Table 2.

Systematic presentation of studies on possible use of natural Nrf2 activators in therapy with possible mechanisms of action connected to Nrf2 activation pathways.

Natural Products
Type of Study	Experimental Model	Treatment Doses and Duration	Observed Mechanism of Action/Effects	Proposed Application in Therapy	Ref.
In vivo	male Albino rats Wistar strain	200 mg/kg dose of curcumin for four consecutive days oral administration	enhanced nuclear translocation and ARE-binding of Nrf2 curcumin protects against dimethylnitrosamine (DMN)-induced hepatic injury	chemopreventive agent	[360]
In vivo	male C57BL/6J mice	daily treated with curcumin at the dose of 50 mg/kg body weight by oral gavage	mediated nuclear translocation of Nrf2, followed by induction of HO-1 curcumin intervention dramatically reversed the defects in Nrf2 signaling induced by high fat diet	improving glucose intolerance	[361]
In vivo	male Sprague–Dawley rats	supplemented with curcumin (1 g/kg diet) for 16 weeks	increased HO-1 expression was increased along with suppressed oxidative stress as well as reduced hepatic fat accumulation and fibrotic changes	attenuating the pathogenesis of fatty liver induced metabolic diseases	[362]
In vivo/In vitro	female specific pathogen-free BALB/c mice mouse macrophage RAW264.7 cells	on day 22, the mice were treated with curcumin (200 mg/kg) 1 h before ovalbumin challenge. cells treated with different concentrations (0, 5, 10, 25, and 50 μmol/L) of curcumin for 24 h or with 50-μmol/L curcumin for different lengths of time (0, 4, 8, 12 and 24 h)	Nrf2 and HO-1 levels in lung tissues were significantly increased heme oxygenase-1 and nuclear Nrf2 levels were enhanced in dose- and time-dependent manners in curcumin-treated RAW264.7 cells.	potentially effective drug in asthma treatment (alleviate airway inflammation in asthma through the Nrf2/HO-1 pathway)	[363]
In vitro	primary cultures of cerebellar granule neurons(CGNs) of rats	pretreated with 5–30 μM curcumin	mediated neuro-protection against hemin induced damage via Nrf2 dependent HO-1 expression	neuroprotective agent	[364]
In vitro	human breast cancer cell line MCF-7	treatment with DMSO (vehicle) or various concentrations of curcumin for 12, 24 or 48 h	inhibition of the proliferation of breast cancer cells through Nrf2-mediated down-regulation of Fen1 expression	chemotherapeutic agent	[365]
In vivo	female Sprague-Dawley rats	food supplemented with resveratrol equivalent to 50, 100, or 300 mg/kg body weight/d	elevated protein and mRNA expression of hepatic Nrf2, attenuation of oxidative stress and suppression of inflammatory response mediated by Nrf2	prevention and intervention of human hepatocellular carcinoma	[366]
In vivo/In vitro	female August Copenhagen Irish rats non-tumorigenic human breast epithelial cell line MCF-10A	resveratrol given as a 50 mg subcutaneous pellet every other month during 8 months to animals subcutaneously treated with 3 mg E2 pellets prepared in cholesterol cells were treated with E2 (10 and 50 nM) and Res (50 µM) for up to 48 h	resveratrol treatment alone or in combination with E2 significantly upregulated expression of nuclear factor erythroid 2-related factor 2 (NRF2) in mammary tissues; increased expression of NRF2-regulated genes codying for antioxidant enzymes (NQO1, SOD3 and OGG1) involved in protection against oxidative DNA damage inhibition of suppression of FMO1 and AOX1 genes expression via regulation of NRF2	a potential chemopreventive agent in the development of therapeutic strategies for the prevention of estrogen-induced breast neoplasia	[367]
In vitro	MCF-10F and MCF-7 cells	retreated with 0.1 to 30 nmol/L TCDD with or without 25 μmol/L resveratrol for 72 h and then incubated with E2 (0.1–10 μmol/L) for 24 h	induction of NAD(P)H quinone oxidoreductase 1 (NQO1) in MCF-10F cells exposed to resveratrol may involve the Nrf2-Keap1-ARE pathway (dissociation of Nrf2 from Keap 1 and translocation of Nrf2 to the nucleus, where it binds to the ARE to activate the transcription of NQO1 mRNA)	a potential chemopreventive agent against estrogen-initiated breast cancer	[368]
