Saffron (Crocus sativus L.) is known as the most expensive spice. C. sativus dried red stigmas, called threads, are used for culinary, cosmetic, and medicinal purposes. The rest of the flower is often discarded, but is now being used in teas, as coloring agents, and fodder. Previous studies have attributed antioxidant, anti-inflammatory, hepatoprotective, neuroprotective, anti-depressant, and anticancer properties to C. sativus floral bio-residues. The aim of this study is to assess C. sativus flower water extract (CFWE) for its effects on hemoglobin, brush boarder membrane (BBM) functionality, morphology, intestinal gene expression, and cecal microbiome in vivo (Gallus gallus), a clinically validated model. For this, Gallus gallus eggs were divided into six treatment groups (non-injected, 18 Ω H₂O, 1% CFWE, 2% CFWE, 5% CFWE, and 10% CFWE) with n~10 for each group. On day 17 of incubation, 1 mL of the extracts/control were administered in the amnion of the eggs. The amniotic fluid along with the administered extracts are orally consumed by the developing embryo over the course of the next few days. On day 21, the hatchlings were euthanized, the blood, duodenum, and cecum were harvested for assessment. The results showed a significant dose-dependent decrease in hemoglobin concentration, villus surface area, goblet cell number, and diameter. Furthermore, we observed a significant increase in Paneth cell number and Mucin 2 (MUC2) gene expression proportional to the increase in CFWE concentration. Additionally, the cecum microbiome analysis revealed C. sativus flower water extract altered the bacterial populations. There was a significant dose-dependent reduction in Lactobacillus and Clostridium sp., suggesting an antibacterial effect of the extract on the gut in the given model. These results suggest that the dietary consumption of C. sativus flower may have negative effects on BBM functionality, morphology, mineral absorption, microbial populations, and iron status.

Quinoa (Chenopodium quinoa Willd.), a gluten-free pseudo-cereal, has gained popularity over the last decade due to its high nutritional value. Quinoa is a rich source of proteins, carbohydrates, fibers, tocopherols (Vitamin E), unsaturated fatty acids and a wide range of polyphenols. The study used Gallus gallus intra-amniotic feeding, a clinically validated method, to assess the effects of quinoa soluble fiber (QSF) and quercetin 3-glucoside (Q3G) versus control. Quercetin is a pharmacologically active polyphenol found in quinoa. Six groups (no injection, 18 Ω H₂O, 5% inulin, 1% Q3G, 5% QSF, 1% Q3G + 5% QSF) were assessed for their effect on the brush border membrane (BBM) functionality, intestinal morphology and cecal bacterial populations. Our results showed a significant (p < 0.05) improvement in BBM morphology, particularly goblet and Paneth cell numbers, in the group administered with quinoa and quercetin. However, there were no significant changes seen in the expression of the genes assessed both in the duodenum and liver between any of the treatment groups. Furthermore, fibrous quinoa increased the concentration of probiotic L. plantarum populations compared to the control (H₂O). In conclusion, quercetin and quinoa fiber consumption has the potential to improve intestinal morphology and modulate the microbiome.

This efficacy trial evaluated the effects of two polyphenolic stilbenes, resveratrol and pterostilbene, mostly found in grapes, on the brush border membrane functionality, morphology and gut microbiome. This study applied the validated Gallus gallus intra-amniotic approach to investigate the effects of stilbene administration versus the controls. Three treatment groups (5% resveratrol; 5% pterostilbene; and synergistic: 4.75% resveratrol and 0.25% pterostilbene) and three controls (18 MΩ H₂O; no injection; 5% inulin) were employed. We observed beneficial morphological changes, specifically an increase in the villus length, diameter, depth of crypts and goblet cell diameter in the pterostilbene and synergistic groups, with concomitant increases in the serum iron and zinc concentrations. Further, the alterations in gene expression of the mineral metabolism proteins and pro-inflammatory cytokines indicate a potential improvement in gut health and mineral bioavailability. The cecal microbiota was analyzed using 16S rRNA sequencing. A lower α-diversity was observed in the synergistic group compared with the other treatment groups. However, beneficial compositional and functional alterations in the gut microbiome were detected. Several key microbial metabolic pathways were differentially enriched in the pterostilbene treatment group. These observations demonstrate a significant bacterial–host interaction that contributed to enhancements in intestinal functionality, morphology and physiological status. Our data demonstrate a novel understanding of the nutritional benefits of dietary stilbenes and their effects on intestinal functionality, morphology and gut microbiota in vivo.

