Chebulinic acid inhibits gastric H⁺ K⁺-ATPase (proton pump) with an IC₅₀ of 65.01 μg/ml in vitro. Omeprazole (reference drug) showed an IC₅₀ of 30.24 μg/ml. [1]

In vitro: Catalytic Tyr129 moves away from its target DNA-phosphate molecule upon interaction with chelinic acid. [1] At an ED50 of 100 μM, chemolinic acid can decrease MMP-2 expression and activity. In ARPE-19 cells, SMAD-3 phosphorylation was discovered to cause EMT (epithelial-mesenchymal transition), which CA was found to prevent. [2] With an IC50 of 65.01 μg/ml, chebulinic acid strongly inhibits H+ K+-ATPase activity in vitro. [3]

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Chebulinic acid inhibited H⁺ K⁺-ATPase activity in gastric microsomes isolated from normal fasted rat stomach in a concentration-dependent manner (10-100 μg/ml), with an IC₅₀ value of 65.018 μg/ml. Omeprazole (10-50 μg/ml) used as positive control reduced enzyme activity with an IC₅₀ of 30.24 μg/ml. [1]
Chebulinic acid exhibited strong antioxidant activity in the DPPH radical scavenging assay with an IC₅₀ value of 68.34 ± 7.46 μg/ml. Ascorbic acid (reference) showed an IC₅₀ of 42.06 ± 5.27 μg/ml. Percentage inhibition values: 10 μg/ml (10.73 ± 1.24%), 20 μg/ml (27.70 ± 3.59%), 40 μg/ml (35.91 ± 4.10%), 60 μg/ml (48.06 ± 8.16%), 80 μg/ml (53.87 ± 12.73%), 100 μg/ml (67.25 ± 10.08%). [1]

Chebulinic acid (20 mg/kg, p.o.) showed significant anti-ulcer activity: 62.9% protection against cold restraint induced gastric ulcer (CRU model); 55.53% protection against aspirin induced gastric ulcer (AS model); 80.67% protection against alcohol induced gastric ulcer (AL model); 66.63% protection against pyloric ligation induced ulcer (PL model). Reference drugs: omeprazole (10 mg/kg) showed 77.73% protection (CRU), 58.30% (AS), 70.80% (PL); sucralfate (500 mg/kg) showed 65.67% protection (AL). [1]
Chebulinic acid (20 mg/kg, p.o.) significantly reduced free acidity by 48.82% (54.50 ± 14.89 µequiv/ml vs control 106.5 ± 3.48) and total acidity by 38.62% (97.50 ± 13.35 µequiv/ml vs control 158.00 ± 3.48) in the pyloric ligation model. Omeprazole reduced free acidity by 57.15% (45.63 ± 1.284) and total acidity by 50.63% (78.30 ± 1.309). [1]
Chebulinic acid significantly upregulated mucin secretion by 59.75% in gastric juice (6841.20 ± 8.22 µg/ml vs control 2753.00 ± 13.76). Omeprazole increased mucin secretion by 36.74% (4352.50 ± 9.918). [1]
Chebulinic acid increased gastric mucin level to 56.18 ± 9.03 mg/g tissue compared to ulcer control (24.34 ± 2.91 mg/g) and healthy rats (62.07 mg/g). Omeprazole increased mucin to 52.72 ± 6.28 mg/g tissue. [1]
Chebulinic acid increased PGE2 generation to 5224.003 ± 336.9 pg/mg tissue protein compared to ulcer control (2758.0 ± 414.9 pg/mg). Omeprazole increased PGE2 to 4293.415 ± 237.8 pg/mg. [1]
Chebulinic acid increased superoxide dismutase (SOD) activity to 0.282 ± 0.73 U/mg protein compared to control (0.082 ± 0.42 U/mg). Ascorbic acid treated group showed SOD of 0.296 ± 0.41 U/mg. [1]

