Antiulcerative and Antinociceptive Activities of Casein and Whey Proteins

Abstract

The aim of this study was to evaluate the antiulcerative and antinociceptive activities of milk proteins using the induced gastric ulcer with ethanol rat model and the acetic acid-induced writhing mouse model. Casein (CN), (100, 300, and 1000 mg kg⁻¹) doses presented antiulcerative activity on a dose-dependent manner with values of 30.8%, 41.4%, and 57.0% of inhibition measured using the ulcerative lesions index (ULI), respectively. Whey protein concentrate (WPC), (100, 300, and 1000 mg kg⁻¹) doses presented antiulcerative activity on a dose-dependent manner with values of 48.9%, 65.5%, and 68.22% of ULI inhibition, respectively. CN, casein hydrolysates (CNH), WPC, and whey protein hydrolysates (WPH), (3, 10, and 30 mg kg⁻¹) doses presented antinociceptive activity using the acetic acid-induced writhing in the mouse model. CN (30 mg kg⁻¹) presented a value of 40% of inhibition writhing, and CNH (30 mg kg⁻¹) presented antinociceptive activity with a value up to 46% of writhing inhibition. WPC (30 mg kg⁻¹) presented a value of 52.50%, and WPH (30 mg kg⁻¹) presented antinociceptive activity with a value up to 88.00% of writhing inhibition. In conclusion, CN and WPC demonstrated in vivo antiulcerative properties and represent a promising alternative to be used as protectors of the gastric mucosa. CNH and WPH demonstrated in vivo antiulcerative properties and represent a promising alternative to be used as natural analgesic.

Introduction

Milk proteins are the most bioactive peptide source described in the scientific literature. Milk contains about 3% protein content, of which caseins (CN), represent 80% of total proteins and 20% whey proteins (WP). CN content α-S₁CN, α-S₂CN, β-CN, and k-CN.¹ WP content β-lactoglobulin (50–55%), α-lactalbumin (20–25%), immunoglobulins (10–15%), bovine serum albumin (5–10%), and lactoferrin (1–2%).² Food proteins can induce a physiological function, directly or after their hydrolysis with enzymes to produce fragments named bioactive peptides.³

Milk is also a known food source of potent bioactive peptides with various activities such as antihypertensive, antimicrobial, antioxidant, opioid, and antiviral.⁴ Whey protein concentrate (WPC) is described presenting antitumor and antimicrobial activities.^5
–7 The biological activities of lactoferrin milk described include antibacterial, antitumor, antiviral, antifungal, and antiparasitic activities.^8

–15

Matsumoto et al. have reported antiulcerative activities of WPC and bovine α-lactalbumin.¹⁶ The protective effect exhibited by WPCs and derivates has been predominantly linked to their content of α-lactoalbumin (alpha-La). Further studies have confirmed the ulcer inhibitory properties of this WP. Matsumoto et al. found a significant inhibition of ulcer formation mediated by the ingestion of a whey protein isolate (WPI), followed by the evaluation of the activities of individual components at representative concentrations, showing that only α-La emulated the observed effect.¹⁶ Tavares et al. reported a WPC hydrolyzed with extracts of Cynara cardunculus, which has demonstrated a protective effect against ulcerative lesions induced by ethanol in rats. The fraction below 3 kDa was the most active.¹⁷

Brantl et al. reported for the first-time analgesic activity/opioid activity of an identified peptide of bovine β–CN, which was named β-casomorphin 7.¹⁸ Then, β-casomorphin peptides from 4 to 13 have been described in the literature as peptides with opioid activity, that is, agonist. Then, other 26 agonist peptides have been described: (8 from β-CN, 8 from α-S₁CN, 3 from k-CN, 1 from β-Lg, and 1 from lactoferrin [Lf]). Among the 26 agonist peptides, 21 were identified from cow's milk, and the other 5 were identified from human milk. Furthermore, only eight additional antagonist peptides have been described in cow's milk.^19
–21

Carrillo et al. reported antinociceptive activity of hen egg-white lysozyme in its native and denatured form and its hydrolysates obtained with pepsin. The activity was evaluated in a classic acetic acid-induced abdominal writhing tests in mice.²² The hydrolyzed native lysozyme, presented 48% of inhibition of abdominal writhing in mice. The aim of this study was to evaluate the antiulcerative and antinociceptive activities of milk proteins and hydrolysates using the rat model of induced gastric ulcer with ethanol and the mouse acetic acid-induced writhing model.

