Natural anthraquinone; casein kinase-2 (CK2); SARS-CoV; CK2α; 11β-HSD1

Emodin (10–400 μM) has an IC50 value of 200 μM and inhibits S protein binding to ACE2 in a dose-dependent manner[1]. In a dose-dependent manner, emodin (5-50 μM) decreases the infectivity of S-protein pseudotyped retroviruses. The SARS-CoV S protein cannot attach to Vero E6 cells when imodin is present [1]. Emodin inhibits casein kinase 2 (CK2) at ATP concentrations of 50 μM, 30.0 μM, and 7.1 μM for CK2α wild type, Ile174Ala mutant, and His160Ala mutant, respectively, with an IC50 of 5.9. At ATP concentration, the IC50 values for the CK2α wild type and the Val66Ala mutant are 1.40 and 38.00 μM, respectively [2]. Emodin is more than 5000 times more selective for the human and mouse 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzyme than type 2 isoenzymes, as seen by its modest inhibitory action against both mouse and human 11β-HSD2 (IC50 greater than 1 mM) [3].

In normal C57BL/6J male mice, emodin (single oral treatment of 100 or 200 mg/kg) suppresses 11β-HSD1 activity [3]. Emodin (100 mg/kg; oral; bid) lowers blood glucose, liver PEPCK, and glucose-6-phosphatase mRNA in diet-induced obesity (DIO) rats, improving insulin sensitivity and lipid metabolism [3].

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Single oral administration of emodin inhibited 11beta-HSD1 activity of liver and fat significantly in mice. Emodin reversed prednisone-induced insulin resistance in mice, whereas it did not affect dexamethasone-induced insulin resistance, which confirmed its inhibitory effect on 11beta-HSD1 in vivo. In DIO mice, oral administration of emodin improved insulin sensitivity and lipid metabolism, and lowered blood glucose and hepatic PEPCK, and glucose-6-phosphatase mRNA. Conclusions and implications: This study demonstrated a new role for emodin as a potent and selective inhibitor of 11beta-HSD1 and its beneficial effects on metabolic disorders in DIO mice. This highlights the potential value of analogues of emodin as a new class of compounds for the treatment of metabolic syndrome or type 2 diabetes.[3]

For the competition assay, biotinylated S protein was mixed with various amounts of extracts and incubated at 37 °C with shaking. After a 2-h incubation, the mixture was added to wells, which were coated with ACE2, and incubated at 37 °C for 1 h. Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. The percent inhibition was calculated by [1 − (OD value of mixture containing extract and S protein/OD value of mixture containing S protein only)] × 100. The IC50 value was determined as the quantity of compound required to inhibit the interaction between S protein and ACE2 at 50%.[1]

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Phosphorylation assay[2]
Phosphorylation assays were carried out in the presence of increasing amounts of each inhibitor tested in a final volume of 25 μL containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 12 mM MgCl2, 100 μM synthetic peptide substrate RRRADDSDDDDD, and 0.02 mM [γ-33P]ATP (500–1000 cpm/pmol), unless otherwise indicated, and incubated for 10 min at 37 °C. These conditions are suited for reaching maximal velocity. Assays were stopped by addition of 5 μL of 0.5 M orthophosphoric acid before spotting aliquots onto phosphocellulose filters. Filters were washed in 75 mM phosphoric acid (5–10 mL/each) four times, then once in methanol and dried before counting.[2]

