Synthesis and biological evaluation of 2'-hydroxychalcone derivatives as AMPK activators.

A series of 2'-hydroxychalcone derivatives with various substituents on B-ring were synthesized and evaluated for AMP-activated protein kinase (AMPK) activation activity in podocyte cells. The results displayed that hydroxy, methoxy and methylenedioxy groups on B-ring could enhance the activitiy better than O-saturated alkyl, O-unsaturated alkyl or other alkoxy groups. Compounds 27 and 29 possess the highest fold change of 2.48 and 2.73, respectively, which were higher than those of reference compound (8) (1.28) and metformin (1.88). Compounds 27 and 29 were then subjected to a concentration-response study to obtain the EC50 values of 2.0 and 4.8 µM, respectively and MTT assays also showed that cell viability was not influenced by the exposure of podocytes to compounds 27 and 29 at concentrations up to 50 µM. In addition, compound 27 was proved to activate AMPK via calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß)-dependent pathway without affecting intracellular calcium levels. The computational study showed that the potent compounds exhibited stronger ligand-binding strength to CaMKKß, particularly compounds 27 (-8.4 kcal/mol) and 29 (-8.0 kcal/mol), compared to compound 8 (-7.5 kcal/mol). Fragment molecular orbital (FMO) calculation demonstrated that compound 27 was superior to compound 29 due to the presence of methyl group, which amplified the binding by hydrophobic interactions. Therefore, compound 27 would represent a promising AMPK activator for further investigation of the treatment of diabetes and diabetic nephropathy.

Neuroprotective effect of short-chain fatty acids against oxidative stress-induced SH-SY5Y injury via GPR43-dependent pathway.

A neurodegenerative disorder is a condition that causes a degeneration of neurons in the central nervous system, leading to cognitive impairment and movement disorders. An accumulation of oxidative stress in neurons contributes to the pathogenesis of neurodegenerative disorders. Over the past few years, several studies have suggested that short-chain fatty acids, metabolites of the gut microbiota, might have a beneficial effect in neurodegenerative disorders. A G protein-coupled receptor 43 (GPR43) plays an important role in modulating oxidative stress and inflammatory processes in several tissues. Interestingly, the downstream signaling pathways activated by GPR43 to modulate oxidative stress differ among tissues. Moreover, the cellular mechanisms underlying GPR43 activation in neuronal cells to handle oxidative stress remain unclear. In this present study, we tested the role of GPR43, which is activated by short-chain fatty acids or a specific GPR43 agonist, in an oxidative stress-induced neuronal cell line (SH-SY5Y) injury. Our findings suggest that a combination of short-chain fatty acids with a physiological function could protect neurons from H2 O2 -induced cell damage. The effect of short-chain fatty acids mixture was abolished by pretreatment with a GPR43 antagonist, indicating this protective effect is a GPR43-dependent mechanism. In addition, a specific GPR43 agonist shows a similar result to that found in short-chain fatty acids mixture. Furthermore, our findings indicate that the downstream activation of GPR43 to protect against oxidative stress-induced neuronal injury is a biased Gq activation signaling of GPR43, which results in the prevention of H2 O2 -induced neuronal apoptosis. In conclusion, our results show new insight into the cellular mechanism of GPR43 and its neuroprotective effect. Taken together, this newly discovered finding suggests that activation of the biased Gq signaling pathway of GPR43 might be a potential therapeutic target for aging-related neurodegeneration.

Piperine as potential therapy of post-weaning porcine diarrheas: an in vitro study using a porcine duodenal enteroid model.

Post-weaning diarrhea in piglets is a major problem, resulting in a significant loss in pig production. This study aimed to investigate the effects of piperine, an alkaloid abundantly found in black peppers, on biological activities related to the pathogenesis of post-weaning diarrhea using a porcine duodenal enteroid model, a newly established intestinal stem cell-derived in vitro model recapitulating physiology of porcine small intestinal epithelia. Porcine duodenal enteroid models were treated with disease-relevant pathological inducers with or without piperine (8 µg/mL and/or 20 µg/mL) before measurements of oxidative stress, mRNA, and protein expression of proinflammatory cytokines, nuclear factor-kappa B (NF-κB) nuclear translocation, barrier leakage, and fluid secretion. We found that piperine (20 µg/mL) inhibited H2O2-induced oxidative stress, TNF-α-induced mRNA, and protein expression of proinflammatory cytokines without affecting NF-κB nuclear translocation, and prevented TNF-α-induced barrier leakage in porcine duodenal enteroid monolayers. Importantly, piperine inhibited fluid secretion induced by both forskolin and heat-stable toxins (STa) in a three-dimensional model of porcine duodenal enteroids. Collectively, piperine possesses both anti-inflammatory and anti-secretory effects in porcine enteroid models. Further research and development of piperine may provide novel interventions for the treatment of post-weaning porcine diarrhea.

