Effects of Anchusa strigosa root aqueous extract on gastric ethanol-induced ulcer in laboratory animals.

Anchusa strigosa Banks et Sol (Boraginaceae) root extracts (ASRE) were prepared by soaking the dry material in boiling water. The clear soluble extract was dried and found to be 0.238 g/g dry roots. A gastric ulcer was induced in fastened animals by oral ingestion of ethanol. Administration of 0.080 g of ASRE prior to ethanol ingestion protected the stomach of the rat from ulcer formation. The ulcer index values, expressed as a percentage of total stomach surface area affected by the ulcer, were lowered from 34.0+/-4.0 to 6.0+/-0.7 and 32.5+/-9.4 to 2.2+/-1.4 by the morphometric and the planimetric methods, respectively. Treatment of the induced ulcer in guinea pigs was achieved by oral administration of ASRE at the therapeutic dose of extract of 0.286 g/kg body weight/day for 24 days. The intraperitoneal LD50 of ASRE in mice was 0.080 g extract/kg body weight. Replacing water intake by ASRE at 75 ml of variable extract concentrations of 2.865, 3.57 and 4.284 g/l per animal per day for 90 days showed no histopathological changes in all organs of the rat. However, a clear depression effect on the central nervous system and anemia were observed particularly with extract of 3.57 g/l or more.

Pepsin inhibitor from roots of Anchusa strigosa.

A pepsin inhibitor of undetermined exact chemical composition was isolated from the aqueous extracts of the roots of Anchusa strigosa. The extract of 1 g dry roots inhibited 9380 +/- 390 micrograms of pepsin. The main steps of the isolation procedure consisted of extraction in boiling water, precipitation at pH 3 and washing the precipitate with ethanol, column chromatography on sephadex G-75, and finally ultrafiltration on UM10 membrane. The specific activity at the last step was 44 units ml-1 solution which measured 1.0 at 287 nm (a one unit of the inhibitor is that amount which inhibited 1 microgram of pepsin under the conditions of the assay). The overall purification was 22-fold. The inhibitor did not bind CM-cellulose, but it did bind DEAE-cellulose irreversibly. A pepsin-inhibitor complex was formed with a KI = 2 x 10(-8) M. The inhibitor inhibited peptic milk-clotting activity at pH 5.3. A solution of the inhibitor exhibited split-peak spectra with two maxima at 280 nm and 287 nm. The epsilon at 287 nm was estimated at 9.4 x 10(5) l mol-1 cm-1 based on a M(r) value which was estimated at 62,500.

Isolation and characterization of camel pepsins.

Camel (Camelus dromedarius) pepsins were precipitated from the extract of the fundic gastric mucosa by ammonium sulfate between 35 and 80% saturation. DEAE--cellulose chromatography of this fraction produced two isoenzymes, I and II, which were further purified to homogeneity on sephadex G-100. Their Km with N-acetyl-L-phenylalanyl-diiodotyrosine were 0.10 and 0.90 mM, and their molecular weights which were determined on sodium dodecylsulfate gel electrophoresis exhibited 35,500 and 34,700, respectively. The two pepsins were essentially free of carbohydrates, but contained 0.3 and 1.0 mol of organic phosphate per 1 mol of protein, respectively. The apparent mobilities of the phospho- and dephosphoforms of each pepsin were indifferent in polyacrylamide gel electrophoresis at pH 8.9. The N-terminal residues of pepsins I and II were found to be alanine and leucine, respectively. Pepsin I was inactivated at a faster rate than that of pepsin II at pH 8 and 0 degrees C, and at pH 7.5 and 37 degrees C; but both were denatured under these conditions. The properties of these enzymes are compared with those of other mammalian and avian pepsins.

Reformation of reduced disulfide bonds of pepsinogen and pepsin: role of the phosphate group.

Reduction of the disulfide bonds in pepsinogen and pepsin with 2-mercaptoethanol eliminated the proteolytic activity of the enzyme and the potential proteolytic activity of the zymogen. Removal of the reducing agent followed by airation resulted in reformation of the disulfide bonds in the phospho-forms of pepsinogen and pepsin, but not in the dephospho-forms. To account for the role of the phosphate group in the stabilization of the native conformation even at reduced states of the zymogen and the enzyme, it is suggested that the group which is linked to the seryl residue is also linked to another amino acid residue in the form of anhydride, hydrogen bond(s), or/and ionic interaction.