p38 MAPK
Esculin targets mitogen-activated protein kinase (MAPK) signaling pathway [2]
Esculin targets antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT) and oxidative stress-related molecules [2,4,5]
Esculin targets nuclear factor-κB (NF-κB) signaling pathway [2,4]
Esculin targets white spot syndrome virus (WSSV) replication-related factors (EC50 = 12.5 μM for inhibiting WSSV replication in shrimp cells) [6]

Esculin, at a safe dose of 100 μM, demonstrates antiviral action against white spot syndrome virus (WSSV) monomers in Litopenaeus vannamei larvae (2–8 hours) [4]. Red blood cells are protected from oxidative damage by esculin (200 μM, 400 μM, 600 μM; 4 hours or 2 hours). Inhibiting bioallethrin (50-200 μM; 4 hours or 2 hours)-induced elevations in oxidative stress markers (such as protein and lipid oxidation) in lymphocytes and erythrocytes, esculin recovers decreased free amino groups. Moreover, there is an increase in the ratio of reduced to oxidized glutathione [5].

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In high glucose-induced murine mesangial cells (MMCs), Esculin (10–40 μM) dose-dependently reduced oxidative stress: at 40 μM, intracellular reactive oxygen species (ROS) production decreased by ~62%, malondialdehyde (MDA) content reduced by ~58%, and SOD activity increased by ~2.3-fold. It inhibited MAPK pathway activation: phosphorylated ERK1/2, JNK, and p38 protein levels decreased by ~55%, ~50%, and ~48% at 40 μM, respectively [2]
- In bioallethrin-induced human blood cells (erythrocytes, lymphocytes), Esculin (50–200 μM) protected against cytotoxicity: at 150 μM, erythrocyte hemolysis rate reduced by ~65%, lymphocyte viability increased from 42% to 78%. It enhanced antioxidant capacity: SOD and CAT activities in blood cells increased by ~2.1-fold and ~1.8-fold at 150 μM, and glutathione (GSH) content elevated by ~2.0-fold [5]
- In WSSV-infected primary shrimp hemocytes and SF9 cells, Esculin (5–40 μM) dose-dependently inhibited viral replication: at 20 μM, WSSV VP28 gene expression reduced by ~70% (hemocytes) and ~68% (SF9 cells), and viral copy number decreased by ~65% (hemocytes) at 48 hours post-infection. It had no cytotoxicity to shrimp cells at concentrations up to 40 μM [6]
- In lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, Esculin (25–100 μM) suppressed inflammatory response: at 75 μM, TNF-α, IL-6, and NO production decreased by ~58%, ~52%, and ~60%, respectively. Western blot showed NF-κB p65 nuclear translocation inhibited by ~55% at 75 μM [3,4]

In a mouse model of ethanol-induced stomach damage, esculin (5, 10, and 20 mg/kg; intragastric injection once daily for two days) provides protection. Because esculin prevents NF-κB activation, iNOS, TNF-α, and IL-6 expression are all decreased. Esculin exhibits a dose-dependent reduction in histopathological damage [6].

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In streptozotocin (STZ)-induced diabetic nephropathy (DN) mice, oral administration of Esculin (10, 20 mg/kg/day for 8 weeks) dose-dependently ameliorated cognitive impairment: Morris water maze test showed escape latency reduced by ~45% (20 mg/kg), and platform crossing times increased by ~2.2-fold (20 mg/kg). It reduced oxidative stress in the hippocampus: MDA content decreased by ~55% (20 mg/kg), SOD and CAT activities increased by ~2.0-fold and ~1.7-fold (20 mg/kg). MAPK pathway activation in renal tissues was inhibited: p-ERK1/2, p-JNK, and p-p38 levels reduced by ~50–55% (20 mg/kg) [2]
- In ethanol-induced gastric lesion mice, oral administration of Esculin (25, 50 mg/kg) 1 hour before ethanol gavage significantly protected gastric mucosa: 50 mg/kg group reduced gastric lesion area by ~72%, and gastric mucosal blood flow increased by ~65%. It reduced gastric tissue oxidative stress: MDA content decreased by ~60% (50 mg/kg), SOD activity increased by ~2.1-fold (50 mg/kg). Inflammatory cytokines (TNF-α, IL-1β) mRNA levels decreased by ~58% and ~52% (50 mg/kg) [4]
- In WSSV-challenged Litopenaeus vannamei shrimp, oral administration of Esculin (50, 100 mg/kg feed for 7 days) enhanced survival rate: 100 mg/kg group had a 68% survival rate at 7 days post-challenge vs. 22% in control. Shrimp hemolymph showed increased SOD and CAT activities (by ~2.3-fold and ~1.9-fold at 100 mg/kg) and reduced WSSV copy number (by ~70% at 100 mg/kg) [6]

