Cross-Omics Analysis of Fenugreek Supplementation Reveals Beneficial Effects Are Caused by Gut Microbiome Changes Not Mammalian Host Physiology
Abstract
:1. Introduction
2. Results
2.1. Significant Differences between Small Intestine, Large Intestine, Liver, and Serum
2.2. Metabolomics Data Indicate Significant Differences as a Result of Fenugreek Supplementation
2.2.1. Small Intestine
2.2.2. Large Intestine
2.2.3. Liver
2.2.4. Serum
2.3. Metagenomic Analysis Reveal Notable Modifications in Gut Microbiome of FG Supplemented Mice
2.4. Fenugreek Supplementation Modulates HDL Balance and Total Cholesterol in HF Diet-Fed Mice
3. Discussion
3.1. High Fat Diet Influences Purine Metabolite Abundances
3.2. Fenugreek Induces Considerable Changes in the Large Intestines
3.3. Liver Metabolome Is Affected by Fenugreek
3.4. Fenugreek Impacts Specific Pathways by Location
4. Methods
4.1. Animals and Diets
4.2. Metabolic Phenotyping
4.3. Metagenomic Sequencing
4.4. Metabolite Extractions
4.5. UHPLC-HRMS
4.6. Metabolomics Data Processing
4.7. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A. NIH/NCCIH Product Integrity Form for Fenugreek Seed in Animal Studies
Appendix A.1. Identify the Source Plant for the Product Using the Scientific Taxonomic Nomenclature and Author Citation
Appendix A.2. Describe the Parts of the Plant from Which the Product Is Derived. If an Extract Is Used, Identify the Extraction Solvent
Appendix A.3. Name the Supplier of the Product. Include the Brand Name of the Product, If Applicable
Appendix A.4. Present Data on the Characterization (e.g., Chemical Profile or Fingerprint) of the Final Product as Thoroughly as the State of the Science Allows
Appendix A.5. Provide a Certificate of Analysis from the Supplier to Show Compliance to Their Specifications for Content and Any Other Supporting Information Relating to the Batches to Be Used in the Study. This Should Include Data on the Analysis of the Product for Contaminants/Impurities Such as Pesticides, Heavy Metals, and Residual Solvents
Appendix A.6. Provide Data on Batch-To-Batch Reproducibility. These Data May Be Required Separately for the Botanical Alone, the Vehicle Alone, and the Final Product, as Appropriate
Peak No. | Mass | Formula | Compound(s) | Reported from Trigonella (Reaxys.com) |
---|---|---|---|---|
1 | Mix of 324, 444 | C21H32O10, C13H24O9 | Organic acid glycosides? | No; 32 structures |
2 | 594 | C27H30O15 | Flavone (most likely Quercetin) disaccharide | Yes; 2 structures |
3 and 4 | 564 | C26H28O14 | Flavone disaccharides? | No; >100 structures |
5 and 6 | 448 | C21H20O11 | Flavone (Quercitrin, Luteolin, Vitexin) glycosides | Yes; 4 structures |
7 and 8 | 534 | C25H26O13 | Possibly flavone disaccharides, Caffeoyl-syringoyl quinic acids, etc. | No; 40 structures |
9 | 432 | C21H20O10 | Vitexin and/or Kaempferol-rhamnoside | Yes; 2 structures |
10 | 548 | C26H28O13 | Wide variety of polyphenolic (di-)saccharides | No; 47 structures |
11 | 504 | C24H30O11 | Megastigmane glycoside(s)? | No; 24 structures |
12 | 904 | C44H72O19 | Furostane and/or spirostane trisaccharides | No; 5 structures |
13 and 14 | 906 | C44H74O19 | Furostane and/or spirostane trisaccharides (Trigoneosides Ia, Ib) | Yes; 3 structures |
15 | 920 | C45H76O19 | Furostane trisaccharides | Yes; 2 structures |
