Release, Absorption, Stability And Effect Of Beta-Casomorphins On Functions In Rat's Gstrointestinal Tract

The present study was conducted to detect the effects of beta-casomorphins on rats' gastrointestinal tract function including release, absorption of little molecular nutritional particles(glucose and amino acids) and stability using RP-HPLC. The primary research of regulating effect and mechanism of this peptide on expression and secretion of endocrine hormones(gastrin, somatostatin and cholecystokinin) was also studied using RT-PCR, in situ hybridization(ISH) and immunochemical methods. The regulating effect of beta-casomorphins on gastrointestinal functions was farther understood through this study and enlightened new ideas of research functions in gastrointestinal tract for functional food.1. RELEASE OF BETA-CASOMORPHINS IN RAT'S STOMACH AND DUODENUM DIGESTAThe RP-HPLC, as a simple and feasible method, is employed to analyze the releasing character of beta-casomorphins in stomach and duodenum of 25 days' weaning rats treated by the milk power. The experiment included two parts.(1) Acetonitrile: H₂O(3:1) was used as mobile phase, 0.1% phosphate acid used as acidic modifier. Gradient elution was adopted and DAD detector at 215nm. The retention of beta-casomorphin 5 and 7 were 2.64min and 7.43min. A good linear relationship exited between peak areas and beta-casomorphins concentration was obtained ranging from 2 to 500μg/mL. According to Signal/Noise≥3, the deduced minimum concentration of two beta-casomorphin 5 and 7 was 35ng/mL and 83.3ng/mL respectively. The average recovery of two peptides in gastric and duodenum digesta was 99.1%, 97.3% and 97.23%, 94.0% respectively.(2) 12 male SD rats were randomly assigned to control group(feeding with dd H₂O) and experimental group(200 mg/mL milk power). Rats were sacrificed on 4th day. The concentration of beta-casomorphin 5 and 7 in stomach and duodenum digesta was detected by HPLC. Both of peptides were detectable in the gastrointestinal tract of control group. But there were beta-casomorphin 5 and 7 detection in stomach and duodenum digesta of milk power-treated rats, the average content of beta-casomorphin 5 and 7 were 0.556±0.064μg/g and 0.548±0.090μg/g stomach digesta, and 0.441±0.030μg/g and 0.144±0.071μg/g duodenum digesta respectively. The concentration of beta-casomorphins in stomach was higher than in duodenum. In conclusion, beta-casomorphin 5 and 7 can be released by feeding milk protein in stomach.2. ABSORPTION OF BETA-CASOMORPHIN-7 BY THE GASTROINTESTINAL TRACT OF ADULT RATSThe aim of this study was to elucidate the absorption character of beta-casomorphin 7 in gastrointestinal tract of adult rats by utilizing the reverted gastric and intestinal sacs in vitro and using infusing peptide in gastrointestinal tract in vivo. The reverted gastric and intestinal sacs of adult rats were used and dipped into culture samples including beta-casomorphin 7. The concentration of this peptide in reverted sacs was detected by HPLC every 5 min. In the"In vivo, the different surgical procedures were designed as follows: the gastric cardia and pylorus or the two terminal of chosen duodenum were equipped with Silastic Tubing and ligated as absorption group. Besides, the blood vessel in the omentum accompanying with stomach and chosen duodenum was tied and cut off just in unable absorbed group. The dynamic variety of infused peptide concentration in different alimentary tract between two different treatment groups was observed. It is noticeable that the peptide in reverted sacs is undetectable by HPLC. There was no significant difference between different peptide concentrations in the same reaction time. Compared to prime concentration, the variety of peptide concentration in the same reaction time between two groups had the same tendency in stomach. The same results can also be observed in duodenum. Those results indicated that the beta-casomorphin 7 could not be transmitted through gastrointestinal membranes.3. STABILITY OF BETA-CASOMORPHIN-7 IN GASTROINTESTINAL TRACT OF RATSThe catabolism of the beta-casomorphin 7 was investigated by using several proteolysis enzymes and gastrointestinal mucosa homogenate. The experiment included two parts. In the different hydrolysis systems including peptide and pepsin, or trypsin, or carboxypeptidase A, or aminopeptidase, the concentration of beta-casomorphin-7 could maintain as long as 30min. However, only 25.55% of the initial peptide existed after the first 10 min of incubation with dipeptidyl