Release of antidiabetic peptides from Stichopus japonicas by simulated gastrointestinal digestion

Highlights

• Peptides were released from sea cucumber by in vitro gastrointestinal digestion.

• Peptides obtained were predicted to be potential inhibitory of DPP-IV.

• Antidiabetic activity was detected in 3T3-L1 cells and Hep G2 cells.

• In silico results indicated that peptides inhibit DPP-IV like anagliptin.

Abstract

Sea cucumber (Stichopus japonicus) is a high-protein food with the potential to release certain peptides through enzymolysis. This work is to explore the characteristics of peptides released from Stichopus japonicus protein in the process of digestion. Hydrolysates were obtained by gastrointestinal digestion and fractioned to <3, 3–10, 10–30 and >30 kDa fractions. Fifty-eight peptides from <3 kDa fraction were characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hydrolysates could improve glucose uptake of 3 T3-L1 cells and high insulin-induced insulin-resistant Hep G2 cells. Molecular docking showed that the released peptides had similar binding mode with anagliptin, a dipeptidyl peptidase IV (DPP-IV) inhibitor. The <3 kDa fraction in gastro and intestinal digestion showed the greatest DPP-IV inhibitory potency (IC₅₀ 0.51 and 0.52 mg/mL, respectively). The results indicated that sea cucumber could be used as a functional food to release antidiabetic peptides through gastrointestinal digestion.

Introduction

Diabetes mellitus (DM), as a complex metabolic disease, has become an important global health problem. Nowadays, the incidence rate of diabetes mellitus is increasing explosively worldwide. Type 1 diabetes mellitus is caused by low yield of insulin, resulting in decrease of glucose uptake (Daneman, 2006). Type 2 diabetes mellitus is along with insulin resistance, in which the tissues have hyposensitivity to insulin. Type 1 diabetes mellitus can be control with primary insulin injection, but the other one is intractable, due to its intricate signaling pathway and the presence of many severe complications, such as retinopathy, nephropathy, cardiovascular disease and peripheral vascular disease (Ming, Bhupathiraju, Mu, Dam, & Hu, 2014). Insulin resistance (IR) is ubiquitous in the development of type 2 diabetes mellitus, especially in peripheral tissues responsive to insulin including liver, adipose tissue and skeletal muscle (Cao et al., 2015, Herrera et al., 2009). Some molecular targets used in the treatment of diabetes mellitus are the inhibition of enzymes dipeptidyl peptidase IV (DPP-IV), α-amylase and α-glucosidase. DPP-IV inhibitors are a new class of oral anti-hyperglycemic agents for the treatment of type 2 diabetes. Due to their chemical structure, inhibitors can interact with amino acids in the catalytic cavity of DPP-IV that is related to diabetes mellitus, and then play the inhibitory effect (see Fig. 1).

Peptides from food proteins are verified to be safer, milder and easily absorbed compared with synthetic drugs. Thus, more and more attention has been focused on the research about peptides with bioactivity from food, like soybean (Fan et al., 2009, Rho et al., 2009), milk (Liu & Pischetsrieder, 2017), oyster protein (Wang et al., 2008), bovine collagen (Fu et al., 2016) and sea cucumber (Lin et al., 2018). Sea cucumber (Stichopus japonicus), rich in protein, is a valuable and traditional species in Chinese food and medicines. About 70% of the total body wall protein of sea cucumber is collagen (Saito, Kunisaki, Urano, & Kimura, 2010). It is known that bromelain and alcalase can be used as proteolytic enzymes to release antihypertensive peptides (Zhao et al., 2007, Zhao et al., 2009, Zhao et al., 2012). Besides, collagenase has been applied for the hydrolysis giant red sea cucumber (Parastichopus californicus) (Liu, Chen, Su, & Zeng, 2011). In addition, some peptides released from sea cucumber were found to possess multiple functions, such as antioxidant activity (Chen et al., 2010, Pérez-Vega et al., 2013, Zhou et al., 2012), angiotensin-I converting enzyme (ACE) inhibitory activity (Liu et al., 2011, Zhao et al., 2007, Zhao et al., 2009, Zhao et al., 2012) and anti-aging activity (Lin et al., 2018). Usually, the released sea cucumber peptides with lower molecular weights were found to have higher bioactivities than those with higher molecular weights (Chen et al., 2010, Liu et al., 2011, Pérez-Vega et al., 2013, Zhao et al., 2007, Zhao et al., 2009, Zhao et al., 2012, Zhou et al., 2012). In this context, the aim of this work is to explore the characteristics of released peptides from Stichopus japonicus protein in the process of digestion. Considering multifunction peptides could be released from sea cucumber through enzymolysis, we envisioned that some bioactive peptides might be generated in situ during the process of digestion. To test this hypothesis, a gastrointestinal digestion model was established here to simulate digestive process, and the released peptides were investigated by LC-MS/MS, cells experiment and in silico molecular interactions.

On the other hand, enzyme concentration, pH and digestive time were well considered in the previous reports on the obtention of bioactive peptides via simulated gastrointestinal digestion (Alemán et al., 2013, Liu and Pischetsrieder, 2017, Pérez-Vega et al., 2013). It is worth mentioning that activity of digestive enzyme can be altered by salts concentrations such as calcium (Minekus, Alminger, Alvito, Ballance, & Bohn, 2014). As the electrolytes concentrations have not attracted enough attention in the above reports about peptides generation in digestive process of sea cucumber, the electrolytes concentrations of the human digestive tract were adopted in this work, which made our simulated GI digestive process more practical in its potential applications.