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Article Cite This: ACS Omega 2018, 3, 17474−17480
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Bioactive Compound Prodigiosin in Vivo Affecting the Nutrient Metabolism of Weaned Rats Peizhou Yang,*,† Jing Qian,† Wei Xiao,† Zhi Zheng,† and Mingshan Zhu*,‡ †
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College of Food and Biological Engineering, Anhui Key Laboratory of Intensive Processing of Agricultural Products, Hefei University of Technology, Hefei 230009 China ‡ School of Environment, Jinan University, Guangzhou 510632 China ABSTRACT: Nuclear magnetic resonance-based metabolomics, intestinal microorganisms, and nutrient absorption were investigated to examine the effects of prodigiosin (PG) on the nutrient metabolism of weaned rats in vivo. Results revealed that the addition of 200 μg of PG kg−1 of body weight each day for 14 days in the diet for weaned rats could effectively affect their nutrient metabolism. Compared with the control group, the contents of albumin (31.79 ± 5.15 g L−1), triglyceride (1.2 ± 0.16 mmol L−1), and high-density lipoprotein (1.7 ± 0.26 mmol L−1) in the blood of the weaned group were significantly higher, and the ammonia content (75.8 ± 9.84 μmol L−1) was significantly lower (P < 0.05). The apparent digestibility of crude protein (85.4 ± 1.7%) and fat (83.4 ± 1.2%) and the amount of Lactobacillus probiotics were also significantly higher than those in the control group (P < 0.05). In addition, the proton NMR (1H NMR) spectrum of the serum samples displayed 48 kinds of metabolites. PG significantly increased valine, isoleucine, and lipids (P < 0.05). In addition, PG significantly decreased methylamine, trimethylamine, and trimethylamino oxide (P < 0.05). Therefore, PG could affect the nutrient metabolism of weaned rats by controlling the digestion of nutrient substances and enhancing the metabolic levels of protein.
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INTRODUCTION The natural active substance Serratia marcescens prodigiosin (PG) is structured as three tri-pyrrole rings, and presents antibacterial,1 antimalarial,2 immunosuppressive,3 and anticancer properties.4,5 Given its nontoxicity to nonmalignant cells in vitro,6−8 PG has promising applications in functional food,8 such as polyolefine colorant,9 food additive,10 and so on. Previous reports mainly focus on the cytotoxicity and regulation mechanism of PG against malignant cells through in vitro approach.4,5,11,12 The in vivo bioactivity of PG can provide a reliable reference for PG application in the food and pharmaceutical industries. Nevertheless, the in vivo effect of PG on the nutrient metabolism of mammals is yet to be explored. The weaned rat is an animal model for investigating the growth and development of target substances in mammals.13,14 Weaning can trigger metabolic syndromes caused by various adverse factors, including oxidative stress15 and neuroendocrine stress.16 These side effects lead to neuropsychiatric disorders,17 gastrointestinal dysfunction, and reduced immunity.16 Enhancing the digestion and absorption of nutrition is an effective strategy to alleviate adverse stresses arising from the environment and diet.18 After feeding, nutrient utilization directly affects the function of tested materials.19 The metabolic analysis of characteristic metabolites and biochemical indexes of blood can also reflect the health level of animals.20 © 2018 American Chemical Society
In this study, various analysis methods were utilized to explore the effects of PG on the nutrient metabolism of weaned rats in vivo. This fundamental work is expected to provide effective support for the regulation of PG in the nutrition metabolism of mammals.