Materials and Methods

Milk proteins

Bovine CN was obtained by isoelectric precipitation using whole cow's milk by adding 2 M HCl (pH 4.6). CN was recovered by centrifugation at 5000 g for 20 min. The CN precipitate was washed three times with acidulated water (pH 4.6), and the remaining fat in the CN precipitate was removed by washing with dichloromethane acidulated water (1:1, v/v). The supernatant (WPC) was retired and lyophilized until its use. This product is referred to as total WPC. The final bovine CN precipitate was then lyophilized. This product is referred to as total CN.²³

Production of milk protein hydrolysates

Both CN and WPC rich in β-lactoglobulin (β-Lg) hydrolysates were prepared using the company Innaves S.A. (Porriño, Pontevedra, Spain). Hydrolysis with food-grade pepsin (Biocatalyst, Cardiff, United Kingdom) of commercial CN (Promilk 85, Arras Cedex, France) was carried out to obtain the CNH (LowPept^®) according to Sánchez et al. ²⁴ In brief, CN at 0.06% (W/W) was digested at 37°C at a pH 2.0 acid with pepsin at a ratio of 1:50 (w/w) (enzyme:substrate), with the addition of the enzyme at the beginning of hydrolysis and after 3 h. Hydrolysis was then inactivated by increasing pH up to 7.0. The supernatant after clarification was collected and spray dried.

The protein content of this hydrolysate determined by the Kjeldahl method was 71.33% ± 1.55% (w/w), as previously assessed.²⁵ To produce the β-Lg hydrolysate, a WPC rich in β-Lg (Friesland Campina Domo, Zwolle, The Netherlands), containing at least 99.0% of β-Lg of protein content, was hydrolyzed with food-grade trypsin (Biocatalyst), as reported by Anadón et al. ²⁶ In brief, WPC was suspended at 5% (w/v) and heated at 90°C for 10 min. After addition of trypsin at a ratio 1:20, hydrolysis was performed at pH 8.0 and 37°C for 3 h and stopped by heat inactivation of trypsin at 95°C for 15 min. The hydrolysate was clarified and spray dried. The protein content for this hydrolysate was 74.07% ± 0.19%.

Animals

Wistar male rats, 250–350 g body weight (bw) and 8-week-old male Swiss and BALB/c mice of 25–35 g bw, were obtained from the Experimental Animal Center (CEMIB) of Campinas University (São Paulo, Brazil).

Before the experiments, animals were kept for at least 7 days at 20°C and under alternative light/dark cycles of 12 h, receiving a commercial standard diet (Nuvital Nutrients, Curitiba, Brazil) and water ad libitum. Animals fasted for 12 h before the experiments.

The studies were carried out in accordance with current guidelines for the veterinary care of laboratory animals and were performed under the consent and surveillance of Unicamp Institute of Biology Ethics Committee for Animal Research (number 2206-2).

Ethanol-induced gastric ulcer model

The protective effect on rat stomach mucosa was studied using the absolute ethanol ulcerogenesis model.²⁷ The Ethics Committee for Animal Research of Campinas University approved the experimental protocol in agreement with the ethical principles of The International Association for the Study of Pain (IASP). Animals were divided in groups of five rats, a group per treatment. Sodium chloride solution (0.9% w/v) at 10 mL kg⁻¹ bw (negative control) and an antiulcerative drug, carbenoxolone (Sigma, St. Louis, MO, USA), at 200 mg kg⁻¹ bw (positive control) were used as negative and positive controls.

Samples were administrated at three different concentrations (100, 300, and 1000 mg kg⁻¹ bw) in a single dose by gastric intubation. One hour after the administration of the sample or the controls, 1 mL of absolute ethanol was provided to each rat. One hour later, animals were sacrificed and their stomachs were extracted and washed with a saline solution for ulcerative lesions analysis.

