Purnima Kaul Tiku

In vitro human gastro-intestinal enzyme digestibility of globulin isolate from oil palm (Elaeis guineensis var. tenera) kernel meal and the bioactivity of the digest

Globulins are the major seed storage proteins (41%) present in oil palm kernel. The study describes the ex vivo digestibility of globulin isolate prepared from oil palm kernel meal. The bioactivity and nutritional value of the ex vivo digest of globulin isolate was also investigated. Globulin isolate (85% protein content) was prepared by a salt extraction method and its digestibility was studied using human gastroduodenal juices (HGDJJ). By SDS-PAGE analysis it was evident that the globulin isolate was completely digested by HGDJ. Easy digestibility of a dietary protein is essential from the nutritional point of view. In the present ex vivo stUdy. the digestion profile and degree of hydrolysis of globulin isolate were observed to be comparable with that of casein. RADVFNPR and KLPLVERIP were the two peptides identified by MS/MS analysis in the final hydrolysate. By pepsinolysis. it was evident that the novel globulin protein preparation was devoid of pepsin resistant proteins that are otherwise considered to have allergenic potential. The ex vivo hydrolysate exhibited potent ACE-inhibitory activity (IC50 µg ml-1 and anticancer activity against human colon epithelial cancer HT-29 cells and hepatocarcinoma HepG2 cells. Easy digestibility. bioactivity, the presence of nutritionally important free essential amino acids and non-protein amino acids (gamma-aminobutyric acid and citrulline) in the gastrointestinal digest suggests that the globulin isolate prepared from palm kernel meal can be used as a nutraceuticaL protein for food applications.

Effect of arginine : lysine ratio in free amino acid and protein form on L-NAME induced hypertension in hypercholesterolemic Wistar rats

Arginine plays an important role in cardiovascular diseases, especially as a nitric oxide precursor leading to vasodilation. The present study focuses, on the effect of the arginine/lysine (Arg : Lys) ratio in the protein form and free amino acid form on hypertension in hypercholesterolemic Wistar rats. Hypertension was induced by administration of L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) orally for 16 weeks in high cholesterol fed Wistar rats. Arginine : lysine ratio is high in Moringa seed protein isolate (MPI) compared to other oilseed proteins. After inducting, the treatment groups were supplemented with diets containing Moringa seed protein isolate (MPI-LN) and free amino acids (FAA-LN) for 6 weeks. A decrease in systolic blood pressure (SBP) was observed after treatment. The MPI-LN and FAA-LN exhibited 126.6 ± 2.2 mmHg and 127.5 ± 0.2 mmHg of SBP respectively, which is less compared to the hypertensive rats (146.7 ± 4.5 mmHg). The hypertensive markers such as angiotensin-I converting enzyme (ACE) and nitrate levels in kidney were analysed. The MPI-LN and FAA-LN exhibited 10.30 ± 3.7 nmoles per mg per min and 37.56 ± 3.7 nmoles per mg per min ACE activity respectively. In both treatment groups, a 50% decrease in ACE activity was observed compared to hypertensive rats. Increase (30%) in nitrate levels was observed in MPI-LN (0.47 ± 0.04 μM g−1) and FAA-LN (0.4 ± 0.06 μM g−1) compared to hypertensive rats. From the results, it is evident that Arg : Lys has a role in hypertension regulation. Lipid profiles of blood plasma showed a decrease in plasma triacylglyceride and liver triacyglyceride levels in the treatment groups. The Arg : Lys ratio in both the protein form (MPI) and free amino acid form strongly affects the metabolic pathways of hypertension with moderate effect on hypercholesterolemia.

Cupincin: A Unique Protease Purified from Rice (Oryza sativa L.) Bran Is a New Member of the Cupin Superfamily.

Cupin superfamily is one of the most diverse super families. This study reports the purification and characterization of a novel cupin domain containing protease from rice bran for the first time. Hypothetical protein OsI_13867 was identified and named as cupincin. Cupincin was purified to 4.4 folds with a recovery of 4.9%. Cupincin had an optimum pH and temperature of pH 4.0 and 60°C respectively. Cupincin was found to be a homotrimer, consisting of three distinct subunits with apparent molecular masses of 33.45 kDa, 22.35 kDa and 16.67 kDa as determined by MALDI-TOF, whereas it eluted as a single unit with an apparent molecular mass of 135.33 ± 3.52 kDa in analytical gel filtration and migrated as a single band in native page, suggesting its homogeneity. Sequence identity of cupincin was deduced by determining the amino-terminal sequence of the polypeptide chains and by and de novo sequencing. For understanding the hydrolysing mechanism of cupincin, its three-dimensional model was developed. Structural analysis indicated that cupincin contains His313, His326 and Glu318 with zinc ion as the putative active site residues, inhibition of enzyme activity by 1,10-phenanthroline and atomic absorption spectroscopy confirmed the presence of zinc ion. The cleavage specificity of cupincin towards oxidized B-chain of insulin was highly specific; cleaving at the Leu15-Tyr16 position, the specificity was also determined using neurotensin as a substrate, where it cleaved only at the Glu1-Tyr2 position. Limited proteolysis of the protease suggests a specific function for cupincin. These results demonstrated cupincin as a completely new protease.

Cupincin: A Novel Cupin Domain Containing Protease from Rice ( Oryza sativa L.) Bran Comprising of Procoagulant and Fibrinogenolytic Activity

The current study was carried out to evaluate the pharmacological properties of cupincin- A novel cupin domain containing metalloprotease with limited proteolysis from rice bran on blood coagulation and hydrolysis of human fibrinogen. Cupincin preferentially hydrolyzed the Aα chain of fibrinogen and then the Bβ-chain, but not the γ-chain. Cupincin reduced the re-calcification time of citrated human plasma dose dependently. Analysis of citrated whole blood in the presence of cupincin by rotem showed a decrease in coagulation time and clot formation time. Sonoclot analysis indicated that cupincin cleaved fibrinogen of whole citrated blood. SDS-PAGE and sonoclot analysis (LI-30) indicated that cupincin lacked plasmin-like activity. Global hemostasis tests like rotem and sonoclot analysis determined cupincin as a procoagulant enzyme. Cupincin did not show any effect on prothrombin time and activated partial thromboplastin time tests suggesting its action on the common pathway of coagulation. The involvement of proteases from rice (Oryza sativa L.) in haemostasis has never been exploited before. This study could provide the basis for the development of new procoagulant agents from a nontoxic source like rice.