Cardiovascular diseases (CVDs) are considered the most important health problem in industrialized countries. These pathologies represent the main cause of death worldwide, with hypertension being one of the main risk factors for developing CVD [1
]. Hypertension is normally treated with drugs, in particular with angiotensin-converting enzyme (ACE) inhibitors. ACE plays an important physiological role in the regulation of blood pressure. Angiotensin-I is transformed to angiotensin-II by the action of ACE, resulting in arterial constriction and blood pressure elevation [2
]. Since synthetic ACE inhibitors frequently produce side effects, research on natural compounds with ACE inhibitory properties can be advisable for decreasing the high values of arterial blood pressure and to replace synthetic drugs [3
]. In addition, there is mounting evidence linking oxidative stress with the development of a large number of diseases, including hypertension [4
]. Antioxidant compounds with properties that inhibit cell oxidation could also be a valuable strategy to improve oxidative stress and related cardiovascular diseases.
In the last few years, the food industry has shown an increased interest in the study of bioactive compounds from natural sources as an alternative in the search for new therapeutic agents and treatments to improve human health. Among all the food components, proteins from animals and plants are considered one of the main sources to obtain functional ingredients. In this context, food-derived peptides are defined as inactive amino acid sequences within the protein but which carry out certain physiological functions in the body after its release by in vivo (gastrointestinal digestion) and in vitro (chemical or enzymatic) hydrolysis. They have demonstrated potential health benefits against several chronic diseases, in particular hypertension [3
]. In fact, they could represent a good and safe strategy for controlling high blood pressure and some related cardiovascular diseases due to their high enzymatic affinity, strong specificity, and low toxicity [7
]. Recently, plant-protein-derived hydrolysates, mainly from soybean, have received special attention to produce bioactive peptides [8
Rice bran is an abundant byproduct in the rice milling industry, which is used to produce edible rice oil and animal feed [10
]. Since rice bran is considered an important source of proteins (12.6–15.4%) [12
], an interesting alternative may be the production of protein concentrates to obtain hydrolysates and peptides with bioactive properties [7
]. Rice bran enzymatic hydrolysates and peptides have exerted various in vitro biological activities, including antioxidant, antidiabetic, anticancer, and ACE inhibition, but very little is known about the in vivo effects in animal models [14
For all these reasons, the present study hypothesized that the production of different bioactive hydrolysates, derived from rice bran, might improve oxidative stress, hypertension, and/or other cardiovascular-related diseases.
The main aims of this work were: (1) to obtain hydrolysates from industrial and laboratory defatted rice bran protein concentrates using different food-grade microbial enzymes, (2) to investigate the in vitro antioxidant and ACE inhibitory properties of these hydrolysates and (3) to evaluate the in vivo activity of the selected rice bran protein hydrolysates in spontaneously hypertensive rats (SHR) and Wistar–Kyoto (WKY) rats.
3. Results and Discussion
Several methods are being used to produce food bioactive peptides, where the enzymatic hydrolysis process is one of the most used methods. Thus, the protein source, type of enzyme, and degree of hydrolysis (DH) define the characteristics and bioactivities of the peptides present in the hydrolysates [20
]. DRBPC-1 and DRBPC-2 samples were subjected to a hydrolysis reaction with two food-grade microbial enzymes: alcalase or flavorzyme. Figure 1
shows the hydrolysis kinetics of the different defatted rice bran protein concentrates with the different enzymes for 10 h.