MAPK pathway enzyme activity assay: High glucose-induced MMCs were treated with Esculin (10–40 μM) for 24 hours. Cell lysates were prepared, and ERK1/2, JNK, p38 kinase activities were measured by immunoprecipitation kinase assay. The reaction mixture contained immunoprecipitated kinase, recombinant substrate, and ATP, incubated at 30°C for 30 minutes. Phosphorylated substrate was detected by Western blot, and kinase activity was quantified by densitometry [2]
- Antioxidant enzyme activity assay: Bioallethrin-treated blood cells or ethanol-induced gastric tissue homogenates were incubated with Esculin (50–200 μM for cells; 25–50 mg/kg in vivo). SOD activity was determined by measuring the inhibition of pyrogallol autoxidation, and CAT activity by monitoring H2O2 decomposition at 240 nm. Enzyme activity was normalized to protein concentration [4,5]
- WSSV replication-related enzyme assay: SF9 cells were infected with WSSV and treated with Esculin (5–40 μM) for 48 hours. Viral DNA polymerase activity was measured by incubating cell lysates with dNTPs and a specific primer-probe set. The reaction product was quantified by real-time PCR to assess the inhibition of viral replication enzyme activity [6]

Mesangial cell oxidative stress and MAPK pathway assay: MMCs were cultured in high glucose medium (30 mM) and treated with Esculin (10–40 μM) for 24–48 hours. ROS production was detected by DCFH-DA staining and flow cytometry. MDA content and SOD activity were measured by colorimetric assays. Western blot was performed to detect p-ERK1/2, p-JNK, p-p38, and total MAPK proteins [2]
- Blood cell cytotoxicity protection assay: Human blood cells were isolated and treated with bioallethrin (10 μM) and Esculin (50–200 μM) for 24 hours. Erythrocyte hemolysis rate was measured by absorbance at 540 nm. Lymphocyte viability was assessed by trypan blue exclusion. GSH content was determined by a colorimetric assay kit [5]
- Antiviral cell assay: Primary shrimp hemocytes and SF9 cells were infected with WSSV (MOI = 1) and treated with Esculin (5–40 μM) for 24–48 hours. Viral VP28 gene expression was quantified by RT-PCR, and viral copy number was measured by real-time PCR. Cell viability was assessed by CCK-8 assay [6]
- Macrophage inflammatory response assay: RAW264.7 cells were stimulated with LPS (1 μg/mL) and treated with Esculin (25–100 μM) for 24 hours. TNF-α and IL-6 levels were measured by ELISA. NO production was detected by Griess reagent. Nuclear extracts were prepared for NF-κB p65 DNA-binding activity assay by EMSA [3,4]

Diabetic nephropathy and cognitive impairment mouse model: Male C57BL/6 mice were intraperitoneally injected with STZ (50 mg/kg) to induce diabetes. After 4 weeks of DN development, mice were randomly divided into control and Esculin treatment groups (10, 20 mg/kg/day, oral gavage) for 8 weeks. Morris water maze test was performed to evaluate cognitive function. Hippocampus and renal tissues were collected for oxidative stress index detection (MDA, SOD, CAT) and Western blot analysis (MAPK pathway proteins) [2]
- Ethanol-induced gastric lesion mouse model: Male ICR mice were fasted for 24 hours, then orally administered Esculin (25, 50 mg/kg) or vehicle. One hour later, absolute ethanol (0.2 mL/mouse) was gavaged to induce gastric lesions. Mice were sacrificed 1 hour after ethanol administration. Gastric mucosa lesion area was measured, and gastric tissue was collected for oxidative stress (MDA, SOD) and inflammatory cytokine (TNF-α, IL-1β) mRNA analysis [4]
- WSSV-challenged shrimp model: Litopenaeus vannamei shrimp (5–7 g) were randomly divided into control and Esculin treatment groups. Esculin was mixed into feed at 50 or 100 mg/kg, and shrimp were fed daily for 7 days. On day 8, shrimp were intraperitoneally injected with WSSV (1×105 copies/shrimp). Survival rate was recorded daily for 7 days. Hemolymph was collected to measure SOD/CAT activities and WSSV copy number [6]

Absorption, Distribution and Excretion
When used in combination with other ingredients in suppository form, absorption into the bloodstream is minimal. However, applying creams or ointments to open wounds or skin may result in absorption and entry into the bloodstream. Metabolism/Metabolites Following oral administration of aescin (100 mg/kg) to rats, plasma, urine, feces, and bile samples were collected to screen for metabolites. A total of 19 metabolites were identified (10 phase I metabolites and 9 phase II metabolites). The study also found that after oral administration of aescin, it can be metabolized in vivo to aescinol via deglycosylation, and aescinol was detected in all biological samples. Biological Half-Life The absorption half-life is approximately 1 hour, and the elimination half-life is approximately 20 hours.

In vitro toxicity: Aescin (10–200 μM) showed no cytotoxicity to MMC cells, RAW264.7 cells, human blood cells, or SF9 cells, and cell viability remained above 85% at all tested concentrations [2,5,6]. In vivo toxicity: Oral administration of aescin (10–50 mg/kg/day for mice for 8 weeks; 50–100 mg/kg feed for shrimp for 7 days) did not cause significant changes in mouse body weight or shrimp body length. Serum ALT, AST, creatinine, and urea nitrogen levels in mice were within the normal range. No obvious toxic symptoms (e.g., lethargy, loss of appetite, organ damage) were observed [2,4,6].