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RESULTS AND DISCUSSION Effects of PG on Blood Biochemical Indexes. The biochemical indexes of the blood from the alcohol group were not significantly different from those in the control group (Table 1). Compared with the control group, the addition of PG in the diet significantly increased the contents of albumin (31.79 ± 5.15 g L−1), total protein (64.61 ± 10.32 g L−1), triglyceride (1.2 ± 0.16 mmol L−1), and high-density lipoprotein (1.7 ± 0.26 mmol L−1) in the blood. At the same time, the addition of PG significantly decreased the content of blood ammonia (75.8 ± 9.84 μmol L−1) compared with that in the control group (P < 0.05). The contents of albumin, total protein, and high-density lipoprotein in the serum were closely related to the metabolism of protein.21 Triglycerides are an intermediate product of fat metabolism.22 Therefore, PG exerts biological activities by regulating the metabolism of protein and fat. Received: September 21, 2018 Accepted: December 3, 2018 Published: December 17, 2018 17474
DOI: 10.1021/acsomega.8b02476 ACS Omega 2018, 3, 17474−17480
ACS Omega
Article
Table 1. Serum Biochemical Profiles of Four Groups of Weaned Ratsa items AST (U L−1) ALT (U L−1) ALP (U L−1) GLU (mmol L−1) ALB (g L−1) TP (g L−1) AMM (μmol L−1) urea (mmol L−1) CHO (mmol L−1) TG (mmol L−1) LDL (mmol L−1) HDL (mmol L−1)
control group 155.50 50.65 182.91 4.29 27.18 54.26 88.04 6.46 2.34 1.02 0.21 1.41
± ± ± ± ± ± ± ± ± ± ± ±
alcohol group
16.62 8.61 21.52 0.51 3.58 7.05 11.25 0.84 0.35 0.14 0.04 0.22
154.22 47.62 166.92 4.23 28.78 56.78 82.11 6.59 2.44 1.02 0.22 1.42
± ± ± ± ± ± ± ± ± ± ± ±
L-PG group
13.12 3.76 11.31 0.29 3.99 2.37 9.26 0.39 0.21 0.11 0.08 0.26
153 46.32 186.32 4.33 28.34 55.78 85.44 6.87 2.44 1.01 0.22 1.43
± ± ± ± ± ± ± ± ± ± ± ±
H-PG group
21.23 2.12 15.33 0.19 1.56 2.98 4.21 0.54 0.12 0.12 0.08 0.14
146.32 46.39 187.37 4.46 31.79 64.61 75.80 6.09 2.42 1.20 0.25 1.70
± ± ± ± ± ± ± ± ± ± ± ±
33.85 11.73 22.76 0.64 5.15* 10.32* 9.84* 0.60 0.30 0.16* 0.05* 0.26*
The statistical values indicated mean ± S.E.; * indicates the significant difference compared to the control group at the level of P < 0.05. The items of biochemistry analysis were AST (aspartate aminotransferase), ALT (alanine aminotransferase), ALP (alkaline phosphatase), GLU (glucose), ALB (albumin), TP (total protein), AMM (blood ammonia), CHO (cholesterol), TG (triglyceride), LDL (low density lipoprotein), HDL (high density lipoprotein), and urea.
a
Table 2. Effects of PG on the Microbiota of Feces in Weaned Rats lg(CFU/g) items Lactobacillus
Bifidobacterium
E. coli
Enterococcus
control group 0 day 7 day 14 day 0 day 7 day 14 day 0 day 7 day 14 day 0 day 7 day 14 day
7.55 7.81 8.32 6.74 6.98 7.25 7.96 7.64 7.21 6.88 6.69 6.31
± ± ± ± ± ± ± ± ± ± ± ±
alcohol group
0.74 0.62 0.65 0.65 0.57 0.61 0.85 0.78 0.70 0.61 0.49 0.53
7.51 8.01 8.43 6.68 7.11 7.35 8.04 7.76 7.65 6.89 6.65 6.38
± ± ± ± ± ± ± ± ± ± ± ±
0.87 0.99 0.65 0.32 0.38 0.88 0.55 0.31 0.44 0.81 0.22 0.64