In vitro	primary rat hepatocytes were obtained from Sprague–Dawley male rats	cells were incubated in the presence of resveratrol for 24 and 48 h ( at concentrations of 25, 50 and 75 µM)	increase in the level of Nrf2 and induction of its translocation to the nucleus, and the increase in the concentration of the coding mRNA for Nrf2	protection of liver cells from oxidative stress induced damage (chemopreventive agents)	[115]
In vitro	human type II alveolar epithelial cell line, A549	cells were treated with various concentrations of native EGCG (5, 10, 20, 40, 60, 80 and 100 μM) and nano EGCG (1, 2, 4, 6, 8, 10 and 12 μM) and allowed to grow for 48 h	increased expression of Nrf2 and Keap1 in native and nano EGCG treated A549 cells that could regulate apoptosis	chemotherapeutic in lung cancer	[369]
In vitro	bovine aortic endothelial cells (BAECs)	cells treated with various concentrations of EGCG	upregulates Nrf2 levels in nuclear extracts and increases ARE-luciferase activity	therapeutic targets in a variety of oxidant- and inflammatory-mediated vascular diseases	[370]
In vivo	male albino Wistar rats; animal model of bleomycin-induced pulmonary fibrosis	intraperitoneally treated with EGCG at a dosage of 20 mg/kg body weight, once daily throughout 28 days	EGCG enhances antioxidant activities and Phase II enzymes involving Nrf2–Keap1 signaling, with subsequent restraint inflammation during bleomycin-induced pulmonary fibrosis no significant change in the expression of Keap1	treatment of diseases such as pulmonary fibrosis	[371]
In vitro	human hepatocytes (HHL5) and hepatoma (HepG2) cells	exposed to various concentrations of sulforaphane for different times with DMSO (0.1%) as control	increased nuclear Nrf2 levels and intracellular GSH levels in both cell lines but with slightly different pattern indicating a potential risk of chemo-resistance of using sulforaphane for chemoprevention	possible induction of pro-survival effects in cancer cells	[128]
In vivo	male BALB/c mice (6 weeks)	5 μmol/animal sulforaphane plus different doses of microcystin-LR	Nrf2 translocation to the nucleus in mouse livers induction of Nrf2 downstream target genes, NQO1 and HO-1, two phase II enzymes	effective in cytoprotection against MC-LR-induced hepatotoxicity	[372]
In vitro	adult rat cardiomyocytes	exposed to 5μM sulforaphane with or without H2O2	upregulation of Nrf2 by sulforaphane occurs at 1 h of incubation increased protein expression of PGC-1α	protective action against oxidative damage, however, timeline of the sulforaphane actions needs to be established	[373]
In vivo	male BALB/c mice (6 weeks)	5 μmol/animal sulforaphane plus different doses of microcystin-LR	Nrf2 translocation to the nucleus in mouse livers induction of Nrf2 downstream target genes, NQO1 and HO-1, two phase II enzymes	effective in cytoprotection against MC-LR-induced hepatotoxicity	[372]
Electrophilic/Covalent
Triterpenoids
In vitro	K562 myeloid leukemia cells	Exposed to 0.05–10 μM CDDO-Me for 24–48 h	Down-regulated Na+/K+ ATPase Arrested cells in G2/M and S phases Increased mitochondria and death receptor-dependent and ER-mediated apoptosis Triggered activation of autophagy	Activated and potentiated the effects of the apoptosis and autophagy pathways to kill K562 cancer cells	[374]
In vitro and In vivo	SKOV3, OVCAR3, A2780, A2780/CP70 and Hey2 ovarian cancer cells	0–50 μM CDDO-Me depending on cell assays 20 mg/kg CDDO-Me in xenograft model using nude mice	Binds to USP7 cells, decreasing MDM2, MDMX and UHRF1 Suppresses tumor growth in a xenograft model	Targets apoptosis-related substrates, increasing apoptosis and reducing growth of ovarian cancer cells	[375]