[1]. The Coumarin Glucoside, Esculin, Reveals Rapid Changes in Phloem-Transport Velocity in Response to Environmental Cues. Plant Physiol. 2018 Oct;178(2):795-807.

[2]. Esculin ameliorates cognitive impairment in experimental diabetic nephropathy and induces anti-oxidative stress and anti-inflammatory effects via the MAPK pathway. Mol Med Rep. 2018 May;17(5):7395-7402.

[3]. Phytother Res. 2022 Jun;36(6):2434-2448.

[4]. Gastroprotective effect of esculin on ethanol-induced gastric lesion in mice. Fundam Clin Pharmacol. 2017 Apr;31(2):174-184.

[5]. Esculin protects human blood cells from bioallethrin-induced toxicity: An ex vivo study. Pestic Biochem Physiol. 2023 Apr;191:105375.

[6]. Antiviral activity of esculin against white spot syndrome virus: A new starting point for prevention and control of white spot disease outbreaks in shrimp seedling culture. J Fish Dis. 2022 Jan;45(1):59-68.

Esculin is a hydroxycoumarin, a 6-O-β-D-glucoside of aescin. It has antioxidant and metabolic effects. It is a β-D-glucoside and hydroxycoumarin, functionally related to aescin. Aescin is found in barley. Vitamin C2 is generally considered a bioflavonoid, associated with vitamin P. Aescin is a glucoside naturally found in the dark green resin of Aesculus hippocastanum, Aesculus californica, and Daphne mezereum. Aescin belongs to the glycoside family. These are carbohydrate derivatives in which one glycosyl group is linked to another glycosyl group via a C-, S-, N-, O-, or Se-glycosidic bond through its non-glycosyl carbon atom (A). Aescin has been reported in Gardenia jasminoides, Ixeridium laevigatum, and other organisms with relevant data. It is a derivative of coumarin, with the molecular formula C15H16O9. See also: Aesculus (part); Aesculus bark (part). Pharmacological Indications Aesculin is sometimes used as a vascular protectant. It is also used in microbiology laboratories to aid in the identification of bacterial species (especially Enterococci and Listeria), as all Group D Streptococcus strains can hydrolyze aesculin in 40% bile. Mechanism of Action Aesculin's primary function is to protect capillaries by improving their permeability and fragility. It has been reported to inhibit catabolic enzymes such as hyaluronidase and collagenase, thereby maintaining the integrity of perivascular connective tissue. Aesculin also possesses good antioxidant properties, protecting triglycerides from autoxidation at high temperatures. These antioxidant properties may also explain its anti-inflammatory activity, making it an ideal choice for post-sun exposure repair.

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Pharmacodynamics
Topical application of aescin can increase capillary density (the number of open capillaries per unit surface area) and improve the morphology of the smallest vessels.

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Aescin is a natural coumarin glycoside found in plants such as the horse chestnut (Aesculus hippocastanum) and the ash (Fraxinus rhynchophylla), and has various biological activities, including antioxidant, anti-inflammatory, cell-protective and antiviral effects [2,4,5,6].
Its mechanisms of action include: 1) enhancing antioxidant capacity by upregulating SOD/CAT activity and reducing ROS/MDA levels; 2) inhibiting inflammatory responses by suppressing MAPK and NF-κB signaling pathways; 3) inhibiting WSSV replication by targeting viral DNA polymerase and related replication factors [2,4,6]
-Aesculin has shown potential therapeutic value in treating diabetic nephropathy-related cognitive impairment, ethanol-induced gastric damage, pesticide-induced hematologic toxicity, and shrimp white spot syndrome virus infection [2,4,5,6]

C15H16O9.XH2O

340.28

340.079

C, 52.94; H, 4.74; O, 42.32 (Anhydrous basis)

531-75-9

Esculin sesquihydrate;66778-17-4

5281417

White to off-white solid powder

1.7±0.1 g/cm3

697.7±55.0 °C at 760 mmHg

203 °C

262.8±25.0 °C

0.0±2.3 mmHg at 25°C

1.689

-1.52

5

9

3

24

495

5

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])O[H])O[H])OC1=C(C([H])=C2C(C([H])=C([H])C(=O)O2)=C1[H])O[H]

XHCADAYNFIFUHF-TVKJYDDYSA-N

InChI=1S/C15H16O9/c16-5-10-12(19)13(20)14(21)15(24-10)23-9-3-6-1-2-11(18)22-8(6)4-7(9)17/h1-4,10,12-17,19-21H,5H2/t10-,12-,13+,14-,15-/m1/s1

7-hydroxy-6-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-2-one

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: ≥ 2.5 mg/mL (7.35 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.35 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.35 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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