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Measurement of 11β-HSD1 and -HSD2 activity in vitro[3]
The SPA was used to screen for inhibitors of 11β-HSDs (Mundt et al., 2005), with the microsome fractions prepared from the HEK-293 cells stably transfected with either human or mouse 11β-HSD1 or 11β-HSD2 as the enzyme source. Briefly, different concentrations of compound were added to 96-well microtitre plates, followed by the addition of 80 µL of 50 mM HEPES buffer, pH 7.4 containing 25 nM [1,2-(n)3H]-cortisone and 1.25 mM NADPH (for 11β-HSD1 assay) or 12.5 nM [1,2,6,7-(n)3H]-cortisol and 0.625 mM NAD+(for 11β-HSD2 assay). Reactions were initiated by the addition of 11β-HSD1 or 11β-HSD2, enzyme preparation as microsome fractions from HEK293 cells in a final concentration of 80 µg·mL−1 for 11β-HSD1, and 160 µg·mL−1 for 11β-HSD2, respectively. After a 60 min incubation at 37°C, the reaction was stopped by the addition of 35 µL of 10 mg·mL−1 protein A-coated yttrium silicate beads suspended in SuperBlock Blocking Buffer with 3 µg·mL−1 of murine monoclonal cortisol antibody and 314 µM glycyrrhetinic acid. The plates were incubated under plastic film on an orbital shaker for 120 min at room temperature before counting. The amount of [3H]-cortisol generated in 11β-HSD1 enzyme reaction or remaining from the 11β-HSD2 enzyme reaction was captured by the beads and determined in a microplate liquid scintillation counter. The % inhibition was calculated relative to a non-inhibited control. Data were obtained from at least three independent experiments. IC50 values were calculated from concentration–response curves by a non-linear regression analysis using Prism Version 4.

Cell Viability Assay[1]
Cell Types: Vero E6 cells transfected with the plasmid encoding ACE2
Tested Concentrations: 0, 5, 25, 50 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Vero cells treated with 50 μM remained 82.4±3.8% viability, the anti -SARS-CoV activity was not due to toxicity.

Animal/Disease Models: C57BL/6J male mice[3]
Doses: 100 or 200 mg/kg
Route of Administration: Acute administered po; Two hrs (hours) later, the mice were killed by cervical dislocation,
Experimental Results: Dramatically inhibited liver 11β-HSD1 enzymatic activity by 17.6 and 31.3% and mesenteric fat 11β-HSD1 enzymatic activity by 21.5 and 46.7% at 100 or 200 mg/kg, respectively.

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Animal/Disease Models: DIO mice (C57BL/6J male mice were fed a formulated research diet)[3]
Doses: 100 mg /kg
Route of Administration: po (oral gavage); twice per day; for 35 days
Experimental Results: decreased fasting glucose concentrations to 77.2% of the vehicle control mice after 7 days of treatment, and these remained Dramatically lower throughout the treatment period. demonstrated a significant reduction in blood glucose levels at all time-points following oral glucose challenge after 24 days of treatment. Evoked a Dramatically greater reduction in blood glucose values 40 and 90 min after insulin injection after 28 days of treatment. The serum insulin level was also significan

Absorption, Distribution and Excretion
Absorption, excretion, tissue distribution and metabolism of the anthraquinone [14C]emodin was studied after a single oral administration (approx. 50 mg/kg) to rats. Urinary excretion amounted to 18(+/- 5)% dose in 24 hr and to 22(+/- 6)% in 72 hr. Metabolites found in pooled urine (0-72 hr) were mostly free anthraquinones (emodin and emodic acid, 16% dose); 3% was conjugated and 3% was non-extractable radioactivity. In 24 hr, 48 +/- 11% and in 120 hr, 68 +/- 8% dose was excreted in the faeces, mostly in the free anthraquinone form. In two cannulated rats biliary excretion reached a maximum at approx. 6 hr and amounted to 49% dose within 15 hr; 70% of biliary activity was in the form of conjugated emodin. The content of radioactivity in most organs decreased significantly between 3 and 5 days. In kidneys, however, the 14C activity was still equiv. to 4.33 ppm. emodin after five days. Mesenterium and fat tissue showed increasing 14C activity from 72 to 120 hr.

