| Kinase Assay: |
| Food Chem Toxicol. 2014 Apr;66:313-20. | | Transport characteristics of isorhamnetin across intestinal Caco-2 cell monolayers and the effects of transporters on it.[Pubmed: 24525098] | Flavonoid Isorhamnetin occurs in various plants and herbs, and demonstrates various biological effects in humans.
This work will clarify the Isorhamnetin absorption mechanism using the Caco-2 monolayer cell model.
METHODS AND RESULTS:
The Isorhamnetin transport characteristics at different concentrations, pHs, temperatures, tight junctions and potential transporters were systemically investigated. Isorhamnetin was poorly absorbed by both passive diffusion and active transport mechanisms. Both trans- and paracellular pathways were involved during Isorhamnetin transport. Active transport under an ATP-dependent transport mechanism was mediated by the organic anion transporting peptide (OATP); Isorhamnetin's permeability from the apical to the basolateral side significantly decreased after estrone-3-sulfate was added (p<0.01). Efflux transporters, P-glycoproteins (P-gp), breast cancer resistance proteins (BCRP) and multidrug resistance proteins (MRPs) participated in the Isorhamnetin transport process. Among them, the MRPs (especially MRP2) were the main efflux transporters for Isorhamnetin; transport from the apical to the basolateral side increased 10.8-fold after adding an MRP inhibitor (MK571).
CONCLUSIONS:
This study details Isorhamnetin's cellular transport and elaborates Isorhamnetin's absorption mechanisms to provide a foundation for further studies. | | Toxicol Appl Pharmacol. 2014 Jan 15;274(2):293-301. | | Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes.[Pubmed: 24211276] | Isorhamentin is a 3'-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of Isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated.
METHODS AND RESULTS:
Here, we investigated whether Isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of Isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, Isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in Isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of Isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of Isorhamnetin to induce HO-1 and GCL. Furthermore, Isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition Isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of Isorhamnetin to protect cells from injury induced by t-BHP.
CONCLUSIONS:
Taken together, our results demonstrate that Isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes. | | Toxicol Appl Pharmacol. 2006 May 15;213(1):18-26. | | Effect of Ginkgo biloba extract on procarcinogen-bioactivating human CYP1 enzymes: identification of isorhamnetin, kaempferol, and quercetin as potent inhibitors of CYP1B1.[Pubmed: 16226778 ] |
METHODS AND RESULTS:
In the present study, we investigated the effect of Ginkgo biloba extracts and some of its individual constituents on the catalytic activity of human cytochrome P450 enzymes CYP1B1, CYP1A1, and CYP1A2. G. biloba extract of known abundance of terpene trilactones and flavonol glycosides inhibited 7-ethoxyresorufin O-dealkylation catalyzed by human recombinant CYP1B1, CYP1A1, and CYP1A2, and human liver microsomes, with apparent Ki values of 2 +/- 0.3, 5 +/- 0.5, 16 +/- 1.4, and 39 +/- 1.2 microg/ml (mean +/- SE), respectively. In each case, the mode of inhibition was of the mixed type. Bilobalide, ginkgolides A, B, C, and J, quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, and isorhamentin 3-O-rutinoside were not responsible for the inhibition of CYP1 enzymes by G. biloba extract, as determined by experiments with these individual chemicals at the levels present in the extract. In contrast, the aglycones of quercetin, kaempferol, and isorhamentin inhibited CYP1B1, CYP1A1, and CYP1A2. Among the three flavonol aglycones, isorhamentin was the most potent in inhibiting CYP1B1 (apparent Ki = 3 +/- 0.1 nM), whereas quercetin was the least potent in inhibiting CYP1A2 (apparent Ki = 418 +/- 50 nM). The mode of inhibition was competitive, noncompetitive, or mixed, depending on the enzyme and the flavonol. G. biloba extract also reduced benzo[a]pyrene hydroxylation, and the effect was greater with CYP1B1 than with CYP1A1 as the catalyst.
