Anti-trypsin and antihypertensive activity of protein extracts from Nigella sativa seeds

Protease inhibitors (PIs) are a ubiquitous, diverse group of molecules present in multiple forms in all organisms. These inhibitors inactivate proteases from predators/pathogens in addition to regulating intracellular proteolysis. In addition to intracellular localization, storage organs of plants are also a potential site of protease inhibitors. Proteins with trypsin inhibitory activity were isolated from Nigella sativa seed extracts by ammonium sulphate precipitation. Extraction conditions were optimized by choosing an optimum solvent, temperature and incubation period. The highest inhibitory activity of protein extracts was achieved by using 50 mM Tris buffer as solvent and overnight incubation of the suspension at 4°C. The crude seed extract fractionated at 60% ammonium sulphate concentration exhibited highest trypsin inhibitory activity, i.e., 60.15 ± 2.95 %, which was comparable to soybean trypsin inhibitor used as positive control. Ammonium sulphate precipitation of crude extract yielded 39.83-fold purification. Partially purified trypsin inhibitor exhibited 2.39±0.23 TIU mg-1. Additionally, Nigella sativa protein extracts were also investigated for their health-promoting effects against two important proteases, α- Dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE). Highest inhibitory activity against ACE was shown by the crude extract of N. sativa. Among AS fractions, 30% ammonium sulphate concentration exhibited highest inhibition activity against ACE and DPP-IV. Our results suggest that the widely believed role of N. sativa in control of hypertension may at least be partially shared by inhibition of ACE. This is the first study conducted to evaluate the biological activity of N. sativa protein extracts suggesting a potential use of N. sativa proteins in management of hypertension as well as an important source of trypsin inhibitor. Further identification, purification and characterization of different bioactive compounds from N. sativa are being carried out.

Ranacyclin-NF, a Novel Bowman–Birk Type Protease Inhibitor from the Skin Secretion of the East Asian Frog, Pelophylax nigromaculatus

Serine protease inhibitors are found in plants, animals and microorganisms, where they play important roles in many physiological and pathological processes. Inhibitor scaffolds based on natural proteins and peptides have gradually become the focus of current research as they tend to bind to their targets with greater specificity than small molecules. In this report, a novel Bowman–Birk type inhibitor, named ranacyclin-NF (RNF), is described and was identified in the skin secretion of the East Asian frog, Pelophylax nigromaculatus. A synthetic replicate of the peptide was subjected to a series of functional assays. It displayed trypsin inhibitory activity with an inhibitory constant, Ki, of 447 nM and had negligible direct cytotoxicity. No observable direct antimicrobial activity was found but RNF improved the therapeutic potency of Gentamicin against Methicillin-resistant Staphylococcus aureus (MRSA). RNF shared significant sequence similarity to previously reported and related inhibitors from Odorrana grahami (ORB) and Rana esculenta (ranacyclin-T), both of which were found to be multi-functional. Two analogues of RNF, named ranacyclin-NF1 (RNF1) and ranacyclin-NF3L (RNF3L), were designed based on some features of ORB and ranacyclin-T to study structure–activity relationships. Structure–activity studies demonstrated that residues outside of the trypsin inhibitory loop (TIL) may be related to the efficacy of trypsin inhibitory activity.

EFFECT OF SOME PROCESSING METHODS ON NUTRIENT CONTENT AND ANTI-NUTRITIONAL FACTORS OF A VARIETY OF DOLICHOS LABLAB (LABLAB PURPUREUS L.) BEANS GROWN IN KENYA

This study aimed to determine the effect of different processing methods on the proximate composition and anti-nutritional factors of Dolichos lablab beans (Lablab purpureus) of Kenya. The seeds of KAT/DL-2 variety,sourced from Kenya Agricultural Livestock and Research Organisation, Katumani Dryland Research Station were sorted, then subjected to different processing methods (soaking, cooking and germination). The samples were analysed for proximate composition, tannins, phytates and trypsin inhibitory activity. The results showed a significant increase (2.0%) in crude protein content for germinated lablab beans while carbohydrates content was high in cooked samples. The variety KAT/DL-2 had high levels of phytates; 723.6 mg/100g and tannins 330.3mg/100g and trypsin inhibitor activity 1.3mg/100g. Cooking achieved the highest reduction of anti-nutrients with 88% reduction in TIU. The results revealed that the anti-nutrients in lablab beans can be reduced using different methods of processing. However, there is need to investigate the effect of combined methods on the nutrients and anti-nutrients.

A Novel Kunitzin-Like Trypsin Inhibitor Isolated from Defensive Skin Secretion of Odorrana versabilis

Protease inhibitors that were identified from amphibian skin secretions with low molecular weights and potent inhibitory activity were thought to be potential candidates for novel peptide drugs. Here, a novel peptide with trypsin inhibitory activity was found in the skin secretion of the Chinese bamboo leaf odorous frog, Odorrana versabilis. Based on the sequence alignments of sequencing results, the novel peptide (ALKYPFRCKAAFC) was named as Kunitzin-OV. The synthetic replicate of Kunitzin-OV was subjected to a series of functional assays, and it exhibited a trypsin inhibitory activity with a Ki value of 3.042 µM, whereas, when Lys-9 at P1 position was substituted by Phe, trypsin inhibitory activity was undetected and the chymotrypsin inhibitory activity was optimized with a Ki value of 2.874 µM. However, its protease-binding loop was catabolized by trypsin during the trypsin cleavage test. In conclusion, Kunizin-OV is a novel peptide with trypsin inhibitory activity as a member of kunitzins, which is a non-typical Kunitz-like trypsin inhibitor with a highly conserved reactive site (K-A) and quite a short sequence.

Enzymatic Pea Protein Hydrolysates Are Active Trypsin and Chymotrypsin Inhibitors

In this work, we report the potency of enzymatic hydrolysates of pea proteins against trypsin and chymotrypsin. Pea protein concentrate was digested with each of alcalase, chymotrypsin, pepsin, and trypsin, followed by membrane separation of the protein hydrolysates into peptide fractions (<1, 1–3, 3–5, and 5–10 kDa). Peptide size profiling with size-exclusion gel chromatography indicated the narrowest size range (0.85–4.98 kDa) for alcalase. Trypsin activity was strongly (p < 0.05) inhibited by the ultrafiltration fractions (mean IC50 = 2.2 mg/mL) obtained from the trypsin hydrolysate when compared to the unfractionated hydrolysate (IC50 = 6.8 mg/mL). Similarly, ultrafiltration also enhanced trypsin inhibition by the alcalase-digested peptides with an IC50 of 21.4 mg/mL for the unfractionated hydrolysate in comparison to 3.1–4.7 mg/mL for the fractions. However, ultrafiltration did not enhance trypsin inhibitory activity of chymotrypsin-digested peptides, while the peptide separation reduced efficacy of pepsin-digested peptides. In contrast, chymotrypsin inhibition by all the enzymatic digests was significantly (p < 0.05) enhanced by ultrafiltration, especially peptide sizes >3 kDa. Kinetics of enzyme inhibition indicate peptides were bound to the enzyme active site in a competitive mode that led to reduced catalysis. We conclude that the pea peptides could function as useful tools to promote human health and as a preservative during food processing and storage.