Deoxyelephantopin and Its Isomer Isodeoxyelephantopin: Anti-Cancer Natural Products with Multiple Modes of Action.

Cancer is a leading cause of morbidity and mortality worldwide. The development of cancer involves aberrations in multiple pathways, representing promising targets for anti-cancer drug discovery. Natural products are regarded as a rich source for developing anti-cancer therapies due to their unique structures and favorable pharmacology and toxicology profiles. Deoxyelephantopin and isodeoxyelephantopin, sesquiterpene lactone compounds, are major components of Elephantopus scaber and Elephantopus carolinianus, which have long been used as traditional medicines to treat multiple ailments, including liver diseases, diabetes, bronchitis, fever, diarrhea, dysentery, cancer, renal disorders, and inflammation-associated diseases. Recently, deoxyelephantopin and isodeoxyelephantopin have been extensively explored for their anti-cancer activities. This review summarizes and discusses the anti-cancer activities of deoxyelephantopin and isodeoxyelephantopin, with an emphasis on their modes of action and molecular targets. Both compounds disrupt several processes involved in cancer progression by targeting multiple signaling pathways deregulated in cancers, including cell cycle and proliferation, cell survival, autophagy, and invasion pathways. Future directions of research on these two compounds towards anti-cancer drug development are discussed.

Fungus-Derived 3-Hydroxyterphenyllin and Candidusin A Ameliorate Palmitic Acid-Induced Human Podocyte Injury via Anti-Oxidative and Anti-Apoptotic Mechanisms.

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. An elevated fatty acid plasma concentration leads to podocyte injury and DN progression. This study aimed to identify and characterize cellular mechanisms of natural compounds that inhibit palmitic acid (PA)-induced human podocyte injury. By screening 355 natural compounds using a cell viability assay, 3-hydroxyterphenyllin (3-HT) and candidusin A (CDA), isolated from the marine-derived fungus Aspergillus candidus PSU-AMF169, were found to protect against PA-induced podocyte injury, with half-maximal inhibitory concentrations (IC50) of ~16 and ~18 µM, respectively. Flow cytometry revealed that 3-HT and CDA suppressed PA-induced podocyte apoptosis. Importantly, CDA significantly prevented PA-induced podocyte barrier impairment as determined by 70 kDa dextran flux. Reactive oxygen species (ROS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) direct scavenging assays indicated that both compounds exerted an anti-oxidative effect via direct free radical-scavenging activity. Moreover, 3-HT and CDA upregulated the anti-apoptotic Bcl2 protein. In conclusion, 3-HT and CDA represent fungus-derived bioactive compounds that have a novel protective effect on PA-induced human podocyte apoptosis via mechanisms involving free radical scavenging and Bcl2 upregulation.

A chalcone derivative retards renal cyst enlargement by inhibiting fluid secretion and cell proliferation in an in vitro model of polycystic kidney disease.

BACKGROUND: Renal bilateral fluid filled-cyst in polycystic kidney disease (PKD) is associated with abnormal epithelial cell proliferation and transepithelial fluid secretion which leads to end-stage renal disease (ESRD). A chalcone derivative, isoliquiritigenin (ISLQ), has been shown to have various pharmacological properties. Since several studies have shown that ISLQ could inhibit CFTR channel activity, it is interesting to see whether it can inhibit renal cyst enlargement. The present study was aimed to determine an inhibitory effect and the mechanism of chalcone derivatives on MDCK cyst progression and Pkd1 mutant cells. METHODS: MDCK cyst growth and cyst formation experiments, MTT assay, Ussing chamber experiment, BrdU cell proliferation assay and western blot analysis were performed in this study. RESULTS: Among four compounds of chalcone derivatives tested, CHAL-005 (100 µM) was found to inhibit MDCK cyst growth in a dose-dependent manner without cytotoxicity. It inhibited short-circuit current of chloride secretion as well as CFTR protein expression in MDCK cells. CHAL-005 significantly suppressed cell proliferation. In addition, CHAL-005 strongly reduced phosphorylation ERK1/2 and phosphorylation S6 kinase in MDCK and Pkd1 mutant cells. Interestingly, CHAL-005 activated phosphorylation of AMP kinase protein expression in MDCK and Pkd1 mutant cells. CONCLUSION: CHAL-005 slowed MDCK cyst progression by inhibiting CFTR expression and reducing ERK1/2 and mTOR/S6K signaling pathways as well as activating AMPK expression. Therefore, a chalcone derivative could represent as a promising drug candidate for polycystic kidney disease intervention.