L-PG group 7.49 8.08 8.68 6.71 7.14 7.56 7.89 7.37 6.86 6.94 6.11 6.12
± ± ± ± ± ± ± ± ± ± ± ±
0.53 0.75 0.60 0.42 0.49 0.59 0.75 0.92 0.86 0.56 0.65 0.76
H-PG group 7.58 8.34 9.08 6.69 7.27 7.73 8.01 7.16 6.12 6.90 6.01 5.50
± ± ± ± ± ± ± ± ± ± ± ±
0.59 0.57 0.69# 0.60 0.65 0.72 0.66 0.76 0.79# 0.48 0.58# 0.56##
Table 3. Effects of PG on the Apparent Digestibility of Crude Protein and Fat (%) items crude protein
fat
control group 0 day 7 day 14 day 0 day 7 day 14 day
66.3 71.4 73.1 68.6 74.8 79.7
± ± ± ± ± ±
alcohol group
1.6 2.7 2.8 1.7 3.2 2.3
66.4 72.9 74.2 69.5 75.3 80.2
± ± ± ± ± ±
2.9 2.1 2.5 3.6 1.3 1.9
L-PG group 65.2 75.6 82.2 68.3 78.5 82.2
± ± ± ± ± ±
2.5 1.3 1.7 2.1 2.5 2.6
H-PG group 66.7 78.2 85.4 68.2 79.5 83.4
± ± ± ± ± ±
2.4 1.6* 1.7** 2.5 1.1 1.2*
Effects of PG on the Apparent Digestibility of Crude Protein and Fat. Table 3 shows the effects of PG on the apparent digestibility of crude protein and fat in weaned rats. The apparent digestibility of crude protein and fat in the alcohol and L-PG groups were not significantly higher than that in the control group (P < 0.05). However, after PG was administered for 14 days, the apparent digestibility of crude protein (85.4 ± 1.7%) and fat (83.4 ± 1.2%) in the H-PG group became significantly higher than that in the control group (P < 0.05). The obtained apparent digestibility of crude protein and fat implied that the addition of PG in the diet promotes nutrient digestion and absorption. Assignment of 1H NMR Signal Peaks of Serum Samples in Weaned Rats. The original 1H NMR spectrum of typical serum samples is shown in Figure 1. Figure 2 shows 1 H NMR profiles of blood samples from the control and H-PG groups. A total of 48 metabolites were identified in the 1H NMR spectrum in the weaned rat serum. These small
Effects of PG on the Microbiota of Feces in Weaned Rats. The effects of PG on the microbiota of feces were investigated by analyzing and comparing the microbiota from three treatment groups and the control group (Table 2). All indexes of the alcohol and L-PG groups were not significantly different from those of the control group. The indexes of H-PG group Bifidobacterium (7.73 ± 0.72) were not significantly different. However, after PG was administered for 14 days, the index of Lactobacillus probiotics (9.08 ± 0.69) exhibited a significant positive correlation, whereas the indexes of Escherichia coli (6.12 ± 0.79) and Enterococcus (5.5 ± 0.56) displayed a significant negative correlation (P < 0.05). The dietary supplement of Lactobacillus probiotics could promote health benefits.23 E. coli and Enterococcus spp. are good indicators of potential health risks.24 Therefore, PG increases the proliferation of Lactobacillus probiotics and inhibits the growth of harmful microorganisms such as E. coli and Enterococcus. 17475
DOI: 10.1021/acsomega.8b02476 ACS Omega 2018, 3, 17474−17480
ACS Omega
Article
Figure 1. Typical 600 MHz 1H NMR original spectra of serum samples.
Figure 2. 1H NMR profiles of blood samples from the control and H-PG groups.
