p38 MAPK

Two human breast cancer cell lines were exposed to asiatic acid, which inhibited cell growth in a concentration-dependent manner, with MCF-7 being more susceptible than MDA-MB-231. For MCF-7 and MDA-MB-231, asiatic acid's IC50 values were 5.95 M and 8.12 M, respectively.

ERK1/2 and p38 MAPK Kinase Activity Assays, as well as Immunoprecipitation/Immunoblot. When MAPK inhibitors were present or absent, cells were exposed to 10 μM asiatic acid for the allotted amount of time. Apoptosis assay kit made by BioVision Inc., Mountain View, California, was used to separate the mitochondrial and cytoplasmic fractions. In order to prepare the cells for immunoblotting, they were lysed on ice for 40 minutes in a solution containing 50 mM Tris, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM Na3VO4, 2 mM EGTA, 12 mM β-glycerol phosphate, 10 mM NaF, and 16 μg/ml benzamidine hydrochloride, 10 μg/ml phenanthroline, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. The supernatant fraction from the 15-minute 14,000g centrifugation of the cell lysate was used for immunoblotting. SDS-polyacrylamide gel electrophoresis was used to resolve equivalent amounts of protein (10–12%), which were then transferred to polyvinylidene difluoride membranes. The membrane was incubated with the desired primary antibody for 1 to 16 hours after blocking for 1 h in 5% nonfat dry milk in Tris-buffered saline. After applying the proper peroxidase-conjugated secondary antibody to the membrane, as directed by the manufacturer, the immunoreactive proteins were found using an enhanced chemiluminescence kit from Amersham Biosciences Inc., Piscataway, NJ.

The sodium 3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT) assay was used to determine how much asiatic acid inhibited cell proliferation. In 96-well culture plates, cells were plated at a density of 1 × 104 per well. The cells were exposed to asiatic acid (0, 2.5, 5, 10, and 20 μM) for 48 hours after a 24-hour incubation period. Each well was then filled with 50 microliters of the XTT test solution, which was created by combining 5 ml of the XTT-labeling reagent with 100 μl of the electron coupling reagent. At a test wavelength of 492 nm and a reference wavelength of 690 nm, absorbance was measured using an ELISA reader (Multiskan EX; Labsystem, Helsinki, Finland) after a 4-h incubation. The formula inhibition % = [100 - (ODt/ODs) × 100] was used to calculate data as the percentage of inhibition. The optical densities of the test substances and the solvent control were indicated, respectively, by ODt and ODs. Based on 48-h absorbance values, the concentration of test substances that cause 50% cellular cytotoxicity of cancer cells (IC50) was determined.

Absorption, Distribution and Excretion
This study employed a novel high-performance liquid chromatography (HPLC) method to determine the pharmacokinetics of asiatic acid following a single oral dose (30 or 60 mg) and a 7-day course (30 or 60 mg twice daily). Twelve healthy volunteers received each treatment in a randomized crossover design, with a 3-week interval between trials. The time to peak plasma concentration was not affected by dose differences or treatment regimens. Differences in peak plasma concentrations and the area under the concentration-time curve (AUC0-24) calculated after a single 30 or 60 mg dose were attributable to the different dosing regimens. However, after prolonged administration of 30 mg and 60 mg, peak plasma concentrations, AUC0-24, and half-life were significantly higher than those after the corresponding single doses. This phenomenon can be explained by the metabolic interaction between asiatic acid and asiaticoside, which is converted to asiatic acid in vivo. This study investigated the steady-state bioavailability of asiatic acid in 12 healthy male and female volunteers, comparing the effects of oral equimolar doses of asiatic acid (12 mg) or its glycoside derivative asiaticoside (24 mg). Both asiatic acid and asiaticoside are components of the commercially available dermatological product Madecassol. Asiaticoside is converted to asiatic acid in vivo via glycosylation. The steady-state AUC0-12hr values of asiatic acid were similar in both dosing regimens (614 ± 250 ng·hr/mL for asiatic acid and 606 ± 316 ng·hr/mL for asiaticoside), indicating comparable bioavailability of asiatic acid in both components at equimolar doses. Since asiatic acid is considered the most therapeutically active component of Madecassol, the current data suggest that the therapeutic effect of asiaticoside may be achieved through its conversion to asiatic acid.