Nicotinamide riboside (NR) acts as a nicotinamide adenine dinucleotide (NAD+) precursor where NR supplementation has previously been shown to be beneficial. Thus, we synthesized and characterized nicotinamide riboside tributyrate chloride (NRTBCl, water-soluble) and nicotinamide riboside trioleate chloride (NRTOCl, oil-soluble) as two new ester derivatives of nicotinamide riboside chloride (NRCl). NRCl and its derivatives were assessed in vivo, via intra-amniotic administration (Gallus gallus), with the following treatment groups: (1) non-injected (control); and injection of (2) deionized H₂O (control); (3) NRCl (30 mg/mL dose); (4) NRTBCl (30 mg/mL dose); and (5) NRTOCl (30 mg/mL dose). Post-intervention, the effects on physiological markers associated with brush border membrane morphology, intestinal bacterial populations, and duodenal gene expression of key proteins were investigated. Although no significant changes were observed in average body weights, NRTBCl exposure increased average cecum weight. NR treatment significantly increased Clostridium and NRCl treatment resulted in increased populations of Bifidobacterium, Lactobacillus, and E. coli. Duodenal gene expression analysis revealed that NRCl, NRTBCl, and NRTOCl treatments upregulated the expression of ZnT1, MUC2, and IL6 compared to the controls, suggesting alterations in brush border membrane functionality. The administration of NRCl and its derivatives appears to trigger increased expression of brush border membrane digestive proteins, with added effects on the composition and function of cecal microbial populations. Additional research is now warranted to further elucidate the effects on inflammatory biomarkers and observe changes in the specific intestinal bacterial populations post introduction of NR and its derivatives.

Approximately $20 billion of apple sales are generated annually in the United States. With an estimated 5 million tons produced yearly in the U.S. within the last decade, apple consumption is considered ubiquitous. Apples are comprised of bioactive constituents such as phytochemicals and prebiotics that may potentiate intestinal health and the gut microbiome. This study aimed to evaluate the effects of Empire apple juice, pomace, and pulp soluble extracts on intestinal functionality, morphology, and the microbiome in vivo (Gallus gallus). There were five treatment groups: non-injected (NI); 18 MΩ H₂O (H₂O); 6% apple juice (AJ); 6% apple pomace (APo); 6% apple pulp (APu). The eggs were treated by intra-amniotic administration of the samples on day 17 of incubation. After hatching, the blood, tissue, and cecum samples were collected for further analyses—including duodenal histomorphology, hepatic and duodenal mRNA expression, and cecal bacterial populations. Crypt depth was significantly (p < 0.5) shortest in AJ when compared to APo and APu. APo and APu soluble extracts significantly improved villi surface area compared to NI and H₂O control groups. The highest count of Paneth cells per crypt was observed in APo as compared to all groups. In addition, the expression of brush border membrane micronutrient metabolism and functional proteins varied between treatments. Lastly, Lactobacillus cecal microbial populations increased significantly in the AJ group, while AJ, APu, and APu increased the abundance of Clostridium (p < 0.5). Ultimately, these results indicate the potential of Empire apple pomace to improve host intestinal health and the gut microbiome.

This is a preliminary study evaluating the effect of different fractions of Concord grapes (Vitis labrusca L.) on the brush border membrane (BBM) morphology, duodenal gene expression, and specific gut bacterial populations. For this study, we utilized a unique intraamniotic approach, wherein, the test substances are administered into the amnion of the Gallus gallus egg (on day 17). The embryo orally consumes the amniotic fluid along with the injected test substance before the hatch. We randomly divided ~50 fertilized eggs into 5 groups including 6% grape (juice, puree, and pomace) along with controls (no injection and diluent—H₂O). The grape juice was prepared by crushing the grapes; the grape residues were used as pomace. The grape puree included the grape skin, endocarp, mesocarp, and juice but not the seeds. On day 21, the hatch day, the blood, pectoral muscle, liver, duodenum, and large intestine were harvested. Our results showed no significant differences in blood glucose, pectoral glycogen level, or body weight. However, significant (p < 0.05) differences in duodenal and liver gene expression were observed between the treatment groups. The grape puree treatment resulted in higher Clostridium numbers and lower Bifidobacterium numbers when compared to all other groups. In summary, the dietary consumption of grape polyphenols has the potential to beneficially modulate aspects of intestinal health provided their concentration is limited.