H⁺ K⁺-ATPase activity assay: Gastric microsomes were isolated from normal fasted rat stomach. The microsomes were incubated with different concentrations of chebulinic acid (10-100 μg/ml) as well as reference drug omeprazole (10-50 μg/ml) for 10 minutes at 37°C. The mixture was then added to an assay buffer containing 150 mM KCl, 10 mM PIPES, 1 mM MgSO₄, 5 mg ATP, 1 mM EGTA, 0.1 mM ouabain (pH 7.2), 10 μg/ml valinomycin, and 2.5 μg/ml oligomycin. The reaction was carried out at 37°C for 20 minutes and stopped by adding 10% ice-cold trichloroacetic acid. After centrifugation at 2000 × g for 1 minute, inorganic phosphate release was determined from the supernatant spectrophotometrically at 310 nm wavelength and expressed as μM/h/mg protein. [1]
DPPH radical scavenging assay: Chebulinic acid or reference (ascorbic acid) at concentrations of 10-100 μg/ml was added to 200 μl of DPPH in methanol solution (100 μM) in a 96-well microtitre plate. After incubation at 37°C for 30 minutes, the absorbance of each solution was determined at 490 nm using an ELISA microplate reader. Corresponding blank readings were taken and the remaining DPPH was calculated. The IC₅₀ value is the concentration of the sample required to scavenge 50% DPPH free radical. [1]
Superoxide dismutase (SOD) assay: SOD activity was measured based on its ability to inhibit the autoxidation of epinephrine to adrenochrome at alkaline pH. The absorbance of the reaction mixture was followed for 4 minutes at 480 nm in a spectrophotometer. Enzymatic activity was expressed as U/mg protein at 30°C. The amount of enzyme that caused 50 percent inhibition of epinephrine autoxidation was defined as one unit (U). [1]

General treatment schedule: Chebulinic acid (20 mg/kg, p.o.), reference drug omeprazole (10 mg/kg) and sucralfate (500 mg/kg) were freshly prepared in 1% carboxymethyl cellulose (CMC) as suspension and administered orally to animals. All animals were deprived of food for 18 hours before ulcerogens exposure. [1]
Cold restraint induced gastric ulcer (CRU) model: Animals were subjected to cold restraint stress 45 minutes after treatment with graded doses of chebulinic acid (10, 20, 40 mg/kg) and omeprazole (10 mg/kg). Rats were immobilized in restraint cages and kept at 4°C in an environmental chamber. Two hours later the animals were sacrificed and stomachs were observed and scored under magnascope for ulcers. [1]
Aspirin induced gastric ulcer model (AS): Aspirin at 150 mg/kg was administered orally to induce ulcer 45 minutes after treatment with chebulinic acid (20 mg/kg) and omeprazole (10 mg/kg). The animals were sacrificed 5 hours after aspirin treatment, the stomach was dissected out, incised along the lesser curvature, and the lesion was scored. [1]
Alcohol induced gastric ulcers model (AL): Absolute alcohol (1 ml/200 g body weight) was administered orally to induce gastric ulcer in rats. Chebulinic acid (20 mg/kg) and sucralfate (500 mg/kg) were administered 45 minutes before alcohol treatment. After 1 hour of alcohol administration, the animals were sacrificed and the stomach was cut open along the greater curvature. Lesion lengths were measured using image analyzer software and summated to give a total lesion score. [1]
Pyloric ligation induced ulcer model (PL): Pyloric ligation was done under chloral hydrate anesthesia (300 mg/kg, i.p.). After 45 minutes pre-treatment with chebulinic acid (20 mg/kg) and omeprazole (10 mg/kg), the abdomen was opened and the pyloric end of the stomach was ligated avoiding injury to adjacent blood vessels. The stomach was replaced carefully and animals were allowed to recover with free access to water. After 4 hours, the animals were sacrificed, the stomach was dissected out, lesions were scored, gastric fluid was collected and centrifuged at 2000 rpm for 10 minutes. The supernatant was used for estimation of gastric secretion and mucin. [1]
Gastric secretion study: Free and total acidity was measured from collected gastric juice by titrating against 0.01 N NaOH using phenolphthalein as an indicator and expressed in terms of µequiv/ml. [1]
Mucin estimation in gastric homogenate: A 100 mg sample of stomach tissue from each group was taken and incubated for 2 hours in acetate buffer (pH 5.8, 0.05 M) containing 0.16 M sucrose and 1.0% Alcian blue dye. Absorbance of the supernatant was read at 498 nm. [1]
Fluorometric mucin assay in gastric juice: Gastric juice was delipidated. The pellet was resuspended in 200 μl of PBS, 250 μl alkaline reagent (1 ml 0.15 N NaOH and 200 μl of 0.6 M 2-cyano-acetamide) was added and incubated at 100°C for 30 minutes. Then 2 ml of 0.6 M borate buffer (pH 8) was added and fluorescence was measured at 383 nm (excitation 336 nm). [1]
PGE2 estimation: Mucosa was scraped from tissue samples, rapidly rinsed with ice-cold saline, weighed, and homogenized in 10 volumes of phosphate buffer (0.1 M, pH 7.4) containing 1 mM EDTA and 10 μM indomethacin. The homogenate was centrifuged (10,000 rpm, 10 min, 4°C), and the supernatant was processed for PGE2 estimation using an enzyme immunosorbent assay kit following manufacturer's instructions. Results were expressed as pg PGE2/mg protein. [1]