Interactions
Asicoside is a pentacyclic triterpenoid compound found in medicinal plants. This study investigated the cytotoxicity of this compound and its enhancing effect on the anticancer drug irinotecan hydrochloride (CPT-11) in the human colon adenocarcinoma cell line HT-29. Results showed that asiatic acid exhibited dose-dependent cytotoxicity against HT-29 cells. DNA fragmentation, annexin-positive apoptotic cells, and caspase-3 activation were all dose-dependent. Caspase-3 inhibitors inhibited DNA ladder band formation in a concentration-dependent manner. Asicoside treatment reduced the expression of Bcl-2 and Bcl-XL proteins. These results indicate that asiatic acid induces apoptosis in HT-29 cells by activating caspase-3. This study further investigated the cytotoxic effects of combined CPT-11 and asiatic acid treatment on HT-29 cells. Simultaneous or sequential treatment with asiatic acid and CPT-11 showed an additive effect. A synergistic effect was observed when cells were first exposed to CPT-11 and then to asiatic acid. These results suggest that asiatic acid could be used to enhance the sensitivity of colon cancer cells to CPT-11 treatment or to reduce the adverse effects of CPT-11. Asiatic acid is a pentacyclic triterpenoid compound that has been reported to induce apoptosis in various human cancer cells. In this study, we evaluated the antitumor-promoting effect of asiatic acid on 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated, 7,12-dimethylbenzo[a]anthracene (DMBA)-induced skin tumor formation in ICR mice. Topical application of asiatic acid before each TPA application significantly reduced skin tumor formation. We also found that pre-application of asiatic acid reduced TPA-induced [3H]thymidine incorporation, a common marker of skin tumor promotion. Furthermore, asiatic acid inhibits TPA-induced nitric oxide (NO) production and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), both of which are known to play important roles in tumor growth, especially in the tumor-promoting phase. Additionally, topical application of the selective iNOS inhibitor aminoguanidine (AG) and another iNOS inhibitor, N(G)-nitro-L-arginine methyl ester (NAME), 30 minutes prior to TPA treatment significantly inhibited TPA-induced COX-2 expression. These results suggest that asiatic acid may exert its antitumor effects by inhibiting the NO and COX-2 signaling pathways.
Anaecolic acid and corosolic acid are two natural products identified as biofilm inhibitors in biofilm inhibition assays. We evaluated the activity of these two compounds against Pseudomonas aeruginosa biofilms co-cultured with tobramycin and ciprofloxacin in a rotating disk reactor (RDR). To determine the stability of our system, we assessed the antibiotic sensitivity of these biofilms using tobramycin and ciprofloxacin. The results showed that the biofilm bacteria generated in the RDR exhibited significant tolerance to 10 μg/ml ciprofloxacin, thus mimicking the tolerance observed in refractory bacterial infections. These studies further demonstrate that non-myxotropic Pseudomonas aeruginosa strains can form biofilms tolerant to clinically relevant concentrations of ciprofloxacin. Neither asiatic acid nor corosolic acid decreased the viable cell density of the Pseudomonas aeruginosa biofilm. However, both compounds increased the sensitivity of the biofilm bacteria to subsequent tobramycin treatment, suggesting that asiatic acid and corosolic acid are compounds that enhance antibiotic activity. Similar statistical interactions were also observed between ciprofloxacin and subsequent tobramycin treatment. This study investigated the protective effects and mechanisms of triterpenoids on primary cultured rat hepatocytes damaged by D-galactosamine (D-GalN) or carbon tetrachloride (CCl4). Rat hepatocytes were isolated using a two-step collagenase perfusion method and cultured in RPMI 1640 medium. The protective effects of asiatic acid (AA) and β-glycyrrhetinic acid (GA) on hepatocytes damaged by D-GalN (2 mmol/L) or CCl4 (10 mmol/L) were evaluated. Cell morphology was observed by optical microscopy, cell viability was measured by the MTT assay, and AST and LDH activities were measured using an automated biochemical analyzer. The levels of reactive oxygen species (ROS), nitric oxide end products (NOx), and reduced glutathione (GSH) were detected by fluorescence assay, and mitochondrial membrane potential (ΔPsim) was measured using JC-1 staining. After D-GalN injury, AA and GA treatments significantly reduced the levels of AST and LDH in the culture medium (P<0.05), and AA enhanced hepatocyte survival (P<0.05). Furthermore, AA and GA significantly reduced ROS and NOx production and improved D-GalN-induced ΔPsim loss. AA also inhibited the decrease in GSH levels induced by D-GalN and CCl4 treatments. Both AA and GA can protect hepatocytes from D-GalN and CCl4 damage, which is associated with reducing intracellular ROS and NOx levels, reversing GSH inhibition, and improving ΔPsim loss. A novel coumaroyl triterpenoid, 3-O-trans-p-coumarylactinic acid (1), and five known triterpenoids were isolated from the ethyl acetate extract of kiwifruit root: ursolic acid (2), 23-hydroxyursolic acid (3), corosolic acid (4), asiatic acid (5), and betulinic acid (6). The structure of compound 1 was determined by spectroscopic data analysis, particularly by extensive one-dimensional and two-dimensional nuclear magnetic resonance (NMR) studies. All isolates (1-6) were evaluated in vitro for their inhibitory activity against pancreatic lipase (PL). Among all isolates, the novel compound 1 exhibited the highest inhibitory activity against PL with an IC50 value of 14.95 μM, followed by ursolic acid (2, IC50 = 15.83 μM). The other four triterpenoids (3-6) also showed significant PL inhibitory activity, with IC50 values ranging from 20.42 to 76.45 μM.

