Asiatic acid (97%) (#546712, Sigma-Aldrich, St Louis, MO, USA) was prepared as a stock solution of 100 mM by dissolving 4.887 mg of Asiatic acid in 100 μL of dimethyl sulfoxide (DMSO). The solution was stored in the dark at −20 °C until use. All experiments contained <0.1% (v/v) DMSO. Cell culture reagents including Roswell Park Memorial Institute medium (RPMI) 1640 (#31800022), Dulbecco’s modified Eagle medium (DMEM; #31600034), fetal bovine serum (FBS; #10270098), trypsin–EDTA (0.25%), phenol red (#25200072), and penicillin–streptomycin (10,000 U/mL) (#15140122) were from Thermo Fisher Scientific (Waltham, MA, USA).
Asiatic acid
Manufactured by Merck Group
Sourced in United States
Asiatic acid is a chemical compound used in laboratory settings. It serves as a reference standard or analytical tool for various scientific applications. The core function of Asiatic acid is to provide a consistent and reliable substance for research, analysis, and quality control purposes.
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Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Corosolic acid, by Merck Group (2 mentions)
Betulinic acid, by Merck Group (2 mentions)
Oleanolic acid, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Arachidonic acid, by Cayman Chemical (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti phospho gsk3β ser9, by Cell Signaling Technology (1 mentions)
Ly294002, by Merck Group (1 mentions)
Maslinic acid, by Merck Group (1 mentions)
Arjunolic acid, by Merck Group (1 mentions)
Dichloromethane, by Merck Group (1 mentions)
Pyridine, by Merck Group (1 mentions)
Z vad fmk, by Beyotime (1 mentions)
Anti phospho akt, by Santa Cruz Biotechnology (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
15 protocols using asiatic acid
1
Asiatic Acid Stock Solution Preparation
2
Asiatic Acid COX-2 Inhibition Assay
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The materials used in this research are asiatic acid (97%) from Centella asiatica (Sigma Aldrich, St. Louis, MO, USA), arachidonic acid (Cayman Chemical, Ann Arbor, MI, USA), COX-2 enzyme-ovine (Cayman Chemical, Ann Arbor, MI, USA), tris-HCl buffer, and a Colorimeter COX Inhibitor Screening Assay Kit 701050 (Cayman Chemical, Ann Arbor, MI, USA). The pieces of software used in this research are the AMBER18 program, ChemDraw 2D Ultra 12.0 program, LiganScout 4.4.3, MM2 by ChemDraw 3D Software, Gaussian03 Software (Gaussian, Wallingford, CT, USA), Accelrys Discovery Studio 2.5 Software (Accelrys Inc., San Diego, CA, USA), Accelrys Discovery Studio Visualizer 3.5 Software (Accelrys Inc.), AMBER 11 Software (Amber, San Francisco, CA, USA), and AmberTools 1.5 Software (Amber). The pieces of hardware used are an is-nodes Linux cluster, each powered by an Intel Xeon 2.4 GHz processor and 2 GB of RAM (Intel, Santa Clara, CA, USA), an incubator (Memert, Schwabach, Germany), and a plate reader.
3
Clostridium difficile Strain Isolation and Asiatic Acid Preparation
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Clostridium difficile strains 630 and R20291 were kindly provided as a gift from Prof. Nigel Minton, University of Nottingham. Human isolates (RA) of C. difficile were previously isolated from diarrheal patients admitted to Ramathibodi Hospital, Thailand during 2010–2011 (Chankhamhaengdecha et al., 2013 (link)). The C. difficile NIH isolates were obtained from the National Institute of Health (NIH), Thailand. Food and animal C. difficile isolates were previously obtained (Ojha et al., 2016 (link)). All isolates were cultivated in brain heart infusion (BHI) medium at 37°C under anaerobic condition, unless otherwise mentioned. Asiatic acid with the purity of 97% was purchased from Sigma-Aldrich (St. Louis, MO, United States). The structure of AA is shown in Figure 1 . The stock AA solution was prepared at 10 mg/ml in dimethyl sulfoxide (DMSO).
4
Asiatic Acid and LY294002 Regulation
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Asiatic acid and LY294002 were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The anti-Akt and anti-phosphor-Akt antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); the anti-GSK-3β and anti-phospho-GSK-3β (Ser9) antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, USA); the anti-GLUT4 and anti-PPARγ antibody was from Boster Biological Technology, Ltd. (Wuhan, China); and the anti-glyceraldehyde phosphate dehydrogenase (GAPDH) antibody was obtained from KangChen Bio-tech Inc. (Shanghai, China).