metabolites were from the control and PG groups, respectively. In addition, the areas and locations of the peaks varied significantly in the PG groups. The shifts and areas of the peaks suggested that PG changed the metabolite contents. Analysis of Small Molecule Metabolites. The chemical shifts, peak multiplicities, and their corresponding 1H NMR signal multiplicities are presented in Table 5. The spectrum information of the serum samples included the resonances of amino acids, sugar, organic acids, albumin, lipids, unsaturated lipids, choline, creatine, and tricarboxylic acid cycle metabolites. A total of 5, 32, and 11 intermediate products were directly associated with the metabolism of lipids, proteins, and sugar, respectively. In addition, 14 small-molecule amino acids associated with the intermediate products of protein were detected. Several biogenic amines, including methylamine, dimethylamine, trimethylamine, and trimethylamine-N-oxide, were identified as well. The biogenic amine derivatives could proliferate rectal cancer cells25 and induce cardiovascular
molecular metabolites are listed in Table 4. The ordinal numbers in Figure 2 correspond to the sequence number of metabolites in Table 4. The NMR spectra were mainly composed of NMR signals of lipoproteins (low-density and very-low-density lipoprotein), amino acids (leucine, isoleucine, valine, alanine, lysine, glutamic acid, arginine, phenylalanine, and tyrosine), organic acids (3-hydroxy butyric acid and acetoacetate), lipid and choline metabolites (choline, trimetlylamine, trimetlylamine oxide, and so forth), glycolysis (lactic acid and pyruvic acid), three carboxylic acid cycle intermediates (citric acid), short chain fatty acids (acetic acid and propionic acid), and glucose. 1 H NMR Spectroscopic Analysis of the Serum. The 1H NMR spectra of weaned rat serum in the control and PG groups were distributed in the region of δ 0.75−8.84 (Figure 2). The NMR signals were assigned as specific metabolites for 1 H resonances, and 48 metabolites were distinctly assigned. The marked 25 and 37 1H NMR spectrum peaks of the serum 17476
DOI: 10.1021/acsomega.8b02476 ACS Omega 2018, 3, 17474−17480
ACS Omega
Article
Table 4. 1H NMR Data of Metabolites in the Serum of Weaned Ratsa key
metabolites
moieties CH3(CH2)n CH3CH2CH2C αCH, βCH2, γCH, δCH3 αCH, βCH, βCH3, γCH2, δCH3
1 2 3
LDL VLDL leucine
4
isoleucine
5
valine
αCH3, βCH, γCH3
6 7 8 9
propionate isobutyrate ethanol 3-hydroxybutyrate
CH3, CH2 CH3 CH3, CH2 αCH2, βCH, γCH3
10
lipids
δ1H (ppm) and multiplicity 0.88(m) 0.90(t) 3.73 (t), 1.72(m), 0.96 (d), 0.91(d) 3.68(d), 1.99(m), 1.01 (d), 1.26(m), 1.45(m), 0.94(t) 3.62(d), 2.28(m), 0.99 (d), 1.04(d) 1.08(t), 2.18(q) 1.12(d) 1.19(t), 3.66(q) 2.31(dd), 2.42(dd), 4.16 (m), 1.21(d) 1.29(m), 1.58(m), 2.02 (m) 2.25(m), 2.77(m)
11
threonine
CH2*CH2CO, CH2−CC CH2−CO, CH−O−CO αCH, βCH, γCH3
12 13 14
lactate alanine citrulline
αCH, βCH3 αCH, βCH3 αCH2, βCH2, γCH2
15
lysine
16 17
CH3
2.14(s)
19
acetate N-acetyl glycoprotein O-acetyl glycoprotein glutamate
αCH, βCH2, γCH2, δCH2 CH2−CO CH3
1.32(d), 4.25(m), 3.58 (d) 1.33(d), 4.11(q) 3.77(q), 1.48(d) 3.70(m), 1.59(m), 3.15 (t) 3.77(t), 1.89(m), 1.73 (m) 1.93(s) 2.05(s)
αCH, βCH2, γCH2
20
methionine
3.75(m), 2.12(m), 2.35 (m) 3.87(t), 2.16(m), 2.65 (t), 2.14(s) 2.26(s) 2.29(s), 3.49(s) 2.38(s)
18
21 22 23
acetone acetoacetate pyruvate
αCH, βCH2, γCH2, S−CH3 CH3 CH3, CH2 CH3
key
metabolites
δ1H (ppm) and multiplicity
moieties
24
glutamine
αCH, βCH2, γCH2