[1]. J Pharmacol Exp Ther . 2005 Apr;313(1):333-44.

Therapeutic Uses
Centella Asiatica titration extract (TECA) contains three main components: asiaticoside (AS), asiatic acid (AA), and hydroxyasiatic acid (MA). These components are known to have clinical efficacy against systemic scleroderma, abnormal scar formation, and keloids. ...
/EXPL THER/Anamic acid (a triterpenoid compound extracted from the umbelliferous plant Centella asiatica) has been patented by Hoechst Aktiengesellschaft for the treatment of dementia and to enhance cognitive function.
/EXPL/Parkinson's disease (PD) is a progressive neurodegenerative disease with a prevalence of 1-2% in people over 50 years of age. PD patients exhibit mitochondrial dysfunction, manifested by a 15-30% reduction in complex I activity. Asiatic acid (AA) is a triterpenoid compound with antioxidant properties and is commonly used to treat depression, but its protective effect against PD-like damage has not been reported. This study aimed to investigate the protective effect of aminopyrine (AA) against H₂O₂ or rotenone-induced damage and mitochondrial dysfunction in SH-SY5Y cells. To explore the possible mechanisms of AA's neuroprotective effect, we examined the expression of mitochondrial membrane potential (MMP) and voltage-dependent anion channels (VDACs) in AA-pretreated and unpretreated cells after cell injury. The results showed that AA pretreatment (0.01–100 nM) protected cells from rotenone- or H₂O₂-induced toxic damage. Furthermore, MMP decreased after rotenone exposure, but AA treatment prevented this decrease. More interestingly, pretreatment with AA inhibited the increase in VDAC mRNA and protein levels induced by rotenone (100 nM) or H₂O₂ (300 μM). These data suggest that AA can protect neurons from mitochondrial dysfunction and hint at its potential development as a preventative or therapeutic drug for Parkinson's disease.
/EXPL/ We tested the potential protective effects of asiaticoside (AS) derivatives against Aβ-induced cell death. Of the 28 AS derivatives tested, asiaticoside (AA), asiaticoside 6 (AS6), and SM2 showed strong inhibitory effects on Aβ-induced B103 cell death at a concentration of 1 μM. We further tested the effects of these three AS derivatives on free radical damage and apoptosis. All three AS derivatives reduced H₂O₂-induced cell death and decreased intracellular free radical concentrations, but AA showed the strongest protective effect. In contrast, SM2 was the most effective compound in blocking astrosporin-induced apoptosis. These results indicate that these three AS derivatives block Aβ toxicity through different cellular mechanisms. When applied to hippocampal sections, AA, SM2, and AS6 did not alter the induction of CA1 region N-methyl-D-aspartate (NMDA) or non-NMDA receptor-mediated synaptic transmission, paired pulse facilitation, or long-term potentiation. These results indicate that these three AS derivatives do not alter hippocampal physiological characteristics at concentrations that block Aβ-induced cell death. Therefore, AS6, AA, and SM2 can be considered reasonable candidates for treating Alzheimer's disease and protecting neurons from Aβ toxicity. For more complete data on the therapeutic uses of ASIATIC ACID (6 in total), please visit the HSDB record page.

C30H48O5

488.70

488.35

C, 73.73; H, 9.90; O, 16.37

464-92-6

119034

Solid powder

1.2±0.1 g/cm3

609.4±55.0 °C at 760 mmHg

270 °C

336.4±28.0 °C

0.0±4.0 mmHg at 25°C

1.579

6.46

4

5

2

35

930

12

O([H])[C@@]1([H])[C@@]([H])(C([H])([H])[C@@]2(C([H])([H])[H])[C@]([H])([C@]1(C([H])([H])[H])C([H])([H])O[H])C([H])([H])C([H])([H])[C@@]1(C([H])([H])[H])[C@]3(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]4(C(=O)O[H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[H])[C@]([H])(C([H])([H])[H])[C@@]4([H])C3=C([H])C([H])([H])[C@@]12[H])O[H]

JXSVIVRDWWRQRT-UYDOISQJSA-N

InChI=1S/C30H48O5/c1-17-9-12-30(25(34)35)14-13-28(5)19(23(30)18(17)2)7-8-22-26(3)15-20(32)24(33)27(4,16-31)21(26)10-11-29(22,28)6/h7,17-18,20-24,31-33H,8-16H2,1-6H3,(H,34,35)/t17-,18+,20-,21-,22-,23+,24+,26+,27+,28-,29-,30+/m1/s1

(1S,2R,4aS,6aR,6aS,6bR,8aR,9R,10R,11R,12aR,14bS)-10,11-dihydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a-hexamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylic 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)

Solubility in Formulation 1: ≥ 2.25 mg/mL (4.60 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 22.5 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: 2.08 mg/mL (4.26 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (4.26 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 20.8 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.)