Ulcerative lesion analysis

The ulcerative lesions were evaluated by visual inspection of rat gastric mucosa. According to Gamberini et al., the ulcerative lesions index (ULI) of each animal was determined by summing the scores associated to several parameters: loss of normal morphology (1 point), mucosa discoloration (1 point), edema (1 point), hemorrhage (1 point), petechial points until 9 mm (2 points), petechial points larger than 10 mm (3 points), ulcers up to 1 mm (n × 2 points), ulcers larger than 1 mm (n × 3 points), and perforated ulcers (n × 4 points), where n is the number of ulcers found.²⁸

Percent decrease of the ULI score was calculated by the Equation (1): \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} \% \ { \rm{ decrease \ ULI}} = \left\{ { \left( {{ \rm{mean \ ULI \ negative \ control}} - { \rm{mean \ ULI \ sample}}} \right) / { \rm{mean \ ULI \ negative \ control}}} \right\} \times 100. \tag{1} \end{align*} \end{document}

The effective dose (ED₅₀), defined as the sample concentration that achieves a decrease of 50% ULI, was estimated by using a logarithmic regression equation for ULI values obtained from 100, 300, and 1000 mg kg⁻¹ treatments.

Antinociceptive activity of CN, WPC, and hydrolysates

The writhing test in mice was carried out using the method of Spindola et al. ²⁹ The mice were divided into four groups (n = 6). The writhes were induced by intraperitoneal injection of 0.8% acetic acid (v/v). One dose of native or modified CN, CNH, WPC, and whey protein hydrolysates (WPH) (3, 10, and 30 mg kg⁻¹ bw) was administered by oral gavage to groups of six animals each, 30 min before chemical stimulus (acetic acid). Sodium chloride 0.9% (10 mL kg⁻¹) was used as negative control and indomethacin (30 mg kg⁻¹) as positive control. The number of muscular contractions was counted over a period of 20 min after acetic acid injection. Contraction of the abdominal muscle concomitant with a stretching of the hind limbs in response to an intraperitoneal injection of acetic acid 0.8% (10 mL kg⁻¹) was measured.

The percentage of protection against acetic acid-induced writhing was calculated using the following equation: percentage of protection = (Wc − Wt)/Wc) × 100. The effective dose (ED₅₀), defined as the sample concentration that achieves a decrease of 50% writhing, was estimated by using a logarithmic regression equation for writhing values obtained from 3, 10, and 30 mg kg⁻¹ treatments.

Statistical analysis

Experimental results were analyzed using a one-way ANOVA method, followed by Tukey's test. GraphPad Prism 4 software was used. Different letters indicate significant differences among dose groups (P < .05). *(P < .05), **(P < .01) and ***(P < .001) express statistical differences between samples and saline control.

Results and Discussion

Antiulcerative activity

Peptic ulcer is a disease with a major health risk in terms of morbidity and mortality. Globally, more than 14 million people are diagnosed with gastric ulcer annually, and about 4 million people would die from related complications.^30,31 This disease is one of the most common disruptions of the mucosal integrity of the stomach (gastric ulcer) and small intestine (duodenal ulcer). In this disease, there is an imbalance between offensive factors versus defensive factors, producing a break in the gastrointestinal tract bathed by acid/pepsin, alcohol, tobacco and caffeine, Helicobacter pylori infection, anti-inflammatory no steroids (NSAID), mucus secretion, bicarbonate production, mucosal blood flow, cellular repair mechanisms, prostaglandin E, and growth factors.^32
–34

In this study, absolute ethanol was used to produce gastric lesions in rats. Ethanol-induced gastric lesion formation may be due to stasis in gastric blood flow, which contributes to the development of the hemorrhage and necrotic aspects of tissue injury. Alcohol rapidly penetrates the gastric mucosa apparently causing cell and plasma membrane damage leading to increased intracellular membrane permeability to sodium and water. The massive intracellular accumulation of calcium represents a major step in the pathogenesis of gastric mucosal injury.^35,36

Many classes of synthetic antiulcer drugs have been used for its treatment, but these drugs present adverse effects and increased incidence of relapses during ulcer therapy. Therefore, search for new antiulcer drug continues and can be extended to proteins and hydrolysates from food proteins for their easy availability, high protection, low cost, and low cytotoxicity.

The CN and WPC antiulcerative activities were studied in a model of ulcer induced by absolute ethanol in rats. The lesions of rat gastric mucosa were analyzed with macroscopic observation. The group treated with saline solution (negative control) presented a high number of lesions with red bands with inflammation. The group treated with carbenoxolone (positive control) presented an absence of inflammation signs. Carbenoxolone is a drug used to prevent and treat gastric ulcers.

