Preliminary investigation on the DNA of winged bean (Psophocarpus tetragonolobus) and cowpea (Vigna unguiculata) with bioactive peptide exhibiting antihypertensive activity

Date

2011

Degree

Bachelor of Science in Chemistry

College

College of Arts and Sciences (CAS)

Request Access

To request access of this material, please email the administrator at uscs-mainlib.uplb@up.edu.ph

Abstract

In silico analysis revealed that winged bean and cowpea contain bioactive peptides in their major storage protein. The antihypertensive bioactive peptides for the winged bean and cowpea were MY and AVP respectively. The genomic DNA was isolated by the method of Cheung et al.- Datta et al.- Dellaporta method as modified by Angeles et al (2005). The genomic DNA was then used as a template for the polymerase chain reaction using a pair of forward and reverse primers specifically designed for the amplification of the target gene for each of the crops. The forward and reverse primers designed for cowpea are 5’-CGCTACTCACTCACCATCATC-3’ and 5’- CTCCTCGTCTTGCTGTTTCTG-3’ respectively. On the other hand, the forward and reverse sequence of winged bean are 5’-CGATGTGGTGGCTACTGGA-3’ and 5’- ATCCACCACAGACCACACA-3’. The cowpea PCR product sequence result revealed that the target sequence corresponding to the tripeptide,AVP, was successfully synthesized along with another antihypertensive peptide, VK. On the other hand, the desired winged bean gene sequence was not synthesized, though ACE inhibitory peptides MY and LA were obtained. The crude protein cowpea was extracted using 50mM borate buffer (pH 8.0) as the extraction buffer. On the other hand, winged bean was isolated using Tris-HCl phosphate buffer saline (0.1 M Tris-HCl buffer, pH 8.3 with 35 mM potassium phosphate buffer and 0.4 M NaCl) as extraction buffer. A protein content determination by Bradford showed that the cowpea protein has an average estimate concentration of 16.65 mg/mL while winged bean has an average estimate concentration of 16.40 mg/mL. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiling revealed that the estimated molecular weight of the protein isolates from the two bands of cowpea were 24 and 30 kDa, while that of the winged bean were about 15 and 25kDa. The protein isolates were then subjected to Angiotensin-1 converting enzyme assay method of Cushman and Cheung (1979) in order to confirm inhibitory potential of the legume seeds. The crude protein of both cowpea

and winged bean were digested with pepsin for 24 hours, and assayed using the ACE pre- extracted from fresh rabbit lungs. The absorbance of the assay mixture was read at

228nm and it was found that the winged bean and cowpea digested proteins had an ACE inhibition of 76.92 and 74.36 percent respectively. This confirmed that winged bean and cowpea have antihypertensive activity.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Document Type

Thesis

Link to Full Text

Share

COinS