5
Quantification of Pentacyclic Triterpenoids
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The standards: asiatic acid (2α,23-dihydroxyursolic acid, 97% purity), arjunolic acid (2α,3β,4α)-2,3,23-trihydroxyolean-12-en-28-oic acid, ≥95 purity), corosolic acid (2α,3β-dihydroxyurs-12-en-28-oic acid, ≥98% purity), maslinic acid (2α,3β-dihydroxyolean-12-en-28-oic acid, ≥98% purity), and (4α)-23-hydroxybetulinic acid ((3β,4α)-3,23-dihydroxylup-20(29)-en-28-oic acid, ≥98% purity) were purchased from Sigma-Aldrich (Spain, Madrid). The standards: madecassic acid (≥90% purity), caulophyllogenin ((3β, 4α, 16α)-3,16,23-trihydroxyolean-12-en-28-oic acid, ≥98.5% purity), and hederagenin ((3β, 4α)-3,23-dihydroxyolean-12-en-28-oic acid, ≥98.5% purity) were purchased from Extrasynthese (France, Paris). The derivatization reagents (pyridine and N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA)) and dichloromethane were acquired from Sigma-Aldrich (Spain).
6
Asiatic Acid and 2-ME Mediated Apoptosis in H9c2 Cells
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H9c2 cell lines were purchased from American Type Culture Collection (ATCC, CRL-1446) (Rockville, MD, USA). Asiatic acid (97% purity) and 2-Methyoxyestradiol (2-ME) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and were prepared as a stock solution by dissolving in dimethyl sulfoxide (DMSO, 0.5%, final concentration). The anti-Bcl-2, anti-Bax, anti-Akt and anti-phospho-Akt antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); the GSK-3β and phospho-GSK-3β (Ser9) antibodies were from Cell Signaling Technology, Inc., (Cell Signaling, Danvers, MA, USA), and the glyceraldehyde phosphate dehydrogenase (GAPDH) antibody was obtained from KangChen Bio-tech Inc., (Shanghai, China). MK2206 was obtained from Biovision (Milpitas, CA, USA), and z-VAD-fmk was from Beyotime Institute of Biotechnology (Haimen, China).
7
Osteoclast Differentiation Protocol
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Recombinant macrophage colony stimulating factor (MCSF) and receptor activator for the nuclear factor-κB ligand (RANKL) were purchased from R&D Systems (Minneapolis, MN, USA). RbCl and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MAPK activator anisomycin and Asiatic acid were purchased from Sigma-Aldrich. Antibodies for western blotting and immunohistochemical analyses were purchased from Cell Signaling Technology (Cambridge, MA, USA).
Osteoclast precursor RAW 264.7 cells and osteoblast MC3T3-E1 cells were cultured in standard α-MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. Primary murine bone marrow monocytes (BMMs) were harvested from the femurs and tibiae of four- to six-week-old C57BL/6 mice and incubated in α-MEM medium with 30 ng/ml MCSF and 10% FBS. All cells were kept in a sterile condition at 5% CO2 of 37°C.
Osteoclast precursor RAW 264.7 cells and osteoblast MC3T3-E1 cells were cultured in standard α-MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. Primary murine bone marrow monocytes (BMMs) were harvested from the femurs and tibiae of four- to six-week-old C57BL/6 mice and incubated in α-MEM medium with 30 ng/ml MCSF and 10% FBS. All cells were kept in a sterile condition at 5% CO2 of 37°C.
8
Cardiovascular Pharmacology Protocols
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Asiatic acid, ethylenediaminetetraacetic acid (EDTA), norepinephrine, acetylcholine, phenylephrine, sodium nitroprusside and capsaicin were obtained from Sigma-Aldrich (St. Louis, MO, USA) (purity >95 %). All other chemicals used were analytical grade quality.
9
Asiatic Acid Stock Preparation
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Asiatic acid (purity > 97.0%; molecular weight 488.70), purchased from Sigma–Aldrich (St. Louis, USA), was dissolved in dimethylsulfoxide (DMSO) as a 2-mM stock solution and stored at 4 °C. Further dilution was done in cell culture medium.
10
Pharmacokinetic Analysis of Centella, Piperine, and Curcumin
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The Centell-S (purity ≥ 86.7%), piperine (purity ≥ 98.0%) and curcumin (purity ≥ 95.0%) used in the pharmacokinetic experiment were provided by Siam Herbal Innovation Co. Ltd. Analytical grade madecassoside (purity ≥ 96.7%) and madecassic acid (purity ≥ 97.5%) were purchased from Chromadex Corp. Analytical standards of asiaticoside (purity ≥ 98.5%) and asiatic acid (purity ≥ 97.0%) were purchased from Sigma-Aldrich Inc. The internal standards glycyrrhizin (purity ≥ 90.0%) and glycyrrhetinic acid (purity ≥ 98.0%) were purchased from Wako Pure Chemical Industries Ltd.
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