The DH increased with hydrolysis time, showing a gradual release of peptide fragments during the process. The curves present a high initial reaction rate followed by a decrease up to the stationary period, in which the degree of hydrolysis became constant. The stationary period was reached faster for the products obtained using flavorzyme, in 210 min (RBPH-2F) and 270 min (RBPH-1F) of hydrolysis. Hydrolysates using alcalase reached their stationary period after 480 min of reaction. At the end of the hydrolysis period, the DH was 10.4%, 13.4%, 4.2%, and 5.9% for RBPH-1A, RBPH-2A, RBPH-1F, and RBPH-2F, respectively. Thamnarathip, Jangchud, Jangchud, and Vardhanabhuti [21
] also presented similar values of DH using rice bran hydrolyzed with alcalase (18.7%) compared to flavorzyme (7.4%) after 360 min of reaction. These results suggest that the specificity of the protease used to release peptides plays a very important role, independent of incubation time with enzymes. The action of the alcalase was more effective in both concentrates. However, both enzymes worked faster with the rice bran protein concentrates defatted by an industrial process (DRBPC-2) than with those obtained by our laboratory extraction process (DRBPC-1). This could be explained by the fact that, in some industries, the crude rice bran is submitted to a pelletization process to preserve the bran, where an agglomeration of milled particles occurs via mechanical processes combined with moisture, heat, and pressure conditions, to stabilize its lipolytic enzymes and to prevent lipid degradation, that could result on rancidity [22
]. The industrial conditions can promote protein changes, modifying its structure produced via denaturation, and it is known that the best action of enzymes is obtained after protein denaturation [23
]. Similar results were obtained by Uraipong and Zhao [7
] after 4 h of hydrolysis of rice bran albumin using alcalase plus flavorzyme, and Sbroggio et al. observed the same behavior after denaturation using okara protein [24
Despite both enzymes being from microbial origin, alcalase is an endopeptidase and has a limited range of specificity of peptide bonds for hydrolysis [25
]. On the other hand, flavorzyme is a mixture of an endopeptidase and exopeptidase produced by Aspergillus oryzae
, which can exert a broader range of action and therefore a higher DH would be expected with this enzyme [25
]. Contrary to expectations, in our study, we obtained higher DH in alcalase hydrolysates.
As mentioned in the introduction section, enzymatic proteolysis may change food properties, such as digestibility, nutritional, and sensory quality, but provides health benefits due to the formation of bioactive peptides. The length and characteristics of the peptides formed, in terms of their constituent amino acids, the presence of polar and ionizable groups, and their hydrophobicity, determine the functional and bioactive properties of food hydrolysates; these depend not only on the degree of hydrolysis but also on the specificity of the enzyme and protein used as a substrate [26
Natural antioxidants from food proteins have been investigated by several research groups because of their low price, high activity, and easy absorption [27
]. Food hydrolysates with potential antioxidant activity are related to its composition, structure, hydrophobicity, and amino acid position in the peptide sequence [28
], and these characteristics also seem to influence the antioxidant mechanism. Despite the mechanisms not being clear, different studies show that some peptides can act as inhibitors of lipid peroxidation [29
], free radical neutralizers [29
], and chelating agents of transition metallic ions [28
]. Another important consequence of protein hydrolysis is the exposition of the active R-groups of the amino acids, and then the increase of its antioxidant power [30
]. ORAC (ROO•) was recommended as a standard method by Prior, Wu, and Schaich [31
] to be used as routine quality control and measurement of food antioxidant capacity. It is a HAT-based (hydrogen-atom-transfer-based) method that uses a controllable source of peroxyl radicals, where it can detect both hydrophilic and hydrophobic antioxidants by altering the radical sources and solvent. For that reason, ORAC (ROO•) is recommended for analyzing the antioxidant capacity of vegetables if only one method is justifiable [32
]. Nevertheless, since there is not only one standardized official method, evaluating this property using different methods and conditions of the oxidation process is suggested [33
]. Therefore, to evaluate the antioxidant capacity of rice bran proteins, the ORAC method and reducing power assays were performed (Table 1
The antioxidant activity obtained using the ORAC method was higher in DRBPC-2 than in DRBPC-1, independent of the enzyme used. The high temperature and pressure used in the industry process might modify the protein structure to obtain different active products compared with native protein [10
]. Ou et al. [34
] studied the antioxidant activity of vegetable sources using the ORAC method and obtained values ranging from 18 µmol/g (for pea) to 160 µmol/g (for green pepper). These values were lower than those obtained from our RBPH.
The antioxidant activity significantly increased after hydrolysis with both food enzymes, and this activity increased by between two and three times in fractions smaller than 3 kDa, which showed the highest antioxidant activities. The ultrafiltration is usually used to enrich biologically food peptides and this process confirms that the antioxidant activity can be mainly attributable to low-molecular-mass peptides. Wattanasiritham et al. [10
] worked with native and denatured protein fractions (albumin, globulin, and glutelin) that were non-hydrolyzed and hydrolyzed with pepsin and papain, and these researchers observed values from 100 µmol eq Trolox/g protein for native protein, 1500 µmol eq Trolox/g protein for hydrolyzed protein with papain, and 1000 µmol eq Trolox/g protein for hydrolysates with pepsin. In our study, a potent antioxidant activity was found using the ORAC method in fractions with a low molecular weight, with means ranging from 434.79 to 1916.15 µmol eq Trolox/g protein.
