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

大黄素 (10–400 μM) 的 IC50 值为 200 μM，并以剂量依赖性方式抑制 S 蛋白与 ACE2 的结合[1]。大黄素 (5-50 μM) 以剂量依赖性方式降低 S 蛋白假型逆转录病毒的感染性。当存在伊莫丁时，SARS-CoV S 蛋白无法附着到 Vero E6 细胞上 [1]。对于 CK2α 野生型、Ile174Ala 突变体和 His160Ala 突变体，大黄素在 ATP 浓度分别为 50 μM、30.0 μM 和 7.1 μM 时抑制酪蛋白激酶 2 (CK2)，IC50 为 5.9。在 ATP 浓度下，CK2α 野生型和 Val66Ala 突变体的 IC50 值分别为 1.40 和 38.00 μM [2]。大黄素对人类和小鼠 2 型 11β-羟基类固醇脱氢酶 (11β-HSD2) 的选择性比 2 型同工酶高 5000 倍以上，这可以从其对小鼠和人类 11β-HSD2 的适度抑制作用看出（IC50 大于 1）毫米）[3]。

在正常 C57BL/6J 雄性小鼠中，大黄素（单次口服治疗 100 或 200 mg/kg）可抑制 11β-HSD1 活性 [3]。大黄素（100 mg/kg；口服；bid）可降低饮食诱导肥胖 (DIO) 大鼠的血糖、肝脏 PEPCK 和葡萄糖 6 磷酸酶 mRNA，改善胰岛素敏感性和脂质代谢 [3]。

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单次口服大黄素可显著抑制小鼠肝脏和脂肪11 - β - hsd1活性。大黄素逆转强的松诱导的小鼠胰岛素抵抗，而对地塞米松诱导的胰岛素抵抗无影响，证实了大黄素在体内对11β - hsd1的抑制作用。在DIO小鼠中，口服大黄素改善胰岛素敏感性和脂质代谢，降低血糖和肝脏PEPCK和葡萄糖-6-磷酸酶mRNA。 结论和意义:本研究揭示了大黄素作为11β - hsd1的有效选择性抑制剂的新作用及其对DIO小鼠代谢紊乱的有益作用。这凸显了大黄素类似物作为一类治疗代谢综合征或2型糖尿病的新型化合物的潜在价值。[3]

在竞争实验中，将生物素化S蛋白与不同量的提取物混合，在37℃下摇匀孵育。孵育2小时后，将混合物加入孔中，用ACE2包被，在37℃下孵育1小时。三次洗涤后，依次加入过氧化物酶偶联的亲和素和着色底物。用ELISA读板仪在405 nm处读取吸光度。用[1−(含提取物和S蛋白混合物的OD值/只含S蛋白混合物的OD值)]× 100计算抑制率。IC50值为抑制S蛋白与ACE2相互作用所需化合物的量(50%)。[1]

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磷酸化实验[2]
磷酸化实验在每种抑制剂用量增加的情况下进行，最终体积为25 μL，含50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 12 mM MgCl2, 100 μM合成肽底物RRRADDSDDDDD和0.02 mM [γ-33P]ATP (500-1000 cpm/pmol)，除非另有说明，并在37℃下孵育10分钟。这些条件适合达到最大速度。加入5 μL 0.5 M正磷酸停止检测，然后将等分液点染到磷酸纤维素过滤器上。过滤器在75 mM磷酸(5-10 mL/个)中洗涤四次，然后在甲醇中洗涤一次，干燥后计数。[2]

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体外测定11β-HSD1和-HSD2活性[3]
使用SPA筛选11β- hsd抑制剂(Mundt et al.， 2005)，以稳定转染人或小鼠11β-HSD1或11β-HSD2的HEK-293细胞制备的微粒体部分为酶源。简单地说，将不同浓度的化合物加入96孔微滴板，然后加入80µL 50 mM HEPES缓冲液，pH 7.4含有25 nM [1,2-(n)3H]-可的松和1.25 mM NADPH(用于11β-HSD1测定)或12.5 nM [1,2,6,7-(n)3H]-皮质醇和0.625 mM NAD+(用于11β-HSD2测定)。11β-HSD1和11β-HSD2分别以80µg·mL−1和160µg·mL−1的浓度从HEK293细胞中提取酶制剂作为微粒体，加入酶制剂引发反应。37℃孵育60 min后，加入35µL 10 mg·mL−1蛋白包被的硅酸钇珠悬浮在SuperBlock阻断缓冲液中，其中含有3µg·mL−1小鼠单克隆皮质醇抗体和314µM甘草次酸，停止反应。在室温下，在轨道摇床上用塑料薄膜孵育120分钟，然后计数。珠子捕获11β-HSD1酶反应中产生的[3H]-皮质醇的量或11β-HSD2酶反应中剩余的[3H]-皮质醇的量，并在微孔板液体闪烁计数器上测定。计算相对于非抑制对照的抑制率。数据来自至少三个独立的实验。采用Prism Version 4对浓度-响应曲线进行非线性回归分析，计算IC50值。

细胞活力测定[1]
细胞类型：用编码 ACE2 的质粒转染的 Vero E6 细胞
测试浓度： 0、5、25、50 μM
孵育时间：24小时
实验结果：用50 μM处理的Vero细胞仍保持82.4±3.8%的活力，即抗SARS-CoV活性并不是因为毒性。

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

94.83

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)

配方 1 中的溶解度: 3.33 mg/mL (12.32 mM) in 0.5% CMC-Na/saline water (这些助溶剂从左到右依次添加，逐一添加),悬浮液； 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 10 mg/mL (37.00 mM) in 0.5% MC 0.5% Tween-80 (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。