In vitro	MiaPaCa-2 and BxPC-3 cell lines 6 week old Scid/Ner mice	0.625–5μM CDDO-Me in cell culture CDDO-Me 7.5 mg/kg × 5 days/wk by oral gavage until day 40 (to treat primary tumor) or day 100 (to treat residual disease)	Decreased proliferation and increased apoptosis in K-ras normal and mutant cells Inhibits antiapoptotic pathways Inhibited tumor growth in mice Increased survival time of mice	Combination of in vitro and in vivo effects demonstrate that CDDO-Me will increase apoptosis in pancreatic ductal adenoma carcinoma cell lines and improve the survival of animals	[376]
In vitro	MDA-MB 435, MDA-MB 231, MDA-MB 468, BT-549, T47D and MCF-7 breast cancer cells	CDDO-Me 1.5 μM for 4 h	Induces endoplasmic reticulum vacuolation Induces apoptotic pathways Increases intracellular calcium and generation of reactive oxygen species	CDDO-Me-induced c-FLIPL downregulation and relationship between Ca2+ influx and ROS generation are keys in controlling breast cancer growth.	[377]
In vitro	HO8910 and SKOV3 ovarian cancer cells	CDDO-Me concentration range of 0–100 μM 5 μM CDDO-Me in assays requiring a single concentration	Upregulates Hsp70 Degrades Hsp90-associated protein—ErbB2 and Akt CDDO-Me reacts with nucleophiles on Hsp90 to form Michael adducts	May provide added insight to CDDO-Me action, with Hsp90 as a novel target	[378]
In vivo	C57BL/6 WT mice LSL-KrasG12D/+; Pdx-1-Cre (KC) mice Polyoma-middle T (PyMT) mice	CDDO-Im (100 mg/kg diet) fed 2 days prior to LPS injections	Reduced the lethal effects of LPS injection in mutant mice Increased migration of CD45+ cells Increased percentage of Gr1+ myeloid-derived suppressor cells CDDO-Im decreased IL-6, CCL-2, VEGF, and G-CSF in mutant mice	Postulating the use of CDDO-Im as prophylaxes in the development of pancreatic cancer within susceptible populations. This is due to the reduction in proinflammatory mediators.	[379]
In vitro	Human Jurkat E6-1 cells	CDDO-Im 1 nM and 10 nM for 30 min prior to activation with anti-CD3/anti-CD28	Inhibited production of IL-2 Suppression of CD25 in Nrf2-dependent manner with no effect on CD69	Nrf2 activation by CDDO-Im reduces IL-2 secretion and CD25 expression suggesting a role in potential anticancer therapy.	[380]
Dithiolethiones			∙
In vitro	Mouse carcinoma Hepa-1c1c7 cells	Oltipraz 5–60 μM anetholedithione (ADT) 3–15 μM 1,2-dithiole-3-thione (D3T) 1–5 μM	Dithiolethiones are reduced in Hepa-1c1c7 cells leading to generation of superoxide radicals Superoxide dismutates to form hydrogen peroxide promoting translocation of Nrf2 to nucleus Translocated Nrf2 results in the upregulation of various Phase II enzyme expression.	Use of D3T and members of this family may be able to modify KEAP-1 activity and upregulate the expression of Phase II enzymes	[381]
In vivo	Male Fischer 344 (100 g)	D3T administered by oral gavage 0.5 mmol/kg (in distilled water with 1% Cremaphor, and 25% glycerol	Increased heme oxygenase (HO-1) activity Increased expression of HO-1 Increased levels of ferritin	Very early paper describing the potential utility of Dithiolethiones, like D3T, may offer protection against pro-carcinogenic compounds that increase oxidative stress	[382]
In vitro	HT29 colon adenocarcinoma cells	30μM D3T in DMSO vehicle (less than 0.1% final DMSO concentration)	Increased expression and activity of multiple reductases—Thioredoxin reductase 1, prostaglandin reductase 1, NAD(P)Quinone oxidoreductase 1 Potentiation of hydroxymethylacylfulvene action in alkylating DNA	D3T was a much stronger inducer of reductases compared to selenite and increased potency of the anticancer drug, hydroxymethylacylfulvene	[383]
In vivo	Male Fisher 344 rats (90–100 g)	Oral gavage of DST at 0.03 to 0.3 mmol/kg body wt at 3 days/week for 3 weeks Also 0.1 mmol/kg for measuring hepatic protein expression	Reduced the pre-cancer potency of aflatoxin B by inhibiting the expression of glutathione S-transferase-placental isoform Blocked aflatoxin-mediated increase in Induced multiple hepatic genes associated with detoxifying aflatoxin—including glutathione S-transferase A5 (GSTA5) and AFB1 aldehyde reductase	D3T is more potent than older Dithiolethiones like oltipraz and could be a probe for measuring anticancer potencies of this drug class	[384]