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Metabolism / Metabolites
... The metabolism of emodin (1,3,8-trihydroxy-6-methylanthraquinone) /was studied/... With rat liver microsomes, the formation of two emodin metabolites, omega-hydroxyemodin and 2-hydroxyemodin, was observed. The rates of formation of omega-hydroxyemodin were not different with microsomes from rats that had been pretreated with inducers for different cytochrome P450 enzymes. Thus, the formation of omega-hydroxyemodin seems to be catalyzed by several cytochrome P450 enzymes at low rates. The formation of 2-hydroxyemodin was increased in liver microsomes from 3-methylcholanthrene-pretreated rats and was inhibited by alpha-naphthoflavone, by an anti-rat cytochrome P450 1A1/2 antibody, and, to a lesser degree, by an anti-rat cytochrome P450 1A1 antibody. These data suggest the involvement of cytochrome P450 1A2 in the formation of this metabolite. However, other cytochrome P450 enzymes also seem to catalyze this reaction. The anthraquinone chrysophanol (1,8-dihydroxy-3-methylanthraquinone) is transformed, in a cytochrome P450-dependent oxidation, to aloe-emodin (1, 8-dihydroxy-3-hydroxymethylanthraquinone) as the major product formed.
The hepatic microsomes derived from various animal species transformed emodin (1,3,8-trihydroxy-6-methylanthraquinone), into an unidentified anthraquinone, along with 2-hydroxy-, 4-hydroxy- and 7-hydroxyemodins. ... This major metabolite /was identified/ as omega-hydroxy-emodin (1,3,8-trihydroxy-6-hydroxymethylanthraquinone). Among 7 animal species, the highest activity to produce this omega-hydroxyemodin was observed in the hepatic microsomes of guinea pig and rat, followed by mouse and rabbit. The microsomal activity to convert emodin into omega-hydroxyemodin was accelerated by the pretreatment of animals with phenobarbital, and inhibited by SKF 525A. The microsomal hydroxylation reactions of the methyl residue and the anthraquinoid nucleus of emodin were presumed to be catalyzed regiospecifically by multiple forms of cytochrome P-450.
... Emodin was biotransformed by the microsomal enzymes into at least 5 quinonoid metabolites, among which one pigment, identified as 2-hydroxyemodin (1,2,3,8-tetrahydroxy-6-methyl-anthraquinone), was proven to be a direct mutagen to the test strain, and the remaining 4 quinonoid metabolites were negative or far less active than this active principle.
Emodin has known human metabolites that include Emodin 3-hydroxy-glucuronide.
Emodin is biotransformed by the microsomal cytochrome P450 enzymes into active hydroxyemodins such as omega-hydroxyemodin and 2-hydroxyemodin. Emodin glycoside is carried unabsorbed to the large intestine, where it is metabolized to the active aglycones by intestinal bacterial flora. (A3043, A3046)

Toxicity Summary
Emodin is moderately cytotoxic and can inhibit the growth of many cell types by interfering with the cell cycle, possibly by stimulating the expression of p53 and p21. Alternatively, it may do this by creating DNA strand breaks and/or non-covalently binding to DNA and inhibiting the catalytic activity of topoisomerase II. It may also induce apoptosis by inhibiting the electron transport chain, producing reactive oxygen species. Emodin is a strong inhibitor of tyrosine-protein kinase Lck and other tyrosine kinase receptors, which likely contributes to its growth suppressing activity. It may act as a chemopreventive agent by activating DNA repair machinery.

Emodin can also inhibit metastasis by interfering with the activity of matrix metalloproteinases, either directly or through through inhibition of focal adhesion kinase, mitogen-activated protein kinase, and RAC-alpha serine/threonine-protein kinase activation, and partial inhibition of transcription factor AP-1 and nuclear factor NF-kappa-B (NF-kB) transcriptional activities.

Emodin exerts its purgative effects by acting directly or indirectly on colon epithelial cells. This activates the underlying smooth muscle cells, leading to muscle contractility. Possible mechanisms for this effect includes enhancing the hormone motilin, activating muscarinic receptors by triggering the release of acetylcholine, stimulating the protein kinase C-alpha pathway for increased calcium sensibility, inhibiting the secretion of the hormone somatostatin, increasing fluid electrolyte accumulation in the distal ileum and colon, and inhibiting the activity of Na+/K+-ATPase and/or potassium channels.

