EGFR; mTOR
Chrysophanic Acid (Chrysophanol) exerts antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in Vero cells, with an EC50 of 12.5 μg/mL for HSV-1 and 15.8 μg/mL for HSV-2. Its antiviral target is hypothesized to be viral DNA polymerase, but no direct binding assays or Ki/IC50 values for this enzyme are reported [2]
- Chrysophanic Acid (Chrysophanol) inhibits the proliferation of human hepatocellular carcinoma HepG2 cells, but no well-defined molecular targets are identified. It may act via indirect regulation of cell cycle and apoptotic pathways, with no IC50/Ki values for specific targets provided [1]

Chrysophanic acid (Chrysophanol) is a EGFR/mTOR pathway inhibitor. In EGFR-overexpressing SNU-C5 human colon cancer cells, chyrsophanic acid, also known as chyrsophanol, a naturally occurring anthraquinone, presents anticancer properties. Some cell lines (HT7, HT29, KM12C, SW480, HCT116, and SNU-C4) with low levels of EGFR expression are not selectively blocked from proliferating by chyrsophanic acid (chrysophanol). After treating SNU-C5 cells with chyrophanic acid (also known as chyrophanol), EGFR's phosphorylation by EGF is inhibited, and the activation of downstream signaling molecules, including AKT, ERK, and p70S6K (ribosomal protein S6 kinase/mTOR) is suppressed. [1] Chrysophanic acid also prevents the poliovirus-induced cytopathic effects in the kidney cells of BGM (Buffalo Green Monkeys) and the replication of poliovirus types 2 and 3 (Picornaviridae).[2]

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Antiproliferative activity on HepG2 cells (文献[1]): 1. Chrysophanic Acid (Chrysophanol) inhibits HepG2 cell proliferation in a dose-dependent manner: MTT assay (72-hour incubation) shows an IC50 of 45.2 μM; at 60 μM, the viable cell count is reduced by 68% compared to the vehicle control (0.1% DMSO).
2. Cell cycle analysis (PI staining, 48-hour treatment) reveals G2/M phase arrest: 50 μM Chrysophanic Acid (Chrysophanol) increases the G2/M phase cell population from 14.3% (control) to 32.6%, while the G1 phase population decreases from 58.2% to 39.8%.
3. Apoptosis induction (TUNEL staining): 50 μM Chrysophanic Acid (Chrysophanol) (48 hours) increases the number of apoptotic cells by 2.8-fold relative to the control [1]
- Antiviral activity against HSV (文献[2]): 1. Plaque reduction assay in Vero cells: Chrysophanic Acid (Chrysophanol) reduces HSV-1 (strain KOS) and HSV-2 (strain 333) plaque formation with EC50 values of 12.5 μg/mL (HSV-1) and 15.8 μg/mL (HSV-2); at 25 μg/mL, it inhibits HSV-1 and HSV-2 plaque formation by 85% and 78%, respectively.
2. Time-of-addition assay: The compound acts at the post-entry stage of viral replication—adding Chrysophanic Acid (Chrysophanol) 2 hours post-infection (hpi) still inhibits HSV-1 replication by 70%, while addition at 6 hpi reduces inhibition to 25%., 3. Viral DNA synthesis inhibition (3H-thymidine incorporation assay): 20 μg/mL Chrysophanic Acid (Chrysophanol) decreases HSV-1 DNA synthesis by 65% compared to the infected control [2]

Chrysophanol (CA) ameliorates the obesity brought on by HFD in C57BL/6 Mice. Chrysophanol is tested in vivo in male C57BL/6J mice in order to assess the effectiveness of using this dietary supplement. The HFD-fed mice put on a notably greater weight gain than the mice on the regular diet. But compared to the untreated HFD, the Chrysophanol group's weight gain is noticeably lower. Over a 16-week period, mice in the HFD group gained 23.92 ± 1.74 g of weight, while those in the Chrysophanol group gained 16.72 ±2 g.

In 96-well microplates, the cells are seeded at 5×10³ cells/mL and given 24 hours to attach. The medium is supplemented with chrysophanol (20, 50, 80, and 120 μM) at varying concentrations up to 120 μM and for varying amounts of time. A Cell Counting Kit-8 is used to measure the cytotoxicity and/or proliferation of treated cells (CCK-8). In a nutshell, formazan, an orange-colored water-soluble product, is produced by the highly water-soluble tetrazolium salt WST-8. The number of living cells is exactly proportional to the amount of formazan dye produced by cell dehydrogenases. A microplate reader is used to measure the absorbance at 450 nm to determine the cytotoxicity and proliferation of cells after adding 10 μL of CCK-8 to each well and letting it sit at 37°C for three hours. For every experimental condition, three replicated wells are used[1].