Reducing power, which measures the transformation of the Fe3+
of a ferricyanide complex to the ferrous form, was determined using the absorbance values of the samples, where a high absorbance is related to a high reducing power. This method argues that the ability to reduce iron has little relationship to the radical quenching processes (HAT) mediated by most antioxidants. However, the radical oxidation or reduction to the ions still stops the radical formation and the reducing power could reflect the ability of compounds to modulate the redox tonus in plasma and tissues [29
]. When analyzing the results (Table 1
), it was found that the products obtained from DRBPC-2 with alcalase after hydrolysis and ultrafiltration processes did not increase the reducing power. However, a significant increase in the absorbance values of the fractions smaller than 3 kDa obtained with flavorzyme was observed, suggesting an improvement in the reducing power. Zhang et al. [35
] also obtained higher reducing power levels in fractionated protein hydrolysates from heat-stable defatted rice bran.
Rice is a rich source of many bioactive compounds, including special phenolic acids (such as ferulic acid, p-coumaric, and diferulate), which are not present in significant quantities in fruits or other vegetables [36
]. These molecules exert antioxidant activity, where this property depends on the number and the position of hydroxyl groups on the phenolic ring [37
]. Previous researchers have postulated the release of phenolic compounds after hydrolysis treatment. In fact, Chanput et al. [13
] mentioned that the crude hordein fraction (a prolamin glycoprotein that presents in barley and some other cereals) showed the lowest phenolic content compared to their hydrolysates, and these researchers considered that this might be because of the unfolding of the protein, exposing the phenolic groups that were originally enclosed, suggesting that phenolic compounds could also be implicated in the high antioxidant activity observed after the hydrolysis of rice bran. To establish where our hydrolysis conditions allowed for releasing phenolic compounds and to know their participation in the antioxidant properties of the hydrolysate, we performed an analysis of the total phenolic compounds (Table 2
Since both amino acids and phenolic compounds can show HAT ability in their molecular structure, the results obtained for the antioxidant capacity using ORAC could be linked not just with the hydrolysis process and ultrafiltration but also with the TPC released in these rice bran products. The positive relation between TPC and ORAC results, with a Pearson’s ratio coefficient of 0.98, could explain the antioxidant mechanisms underlying these products.
shows the ACE inhibitory activity of the fractions smaller than 3 kDa obtained from the two types of DRBPC, hydrolyzed with either alcalase or flavorzyme, followed by the ultrafiltration process (RBPH-1A<3, RBPH-2A<3, RBPH-1F<3, RBPH-2F<3).
RBPH-1A<3 showed higher inhibitory activity than those hydrolyzed with flavorzyme. The IC50
value observed in RBPH-1A<3 was low (0.15 ± 0.02 mg/mL) and was within the range reported in the literature for other ACE inhibitory food hydrolysates with antihypertensive activity demonstrated in experimental models [38
]. Regarding rice-derived hydrolysates, Chen et al. [39
] worked with hydrolysates of rice prepared with alcalase and trypsin, where they obtained IC50
values of 0.28 mg/mL. These researchers, obtained fractions using macroporous resin, and the elution with ethanol 50% presented a lower IC50
value, reaching a value of 0.17 mg/mL, similar to that presented by the fraction RBPH-1A<3 [38
]. Wang et al. [40
] evaluated hydrolyzed from rice bran protein prepared with trypsin, showing IC50
= 0.30 mg/mL. These results suggest that an RBPH-1A<3 fraction could be a promising source of peptide sequences with antihypertensive properties.
Some physiological processes, such as changes in structure and function of the peptides during ingestion, digestion, and/or absorption, could be occurring, and the bioactive sequences could be destroyed by gastrointestinal proteases to produce inactive peptides or to become more biologically active. For this reason, in this study, we also conducted experiments on SHR to explore the in vivo antihypertensive activity of the hydrolysates of the industrial by-product of rice-milling.