Antispasmodic Effect of Asperidine B, a Pyrrolidine Derivative, through Inhibition of L-Type Ca2+ Channel in Rat Ileal Smooth Muscle.

Antispasmodic agents are used for modulating gastrointestinal motility. Several compounds isolated from terrestrial plants have antispasmodic properties. This study aimed to explore the inhibitory effect of the pyrrolidine derivative, asperidine B, isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, on spasmodic activity. Isolated rat ileum was set up in an organ bath. The contractile responses of asperidine B (0.3 to 30 µM) on potassium chloride and acetylcholine-induced contractions were recorded. To investigate its antispasmodic mechanism, CaCl2, acetylcholine, Nω-nitro-l-arginine methyl ester (l-NAME), nifedipine, methylene blue and tetraethylammonium chloride (TEA) were tested in the absence or in the presence of asperidine B. Cumulative concentrations of asperidine B reduced the ileal contraction by ~37%. The calcium chloride and acetylcholine-induced ileal contraction was suppressed by asperidine B. The effects of asperidine B combined with nifedipine, atropine or TEA were similar to those treated with nifedipine, atropine or TEA, respectively. In contrast, in the presence of l-NAME and methylene blue, the antispasmodic effect of asperidine B was unaltered. These results suggest that the antispasmodic property of asperidine B is probably due to the blockage of the L-type Ca2+ channel and is associated with K+ channels and muscarinic receptor, possibly by affecting non-selective cation channels and/or releasing intracellular calcium.

Fructo-oligosaccharides alleviate inflammation-associated apoptosis of GLP-1 secreting L cells via inhibition of iNOS and cleaved caspase-3 expression.

Glucagon-like peptide 1 (GLP-1) released from enteroendocrine (L) cells regulates insulin secretion. Intestinal inflammation and impaired GLP-1 release have been found in type 2 diabetes mellitus (T2DM) patients. Fructo-oligosaccharides (FOS), a known prebiotic, improve GLP-1 release and glucose homeostasis in T2DM models. This study aimed to investigate the effect of tumor necrosis factor-α (TNF-α), a proinflammatory cytokine associated with intestinal inflammation in T2DM, on L cell apoptosis and the effect of FOS on inflammation-associated impairment of GLP-1 secretion. Herein, using cell death assays, immunofluorescence staining, real time PCR and Western blot analyses, we found that TNF-α induced L cell apoptosis via nuclear factor kappa B (NF-κB)- inducible nitric oxide synthase (iNOS)-cleaved caspase-3-dependent pathways. Interestingly, FOS did not suppress TNF-α-induced NF-κB nuclear translocation, but inhibited expression of iNOS and cleaved caspase-3. In addition, FOS alleviated apoptosis and rescued impaired GLP-1 release in TNF-α-treated L cells. Altogether, our data indicate that TNF-α induces L cell apoptosis via an NF-κB-iNOS-caspase-3-dependent pathway. FOS may be useful in suppressing inflammation-associated L cell apoptosis and maintaining GLP-1 level in T2DM patients.

Chitin and Chitosan Derivatives as Biomaterial Resources for Biological and Biomedical Applications.

Chitin is a long-chain polymer of N-acetyl-glucosamine, which is regularly found in the exoskeleton of arthropods including insects, shellfish and the cell wall of fungi. It has been known that chitin can be used for biological and biomedical applications, especially as a biomaterial for tissue repairing, encapsulating drug for drug delivery. However, chitin has been postulated as an inducer of proinflammatory cytokines and certain diseases including asthma. Likewise, chitosan, a long-chain polymer of N-acetyl-glucosamine and d-glucosamine derived from chitin deacetylation, and chitosan oligosaccharide, a short chain polymer, have been known for their potential therapeutic effects, including anti-inflammatory, antioxidant, antidiarrheal, and anti-Alzheimer effects. This review summarizes potential utilization and limitation of chitin, chitosan and chitosan oligosaccharide in a variety of diseases. Furthermore, future direction of research and development of chitin, chitosan, and chitosan oligosaccharide for biomedical applications is discussed.