The group treated with CN (1000 mg kg⁻¹) and WPC (1000 mg kg⁻¹) presented a few lesions induced by absolute ethanol. This fact indicates that the sample was effective to prevent the lesions induced by absolute ethanol (Fig. 1). On the contrary, the percentage of decrease of ULI was calculated with the [Eq. (1)]. We found that the saline solution presented an absence of antiulcerative effect compared with the carbenoxolone (positive control). Carbenoxolone presented a 92.7% of ULI inhibition. CN (100, 300, and 1000 mg kg⁻¹) samples presented antiulcerative effect on a dose-dependent manner with values of 30.8%, 41.4%, and 57.0% of ULI inhibition, respectively (Fig. 2). WPC (100, 300, and 1000 mg kg⁻¹) samples presented antiulcerative effect on a dose-dependent manner with values of 48.9%, 65.5%, and 68.22% of ULI inhibition, respectively (Fig. 2). WPC samples were more active than CN samples. CN and WPC samples presented statistical differences (P < .05). The values of ED₅₀ obtained were of 177 mg kg⁻¹ for CNH and 729 mg kg⁻¹ for WPH. The ED₅₀ to WPC is very low compared with CN samples.

FIG. 1.

Images of ethanol-induced ulcers in rat stomachs with caseins and whey protein treatments: saline solution 10 mL kg⁻¹ bw (A), carbenoxolone 200 mg kg⁻¹ bw (B), casein (CN) 1000 mg kg⁻¹ bw (C), WPC 1000 mg kg⁻¹ bw (D). bw, body weight; WPC, whey protein concentrate.

FIG. 2.

Antiulcerative activities of caseins and whey protein. Effect of single dose of casein (CN) (A) and WPC (B) on the protective effect in rat stomachs against absolute ethanol-induced ulcers, in terms of the ULI. Vertical bars represent mean ± standard deviation (n = 5) of ULI. Data analyzed by a one-way ANOVA and followed by Tukey's test. Different letters indicate significant differences between samples and saline solution group (P < .05). ULI, ulcerative lesions index.

Mezzaroba et al. ³⁷ reported antiulcerative activity of commercial α-lactalbumin with a value of 44% of ULI inhibition and α-lactalbumin obtained in laboratory with a value of 12% of ULI inhibition. Matsumoto et al. ¹⁶ reported antiulcerative activity of WPI (200 mg kg⁻¹) with a value of 47% of ULI inhibition, while CN only presented a value of 9% of ULI inhibition. Furthermore, α-La (5, 10, 200, and 1000 mg kg⁻¹) presented 27%, 47%, 82%, and 98% of ULI inhibition, respectively. They found that the protect effect of WPI is due to the presence of α-lactalbumin.

The WPC used in this study has a content of 99% of β-lactoglobulin, which indicates that the antiulcerative activity is due to the presence of this protein in the WPC.¹⁶ We are reporting antiulcerogenic activity of bovine α-lactalbumin with a highest value of 68.22% of ULI inhibition at a concentration of 1000 mg kg⁻¹. We are also reporting antiulcerative activity of bovine caseins with values ranging from 30.8% to 57% of ULI inhibition. In the assays of Matsumoto et al.,¹⁶ the samples were incubated for 30 min; in this study, samples were incubated for 60 min. Matsumoto et al. ¹⁶ induced the ulcers with 60% ethanol with HCL; in this study, the ulcers were induced with absolute ethanol.

Furthermore, antiulcerative activity of hydrolysates from milk proteins has also been reported. For example, Tavares et al. ¹⁷ reported antiulcerative activity of hydrolysate (fraction below of 3 kDa) obtained of WPC in the ulcer induced for ethanol model. Pacheco et al. reported antiulcerative activity of hydrolysate obtained of WPC with pancreatin enzyme with a value of 65.5% of ULI inhibition at a concentration of 1000 mg kg⁻¹.³⁸

Antinociceptive activity

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most frequently prescribed medicines such as ibuprophen, naproxen, and diclofenac. It is known, that NSAIDs act by inhibiting cyclooxygenases (COX-1 and COX-2) blocking the biosynthesis of proinflammatory prostaglandins, which contribute to pain and inflammation.^39,40 However, these prostaglandins also serve to protect the gastric mucosa and kidney function. Prolonged consumption of NSAIDs is associated to gastrointestinal damage and renal toxicity. For this reason, much research has been done to find new molecules with analgesic capacity that can protect the gastric mucosa with mechanisms of protection different to conventional analgesics.⁴¹ In this context, proteins and their hydrolysates may play a promising role as new drugs with antiulcerogenic and antinociceptive activity.