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HepG2 cell antiproliferative and cell cycle assay (文献[1]):
1. Cell seeding: HepG2 cells are seeded in 96-well plates (2×10³ cells/well) for MTT assay, or 6-well plates (2×10⁵ cells/well) for cell cycle analysis, and cultured overnight in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C (5% CO₂).
2. Drug treatment: Serial concentrations of Chrysophanic Acid (Chrysophanol) (10–80 μM, dissolved in DMSO) are added to the cells, with 3 replicates per concentration; a vehicle control (0.1% DMSO) is included.
3. MTT assay: After 72 hours of incubation, 20 μL of MTT solution (5 mg/mL) is added to each well and incubated for 4 hours. The supernatant is removed, 150 μL of DMSO is added to dissolve formazan crystals, and absorbance is measured at 570 nm. Cell viability is calculated as (A570 of sample / A570 of control) × 100%, and IC50 is determined using GraphPad Prism.
4. Cell cycle analysis: After 48 hours of treatment, cells are harvested by trypsinization, washed twice with cold PBS, and fixed with 70% ethanol at 4°C overnight. Fixed cells are treated with RNase A (100 μg/mL) at 37°C for 30 minutes, stained with propidium iodide (PI, 50 μg/mL) in the dark for 15 minutes, and analyzed by flow cytometry (BD FACSCanto) [1]
- HSV plaque reduction assay (文献[2]):
1. Cell preparation: Vero cells are seeded in 6-well plates (5×10⁵ cells/well) and cultured overnight to form a confluent monolayer at 37°C (5% CO₂).
2. Viral adsorption: HSV-1 (KOS strain) or HSV-2 (333 strain) is diluted to 100 plaque-forming units (PFU)/well, added to the cell monolayer, and incubated for 1 hour at 37°C to allow viral adsorption.
3. Drug treatment: Unbound virus is removed, and serial concentrations of Chrysophanic Acid (Chrysophanol) (5–40 μg/mL, dissolved in DMSO) mixed with 1% methylcellulose (in minimal essential medium, MEM) are overlaid onto the cells; a vehicle control (0.1% DMSO in 1% methylcellulose) is included.
4. Plaque counting: Plates are incubated for 72 hours at 37°C, then cells are fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and plaques are counted. Plaque reduction rate is calculated as [(plaques in control - plaques in sample) / plaques in control] × 100%, and EC50 is determined [2]

5 mg/kg Mice: One week is spent in maintenance before experiments begin with 4-week-old male C57BL/6J mice. In a pathogen-free animal facility, mice are kept on a 12-hour light/dark cycle and given unlimited access to water and a laboratory diet. The mice receive a high-fat, high-calorie diet (HFD) to induce obesity. Commercial standard chow is fed to the control group (C). Mice in the HFD group (HFD) are given only HFD. HFD + CA group (CA): Four weeks of HFD are given to the mice prior to the administration of 5 mg/kg/day of chyrsophanol. For sixteen weeks, the mice are split into three groups (n = 5) and fed three different diets: chow diet, HFD, and HFD plus Chrysophanol. Three times a week, food intake and body weight are measured.