Emodin's antiinflammatory action is due to its specific inhibition of the transcription factor NF-kB. It also regulates angiogenesis by inhibiting the enzymes casein kinase II and nitric oxide synthase and has shown potent estrogen receptor binding affinity. Emodin can induce the microsomal enzyme cytochrome P-450 1A1, perpetuating its own metabolic activation. (A3043, A3044, A3045, A3046, A3047, A3048, A3049, A3050, A3051, A3052, A3053, A3054)

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Toxicity Summary
Emodin is moderately cytotoxic and can inhibit the growth of many cell types by interfering with the cell cycle, possibly by stimulating the expression of p53 and p21. Alternatively, it may do this by creating DNA strand breaks and/or non-covalently binding to DNA and inhibiting the catalytic activity of topoisomerase II. It may also induce apoptosis by inhibiting the electron transport chain, producing reactive oxygen species. Emodin is a strong inhibitor of tyrosine-protein kinase Lck and other tyrosine kinase receptors, which likely contributes to its growth suppressing activity. It may act as a chemopreventive agent by activating DNA repair machinery. Emodin can also inhibit metastasis by interfering with the activity of matrix metalloproteinases, either directly or through through inhibition of focal adhesion kinase, mitogen-activated protein kinase, and RAC-alpha serine/threonine-protein kinase activation, and partial inhibition of transcription factor AP-1 and nuclear factor NF-kappa-B (NF-kB) transcriptional activities. Emodin exerts its purgative effects by acting directly or indirectly on colon epithelial cells. This activates the underlying smooth muscle cells, leading to muscle contractility. Possible mechanisms for this effect includes enhancing the hormone motilin, activating muscarinic receptors by triggering the release of acetylcholine, stimulating the protein kinase C-alpha pathway for increased calcium sensibility, inhibiting the secretion of the hormone somatostatin, increasing fluid electrolyte accumulation in the distal ileum and colon, and inhibiting the activity of Na+/K+-ATPase and/or potassium channels. Emodin's antiinflammatory action is due to its specific inhibition of the transcription factor NF-kB. It also regulates angiogenesis by inhibiting the enzymes casein kinase II and nitric oxide synthase and has shown potent estrogen receptor binding affinity. Emodin can induce the microsomal enzyme cytochrome P-450 1A1, perpetuating its own metabolic activation.

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Non-Human Toxicity Values
LD50: 35 mg/kg (Intraperitoneal, Mouse) (L135)

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Toxicity Data
LD50: 35 mg/kg (Intraperitoneal, Mouse) (L135)

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Non-Human Toxicity Values
LD50 Mouse ip 35 mg/kg

[1]. Emodin blocks the SARS coronavirus spike protein and angiotensin-converting enzyme 2 interaction. Antiviral Res. 2007 May;74(2):92-101.

[2]. Toward the rational design of protein kinase casein kinase-2 inhibitors. Pharmacol Ther. Feb-Mar 2002;93(2-3):159-68.

[3]. Emodin, a natural product, selectively inhibits 11beta-hydroxysteroid dehydrogenase type 1 and ameliorates metabolic disorder in diet-induced obese mice. Br J Pharmacol. 2010 Sep;161(1):113-26.