The acetic acid-induced writhing response is a sensitive test used for screening analgesic activity, regardless of the central or peripheral causes. Acetic acid is an irritant which causes the synthesis and release of proinflammatory mediators such as prostaglandins (PGE and PGF2) and sympathetic nervous system mediators that provoke pain nerve endings.⁴² In this study, acetic-induced writhing in mice was used to evaluate the antinociceptive activity of milk proteins and their hydrolysates.

The antinociceptive activity of CN and CNH is shown in Figure 3. The mice treated with indomethacin showed a decrease of 67% in the number of writhes, which was significantly higher than the ones obtained with mice treated with CN and CNH on a dose-dependent manner. CN only presented antinociceptive activity at a concentration of 30 mg kg⁻¹ with a value of 40% of inhibition of writhes (Fig. 3A).

FIG. 3.

Analgesic effects of the CN (caseins) and CNH (hydrolysate of caseins). (A) Antinociceptive activity of CN and (B) antinociceptive activity of CNH. Vertical bars represent mean ± SEM of writhings. Different letters are statistically different from the control group (P < .05) ANOVA and Tukey's test. Data inside the bars show percentage of inhibition. Positive control (IND, indomethacin).

No significant differences were found between the antinociceptive activity of CNH at 3, 10, and 30 mg kg⁻¹. CNH at 3, 10, and 30 mg kg⁻¹ presented antinociceptive activity with values of 16%, 29%, and 46%, respectively (Fig. 3B).

WPC at 3, 10, and 30 mg kg⁻¹ presented high antinociceptive activity with an effect dependent on doses, with values of 13.66%, 30.93% and 52.50%, respectively (Fig. 4A). WHP at 3, 10, and 30 mg kg⁻¹ presented high antinociceptive activity with an effect dependent on doses, with values of 38%, 42%, and 88%, respectively (Fig. 4B). WPH at 30 mg kg⁻¹ presented a higher antinociceptive activity compared to indomethacin used as positive control. A high number of peptides from milk proteins with opiod activity have been described so far. The values of ED₅₀ obtained for WPC were of 27.30 mg kg⁻¹ and for WPH, 11.14 mg kg⁻¹.

FIG. 4.

Analgesic effects of the WPC and WPH. (A) Antinociceptive activity of WPC and (B) antinociceptive activity of WPH. Vertical bars represent mean ± SEM of writhings. Different letters are statistically different from the control group (P < .05) ANOVA and Tukey's test. Data inside the bars show percentage of inhibition. Positive control (IND, indomethacin). WPH, whey protein hydrolysates.

The ED₅₀ to WPH is very low compared to the WPC sample. The value of ED₅₀ for CNH was of 18.70 mg kg⁻¹. For the CN sample, it was impossible to calculate the ED_50, as this sample only presented activity for the 1000 mg kg⁻¹ dose. WPH presented a value of ED₅₀ lower than CNH.

Yamaguchi et al. reported that orally administered α-LA protein showed an inhibition of writhing induced by acetic acid in the mice model. The suppression of nociception and inflammation in rat footpads was caused by the carrageenan in the rats.⁴³ Hayashida et al. ⁴⁴ reported a reduction of nociception in various pain models, as shown by the formalin test, hot plate test, and acetic acid writhing test in rats. Intraperitoneal (i.p.) administration of bovine lactoferrin significantly inhibited nociception in these pain models. WPC contents α-La and Lf proteins, described with antinociception activity in different pain models. For this reason, our results are in accordance to the bibliography. Sasaki et al. described that bovine lactoferrin (100 mg kg⁻¹) presented antinociceptive effects in a rat lumbar disc herniation model with a permanent effect continuing during the 21 days of the experiment.⁴⁵

Bovine caseins are precursors of a significant number of opioid peptides described as β-casomorphins. These opioids peptides have been evaluated in different animal and human models, demonstrating their efficacy against pain.⁴⁶ When β-casomorphins are injected in the blood stream, they induce an analgesic and sedative effect due to their action on the central nervous system.⁴⁷ β-Casomorphin-4, β-casomorphin-5, and β-casomorphin-6 cause analgesia for up to 45 min, whereas β-casomorphin-7 causes analgesia for more than 90 min.⁴⁸