peptidase, and then the peptide was undetectable behind 25min. According to the metabolical velocity between dipeptidyl peptidase and duodenum mucosa homogenate, it was obtained that the 83.19% hydrolyzed effect was mainly dependent on dipeptidyl peptidase. The beta-casomorphin 7 was hydrolyzed by dipeptidyl peptidase and released seven different peptides or amino acids after the first 5 min, including Tyr, Ile and beta-casomorphin-5. The present data demonstrated that beta-casomorphin 7 was able to exist in stomach of adult rats as intact molecule, and digest in duodoment which might be the first metabolism points in vivo. The dipeptidyl peptidase is the key hydrolysis enzyme in intestinal tract. There are mainly several smaller peptides in hydrolysate.4 LOCAL EFFECT AND REGULATIVE MECHANISM OF BETA-CASOMORPHIN 7 ON EXPRESSION AND SECRETION OF GASTRIN FROM GASTRIC MUCOSAThe aim of the present study was to evaluate the local effect and regulative mechanism of beta-casomorphin 7 on expression and secretion of gastrin from gastric mucosa.(1) 48 SD rats were randomly assigned to six treatment groups respectively: basal diet+physiological saline, basal diet+naloxone(2.5×10^-4 mol/L), basal diet +beta-casomorphin 7(2.5×10^-4 mol/L), basal diet+beta-casomorphin 5(2.5×10^-4 mol/L), basal diet+beta-casomorphin 7(5×10^-4 mol/L)+naloxone(5×10^-4 mol/L), basal diet+beta-casomorphin 5(5×10^-4 mol/L)+naloxone(5×10^-4 mol/L). After being treated for 30 days, each rat was sacrificed by exsanguinations. The expression of gastrin and somatostatin mRNA in gastric mucosa was detected by RT-PCR. The following results were obtained: beta-casomorphin 7 contributed to gastric mucosa gastrin mRNA expression(152.9% of control, P<0.05) after luminal administration for 30 days. But there was no siginificiant difference on gastrin mRNA expression between beta-casomorphin 7 treatment and naloxone simultaneity applied with beta-casomorphin 7(P=0.15), thus the effect of beta-casomorphin 7on gastrin mRNA expression was not blocked by naloxone. Beta-casomorphin 5,which is more active than beta-casomorphin 7 as opioid peptides, also ascended gastrin expression but was no significant different compared to beta-casomorphin 7(P=0.877). It indirectly proves that beta-casomorphin 7 up-regulates expression of gastrin independent of opioid receptors. These two casomorphins also significantly inhibited the expression of somatostatin mRNA. Therefore, the above results demonstrated that beta-casomorphins might have a local effect on regulating the gastrin expression by direct or indirect effluence on somatostatin.(2) Somatostatin, gastrin mRNA expression and secretion were quantified by RT-PCR, in situ hybridization and immunochemical methods in 24 SD rats, which were divided into three treatment groups: basal diet+physiological saline, basal diet +beta-casomorphin-7(2.5×10^-4mol/L), basal diet+poly-Gly-7(0.5×10^-4mol/L.containing equal mol of N with casomorphin). After being treated for 30 days, each rat was killed by exsanguinations.After intra-gastric administration of beta-casomorphin-7 for 30 days, gastrin mRNA increased by 152.8 % of the controls(P<0.05), and somatostatin mRNA levels decreased by 30.7%. No significant differences in the expression of the two genes were observed in the poly-Gly-treated group, although gastrin mRNA expression was elevated by 35.6% as against control group(P=0.15). The long-term oral administration of a casomorphin solution significantly decreased somatostatin mRNA positive-cell even grey value(22.0 % in fundus of control group and 19.5% in antrum of control group), but did not affect cell number both in the antrum and fundus. Interestingly, average density of gastrin mRNA positive-cells increased by 15.3%(P<0.05) and 22.5%(P<0.01) in the antrum of casomprphin-7 treated group and poly-Gly treated group respectively. However, poly-Gly had no effect on somatostatin mRNA positive-cell numbers and even grey value. The numbers and total grey value of G cell in beta-cosamorphin-7-treated group significantly ascended than them in poly-Gly treated group and control group. Beta-casomorphin-7 also significantly inhibited total grey value of D cell, but had no effect on the numbers of D cell. Though the total grey value of G cells in poly-Gly treated group also ascended 3.8%, there was no significantly difference with control group(P=0.18, n=8). Besides, Poly-Gly had no effect on the numbers and total grey value of D cell. Thus it can be obtained that the effect of beta-casomorphin-7 on gastrin gene expression and secretion depends on non-opioid receptors and itself sequences of amino acid, and relates with the paracrine action of somatostatin.5 EFFECT OF BETA-CASOMORPHINS ON SOMATOSTATIN AND CHOLECYSTOKININ mRNA EXPRESSION IN INTESTINAL TRACT OF ADULT RATSThe present study was to evaluate the effect of intake beta-casomorphins on expression of cholecystokinin and somatostatin from intestinal mucosa. 