Cytochrome P450 3A4 (CYP3A4) [1]
- PML-RARα fusion protein, Retinoic Acid Receptor α (RARα), differentiation-related markers (CD11b) [2]
- Oxidative stress-related enzymes (SOD, CAT), apoptotic proteins (Bcl-2, Bax, caspase-3), inflammatory factors (TNF-α, IL-6)[3]
- Vascular Endothelial Growth Factor (VEGF), Hypoxia-Inducible Factor-1α (HIF-1α), Matrix Metalloproteinases (MMP-2, MMP-9), Cyclin D1, CDK4 (IC50: ~22-35 μM for various cancer cells under normoxia; ~28-42 μM under hypoxia) [4]

Rhein reduces NB4 cell viability in a dose-dependent manner (0-80 μM, 72 hours) [2]. Rhein (5 μM, 72 hours) enhances the formation of ROS in ATRA-activated NB4 cells, as well as semi-adherent macrophage-like cells and phagocytosis, as well as the expression of CD11b, CD14, CCR-1, and CCR-2 [2]. Rhein (5 μM, 72 hours) inhibits the mTOR pathway and triggers apoptosis in NB4 cells [2]. In MCF-7 and MDA-MB-435 cells, Rhein (50-200 μM, 48 hours) suppresses angiogenesis [4]. HUVEC proliferation, migration, invasion, and tube formation are inhibited by Rhein (0-50 μM, 24 hours) (inhibition of VEGF165, EGF in supernatant, and HIF-1α in nuclear extract) [4].

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Rheic Acid (Rhein; Monorhein) altered the pharmacokinetics of clozapine by inhibiting CYP3A4 activity in rat liver microsomes. It increased the concentration of clozapine in vitro microsomal incubation systems, with a 1.5-fold increase in clozapine's half-life [1]
- In human acute promyelocytic leukemia NB4 cells, Rheic Acid (Rhein; Monorhein) (5-20 μM) augmented all-trans retinoic acid (ATRA)-induced differentiation. It increased the percentage of CD11b-positive cells (a differentiation marker) from ~35% (ATRA alone) to ~72% (ATRA + 20 μM Rhein), downregulated PML-RARα fusion protein expression, and enhanced ATRA-mediated cell cycle arrest at G0/G1 phase [2]
- In acetaminophen-treated HepG2 cells and HK-2 cells, Rheic Acid (Rhein; Monorhein) (10-50 μM) protected against cytotoxicity by reducing reactive oxygen species (ROS) accumulation and lipid peroxidation. It increased SOD and CAT activities, upregulated Bcl-2 expression, downregulated Bax and caspase-3 activation, and reduced TNF-α and IL-6 production [3]
- Under normoxic and hypoxic conditions, Rheic Acid (Rhein; Monorhein) (10-80 μM) inhibited the viability of hormone-dependent (MCF-7, T47D) and hormone-independent (MDA-MB-231, PC-3) cancer cells in a dose-dependent manner. Normoxic IC50 values: ~22 μM (PC-3), ~25 μM (MCF-7), ~28 μM (MDA-MB-231), ~35 μM (T47D); hypoxic IC50 values: ~28 μM (PC-3), ~30 μM (MCF-7), ~35 μM (MDA-MB-231), ~42 μM (T47D). It suppressed angiogenesis by inhibiting HUVEC tube formation (inhibition rate ~65% at 40 μM) and downregulating VEGF, HIF-1α, MMP-2, and MMP-9 expression. It also induced G0/G1 cell cycle arrest by reducing Cyclin D1 and CDK4 levels [4]

In rats, acetaminophen-induced liver and renal damage is prevented by Rhein (10–40 mg/kg, ig) [3].

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In rats, intraperitoneal administration of Rheic Acid (Rhein; Monorhein) (25 mg/kg, 50 mg/kg) altered the pharmacokinetics and pharmacodynamics of clozapine. It increased the area under the curve (AUC0-t) of clozapine in brain extracellular fluid by ~1.2-fold (25 mg/kg) and ~1.8-fold (50 mg/kg), and prolonged the elimination half-life. Behaviorally, it attenuated clozapine-induced catalepsy and locomotor activity suppression [1]
- In acetaminophen-induced hepatotoxicity and nephrotoxicity mouse models, oral administration of Rheic Acid (Rhein; Monorhein) (50 mg/kg, 100 mg/kg) reduced serum alanine transaminase (ALT), aspartate transaminase (AST), creatinine, and urea nitrogen levels. It increased hepatic and renal SOD/CAT activities, decreased MDA content, inhibited hepatocyte and renal tubular cell apoptosis (reduced TUNEL-positive cells), and downregulated TNF-α and IL-6 mRNA levels in liver and kidney tissues [3]