Absorption, Distribution and Excretion
This study compared the oral pharmacokinetics of five anthraquinone compounds (aloe-emodin, rhein, chrysophanol, hypericin, and chrysophanol) from Rheum palmatum extract in normal rats and rats with a thrombotic focal cerebral ischemia (TFCI) model. Plasma samples were clarified by solid-phase extraction, and the anthraquinone compounds were simultaneously determined using a validated high-performance liquid chromatography-fluorescence (HPLC-FIG) system. Results showed that the Cmax, t_1/2, and AUC_0-t values of aloe-emodin, rhein, chrysophanol, and hypericin in TFCI model rats were almost twice that of normal rats, while the CL value was significantly reduced (p < 0.05). The plasma drug concentration-time data of the five anthraquinone drugs in rats conformed to a two-compartment open model. All five anthraquinone drugs showed rapid absorption and slow elimination in the plasma of both groups of rats. These results contribute to the evaluation of the efficacy and safety of this drug in clinical applications.
Pharmacological Significance: Quyu Qingre Granules (QYQRGs) are a commonly used traditional Chinese medicine compound preparation for treating blood stasis syndrome. Comparing the pharmacokinetic differences of various components after administration of QYQRG to normal rabbits and rabbits with blood stasis syndrome can provide valuable information. The main purpose of this study was to compare the pharmacokinetics of emodin and hyperoside in normal rabbits and rabbits with acute blood stasis model after oral administration of 2.0 g/kg body weight of QYQRG. Materials and Methods: Blood samples were collected at 5, 10, 15, 20, 30, 45, 60, 75, 90, 120, 240, 360, and 480 minutes after oral administration of QYQRG. The concentrations of emodin and hyperoside in rabbit plasma were determined by high performance liquid chromatography (HPLC), and the main pharmacokinetic parameters were obtained. Results: The pharmacokinetic parameters AUC(0-∞), T(lag), Cmax, and K21 of emodin and hyperoside in the plasma of rabbits with acute blood stasis model were significantly different from those of normal rabbits. Furthermore, significant differences were observed in the A, β, MRT, and T(1/2β) of emodin and the α and T1/2a of hyperoside between normal rabbits and rabbits with acute blood stasis. Conclusion: In the rabbit model of acute blood stasis, the absorption time and amount of emodin and hyperoside were accelerated, suggesting that emodin and hyperoside may be two active ingredients in QYQRG.
Research Objective: This study compared the tissue distribution of rhubarb anthraquinone derivatives (AQs) in normal rats and rats with carbon tetrachloride (CCl4)-induced liver injury to explore whether there were differences in their absorption and to investigate the possible reasons for the different toxicity of AQs at the tissue distribution level in pathological model rats and normal rats. Materials and Methods: Total rhubarb extract (based on crude extract amount, 14.49 g/kg body weight daily) was administered to normal and model rats by gavage for 12 weeks. The concentration of free anthraquinone compounds (AQs) in tissues was quantitatively analyzed by liquid chromatography-tandem mass spectrometry (LC-MS). Four weeks after drug withdrawal, tissue distribution was measured again. Results: Five free anthraquinones—aloe-emodin, rhein, emodin, hyperoside, and chrysophanol—were detected in the liver, kidney, and spleen, but only the concentrations of rhein, aloe-emodin, and emodin reached the limits of quantification. The concentrations of rhein (p < 0.001), aloe-emodin (p < 0.001), and emodin (p < 0.05) in normal rat tissues were higher than those in model rats. The concentration order of rhein in kidney and spleen tissues was rhein > aloe-emodin > emodin, while in liver tissue it was rhein > rhein > emodin. Four weeks after drug withdrawal, free anthraquinones were not detected in tissues. Conclusion: These results indicate that the tissue toxicity of anthraquinone compounds in normal animals is higher than that in pathological model animals, and the cumulative toxicity of rhubarb is relatively low. This result is consistent with the "You Gu Gu Yun" theory recorded in the traditional Chinese medicine classic Suwen.

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Metabolic/Metabolic Products
This study aims to elucidate the enzymes involved in the biotransformation of naturally occurring 1,8-dihydroxyanthraquinone and to explore whether the biotransformation of 1,8-dihydroxyanthraquinone constitutes a bioactivation pathway. We first investigated the metabolism of emodin (1,3,8-trihydroxy-6-methylanthraquinone), a compound found in pharmaceutical preparations. In rat liver microsomes, we observed the formation of two emodin metabolites—ω-hydroxyemodin and 2-hydroxyemodin. The rate of ω-hydroxyemodin formation in rat liver microsomes pretreated with different cytochrome P450 enzyme inducers did not differ. Therefore, the formation of ω-hydroxyemodin appears to be catalyzed by several cytochrome P450 enzymes at a relatively low rate.
Pretreatment with 3-methylcholanthrene increased the production of 2-hydroxyemodin in rat liver microsomes, which was inhibited by α-naphthoflavone, anti-rat cytochrome P450 1A1/2 antibody, and (to a lesser extent) anti-rat cytochrome P450 1A1 antibody. These data suggest that cytochrome P450 1A2 is involved in the production of this metabolite. However, other cytochrome P450 enzymes also appear to catalyze this reaction. The anthraquinone compound emodin (1,8-dihydroxy-3-methylanthraquinone) is oxidized in a cytochrome P450-dependent manner to aloe-emodin (1,8-dihydroxy-3-hydroxymethylanthraquinone), which is the major metabolite. The mutagenicity of the parent dihydroxyanthraquinone and its metabolites was compared in an in vitro micronucleus assay in mouse lymphoma L5178Y cells. Compared to emodin, 2-hydroxyemodin induced a significantly higher frequency of micronuclei; compared to emodin, ω-hydroxyemodin induced a lower frequency of micronuclei; and aloe-emodin induced a significantly higher frequency of micronuclei than chrysophanol. These data suggest that cytochrome P450-dependent biotransformation of emodin and hypericin may be the bioactivation pathway for these compounds. Hypericin is a major anthraquinone component in many traditional Chinese medicines and is considered an important active ingredient with various pharmacological effects, including antibacterial and anticancer properties. Previous studies have shown that exposure to hypericin induces cytotoxicity, but the mechanism of toxicity remains unclear. In this metabolic study, three oxidative metabolites (M1-M3, aloe-emodin, 7-hydroxyemodin, and 2-hydroxyemodin) and five GSH conjugates (M4-M8) were detected in hypericin-supplemented rat and human liver microsomal incubation media containing glutathione (GSH). Except for M4 and M5, the generation of the other metabolites was dependent on NADPH. M4 and M5 are directly derived from the parent compound rhein, M6 from M2, and M7 and M8 are derived from the oxidation of M4 and M5. Metabolites M5 and M6 were also observed in the bile of rats exposed to rhein; M1-M3 and an NAC conjugate (M9) were detected in the urine of rats given rhein, with urinary metabolite M9 derived from the degradation of the GSH conjugate M6 in bile. Recombinant P450 enzyme incubation and microsomal inhibition experiments showed that P450 1A2 is the main enzyme responsible for the metabolic activation of rhein, while P450 2B6 and P450 3A4 also participate in the generation of oxidative metabolites.

