Betulinic acid

Standards quinic acid (1), caffeic acid (2), gallic acid (4), vanillic acid (5), catechin (7), epicatechin (9), ferulic acid (13), chrysin (15), rutin (16), quercetin-3,4′-di-O-glucoside (26), kaempferol-3-O-rutinoside (28), ellagic acid (35), coumaric acid (37), eriodictyol (39), methy-O-ellagic acid (42), protocatechuic acid (44), quercetin (45), luteolin (46), kaempferol (48) betulinic acid (50) and oleanolic acid (51) were purchased from Sigma-Aldrich (St. Louis, MO, USA) (Fig. 1). LC–MS grade solvents (acetonitrile methanol and formic acid) were also purchased from Sigma–Aldrich (St. Louis, MO, USA) and used throughout the study. Ultra-pure water was produced by Milli-Q Advantage system (Millipore, Milford, MA, USA). AR grade ethanol (Merck, Darmstadt, Germany) was used in the preparation of the ethanolic extracts.Fig. 1

Chemical structures of standard compounds.

Fig. 1.

In the experimental study 70 male NMRI mice (5 weeks old, 25-35 g) were purchased from animal center of Ahvaz Jundishapur University of Medical Sciences (AJUMS). The Ethic Committee for Animal Experiments approved the study (IR.AJUMS.REC.1394.145-6794) and all experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996. Mice were housed in cages under controlled conditions at 25 °C and a12 h light–dark cycle and 10% humidity. Mice were receiving food pellets and water ad libitum. Betulinic acid (Sigma-Aldrich, USA) and metformin (Sigma-Aldrich, USA) were orally administered 2 weeks after confirmed diabetes induction. The mice were randomly divided into seven groups of 10 animals each as follows: group 1, normal control; group 2, sham (0.1 mg/kg saline intraperitoneal, i.p.); group 3, untreated type 2 diabetic (STZ 50 mg/kg i.p., 15 min later a single dose of NA 120 mg/kg i.p.); group 4 to 6, type 2 diabetic animals received BA at 10, 20 and 40 mg/kg by gavage) (16 (link)); group 7, type 2 diabetic animals received metformin (200 mg/kg, by gavage) (13 (link)).

Already shed Carica papaya leaves (CPL) were collected from the commercial farm of Michael Okpara University of Agriculture Umudike, Abia state, Nigeria. The CPL were rinsed in running water to remove all the adhering dirt. The rinsed CPL were spread on a tray and air dried in Chemical Engineering laboratory for two weeks at room temperature of approximately 25 °C to attain a stable weight. After the sample drying, the CPL were pulverized, using an electric blender, screened with sieve aperture of 0.105 mm and kept in an airtight black polythene bag at 4 °C before the extraction experiment commenced. The moisture content of the air dried CPL sample was determined using the oven drying method and was found to be approximately 9% (w/w). The chemicals used in the course of experimentation such as sodium carbonate (Na₂CO₃) and Folin Ciocateu phenol reagent were purchased from Sigma Aldrich, Poole, England. Other HPLC grade phenolic standards were gallic acid, betulinic acid, chlorogenic acid, ellagic acid, ferulic acid, caffeic acid, quercetin and rutin and were procured from GFS Chemicals, Inc. USA (Sigma Aldrich, Germany). The distilled water was obtained from the Chemical Analysis Laboratory of Chemical Engineering Department, Michael Okpara University of Agriculture Umudike. All chemicals used were of analytical grades.