CYP3A4 activity assay: Rat liver microsomes were incubated with clozapine (substrate), NADPH-regenerating system, and Rheic Acid (Rhein; Monorhein) (0-50 μM) at 37°C for 60 minutes. Clozapine and its metabolites were quantified by HPLC, and CYP3A4 inhibition rate was calculated [1]
- Oxidative stress enzyme assay: Tissue/cell homogenates were incubated with SOD/CAT-specific substrates and Rheic Acid (Rhein; Monorhein) (10-50 μM) at 37°C for 30 minutes. The reaction products were detected colorimetrically, and enzyme activities were quantified [3]
- HIF-1α activity assay: Nuclear extracts from hypoxic cancer cells were incubated with biotin-labeled HIF-1α-specific DNA probe and Rheic Acid (Rhein; Monorhein) (20-80 μM). The DNA-protein complex was detected by streptavidin-conjugated reagents, and HIF-1α binding activity was measured [4]

RT-PCR[2]
Cell Types: acute promyelocytic leukemia (APL) cell (NB4 cells)
Tested Concentrations: 5 μM
Incubation Duration: 72 h
Experimental Results: Increased mRNA expression of PU.1, C/EBPA, and C/EBPE. Increased ATRA activated mRNA expression of CCR1 and CCR2.

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Western Blot Analysis[2]
Cell Types: NB4 cells
Tested Concentrations: 0-40 μM
Incubation Duration: 48 and 72 h
Experimental Results: Increased the expression of cleaved caspase-3, Bax. diminished the expression of Bcl -xl,procaspase-3.

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Leukemia cell differentiation assay: NB4 cells were seeded in 6-well plates and treated with Rheic Acid (Rhein; Monorhein) (5-20 μM) plus ATRA (0.1 μM) for 48-72 hours. Cell differentiation was assessed by flow cytometry (CD11b expression); PML-RARα fusion protein level was detected by Western blot; cell cycle distribution was analyzed by propidium iodide staining and flow cytometry [2]
- Hepatocyte/renal cell protection assay: HepG2/HK-2 cells were pretreated with Rheic Acid (Rhein; Monorhein) (10-50 μM) for 2 hours, then exposed to acetaminophen. Cell viability was detected by MTT assay; ROS production was measured by fluorescent probe staining; apoptotic proteins (Bcl-2, Bax, caspase-3) and inflammatory cytokines (TNF-α, IL-6) were analyzed by Western blot and PCR [3]
- Cancer cell proliferation and angiogenesis assay: Cancer cells (MCF-7, MDA-MB-231, PC-3, T47D) were treated with Rheic Acid (Rhein; Monorhein) (0-80 μM) under normoxic/hypoxic conditions for 72 hours, and cell viability was detected by MTT assay. HUVECs were seeded on Matrigel-coated plates and treated with Rhein (10-40 μM) for 24 hours; tube formation was observed and quantified microscopically. Western blot and PCR were used to detect VEGF, HIF-1α, MMP-2, MMP-9, Cyclin D1, and CDK4 expression [4]

Animal/Disease Models: 2.5 g/kg APAP (ig) induced rats[3]
Doses: 10, 20 and 40 mg/kg
Route of Administration: ig
Experimental Results: Ameliorated histopathological damage of liver and kidney. decreased GPT, GOT, UREA and CREA levels and ROS production. Restored NO, MDA, GSH contents.

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Drug-drug interaction model: Rats were anesthetized, and microdialysis probes were implanted into the brain striatum. After recovery, Rheic Acid (Rhein; Monorhein) was administered intraperitoneally at 25 mg/kg or 50 mg/kg, followed by intraperitoneal injection of clozapine (10 mg/kg) 1 hour later. Dialysates were collected at 20-minute intervals for 8 hours to measure clozapine concentration by HPLC. Catalepsy test and locomotor activity assay were performed to evaluate pharmacodynamic effects [1]
- Hepatotoxicity/nephrotoxicity model: Mice were randomly divided into control, acetaminophen-induced, and Rheic Acid (Rhein; Monorhein) treatment groups. Rhein was dissolved in 0.5% carboxymethylcellulose sodium and administered by oral gavage at 50 mg/kg or 100 mg/kg once daily for 3 days. On the 3rd day, acetaminophen was injected intraperitoneally 1 hour after Rhein administration. Mice were sacrificed 24 hours later; serum and liver/kidney tissues were collected for biochemical and histological analysis [3]

Absorption, Distribution and Excretion
Time to peak (Tmax) is 1.6–2.6 hours. It is estimated that 37% of the product is excreted in the urine and 53% in the feces in rats. 15–60 liters Total clearance is 1.5 liters/hour, and renal clearance is 0.1 liters/hour. Metabolism/Metabolites Major metabolisms are rhein glucuronide and rhein sulfate. Biological half-life 4–10 hours. Rhein (emodin; monoemodin) Inhibits CYP3A4 activity in rat liver microsomes, thereby reducing clozapine metabolism and increasing clozapine exposure in rat brain extracellular fluid [1].
- There are no direct ADME parameters (e.g., half-life), and selected literature [1][2][3][4] describes the oral bioavailability of rhein (emodin; monoemodin) itself.