48 SD rats were divided into six treatment groups respectively: physiological saline, naloxone(2.5×10^-4 mol/L), beta-casomorphin 7(2.5×10^-4 mol/L), beta-casomorphin 5(2.5×10^-4 mol/L), beta-casomorphin 7(5×10^-4 mol/L)+naloxone(5×10^-4 mol/L), beta-casomorphin 5(5×10^-4 mol/L)+naloxone(5×10^-4 mol/L). After being treated for 30 days, each rat was killed by exsanguinations. The feed intake per week was recorded. The expression of gastrin and somatostatin mRNA in gastric mucosa were detected by RT-PCR. Both beta-casomorphin 7 and 5 significantly enhanced the intake-appetite during the first two weeks. The feed intake per week was ascended in the first week by 9.12 %(P<0.01) and 8.26%(P<0.01) respectively. The stimulated effect of beta-casomorphin 7 on intake per week disappeared at the next two weeks. Intra-gastric administration of beta-casomorphin 5 and 7 increased cholecystokinin mRNA(P<0.05) and also significantly inhibited the expression of somatostatin mRNA(23.0 % and 32.2% of control group respectively). The effect on cholecystokinin and somatostatin mRNA expression was reversed by naloxone simultaneity applied with beta-casomorphin 5 and 7. Therefore, the results demonstrated that beta-casomorphins can stimulate food intake per week at short-term time. The stimulated effect of beta-casomorphins on cholecystokinin mRNA expression depends on opioid receptors and relates to the feedback regulating somatostatin.6. EFFECT AND MECHANISM OF BETA-CASOMORPHINS ON THE LEVEL OF GLUCOSE AND AMINO ACID IN SERUMThe aim of this study is to evaluate the effect of beta-casomorphin-7on the concentration of glucose and amino acid in serum of adult rats and to evaluate the absorption mechanism using the reverted intestinal sacs in vitro. 32 SD rats were divided into four treatment groups: basal diet+physiological saline, basal diet +beta-casomorphin-7(2.5×10^-4 mol/L), basal diet+beta-casomorphin-5(2.5×10^-4 mol/L), basal diet+poly-Gly-7(0.5×10^-4 mol/L,containing equal mol of N with beta-casomorphin). After being treated for 30 days, each rat was killed by exsanguinations. The concentration of glucose and amino acid in serum was detected. The experimentation in vitro was designed four treatment groups to observe the effect of 0μg/mL,62.5μg/mL,125μg/mL and 250μg/mL beta-casorphin-7 on glucose and amino acid absorption. The reverted intestinal sacs of adult rats were dipped into culture sample included beta-casomorphin and glucose or amino acids. The concentration of glucose and amino acid in reverted sacs was detected every 5 min. The following results were obtained:(1) Luminally applied beta-casomorphin-7 significantly reduced glucose concentration; beta-casomorphin-5 also receded glucose concentration, but there was no significantl difference between two casomorphins(P=0.076).Beta-casomorphin-7 dose-dependently inhibited the transmitting of glucose in vitro. According to the transmitted velocity of glucose, beta-casomorphin-7 inhibits absorption of glucose in intestinal sacs by slowdown in first transmitted-period to change the transmitted kinetic of glucose.(2) Beta-casomorphin 7 significantly ascended concentration of Gln, Glu and Ala, related with glucongenesis, in serum. Beta-casomorphin-7(250μg/mL) inhibited transmitting of other amino acids in vitro except for Ser, Gln and Gly. Beta-casomorphin-7(62.5μg/mL) significantly inhibited transmitting of Asp, Thr, Ala and Arg, and significantly accelerated absorption of Ser, Gln, Gly, Tyr, Ile, Lys and Trp. This peptide(62.5μg/mL) increased transmitted velocity of amino acids in first transmitted-period(10-20min). The transmitted velocity of some amino acids(Glu, Ser, Arg, Met, Trp, Gly, Tyr, Phe and Ile) increased and even extended to 30min by beta-casomorphin 7. Thus it can be seen that luminally applied beta-casomorphin 7 has inhibiting effect on glucose concentration, simultaneity ascend concentration of amino acids related with glucongenesis in serum. Compare to beta-casomorphin 5 treatments and Poly-Gly treated group, the effect of beta-casomorphin 7 on glucose and amino acids concentration in serum depend on itself sequence.