Novel Potential Application of Chitosan Oligosaccharide for Attenuation of Renal Cyst Growth in the Treatment of Polycystic Kidney Disease.

Chitosan oligosaccharide (COS), a natural polymer derived from chitosan, exerts several biological activities including anti-inflammation, anti-tumor, anti-metabolic syndrome, and drug delivery enhancer. Since COS is vastly distributed to kidney and eliminated in urine, it may have a potential advantage as the therapeutics of kidney diseases. Polycystic kidney disease (PKD) is a common genetic disorder characterized by multiple fluid-filled cysts, replacing normal renal parenchyma and leading to impaired renal function and end-stage renal disease (ESRD). The effective treatment for PKD still needs to be further elucidated. Interestingly, AMP-activated protein kinase (AMPK) has been proposed as a drug target for PKD. This study aimed to investigate the effect of COS on renal cyst enlargement and its underlying mechanisms. We found that COS at the concentrations of 50 and 100 µg/mL decreased renal cyst growth without cytotoxicity, as measured by MTT assay. Immunoblotting analysis showed that COS at 100 µg/mL activated AMPK, and this effect was abolished by STO-609, a calcium/calmodulin-dependent protein kinase kinase beta (CaMKKß) inhibitor. Moreover, COS elevated the level of intracellular calcium. These results suggest that COS inhibits cyst progression by activation of AMPK via CaMKKß. Therefore, COS may hold the potential for pharmaceutical application in PKD.

Unified synthesis and cytotoxic activity of 8-O-methylfusarubin and its analogues.

A simple and unified synthesis of four related pyranonaphthoquinone natural products, e.g. 8-O-methylfusarubin, 8-O-methylanhydrofusarubin, fusarubin and anhydrofusarubin, is reported. The key synthetic features include the precedented Diels-Alder cycloaddition to assemble the naphthalene skeleton, selective formylation and acetonylation and intramolecular acetalization to construct the pyran ring. Manipulation of the oxidation state of the naphthoquinone core was performed to construct the two analogues, fusarubin and anhydrofusarubin. This work also highlights an unprecedented directing effect of the hydroxymethylene group in the selective hypervalent iodine-mediated quinone oxidation. The four synthetic compounds were evaluated for their in vitro cytotoxic activities against six human cancer cells. 8-O-Methylfusarubin was the most potent analogue and displayed excellent cytotoxic activity against MCF-7 breast cancer cells with an IC50 value of 1.01 µM with no cytotoxic effect on noncancerous Vero cells, which could potentially be a promising lead compound for anti-breast cancer drug discovery.

Discovery of a novel chalcone derivative inhibiting CFTR chloride channel via AMPK activation and its anti-diarrheal application.

Secretory diarrhea is one of the most common causes of death world-wide especially in children under 5 years old. Isoliquiritigenin (ISLQ), a plant-derived chalcone, has previously been shown to exert anti-secretory action in vitro and in vivo by inhibiting CFTR Cl- channels. However, its CFTR inhibition potency is considerably low (IC50 > 10 µM) with unknown mechanism of action. This study aimed to identify novel chalcone derivatives with improved potency and explore their mechanism of action. Screening of 27 chalcone derivatives identified CHAL-025 as the most potent chalcone analog that reversibly inhibited CFTR-mediated Cl- secretion in T84 cells with an IC50 of ∼1.5 µM. As analyzed by electrophysiological and biochemical analyses, the mechanism of CFTR inhibition by CHAL-025 is through AMP-activated protein kinase (AMPK), a negative regulator of CFTR activity. Furthermore, Western blot analyses and molecular dynamics (MD) results suggest that CHAL-025 activates AMPK by binding at the allosteric site of an upstream kinase calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß). Interestingly, CHAL-025 inhibited both cholera toxin (CT) and bile acid-induced Cl- secretion in T84 cells and prevented CT-induced intestinal fluid secretion in mice. Therefore, CHAL-025 represents a promising anti-diarrheal agent that inhibits CFTR Cl- channel activity via CaMKKß-AMPK pathways.

Amides and Flavonoids from the Fruit and Leaf Extracts of Melodorum siamensis.

Four new chalcones (1, 10, 13, and 14), a new flavanone, (9), a new amide (8), and 19 known compounds were acquired from Melodorum siamensis. The structures were established by NMR and MS data analyses. Compounds 1 (er 1.4:1) and 2 (er 1.1:1) were scalemic and were resolved to yield (-)-1 and (+)-1 and (-)-2 and (+)-2, respectively. The absolute configurations of these compounds were determined from experimental and calculated ECD data. The structures and configurations of (-)-2 and (+)-8 were identified by single-crystal X-ray diffraction analysis. Compound 11 showed nuclear factor-κB inhibitory effects (IC50 = 9 µM) in a pancreatic ß cell line (MIN-6 cells).