In vitro toxicity (Reference [1]): For normal human hepatocytes (LO2 cells), 50 μM rhein (chrysophanol) (incubated for 72 hours) maintained cell viability above 80% (MTT method), indicating low intrinsic cytotoxicity [1]
- In vitro toxicity (Reference [2]): For Vero cells, the half-maximal cytotoxicity concentration (CC50) of rhein (chrysophanol) was 85.3 μg/mL. The therapeutic index (TI = CC50/EC50) for HSV-1 was 6.8, and the therapeutic index for HSV-2 was 5.4 [2]
- In vivo toxicity data (e.g., LD50, hepatotoxicity, nephrotoxicity, plasma protein binding) of rhein (chrysophanol) were not reported in [1] or [2] [1,2]

[1]. Phytother Res . 2011 Jun;25(6):833-7.

[2]. Antiviral Res . 2001 Mar;49(3):169-78.

Chrysophanic acid is a golden-yellow flake or brown powder with a melting point of 196°C, slightly soluble in water. A pale yellow aqueous solution turns red in the presence of alkali, and a concentrated sulfuric acid solution turns red. (NTP, 1992)
Rhein is a trihydroxyanthraquinone, a calendula-based compound with a methyl group substituted at the C-3 position. It has been isolated from aloe and possesses antiviral and anti-inflammatory activities. It can be used as an antiviral agent, anti-inflammatory agent, and plant metabolite. It is functionally related to strychnine.
Strychnine has been reported in Talaromyces islandicus, Ramularia uredinicola, and other organisms with relevant data.
See also: Frangula pulchella bark (partial).
Strychnine (strychnine alcohol) is a natural anthraquinone derivative isolated from medicinal plants of the Rheum genus (e.g., rhubarb, commonly known as rhubarb) and other plant species. It has a long history of use in traditional medicine due to its anti-inflammatory and laxative effects [1,2]
- Antiviral mechanism (reference [2]): The antiviral activity of rhein (chrysophanol) is thought to involve the inhibition of viral DNA synthesis, possibly through interference with the activity of HSV DNA polymerase, but no direct experimental evidence (e.g., enzyme inhibition assay) has been provided [2]
- Antiproliferative mechanism (reference [1]): The antiproliferative effect of rhein (chrysophanol) on HepG2 cells is related to cell cycle arrest in the G2/M phase and the induction of apoptosis, but this study did not explore the specific signaling pathways involved (e.g., p53, MAPK) [1]
- Clinical development status: No clinical development data of rhein (chrysophanol) have been reported (e.g., for the treatment of cancer or viral infection); its bioactivity is currently limited to preclinical in vitro studies [1,2]

C15H10O4

254.24

254.057

C, 70.86; H, 3.96; O, 25.17

481-74-3

10208

Yellow to orange solid powder

1.5±0.1 g/cm3

489.5±45.0 °C at 760 mmHg

194-198 °C

263.9±25.2 °C

0.0±1.3 mmHg at 25°C

1.710

5.03

2

4

0

19

405

0

O([H])C1=C([H])C(C([H])([H])[H])=C([H])C2C(C3C([H])=C([H])C([H])=C(C=3C(C=21)=O)O[H])=O

LQGUBLBATBMXHT-UHFFFAOYSA-N

InChI=1S/C15H10O4/c1-7-5-9-13(11(17)6-7)15(19)12-8(14(9)18)3-2-4-10(12)16/h2-6,16-17H,1H3

1,8-dihydroxy-3-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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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