Tani et al. reported the opiod peptide named β-Lactorphin f(102–105) Tyr-Leu-Leu-Phe with an agonist effect. This peptide was isolated from bovine β-lactoglobulin. This group also reported the opioid peptides named Lactoferroxin A f(318–323) Tyr-Leu-Gly-Ser-Gly-Tyr, Lactoferroxin B f(536–540) Arg-Tyr-Tyr-Gly-Tyr, and Lactoferroxin C f(673–679) Lys-Tyr-Leu-Gly-Pro-Gln-Tyr with antagonist effects. These peptides were obtained from the human lactoferrin protein.⁴⁹ We suggest that WPH can content peptides with opioid ability to protect against pain. Future research can identify the sequence of the peptides with antinociceptive activity in the mouse model.

With the results obtained in this study, we can conclude that CN and WPC can be used as a natural protection of the gastric mucosa and can be used to elaborate functional ingredients to prevent gastric ulcers. CNH and WPH present a high antinociceptive activity in acetic acid-induced writhing in the mouse model. These peptides bioactive can be used to elaborate natural analgesics with the ability to protect the gastric mucosa. Future research can be developed to identify the sequences with antinociceptive activity and its potential pharmaceutical and medical uses.

Footnotes

Acknowledgments

This work has received financial support from the projects CYTED110AC0386 (IBEROFUN), AGL2011-24643, FEDER-INNTERCONECTA-GALICIA (ENVELLEFUN), FP7-SME-2012-315349 (FOFIND), and CNPq (Brazil). The authors are participants in the FA1005 COST Action INFOGEST on food digestion. The English edition has been reviewed by Emilio Labrador.

Author Disclosure Statement

No competing financial interests exist.

References

Gaspard

, Auty

, Kelly

, O'Mahony

, Brodkorb

: Isolation and characterisation of κ-casein/whey protein particles from heated milk protein concentrate and role of κ-casein in whey protein aggregation. Int Dairy J, 2017; DOI: https://doi.org/10.1016/j.idairyj.2017.05.012.

Keri Marshall

: Therapeutic applications of whey protein. Altern Med Rev, 2004; 9:136–156.

Tidona

, Criscione

, Guastella

, Zuccaro

, Bordonaro

, Marletta

: Bioactive peptides in dairy products. Ital J Anim Sci, 2009; 8:315–340.

Pritchard

: Isolation and Characterization of Bioactive Peptides Derived from Milk and Cheese. University of Western Sydney, Australia, 2012, pp. 123–124.

Bounous

: Whey protein concentrate (WPC) and glutathione modulation in cancer treatment. Anticancer Res, 2000; 20:4785–4792.

Early

, Hardy

, Forde

, Kane

: Bactericidal effect of a whey protein concentrate with anti-Helicobacter pylori activity. J Appl Microbiol, 2001; 90:741–748.

Watanabe

, Okada

, Shimizu

, Wakabayashi

, Higuchi

, Niiya

, Kondoh

: Nutritional therapy of chronic hepatitis by whey protein (non-heated). J Med, 2000; 31:283–302.

Conesa

, Rota

, Castillo

, Perez

, Calvo

, Sanchez

: Antibacterial activity of recombinant human lactoferrin from rice: Effect of heat treatment. Biosci Biotechnol Biochem, 2009; 73:1301–1307.

Bellamy

, Wakabayashi

, Takase

, Shimamura

, Tomita

: Role of cell-binding in the antibacterial mechanism of lactoferricin B. J Appl Microbiol, 1993; 75:478–484.

10.

Yoo

, Watanabe

, Hata

, Shimazaki

, Azuma

: Bovine lactoferrin and lactoferricin, a peptide derived from bovine lactoferrin, inhibit tumor metastasis in mice. Cancer Sci, 1997; 88:184–190.

11.

Arnold

, Brewer

, Gauthier

: 1980. Bactericidal activity of human lactoferrin: Sensitivity of a variety of microorganisms. Infect Immun, 1980; 28:893–898.

12.

Van der Strate

BWA

, Beljaars

, Molema

, Harmsen

, Meijer

DKF

: Antiviral activities of lactoferrin. Antiviral Res, 2001; 52:225–239.

13.

Omata

, Satake

, Maeda

, Saito

, Shimazaki

, Yamauchi

, Uzaka

, Tanabe

, Sarashina

, Mikami

: Reduction of the infectivity of Toxiplasma gondii and Eimeria stedai sporozites by treatment with bovine lactoferricin. J Vet Med Sci, 2001; 63:187–190.