CONCLUSIONS:
Overall, our novel findings indicate that G. biloba extract and the flavonol aglycones Isorhamnetin, kaempferol, and quercetin preferentially inhibit the in vitro catalytic activity of human CYP1B1. |
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| Cell Research: |
| J Cell Biochem. 2012 Feb;113(2):473-85. | | Isorhamnetin inhibits H₂O₂-induced activation of the intrinsic apoptotic pathway in H9c2 cardiomyocytes through scavenging reactive oxygen species and ERK inactivation.[Pubmed: 21948481 ] | As a traditional Chinese medicine, the sea buckthorn (Hippophae rhamnoides L.) has a long history in the treatment of ischemic heart disease and circulatory disorders. However, the active compounds responsible for and the underlying mechanisms of these effects are not fully understood.
METHODS AND RESULTS:
In this article, Isorhamnetin pretreatment counteracted H(2)O(2)-induced apoptotic damage in H9c2 cardiomyocytes. Isorhamnetin did not inhibit the death receptor-dependent or extrinsic apoptotic pathways, as characterized by its absence in both caspase-8 inactivation and tBid downregulation along with unchanged Fas and TNFR1 mRNA levels. Instead, Isorhamnetin specifically suppressed the mitochondria-dependent or intrinsic apoptotic pathways, as characterized by inactivation of caspase-9 and -3, maintenance of the mitochondrial membrane potential (ΔΨm), and regulation of a series of Bcl-2 family genes upstream of ΔΨm. The anti-apoptotic effects of Isorhamnetin were linked to decreased ROS generation. H(2)O(2) activated ERK and p53, whereas Isorhamnetin inhibited their activation. ERK overexpression overrode the Isorhamnetin-induced inhibition of the intrinsic apoptotic pathway in H9c2 cardiomyocytes, which indicated that an ERK-dependent pathway was involved. Furthermore, N-acetyl cysteine (a potent ROS scavenger) could attenuate the H(2)O(2)-induced apoptosis. However, PD98059 (an ERK-specific inhibitor) could not effectively antagonize ROS generation, which indicates that ROS may be an upstream inducer of ERK.
CONCLUSIONS:
In conclusion, Isorhamnetin inhibits the H(2)O(2)-induced activation of the intrinsic apoptotic pathway via ROS scavenging and ERK inactivation. Therefore, Isorhamnetin is a promising reagent for the treatment of ROS-induced cardiomyopathy. |
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| Animal Research: |
| J Nutr Biochem. 2014 Sep;25(9):923-33. | | Plant flavonol isorhamnetin attenuates chemically induced inflammatory bowel disease via a PXR-dependent pathway.[Pubmed: 24913217] | Isorhamnetin is an O-methylated flavonol present in fruit and vegetables. We recently reported the identification of Isorhamnetin as an activator of the human pregnane X receptor (PXR), a known target for abrogating inflammation in inflammatory bowel disease (IBD).
The current study investigated the role of Isorhamnetin as a putative mouse PXR activator in ameliorating chemically induced IBD.
CONCLUSIONS:
Using two different models (ulcerative colitis like and Crohn's disease like) of experimental IBD in mice, we demonstrated that Isorhamnetin abrogated inflammation through inhibiting the activity of myeloperoxidase, the levels of TNF-α and IL-6, the mRNA expression of proinflammatory mediators (iNOS, ICAM-1, COX2, TNF-α, IL-2 and IL-6) and the phosphorylation of IκBα and NF-κB p65. PXR gene overexpression inhibited NF-κB luciferase activity, and the inhibition was potentiated by Isorhamnetin treatment. PXR knockdown by siRNA demonstrated the necessity for PXR in Isorhamnetin-mediated up-regulation of xenobiotic metabolism genes. Ligand pocket-filling mutants (S247W/C284W and S247W/C284W/S208W) of human PXR weakened the effect of Isorhamnetin on PXR activation. Molecular docking studies and time-resolved fluorescence resonance energy transfer competitive binding assays confirmed the ligand (Isorhamnetin)-binding affinity.
CONCLUSIONS:
These results clearly demonstrated the ameliorating effect of Isorhamnetin on experimental IBD via PXR-mediated up-regulation of xenobiotic metabolism and down-regulation of NF-κB signaling. The novel findings may contribute to the effective utilization of Isorhamnetin or its derivatives as a PXR ligand in the treatment of human IBD. |
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