25 26 27 28 29 30 31 32
citrate methylamine dimethylamine trimethylamine albumin creatine creatinine choline
33
phosphorylcholine
34
GPC
35 36 37 38
TMAO taurine betaine proline
CH2 CH3 CH3 CH3 Lysyl-CH2 N−CH3, CH2 CH3, CH2 N−(CH3)3, αCH2, βCH2 N−(CH3)3, OCH2, NCH2 N−(CH3)3, OCH2, NCH2 CH3 N−CH2, S−CH2 CH3, CH2 βCH2, γCH2, δCH2
39 40 41
glycine ornithine myo-inositol
42 43 44
β-glucose α-glucose unsaturated lipids
45 46 47
tyrosine 1-methylhistidine phenylalanine
48
formate
CH2 CH2, αCH 5-CH, 4,6-CH, 2-CH 2-CH, 1-CH 1-CH C−CH2−C, −CHCH− 2,6-CH, 3,5-CH 4-CH, 2-CH 2,6-CH, 3,5-CH, 4-CH CH
3.68(t), 2.15(m), 2.45 (m) 2.58(d), 2.75(d) 2.66(s) 2.72(s) 2.92(s) 3.02(s) 3.05(s), 3.93(s) 3.05(s), 4.05(s) 3.21(s), 4.05(t), 3.51(t) 3.23(s), 4.21(t), 3.61(t) 3.24(s), 4.33(t), 3.51(t) 3.26(s) 3.27(t), 3.43(t) 3.29(s), 3.90(s) 2.02−2.33(m), 2.00(m), 3.36(t) 3.57(s) 3.80(s), 3.79(t) 3.30(t), 3.63(t), 4.06(t) 3.25(dd), 4.65(d) 5.26(d) 5.19(m), 5.34(m) 7.19(dd), 6.90(d) 7.04(s), 7.75(s) 7.32(m), 7.42(m), 7.37 (m) 8.46(s)
a
LDL, low density lipoprotein; VLDL, very low-density lipoprotein; TMAO, trimethylamine oxide; GPC, glycerophosphorylcholine; S, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; and m, multiplet.
disease,26 obesity,27 hepatocellular carcinoma, and sclerosing cholangitis.28 The present study shows that PG could increase the levels of lysine and glutamine and decrease the levels of the adverse amines of methylamine, trimethylamine, and trimethylamine oxide. Thus, PG potentially exhibits strong functional activity in vivo. Multiple molecular conformations of alkane groups (αCH, βCH, βCH3, γCH2, and δCH3) and functional groups of small molecules (CH2*CH2CO, CH2−CC, CH2−CO, and CH−O−CO) were marked based on the 1H NMR data. Furthermore, PG could significantly increase the levels of LDL/VLDL, valine, isoleucine, and lipids in weaned rat serum. The formation of small molecular compounds related to lipid and protein metabolism indicated that PG could regulate health by controlling the hydrolysis products of lipids and proteins in the serum. Therefore, PG changed the levels of small molecule metabolites associated with proteins, fat, and sugar. Data Analysis of 1H NMR. The analysis results of the 1H NMR are shown in Figure 3. The 1H NMR spectral datasets were initially analyzed by principal component analysis (PCA), which scored the plots of a serum and revealed that the separation of metabolic profiles was not distinct between the control and PG groups (Figure 3A). Then, partial least-squares
discriminant analysis (PLS-DA) was employed to distinguish the differences of the metabolic patterns. PLS-DA-scored plots could effectively classify these data into two regions (Figure 3B). The parameters of the models (R2X = 0.513, R2Y = 0.784, and Q2 = 0.589) and the validated models (permutation number: 200) indicated that an overfitting does not exist between R2 and Q2 (Figure 3C). The spectral datasets were also analyzed by orthogonal projection to latent structure DA (OPLS-DA) to maximize the discrimination between the two experimental groups (Figure 3D,E). The reliability of the PLSDA and OPLS-DA models depended on the R2 and Q2 values. In this study, most of the R2 and Q2 values were higher than 0.5, suggesting that the models were reliable and exhibited an excellent fitness and prediction function. The VIP statistics of the initial components of the OPLS-DA model (threshold >1.00) and the P value of independent sample t-test (threshold