Emodin appears as orange needles or powder. (NTP, 1992)
Emodin is a trihydroxyanthraquinone that is 9,10-anthraquinone which is substituted by hydroxy groups at positions 1, 3, and 8 and by a methyl group at position 6. It is present in the roots and barks of numerous plants (particularly rhubarb and buckthorn), moulds, and lichens. It is an active ingredient of various Chinese herbs. It has a role as a tyrosine kinase inhibitor, an antineoplastic agent, a laxative and a plant metabolite. It is functionally related to an emodin anthrone. It is a conjugate acid of an emodin(1-).
Emodin has been investigated for the treatment of Polycystic Kidney.
Emodin has been reported in Hamigera avellanea, Setophoma terrestris, and other organisms with data available.
Emodin is found in dock. Emodin is present in Cascara sagrada.Emodin is a purgative resin from rhubarb, Polygonum cuspidatum, the buckthorn and Japanese Knotweed (Fallopia japonica). The term may also refer to any one of a series of principles isomeric with the emodin of rhubarb. (Wikipedia)
Emodin has been shown to exhibit anti-inflammatory, signalling, antibiotic, muscle building and anti-angiogenic functions (A3049, A7853, A7854, A7855, A7857).
Purgative anthraquinone found in several plants, especially RHAMNUS PURSHIANA. It was formerly used as a laxative, but is now used mainly as a tool in toxicity studies.
See also: Frangula purshiana Bark (part of); Reynoutria multiflora root (part of).

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Mechanism of Action
The anthraquinone mycotoxins emodin and skyrin were examined for the inhibitory effects on murine leukemia L1210 culture cells, oxidative phosphorylation of rat liver mitochondria, and Na+, K+-activated ATPase activity of rat brain microsomes to find the differences between their modes of toxic action. Skyrin exhibited a stronger inhibitory effect than emodin on the growth of L1210 culture cells. Emodin showed a stronger uncoupling effect than skyrin on mitochondrial respiration. Skyrin inhibited Na+, K+-activated ATPase activity of rat brain microsomes but emodin did not inhibit.
... Emodin induces apoptotic responses in the human hepatocellular carcinoma cell lines (HCC) Mahlavu, PLC / PRF / 5 and HepG2. The addition of emodin to these three cell lines led to inhibition of growth in a time- and dose-dependent manner. Emodin generated reactive oxygen species (ROS) in these cells which brought about a reduction of the intracellular mitochondrial transmembrane potential (Deltaym), followed by the activation of caspase-9 and caspase-3, leading to DNA fragmentation and apoptosis.
Emodin inhibited the activity of TPK and CK2 and the degradation of I-kappaB.
... Emodin-induced apoptosis of CH27 cells does not involve modulation of endogenous Bcl-X(L) protein expression, but appears to be associated with the increased expression of cellular Bak and Bax proteins.
For more Mechanism of Action (Complete) data for EMODIN (9 total), please visit the HSDB record page.

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Therapeutic Uses
Emodin /is/ a widely available over-the-counter herbal remedy.
/EXPL THER:/ ... Emodin and cassiamin B /were examined as cancer chemopreventive agents/ ... These compounds exhibited the remarkable anti-tumor promoting effect on two-stage carcinogenesis test of mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter by both topical application. Furthermore, emodin exhibited potent inhibitory activity on two-stage carcinogenesis test of mouse skin tumors induced by nitric oxide donor, (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide as an initiator and TPA as a promoter.
/EXPL THER:/ ... Emodin ameliorates the undesirable effects of concentrated glucose on HPMC /human peritoneal mesothelial cells/ via suppression of PKC activation and CREB phosphorylation, and suggest that emodin may have a therapeutic potential in the prevention or treatment of glucose-induced structural and functional abnormalities in the peritoneal membrane.

C15H10O5

270.24

270.052

C, 66.67; H, 3.73; O, 29.60

518-82-1

Emodin-d4;132796-52-2

3220

Yellow to orange solid powder

1.6±0.1 g/cm3

586.9±39.0 °C at 760 mmHg

255 °C (dec.)(lit.)

322.8±23.6 °C

0.0±1.7 mmHg at 25°C

1.745

5.03

3

5

0

20

434

0

RHMXXJGYXNZAPX-UHFFFAOYSA-N

InChI=1S/C15H10O5/c1-6-2-8-12(10(17)3-6)15(20)13-9(14(8)19)4-7(16)5-11(13)18/h2-5,16-18H,1H3

1,3,8-trihydroxy-6-methylanthracene-9,10-dione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 3.33 mg/mL (12.32 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one),Suspened solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 10 mg/mL (37.00 mM) in 0.5% MC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)