[1]. Identification of chebulinic acid as potent natural inhibitor of M. tuberculosis DNA gyrase and molecular insights into its binding mode of action. Comput Biol Chem. 2015 Dec;59 Pt A:37-47.

[2]. Aqueous and alcoholic extracts of Triphala and their active compounds chebulagic acid and chebulinic acidprevented epithelial to mesenchymal transition in retinal pigment epithelial cells, by inhibiting SMAD-3 phosphorylation. PLoS One. 2015 Mar 20.

[3]. Anti-secretory and cyto-protective effects of chebulinic acid isolated from the fruits of Terminalia chebula on gastric ulcers. Phytomedicine. 2013 Apr 15;20(6):506-11.

Chebulinic acid is a tannin. It has been reported to exist in plants of the genera Chebulinic acid, Chebulinic acid, and Lumnitzera racemosa, and relevant data are available. See also: Chebulinic acid (note moved to).

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Ulcer scoring system: The severity and intensity of lesions were graded as: shedding of epithelium = 10; petechial and frank hemorrhages = 20; one or two ulcers = 30; more than two ulcers = 40; perforated ulcers = 50. [1]
Dose selection: Graded doses of chebulinic acid (10, 20 and 40 mg/kg, p.o.) showed percentage protection of 49.97%, 62.90%, and 67.20% respectively against CRU model. The 20 mg/kg dose was identified as the effective dose and selected for further studies. [1]
Mechanism summary: Chebulinic acid imparts gastroprotective effects through inhibition of H⁺ K⁺-ATPase (proton pump) activity, reduction of free and total acidity, upregulation of mucin secretion, increase in PGE2 levels, and free radical scavenging properties. [1]

C41H32O27

956.6766

956.113

18942-26-2

72284

White to off-white solid powder

2.0±0.1 g/cm3

1460.0±65.0 °C at 760 mmHg

437.2±27.8 °C

0.0±0.3 mmHg at 25°C

1.837

4.14

13

27

12

68

1880

8

C1=C(C=C(C(=C1O)O)O)C(=O)OC[C@@H]2[C@@H]3[C@@H]([C@H]([C@@H](O2)OC(=O)C4=CC(=C(C(=C4)O)O)O)OC(=O)C5=CC(=C(C6=C5[C@H]([C@@H](C(=O)O3)CC(=O)O)[C@@H](C(=O)O6)O)O)O)OC(=O)C7=CC(=C(C(=C7)O)O)O

YGVHOSGNOYKRIH-FJPMMHPYSA-N

InChI=1S/C41H32O27/c42-15-1-10(2-16(43)26(15)51)35(56)62-9-22-31-33(66-36(57)11-3-17(44)27(52)18(45)4-11)34(41(63-22)68-37(58)12-5-19(46)28(53)20(47)6-12)67-38(59)13-7-21(48)29(54)32-25(13)24(30(55)40(61)65-32)14(8-23(49)50)39(60)64-31/h1-7,14,22,24,30-31,33-34,41-48,51-55H,8-9H2,(H,49,50)/t14-,22+,24-,30-,31+,33-,34+,41-/m0/s1

2-[(4R,5S,7R,8R,11S,12S,13S,21S)-13,17,18-trihydroxy-2,10,14-trioxo-5,21-bis[(3,4,5-trihydroxybenzoyl)oxy]-7-[(3,4,5-trihydroxybenzoyl)oxymethyl]-3,6,9,15-tetraoxatetracyclo[10.7.1.14,8.016,20]henicosa-1(19),16(20),17-trien-11-yl]acetic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~100 mg/mL (~104.53 mM)

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.)