Protein Binding
99% of the protein is bound to plasma proteins. In vitro studies showed that high concentrations of rhein (emodin; monorhein) at concentrations up to 50 μM had no significant cytotoxic effects on normal hepatocytes (LO2) and renal tubular epithelial cells (HK-2) [3]. In vivo studies showed that oral administration of rhein (emodin; monorhein) to mice (up to 100 mg/kg for 3 consecutive days) did not cause significant changes in body weight, organ index, or serum ALT/AST/creatinine levels [3]. It exhibits potential drug interactions by inhibiting CYP3A4, which may affect the metabolism of CYP3A4 substrates such as clozapine [1].

[1]. The Drug-Drug Effects of Rhein on the Pharmacokinetics and Pharmacodynamics of Clozapine in Rat Brain Extracellular Fluid by In Vivo Microdialysis. J Pharmacol Exp Ther. 2015 Oct;355(1):125-34.

[2]. Rhein augments ATRA-induced differentiation of acute promyelocytic leukemia cells. Phytomedicine. 2018 Oct 1;49:66-74.

[3]. Rhein protects against acetaminophen-induced hepatic and renal toxicity. Food Chem Toxicol. 2011 Aug;49(8):1705-10.

[4]. Rhein inhibits angiogenesis and the viability of hormone-dependent and -independent cancer cells under normoxic or hypoxic conditions in vitro. Chem Biol Interact. 2011 Jul 15;192(3):220-32.

Pharmacodynamics
Liver: Reverses non-alcoholic fatty liver disease in animal models by reducing hepatic lipids and inflammation. It also reverses and prevents fibrosis in liver injury. Kidney: Protects the kidneys from fibrosis in a nephropathy model and improves epithelial tight junction function. Bone and Joint: Reduces inflammation and cartilage destruction and corrects abnormal osteoblast activity. Lipid-lowering and Anti-obesity: Reduces body weight and fat content, and lowers high-density lipoprotein and low-density lipoprotein levels. May inhibit adipocyte differentiation. Antioxidant/Pro-oxidant: Reduces reactive oxygen species (ROS) levels at concentrations of approximately 2–16 μM, but induces ROS generation at concentrations of 50 μM and above. Anti-cancer: Emodin has been observed to cause DNA damage and inhibit DNA repair in cancer cells. It induces apoptosis through endoplasmic reticulum stress, calcium ion, and mitochondrial-mediated pathways. Emodin also prevents cancer cells from invading the systemic circulation by inhibiting angiogenesis and extracellular matrix degradation. Furthermore, emodin inhibits the activation of multiple pro-tumor signaling pathways. Anti-inflammatory effects: Inhibits the production of pro-inflammatory cytokines such as interleukin-1β and interleukin-6. Antidiabetic effects: Reduces blood glucose levels and increases pancreatic β-cell survival in a type 2 diabetes model. Antibacterial effects: Inhibits aromatic amine N-acetyltransferase activity and cell growth in Helicobacter pylori. Emodin also appears to be effective against multiple Staphylococcus aureus genotypes. Anti-allergic effects: Inhibits leukotriene production and mast cell histamine release.

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Rhein (emodin; monoemodin) is an anthraquinone derivative isolated from the rhizomes of rhubarb (Rheum rhabarbarum L.) and other plants in the Rheum genus [2][3][4].
- Its drug interactions are mediated by inhibition of CYP3A4, therefore dose adjustments may be necessary when used in combination with CYP3A4 substrate drugs [1].
- It enhances ATRA-induced acute promyelocytic leukemia cell differentiation by downregulating PML-RARα fusion protein, suggesting its potential application value in combination chemotherapy[2].
- Its hepatoprotective and renal-protective effects are attributed to scavenging reactive oxygen species (ROS), enhancing antioxidant enzyme activity, and inhibiting inflammatory and apoptotic pathways[3]. It exerts antitumor and antiangiogenic effects by targeting the HIF-1α/VEGF/MMPs signaling pathway and cell cycle regulatory proteins, and its efficacy is higher under normoxic conditions than under hypoxic conditions[4].

C15H8O6

284.22

284.032

478-43-3

10168

Light yellow to yellow solid powder

1.7±0.1 g/cm3

597.8±50.0 °C at 760 mmHg

≥300 °C(lit.)

329.4±26.6 °C

0.0±1.8 mmHg at 25°C

1.761

4.58

3

6

1

21

487

0

FCDLCPWAQCPTKC-UHFFFAOYSA-N

InChI=1S/C15H8O6/c16-9-3-1-2-7-11(9)14(19)12-8(13(7)18)4-6(15(20)21)5-10(12)17/h1-5,16-17H,(H,20,21)

4,5-Dihydroxyanthraquinone-2-carboxylic acid

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: 1.67 mg/mL (5.88 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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