In vitro	HepG2 hepatic and LS180 colon cells	S-diclofenac and S-sulindac in range of 0–100 μM	Inhibited activity and expression of CYP1A1, 1B1 and 1A2 Regulates aryl hydrocarbon receptor pathway Blocked binding of aryl hydrocarbon to the responsive element Increased expression of anticancer enzymes, glutathione S transferase, glutamate cysteine ligase, and glutathione reductase	The NSAIDs with the dithiolethione group, S-diclofenac and S-sulindac, may function as effective anticancer agents	[385]
In vitro	HT29 and HCT116 colon adenocarcinoma	100 μM Oltipraz with 0.2% MeSO as a solvent control	Induction of p65, IκB kinase α (IKKα), IκB kinase β (IKKβ), and NF-κB-inducing kinase (NIK) IκBα phosphorylation was also induce Induction of protein binding to a consensus NF-κB element Transcriptional activation of QR was decreased Both MEKK3 and NIK exert effects on IKKα/β activation via different pathways	This is a novel pathway involved in QR gene regulation and may provide insight to the actions of oltipraz as an anticancer agent	[386]
Non-Electrophilic/Non-Covalent
In vivo and in vitro	Male C57/Bl6 mice (22 g) and bone marrow-derived mouse macrophage cells	RA839 was dissolved in a vehicle of 95% (v/v) hydroxyethyl cellulose (0.5% (w/v))/5% (v/v) solutol—injected IP at 30 mg/kg. General RA839 interactions measured at a concentration of 10 μM	Binds to KELCH domain with affinity of 6 μM Blocks Nrf2-Keap-1 protein-protein interaction Modified activity of multiple genes in mouse macrophage cells Reduced the induction of nitric oxide release and inducible nitric oxide synthase activity Induced Nrf2 gene expression in mouse liver	RA839 may be a useful tool in developing anticancer drugs that target the prevention of Nrf2-Keap1 protein interaction.	[80]
In vitro	THP-1 cells	Cells were exposed to TAT14 using a concentration range of 0–75 μM	Activates heme oxygenase 1 expression and activity Did not alter Nrf2 mRNA expression Reduced LPS-induced TNF expression	The 14-mer TAT fragment interacts with Keap1, preventing Nrf2-Keap1 association, allowing Nrf2 to activate mediators downstream	[387]
In vitro	HepG2 hepatic and U2OS bone cell lines	ML334 and its isomers in a concentration range of 0–100 μM	Binds with high affinity to KEAP1 Strong inducer of ARE activity in both hepatic and bone cell lines	First description of ML334 as a potent inhibitor of Nrf2-Keap1 interaction. Highly potent.	[388]
In vitro	Immortalized baby mouse kidney epithelial cells (iBMK) and MDA-MB-231 breast cancer cells	Geopyxin F and other “geopyxin” isomers were used in a concentration range of 0–70 μM depending on assay	Geopyxin F was the most potent of Geopyxin compounds at inducing Nrf2 activity Geopyxin F displayed almost not toxicity/lethality in MDA-MB-231 cells Geopyxin F increased autophagosome formation in iBMK cells Geopyxin F prevents ubiquitination and stabilizes Nrf2 (KEAP1-dependent)—but is independent of interactions at the Cys151 in KEAP1	Geopyxin F, demonstrated a higher level of protection compared to electrophilic Nrf2 activators. Heightened potency suggests that Geopyxin F may be a useful anticancer compound.	[78]
In vitro	MCF-7 breast cancer cells	Multiple drugs were used as “off-label” activators of Nrf2 Astemizole 8 μM Tamoxifen 1 μM Trifluoperazine 10 μM	Astemizole, Tamoxifen and Trifluoperazine increased expression of the NQO1, HO-1, and GCLM genes After 24 h, the stimulated gene expression to basal was highest in Astemizole compared to sulforaphane The genes for the detoxifying enzymes—NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1) where most sensitive to upregulation	After large-scale screening, select drugs were chosen based on their ability to activate Nrf2. This shows that ‘off-label’ mechanisms may have benefit as anticancer drugs. Astemizole was the best candidate.	[79]