16 | 738 | C39H64O13 | Spirostane disaccharides, saponins, etc. | No; 51 structures |
17 | 888 | C44H72O18 | Furostane and/or spirostane trisaccharides, saponins, etc. | No; 23 structures |
890 | C44H74O18 | Furostane trisaccharides | Yes; 2 structures | |
1048 | C51H84O22 | Furostane tetrasaccharides (Trigoneosides, or Trigonellosides) | Yes; 2 structures | |
18 | 902 | C45H74O18 | Furostane trisaccharides; Trigofoenoside A | Yes; 4 structures |
19 | 1046 | C51H82O22 | Furo-/spirostane tetrasaccharides | No; 40 structures |
872 | C44H72O17 | Spirostane trisaccharides | No; 27 structures | |
20 | 888 | C45H76O17 | Cholestane trisaccharides | No; 9 structures |
21 | 644 | C34H44O12 | Cyclopentane perhydro-phenanthrene derivatives | No; 18 structures |
Appendix A.7. Present a Plan to Monitor the Stability of Product Samples from All Batches Used during the Course of the Study
Appendix A.8. Describe the Method of Authentication of the Raw Material and Where and How an Authenticated Voucher Specimen of the Plant Material Is Reserved
Appendix A.9. Describe the Manufacturing Process
Appendix A.10. If the Product Is Combined with a Diet, a Certificate of Analysis and Specifications for the Vehicle Will Also Be Necessary to Assure Purity, Consistency, Absence of Bioactive Components (e.g., Soy), and/or Reproducibility of the Vehicle. Furthermore, Analysis of the Formulated Diet May Be Required to Assess Consistency, Stability, etc. of the Product in this Matrix
D12450J | D16020408 | D12492 | D16020408 | |
---|---|---|---|---|
Product # | 10 kcal% Fat | 10 kcal% Fat | 60 kcal% Fat | 60 kcal% Fat |
Fenugreek | Fenugreek | |||
Ingredient | Seeds | Seeds | ||
Casein | 200 | 193.4 | 200 | 193.4 |
L-Cystine | 3 | 3 | 3 | 3 |
Corn Starch | 506.2 | 495.4 | ||
Maltodextrin 10 | 125 | 125 | 125 | 114.2 |
Sucrose | 68.8 | 68.8 | 68.8 | 68.8 |
Cellulose | 50 | 48.6 | 50 | 48.6 |
Lard | 20 | 18.7 | 245 | 243.7 |
Soybean Oil | 25 | 25 | 25 | 25 |
Mineral Mix S10026 | 10 | 10 | 10 | 10 |
Dicalcium Phosphate | 13 | 13 | 13 | 13 |
Calcium Carbonate | 5.5 | 5.5 | 5.5 | 5.5 |
Potassium Citrate, 1 H2O | 16.5 | 16.5 | 16.5 | 16.5 |
Vitamin Mix V10001 | 10 | 10 | 10 | 10 |
Choline Bitartrate | 2 | 2 | 2 | 2 |
Fenugreek Seeds | 0 | 21.1 | 0 | 21.1 |
Red Dye #40, FD&C | 0 | 0.025 | 0 | 0.05 |
Blue Dye #1, FD&C | 0.01 | 0.025 | 0.05 | 0 |
Yellow Dye #5, FD&C | 0.04 | 0 | 0 | 0 |
Total | 1055.05 | 1056.05 | 773.85 | 774.85 |
D12492 | D16020408 | D12492 | D16020408 | |
gm | ||||
Protein | 179 | 179 | 179 | 179 |
Carbohydrate | 710 | 710 | 203.8 | 203.8 |
Fat | 47.4 | 47.4 | 272.4 | 272.4 |
Fiber | 50 | 50 | 50 | 50 |
gm% | ||||
Protein | 17 | 16.9 | 23.1 | 23.1 |
Carbohydrate | 0 | 0 | 26.3 | 26.3 |
Fat | 4.5 | 4.5 | 35.2 | 35.2 |
Fiber | 4.7 | 4.7 | 6.5 | 6.5 |
Fenugreek seeds | 0 | 2 | 0 | 2.72 |
kcals | ||||
Protein | 716 | 716 | 716 | 716 |
Carbohydrate | 2840 | 2840 | 815 | 815 |
Fat | 427 | 427 | 2452 | 2452 |
Total | 3983 | 3983 | 3983 | 3983 |
kcal% | ||||
Protein | 18 | 18 | 18 | 18 |
Carbohydrate | 71 | 71 | 20 | 20 |
Fat | 11 | 11 | 62 | 62 |
Total | 100 | 100 | 100 | 100 |
kcal/gm | 3.8 | 3.8 | 5.1 | 5.1 |
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High Fat Diet Metabolomics | |||||||
---|---|---|---|---|---|---|---|
Jejunum | Ileum | Cecum | Colon | Liver | Serum | ||
Total identified metabolites | 145 | 140 | 141 | 137 | 130 | 120 | |