14.

Ikeda

, Sugiyama

, Tanaka

, Sekihara

, Shimotohno

, Kato

: Lactoferrin markedly inhibits hepatitis C virus infection in cultured human hepatocytes. Biochem Biophys Res Commun, 1998; 245: 549–553.

15.

Tanaka

, Ikeda

, Nozaki

, Kato

, Tsuda

, Sait

, Sekihara

: Lactoferrin inhibits hepatitis C virus viremia in patients with chronic hepatitis C: A pilot study. Jpn J Cancer Res, 1999; 90:367–371.

16.

Matsumoto

, Shimokawa

, Ushida

, Toida

, Hayasawa

: New biological function of bovine α-lactalbumin: Protective effect against ethanol and stress-induced gastric mucosal injury in rats. Biosci Biotechnol Biochem, 2001; 65:1104–1111.

17.

Tavares

, Monteiro

, Possenti

, Pintado

, Carvalho

, Malcata

: Antiulcerogenic activity of peptide concentrates obtained from hydrolysis of whey proteins by proteases from Cynara cardunculus . Int Dairy J, 2011; 21:934–939.

18.

Brantl

, Teschemacher

, Hemschem

, Lottspeich

: Novel opioid peptides derived from casein (β-casomorphins). Physiol Chem, 1979; 360:1211–1216.

19.

Brandt

, Barth

, Höltje

: Investigations of structure-activity relationships of β-casomorphins and further opioids using methods of molecular graphics. In: β-Casomorphins and Related Peptides: Recent Developments ( Brantl

, Teschemacher

, ed.). VCH, Weinheim, 1994, pp. 93–104.

20.

Kostyra

, Sienkiewicz-Szłapka

, Jarmołowska

, Krawczuk

, Kostyra

: Opioid peptides derived from milk proteins. Pol J Food Nutr Sci, 2004; 13:25–35.

21.

Meisel

: Biochemical properties of bioactive peptides derived from milk proteins: Potential nutraceuticals for food and pharmaceutical applications. Livest Prod Sci, 1997; 50:125–138.

22.

Carrillo

, Spindola

, Ramos

, Recio

, Carvalho

: Anti-inflammatory and anti-nociceptive activities of native and modified hen egg white lysozyme. J Med Food, 2016; 19:978–982.

23.

Rodríguez Saint-Jean

, las Heras

, Carrillo

, Recio

, Ortiz-Delgado

, Ramos

, Pérez-Prieto

: Antiviral activity of casein and αs₂ casein hydrolysates against the infectious haematopoietic necrosis virus, a rhabdovirus from salmonid fish. J Fish Dis, 2013; 36:467–481.

24.

Sánchez

, Kassan

, Contreras

MDM

, Carrón

, Recio

, Montero

, Sevilla

: Long-term intake of a milk casein hydrolysate attenuates the development of hypertension and involves cardiovascular benefits. Pharmacol Res, 2011; 63:398–404.

25.

International Dairy Federation: Determination of Nitrogen Content, Kjeldalh Method 20B, 1993. Joint IDF/ISO/AOAC International Standard. IDF, Brussels, Belgium.

26.

Anadón

, Martínez

, Ares

, Castellano

, Martínez-Larrañaga

, Corzo

, Reglero

: Acute and repeated dose (28 days) oral safety studies of alibird in rats. J Food Prot, 2013; 76:1226–1239.

27.

Robert

: Cytoprotection by prostaglandins. Gastroenterology, 1979; 77:761–767.

28.

Gamberini

, Skorupa

, Souccar

, Lapa

: Inhibition of gastric secretion by a water extract from Baccharis triptera, mart. Memo Inst Oswaldo Cruz, 1991; 86:137–139.

29.

Spindola

, Vendramini-Costa

, Rodrigues

, Foglio

, Pilli

, Carvalho

: The antinociceptive activity of harmicine on chemical-induced neurogenic and inflammatory pain models in mice. Pharmacol Biochem Behav, 2012; 102:133–138.

30.

Srikanta

, Siddaraju

, Dharmesh

: A novel phenol-bound pectic polysaccharide from Decalepis hamiltonii with multi-step ulcer preventive activity. World J Gastroenterol, 2007; 13:5196–5207.

31.