Essential hypertension is one of the most common risk factors for the development of cardiovascular diseases [41
]. SHR are known as a genetic model of hypertension, and, since its development in 1963 by Okamoto and Aoki, these animals are one of the most used experimental models for evaluating the antihypertensive properties of different compounds and to investigate the mechanism of action underlying their antihypertensive effect. In vivo effects are usually tested in this strain because the hypertension presented in this animal model is similar to the essential hypertension that occurs in humans [42
]. Hypertension appears in both humans and SHR at an early age, the risk increases with a family history of hypertension, and the disease gets worse with the consumption of a sodium-rich diet [45
]. This experimental model acquires arterial hypertension after five weeks old, presenting a level of pressure that is considered to be spontaneous hypertension between weeks 7 and 15. The plateau is reached between weeks 20 and 28, presenting no difference between the sexes [42
The oral administration of distilled water to SHR in this study did not change the values of the SBP (Figure 2
The greatest decreases in the SBP were obtained after the administration of 50 mg/kg of Captopril to the rats. The maximal decreases were observed 6 h after the administration of Captopril, and this variable returned to baseline 48 h after the administration (Figure 2
). It was not surprising to find a clear decrease in the SBP when we administered 50 mg/kg of Captopril to the SHR because this drug is a potent antihypertensive and ACE-inhibitor with an IC50
value of 0.02 μM [46
After a single oral administration of 80 mg/kg of the different RBPH fractions lower than 3 kDa, only RBPH-1A<3 and RBPH-2F<3 showed significant decreases of the SBP in SHR. The maximum decrease of the SBP values after administration of RBPH-1A<3 was reached 8 h after administration, and the SBP returned to baseline 24 h afterward (Figure 2
). The maximum decrease caused by RBPH-2F<3 was observed 6 h after administration but the SBP returned to baseline 8 h afterward. The antihypertensive effect of RBPH-1A<3 and RBPH-2F<3 compared favorably with the blood-pressure-lowering effect of other food protein hydrolysates. The antihypertensive effect of the administration of 600 mg/kg rice bran protein hydrolysate with alcalase in SHR showed a maximum reduction of blood pressure (25 mm Hg) 6 h after oral administration [39
]. In our study, we found a reduction of 30 mm Hg approximately 8 h after the oral administration of RBPH-1A<3 at much lower doses (80 mg/kg). In previous studies, we used different doses of egg white hydrolysate fractions with a 3 kDa cut-off membrane (25, 50, and 100 mg/kg) and we also found similar reductions (28 mm Hg) in the SBP in SHR [47
]. Chen et al. [48
] also evaluated different doses (1, 10, and 50 mg/kg) of the rice dreg protein hydrolyzed with trypsin. A decrease in the maximum blood pressure was observed 1 h after the oral administration of the 50 mg/kg dose (29 mm Hg), returning to baseline values after 7 h.
It is well known that blood pressure variability may contribute to organ damage [49
]; therefore, the use of strategies with long-lasting antihypertensive effects is always desirable. In this context, it is important to highlight that the decrease in arterial blood pressure caused by RBPH-1A<3 lasted for a longer period than the antihypertensive effect observed with RBPH-2F<3. In accordance with this idea, the antihypertensive properties of RBPH-1A<3 might be more favorable for controlling high blood pressure in hypertensive patients. Moreover, regarding the in vitro assays, our results suggest that the antihypertensive effect of RBPH-1A<3 could be related to their potent ACE inhibitory and antioxidant activity, while RBPH-2F<3 only showed antioxidant properties. Nevertheless, further studies are needed to determine whether other mechanisms could also be established regarding the blood-pressure-lowering effect of both hydrolysates.
To evaluate whether the blood-pressure-lowering effect produced by RBPH-1A<3 was dependent on the hypertensive condition, we also evaluated the effect of this hydrolysate in normotensive WKY rats (Figure 3
). These animals are frequently used like the normotensive control of the SHR strain because they present the same origin and genetic charge [50
The administration of 80 mg/kg RBPH-1A<3 did not modify the SBP in normotensive animals when compared to the control group, suggesting that this hydrolysate would not exert an hypotensive effect in normotensive subjects.
In this study, we have demonstrated that hydrolysates from rice bran proteins possess potent in vitro antioxidant and ACE inhibitory properties, as well as in vivo activity, to decrease arterial blood pressure in hypertensive rats. These properties can be attributed to the presence of peptides with a low molecular mass, in addition to the phenolic compounds released during hydrolysis processes. These findings provide new evidence of the potential of the industrial by-product of rice milling to be used as a source of bioactive compounds that can improve cardiovascular health and alleviate other related diseases. It is important to note that the potential peptide sequences responsible for the antihypertensive effect have not been identified yet, but, although the majority of studies conducted in recent years have focused on the isolation of peptide sequences released during hydrolysis, it has recently been shown that the administration of complete hydrolysates could be more relevant than the administration of a single isolated peptide, since a greater biological effect could be achieved. Although we consider that hydrolysates could be more interesting products for the development of functional foods from a technological and organoleptic point of view [51
], further investigation is needed to identify the potential sequences responsible for the bioactivity and the antihypertensive mechanisms and pathways implicated in the effect produced by these hydrolysates.