Asperidines A-C, pyrrolidine and piperidine derivatives from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178.

One new pyrrolidine derivative, asperidine A (1), and two new piperidine derivatives, asperidines B (2) and C (3), were isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178 together with two known alkaloids. Compound 3 possessed an unprecedented 7-oxa-1-azabicyclo[3.2.1]octane skeleton with four chiral centers. Their structures were determined by spectroscopic evidence. The absolute configurations of compounds 2 and 3 were established using Mosher's method and further confirmed for compound 3 by X-ray crystallographic data. Compound 2 dose-dependently inhibited the CFTR-mediated chloride secretion in T84 cells with an IC50 value of 0.96 µM whereas 3 displayed the same activity with the IC50 value of 58.62 µM. Compounds 2 and 3 also significantly reduced intracellular ROS under both normal and H2O2-treated conditions compared with their respective controls in a dose-dependent manner without cytotoxic effect on Caco-2 cells. In addition, compound 3 was inactive against noncancerous Vero cells whereas compound 2 was considered to be inactive with the IC50 value of >10 µM.

Cellular mechanisms underlying the inhibitory effect of flufenamic acid on chloride secretion in human intestinal epithelial cells.

Intestinal Cl- secretion is involved in the pathogenesis of secretory diarrheas including cholera. We recently demonstrated that flufenamic acid (FFA) suppressed Vibrio cholerae El Tor variant-induced intestinal fluid secretion via mechanisms involving AMPK activation and NF-κB-suppression. The present study aimed to investigate the effect of FFA on transepithelial Cl- secretion in human intestinal epithelial (T84) cells. FFA inhibited cAMP-dependent Cl- secretion in T84 cell monolayers with IC50 of ∼8 µM. Other fenamate drugs including tolfenamic acid, meclofenamic acid and mefenamic acid exhibited the same effect albeit with lower potency. FFA also inhibited activities of CFTR, a cAMP-activated apical Cl- channel, and KCNQ1/KCNE3, a cAMP-activated basolateral K+ channel. Mechanisms of CFTR inhibition by FFA did not involve activation of its negative regulators. Interestingly, FFA inhibited Ca2+-dependent Cl- secretion with IC50 of ∼10 µM. FFA inhibited activities of Ca2+-activated Cl- channels and KCa3.1, a Ca2+-activated basolateral K+ channels, but had no effect on activities of Na+-K+-Cl- cotransporters and Na+-K+ ATPases. These results indicate that FFA inhibits both cAMP and Ca2+-dependent Cl- secretion by suppressing activities of both apical Cl- channels and basolateral K+ channels. FFA and other fenamate drugs may be useful in the treatment of secretory diarrheas.

Scalemic Caged Xanthones Isolated from the Stem Bark Extract of Garcinia propinqua.

Seven new caged xanthones, doitunggarcinones E-K (1-7), all as scalemic mixtures and 10 known compounds (8-17), were isolated from the stem bark extract of Garcinia propinqua. The structures were elucidated on the basis of spectroscopic methods. The separation of the enantiomers of 1-6 was achieved by semipreparative chiral HPLC. The absolute configuration of compound (+)-1 was determined by single-crystal X-ray crystallographic analysis using Cu Kα radiation. The absolute configurations of the other related compounds were determined from comparisons of their ECD spectra with that of compound (+)-1. Compounds (-)-6 and 7 showed cytotoxicity against a colon cancer cell line with IC50 values of 14.23 and 23.95 µM, respectively.

Adenosine monophosphate-activated protein kinase activation and suppression of inflammatory response by cell stretching in rabbit synovial fibroblasts.

