1. Lineweaver-Burk plots indicated that 5-CQA exerts a mixed type inhibition as km increased and Vmax decreased. The total polyphenol content (TPC) of peeled tuber tissue ranged from 320.59 to 528.94mg 100g−1dry weight (DW) in raw tubers and 282.03–543.96mg 100g−1DW in cooked tubers. With the exception of Désirée, TPC and 5-CQA levels decreased after cooking. Principle component analysis indicated that digestibility is affected by multiple factors including phenolic, dry matter and starch content.

Inhibitory effect of chlorogenic acid on senescence of human skin fibroblasts

Objective To evaluate the inhibitory effect of chlorogenic acid on senescence of human skin fibroblasts (HSFs). Methods Fibroblasts isolated from human foreskin were treated with 1 mmol/L glyoxal in vitro to develop a model for cellular senescence. In order to select effective concentrations of chlorogenic acid, some HSFs were treated with 1 mmol/L glyoxal alone or in combination with chlorogenic acid at different concentrations (5, 10, 20, 40, 80 μmol/L) for 3 days, with those receiving no treatment serving as the blank control group. Then, methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of HSFs. Some HSFs were divided into 5 groups to be cultured alone (blank control group) , or treated with 1 mmol/L glyoxal (glyoxal group) or the combination of 1 mmol/L glyoxal and chlorogenic acid at effective concentrations of 10, 20 and 40 μmol/L (glyoxal+ chlorogenic acid groups). Senescence associated β-galactosidase (SA-β-gal) staining and real-time fluorescence-based quantitative PCR were conducted to determine the percentage of senescent cells and expression level of p16INK4a mRNA respectively. Statistical analysis was carried out by one-way analysis of variance followed by the least significant difference (LSD) -t test. Results Compared with the blank control group, the glyoxal group showed significantly decreased cellular proliferative activity of HSFs (55.65% ± 2.00% vs. 100% ± 6.90%, P 0.05). Therefore, 10- 40 μmol/L was selected as the effective concentrations of chlorogenic acid. The glyoxal group showed significant increases in the percentage of senescent (SA-β-gal-positive) cells (35.65% ± 2.24% vs. 13.00% ± 2.22%, P< 0.01) and expression level of p16INK4a mRNA (2-ΔΔCt: 1.00 ± 0.06 vs.0.26 ± 0.05, P< 0.01) compared with the blank control group, while the glyoxal+ 10-, 20-, 40-μmol/L chlorogenic acid groups showed significantly decreased percentage of senescent cells (31.50% ± 2.13%, 22.31% ± 3.11% and 19.32% ± 3.01% respectively) and expression level of p16INK4a mRNA (2-ΔΔCt: 0.88 ± 0.08, 0.73 ± 0.06 and 0.68 ± 0.04 respectively) compared with the glyoxal group (all P< 0.05). Additionally, the percentage of senescent cells decreased with the increase in chlorogenic acid concentrations in the glyoxal+ chlorogenic acid groups. Conclusion Chlorogenic acid can protect HSFs from glyoxal-induced senescence. Key words: Skin aging; Fibroblasts; Glyoxal; Chlorogenic acid; Cell aging; Cell proliferation; p16INK4a mRNA

Inhibitory effect of chlorogenic acid on fruit russeting in ‘Golden Delicious’ apple

Analysis of the secondary metabolites in apple peels showed that the levels of total flavonoids and chlorogenic acid (CA) during 15–60 days after full bloom (DAFB) were significantly higher in cultivar ‘Feng Shuai’, a spontaneous non-russeting mutant of ‘Golden Delicious’ apple, than in its maternal plant, which indicated higher level of endogenous CA may be associated with the inhibition of russet formation. Application of 0.5mmolL−1 exogenous CA solution to ‘Golden Delicious’ fruits at 30 DAFB effectively inhibited fruit russet formation. It also increased the levels of CA and total flavonoids in fruit peels at the 7th day after treatment, but depressed the lignin content. Furthermore, CA treatment induced the activity of phenylalnine ammonialyase (PAL) and cinnamate-4-hydrolase (C4H), but depressed activity of polyphenol oxidase (PPO) and peroxidase (POD). These effects were further supported by RT-PCR, where the expressions of PAL, C4H, 4CL, CHS, and CHI were up-regulated while those of CAD, POD and PPO were down-regulated by CA treatment. Interestingly, the up-regulated genes are involved in the biosynthesis of chlorogenic acid and flavonoids while the down-regulated ones are involved in lignin biosynthesis. Therefore, it can be deduced that the inhibition of russet formation in ‘Golden Delicious’ apple by CA is associated with the inhibition of lignin biosynthesis, not the enhancement of total flavonoids biosynthesis.

Docking Studies of Chlorogenic Acid against Aldose Redutcase by using Molgro Virtual Docker Software

Docking studies of chlorogenic acid against aldose reductase, an enzyme involved in diabetic complications, have been performed, in order to evaluate the inhibitory effects of chlorogenic acid on this enzyme. The docking studies were performed using Molgro Virtual Docker (MVD) software. From the several available alternative methods to incorporate protein flexibility in docking studies, the use of multiple crystal structures with different bound ligands was applied here, thus, of the available crystal structures of the non- mutated aldose reductase enzyme from Homo sapiens, five were selected for the final docking studies. The docking results of chlorogenic acid with selected aldose reductase crystal structures shows that it forms hydrogen bonds with at least two out of three key active site residues (Tyr48, His110 and Trp111). It also form hydrogen bonds to other active site residues, in particular Thr113. The average MolDock score and the MolDock Re-rank score for cholorogenic acid are -119.34 Kcal/mol and -114.92 Kcal/mol respectively. The docking results show that chlorogenic acid fits well in the active site of aldose reductase and interact with the residues in the active site which are crucial for their biological activity, thus, it could a potent inhibitor of aldose reductase enzyme and thus be used for prevention/treatment of diabetic complications.

Inhibitory effects of chlorogenic acid on aldose reductase activity in vitro and cataractogenesis in galactose-fed rats

Chlorogenic acid (5-O-caffeoylquinic acid, CA), a phenolic compound found ubiquitously in plants, has antidiabetic effect in diabetic animal models. In this study, we investigated the inhibitory effect of CA on diabetic cataractogenesis. We evaluated the aldose reductase (AR) activity during cataract development in 50% galactose-fed rats, an animal model of sugar cataract. Galactose-fed rats were treated orally with CA (10 and 50 mg/kg body weight) once a day for 2 weeks. In vehicle-treated galactose-fed rats, lens opacity was increased, and lens fiber swelling and membrane rupture were observed. In addition, AR protein was highly expressed in lens epithelial cells and lens cortical fibers of galactose-fed rats. However, CA inhibited the rat AR activity in vitro, and the administration of CA prevented the development of sugar cataract through the inhibition of AR activity. These observations suggest that CA is useful for the treatment of sugar cataract.