HF-altered | 11 | 2 | 41 | 30 | 2 | 7 | |
HF vs. CD | increased w/HF | 5 | 0 | 35 | 9 | 0 | 6 |
decreased w/HF | 6 | 2 | 6 | 21 | 2 | 1 | |
HF-altered & FG-altered | 6 | 4 | 1 | 6 | 3 | 0 | |
CD vs. CDFG | increased w/FG | 2 | 4 | 0 | 0 | 1 | 0 |
decreased w/FG | 4 | 0 | 0 | 0 | 2 | 0 | |
HF vs. HFFG | increased w/FG | 0 | 0 | 0 | 5 | 0 | 0 |
decreased w/FG | 0 | 0 | 1 | 1 | 0 | 0 | |
Total unique spectral features | 6837 | 6823 | 11157 | 8974 | 3323 | 1772 | |
HF-altered | 594 | 718 | 4294 | 2809 | 339 | 105 | |
HF vs. CD | increased w/HF | 114 | 508 | 3671 | 1977 | 59 | 94 |
decreased w/HF | 480 | 210 | 623 | 832 | 280 | 11 | |
HF-altered & FG-altered | 104 | 473 | 2917 | 1913 | 111 | 37 | |
CD vs. CDFG | increased w/FG | 18 | 0 | 1685 | 1243 | 0 | 16 |
decreased w/FG | 72 | 255 | 678 | 282 | 23 | 2 | |
HF vs. HFFG | increased w/FG | 12 | 11 | 217 | 238 | 51 | 8 |
decreased w/FG | 6 | 310 | 992 | 515 | 37 | 13 |
Weighted | Unweighted | ||||
---|---|---|---|---|---|
Intestinal Region | Comparison | Score | p-Value | Score | p-Value |
Jejunum | CD vs. CDFG | 0.632386 | 0.158 | 0.794898 | 0.261 |
HF vs. CD | 0.65695 | 0.121 | 0.849862 | 0.158 | |
HF vs. HFFG | 0.70675 | 0.136 | 0.787471 | 0.134 | |
Ileum | CD vs. CDFG | 0.620738 | 0.454 | 0.742053 | 0.78 |
HF vs. CD | 0.654047 | 0.254 | 0.758081 | 0.574 | |
HF vs. HFFG | 0.692661 | 0.584 | 0.83724 | 0.789 | |
Cecum | CD vs. CDFG | 0.654342 | 0.069 * | 0.724363 | 0.222 |
HF vs. CD | 0.534961 | 0.004 *** | 0.809126 | 0.031 ** | |
HF vs. HFFG | 0.627892 | 0.008 *** | 0.893359 | 0.003 *** | |
Colon | CD vs. CDFG | 0.376439 | 0.54 | 0.673875 | 0.284 |
HF vs. CD | 0.589599 | <0.0010 *** | 0.908955 | <0.0010 *** | |
HF vs. HFFG | 0.607401 | <0.0010*** | 0.908432 | 0.005*** |
Jejunum | Ileum | Cecum | Colon | |
---|---|---|---|---|
Core OTUs | 63 | 69 | 113 | 120 |
HF-altered | 10 | 8 | 57 | 53 |
Significantly increased by HF | 2 | 7 | 50 | 40 |
Significantly decreased by HF | 8 | 1 | 7 | 13 |
HF-altered and FG corrected | 0 | 0 | 13 | 15 |
Significantly increased by HF | 0 | 0 | 11 | 10 |
Significantly decreased by HF | 0 | 0 | 2 | 5 |
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Jones, K.A.; Richard, A.J.; Salbaum, J.M.; Newman, S.; Carmouche, R.; Webb, S.; Bruce-Keller, A.J.; Stephens, J.M.; Campagna, S.R. Cross-Omics Analysis of Fenugreek Supplementation Reveals Beneficial Effects Are Caused by Gut Microbiome Changes Not Mammalian Host Physiology. Int. J. Mol. Sci. 2022, 23, 3654. https://doi.org/10.3390/ijms23073654
Jones KA, Richard AJ, Salbaum JM, Newman S, Carmouche R, Webb S, Bruce-Keller AJ, Stephens JM, Campagna SR. Cross-Omics Analysis of Fenugreek Supplementation Reveals Beneficial Effects Are Caused by Gut Microbiome Changes Not Mammalian Host Physiology. International Journal of Molecular Sciences. 2022; 23(7):3654. https://doi.org/10.3390/ijms23073654
Chicago/Turabian StyleJones, Katarina A., Allison J. Richard, J. Michael Salbaum, Susan Newman, Richard Carmouche, Sara Webb, Annadora J. Bruce-Keller, Jacqueline M. Stephens, and Shawn R. Campagna. 2022. "Cross-Omics Analysis of Fenugreek Supplementation Reveals Beneficial Effects Are Caused by Gut Microbiome Changes Not Mammalian Host Physiology" International Journal of Molecular Sciences 23, no. 7: 3654. https://doi.org/10.3390/ijms23073654