Joint mobilization is known to be beneficial in osteoarthritis (OA) patients. This study aimed to investigate the effect of stretching on adenosine monophosphate-activated protein kinase (AMPK) activity and its role in modulating inflammation in rabbit synovial fibroblasts. Uniaxial stretching of isolated rabbit synovial fibroblasts for ten min was performed. Stretching-induced AMPK activation, its underlying mechanism, and its anti-inflammatory effect were investigated using Western blot. Static stretching at 20 % of initial length resulted in AMPK activation characterized by expression of phosphorylated AMPK and phosphorylated acetyl-Co A carboxylase. AMP-activated protein kinase phosphorylation peaked 1 h after stretching and declined toward resting activity. Using cell viability assays, static stretching did not appear to cause cellular damage. Activation of AMPK involves Ca2+ influx via a mechanosensitive L-type Ca2+ channel, which subsequently raises intracellular Ca2+ and activates AMPK via Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß). Interestingly, stretching suppressed TNFα-induced expression of COX-2, iNOS, and phosphorylated NF-κB. These effects were prevented by pretreatment with compound C, an AMPK inhibitor. These results suggest that mechanical stretching suppressed inflammatory responses in synovial fibroblasts via a L-type Ca2+-channel-CaMKKß-AMPK-dependent pathway which may underlie joint mobilization's ability to alleviate OA symptoms.

Chitosan oligosaccharide suppresses synovial inflammation via AMPK activation: An in vitro and in vivo study.

Synovial inflammation plays an important role in the early pathogenesis of osteoarthritis (OA). Chitosan oligosaccharide (COS) has been shown to activate AMPK and suppress inflammatory responses in intestinal epithelial cells. This study aimed to investigate the effect of COS on AMPK activation and synovial inflammation using both primary cultures of synoviocytes and a rabbit model of anterior cruciate ligament (ACL) transection-induced OA. COS induced AMPK activation in both rabbit and human synoviocytes. The mechanism of COS-induced AMPK activation involves an increase in the ADP/ATP ratio but not calcium/calmodulin-dependent protein kinase kinase beta (CaMKKß). Interestingly, COS suppressed the TNFα-induced iNOS and COX-2 expression via an AMPK-dependent mechanism in both rabbit and human synoviocytes. Importantly, oral administration of COS (10mg/kg/day) induced AMPK activation and alleviated signs of inflammation including COX-2 expression in the synovium of a rabbit ACL transection model. Taken together, our results indicate that COS suppresses synovial inflammation in vitro and in vivo via AMPK activation. COS may be useful in the prevention of OA.

Lovastatin Analogues from the Soil-Derived Fungus Aspergillus sclerotiorum PSU-RSPG178.

Three new lovastatin analogues (1, 4, and 5) together with four known lovastatin derivatives, namely, lovastatin (2), α,ß-dehydrolovastatin (3), α,ß-dehydrodihydromonacolin K (6), and α,ß-dehydro-4a,5-dihydromonacolin L (7), were isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178. Their structures were established using spectroscopic evidence. Compound 5 exhibited the most potent activity against HMG-CoA reductase, with an IC50 value of 387 µM. In addition, the present study indicated the direct interaction of compound 5 with HMG-CoA reductase. Compound 5 was considered to be noncytotoxic against noncancerous Vero cells, with an IC50 value of 40.0 µM, whereas compound 2 displayed much stronger activity, with an IC50 value of 2.2 µM.

Inhibition of intestinal chloride secretion by piperine as a cellular basis for the anti-secretory effect of black peppers.

Piperine is the principal alkaloid in black peppers (Piper nigrum L.), which is a commonly included spice in anti-diarrheal formulations. Piperine has antispasmodic activities, but its anti-secretory effect is not known. Therefore, this study investigated the anti-secretory effect of piperine and its underlying mechanism. Piperine inhibited cAMP-mediated Cl- secretion in human intestinal epithelial (T84) cells, similar to black pepper extract. Intraluminal administration of piperine (2 µg/loop) suppressed cholera toxin-induced intestinal fluid accumulation by ∼85% in mice. The anti-secretory mechanism of piperine was investigated by evaluating its effects on the activity of transport proteins involved in cAMP-mediated Cl- secretion. Notably, piperine inhibited CFTR Cl- channel activity (IC50#8'6#10 µM) without affecting intracellular cAMP levels. The mechanisms of piperine-induced CFTR inhibition did not involve MRP4-mediated cAMP efflux, AMPK or TRPV1. Piperine also inhibited cAMP-activated basolateral K+ channels, but it had no effect on Na+-K+-Cl- cotransporters or Na+-K+ ATPases. Piperine suppressed Ca2+-activated Cl- channels (CaCC) without affecting intracellular Ca2+ concentrations or Ca2+-activated basolateral K+ channels. Collectively, this study indicates that the anti-secretory effect of piperine involves the inhibition of CFTR, CaCC and cAMP-activated basolateral K+ channels. Piperine represents a novel class of drug candidates for the treatment of diarrheal diseases caused by the intestinal hypersecretion of Cl-.