Zhao

, Xu

, Chen

, Guo

, Wang

, Meng

: Veronicastrum axillare alleviates ethanol-induced injury on gastric epithelial cells via downregulation of the NF-kB signaling pathway. Gastroenterol Res Pract, 2017. DOI: 10.1155/2017/7395032.

32.

Salama

, El Gayar

, Georgy

, Hamza

: Equivalent intraperitoneal doses of ibuprofen supplemented in drinking water or in diet: A behavioral and biochemical assay using antinociceptive and thromboxane inhibitory dose–response curves in mice. PeerJ, 2016; 4:2239. DOI: 10.7717/peerj.2239.

33.

Kayani

, Dilshad

, Ahmed

, Ismail

, Mirza

: Evaluation of Ajuga bracteosa for antioxidant, anti-inflammatory, analgesic, antidepressant and anticoagulant activities. BMC Complement Altern Med, 2016; 16:375.

34.

Campos

, Costa

, Lima

, Silva

, Lobo

, Monteiro-Moreira

ACO

, Oliveira

: First isolation and antinociceptive activity of a lipid transfer protein from noni (Morinda citrifolia) seeds. Int J Biol Macromol, 2016; 86:71–79.

35.

Bhoumik

, Berhe

, Mallik

: Evaluation of gastric anti-ulcer potency of ethanolic extract of Sesbania Grandiflora Linn leaves in experimental animals. Am J Phytomed Clin Therap, 2016; 4:174–182.

36.

Gupta

, Kumar

, Gupta

: Evaluation of gastric anti-ulcer activity of methanolic extract of Cayratia trifolia in experimental animals. Asian Pac J Trop Dis, 2012; 2:99–102.

37.

Mezzaroba

LFH

, Carvalho

, Ponezi

, Antônio

, Monteiro

, Possenti

, Sgarbieri

: Anti-ulcerative properties of bovine α-lactalbumin. Int Dairy J, 2006; 16:1005–1012.

38.

Pacheco

MTB

, Bighetti

, Antônio

, Carvalho

, Possenti

, Sgarbieri

: Antiulcerogenic activity of fraction and hydrolysate obtained from whey protein concentrate. Braz J Food Technol, 2006; 3:15–22.

39.

Hamza

, Dionne

: Mechanisms of non-opioid analgesics beyond cyclooxygenase enzyme inhibition. Curr Mol Pharmacol, 2009; 2:1–14.

40.

Jalbert

, Castonguay

: Effects of NSAIDs on NNK-induced pulmonary and gastric tumorigenesis in A/J mice. Cancer Lett, 1992; 66:21–28.

41.

Zhao

, Li

, Yang

, Zhang

, Wang

: Anti-nociceptive effect of total alkaloids isolated from the seeds of Areca catechu L (Arecaceae) in mice. Trop J Pharm Res, 2017; 16:363–369.

42.

Wang

, DuBois

: The role of prostaglandin E 2 in tumor-associated immunosuppression. Trends Mol Med, 2016; 22:1–3.

43.

Yamaguchi

, Yoshida

, Uchida

: Novel functions of bovine milk-derived α-lactalbumin: Anti-nociceptive and anti-inflammatory activity caused by inhibiting cyclooxygenase-2 and phospholipase A2. Biol Pharm Bull, 2009; 32:366–371.

44.

Hayashida

, Takeuchi

, Shimizu

, Ando

, Harada

: Novel function of bovine milk-derived lactoferrin on anti-nociception mediated by μ-opioid receptor in the rat spinal cord. Brain Res, 2003; 965:239–245.

45.

Sasaki

, Sekiguchi

, Kikuchi

, Konno

: Anti-nociceptive effect of bovine milk-derived lactoferrin in a rat lumbar disc herniation model. Spine, 2010; 35:1663–1667.

46.

Meisel

, Schlimme

: Milk proteins: Precursors of bioactive peptides. Trends Food Sci Technol, 1990; 1:41–43.

47.

Paroli

: Opioid peptides from food (exorphins) Wld. Rev Nut Diet, 1988; 55:58–63.

48.

Matthies

, Stark

, Hartrodt

: Derivatives of β-casomorphins with high analgesic potency. Peptides, 1984; 5:463–470.

49.

Tani

, Iio

, Chiba

, Yoshikawa

: Isolation and characterization of opioid antagonist peptides derived from human lactoferrin. Agr Biol Chem, 1990; 54:1803–1810.