Modulation of Small-Intestine Morphology in Mice by a Novel Supplement Containing Silybum marianum, Yeast β-Glucan, Prebiotics, and Minerals

Introduction

The investigation of natural compounds for nonpharmacological interventions aimed at promoting health has gained momentum in the pursuit of an extended lifespan coupled with an improved quality of life.¹ Notably, numerous studies have demonstrated the potential of nutraceuticals as effective therapeutic approaches for the prevention and management of various inflammatory and metabolic disorders, including but not limited to obesity, steatosis, and type-2 diabetes mellitus.²³⁴

Silymarin (Silybum marianum) exerts hepatoprotective effects through various mechanisms, including antioxidant activity and stabilization of the hepatocellular membrane.⁵ Furthermore, silymarin has demonstrated the ability to decrease plasma cholesterol and low-density lipoprotein levels in hyperlipidemic animals.⁶ Moreover, experimental studies have reported that specific flavonoids present in silymarin, such as silibinin, exhibit inhibitory effects on renal toxicity induced by cisplatin.⁷

Silymarin and its flavonoid constituents, particularly silibinin, have demonstrated emerging potential in the inhibition of tumor growth.⁸ A recent study revealed a significant dose-dependent reduction in viability and migration of gastric cancer cells following silymarin administration.⁹ Furthermore, silibinin has exhibited strong inhibitory effects on various epithelium-derived cancers, including prostate, colorectal, bladder, and lung cancer.¹⁰¹¹

Understand the effects of phytocompounds on intestinal morphology is of paramount importance to elaborate supplements for a better quality of life in a variety of health conditions.¹² Thus, this study aimed to analyze the effects of a nutraceutical supplementation with silymarin on small intestine morphology.

Materials and Methods

Quantitative Analysis

Morphological parameters were assessed using ImageJ software (National Institutes of Health, United States) to analyze both structure density (%) and numerical quantity. The color deconvolution tool in ImageJ was employed to unmix the brightfield images into channels representing the absorbance of individual dyes.¹⁵¹⁶ After channel splitting, the images were converted to grayscale to measure the area fraction of stained structures in contrast to the white background. This quantitative analysis allowed for the evaluation of the following parameters: acid mucin (Alcian blue), basic mucin (periodic acid-Schiff), and collagen deposition (Masson's trichrome staining). Moreover, the numerical quantity of villi per field was determined using the cell counter tool in ImageJ on sections stained with Masson's trichrome. Morphometric analysis (Fig. 1) was performed on approximately five fields per animal, using AxioVision software (Carl Zeiss, United States), to measure the following parameters (approximately 5 structures per field for each parameter) on Masson's trichrome staining (µm): crypt depth, mucosal thickness, villus length, and villus spacing.¹⁷

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Fig. 1 Schematic representation of measurements of intestinal morphology parameters. The vertical red lines correspond to mucosal thickness, yellow lines indicate villus length, blue lines indicate crypt depth and horizontal green lines demonstrate how villus spacing was determined.

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Results

In Fig. 2, we can see the quantitative analysis, as well as the representative images for each group. Regarding morphometric analysis, animals submitted to the supplementation had a significant decrease of crypt depth (71.61 ± 2.09 vs. 64.08 ± 1.03µm), mucosal thickness (446.5 ± 11.6 vs. 321.2 ± 7.314µm), villus length (330.7 ± 11.56 vs. 210.2 ± 6.78µm), and villus spacing (109.3 ± 4.78 vs. 22.2 ± 0.73µm). In contrast, we observed a significant increase of villus per field (10.5 ± 1.25 vs. 20.1 ± 1.19 villus/field). However, color deconvolution did not show significant difference in collagen deposit or acid mucins between the groups. On the other hand, basic mucin was decreased in the experimental group (2.84 ± 0.23 vs. 1.87 ± 0.17%).

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Fig. 2 Quantitative analysis between the groups. (A) Acid mucins (%); (B) basic mucins (%); (C) collagen density (%); (D) quantity of villus per field; (E) crypt depth (µm); (F) mucosal thickness (µm); (G) villus length (µm); (H) villus spacing (µm). Representative images are shown above for both Masson's trichrome and periodic acid-Schiff with Alcian blue (PAS + AB). Scale bar = 50µm.

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Discussion

Our study aimed to investigate the effects of a novel nutraceutical supplementation on the components of the small intestine in mice. We observed an increase in the number of villi per field, suggesting an enlargement of the absorption area. This result was accompanied by a reduction in villus spacing, length, crypt depth, and mucosal thickness.

Previous research has shown that only a fraction of orally administered silymarin is absorbed from the gastrointestinal tract, as it undergoes extensive enterohepatic circulation.¹⁸ However, an experimental study in mice demonstrated that flavonoids from silymarin, in both free and conjugated forms, exhibited good distribution in various examined tissues.¹⁹ The observed phenomenon in our study can be partially explained by the formulation of the supplement. Silymarin has low permeability across intestinal epithelial cells, low aqueous solubility, and is rapidly excreted in bile and urine, resulting in low bioavailability. Experimental studies often combine its supplementation with compounds such as phospholipids,²⁰ liposomes,²¹ and β-cyclodextrins²² to enhance its bioavailability.

Dietary minerals can be absorbed through the epithelial cells lining the gastrointestinal tract, enabling transcellular mineral transport even at low concentrations in the intestinal lumen.²³ Therefore, mineral conjugates are utilized to enhance gastrointestinal absorption. Prebiotics, which promote the growth of beneficial bacteria in the gastrointestinal tract, have been shown to enhance the absorption capacity when combined with minerals.²⁴ Furthermore, the addition of β-glucan, a dietary fiber found in various sources including yeast, has demonstrated antioxidant activity and glucose control due to its bioavailability.²⁵

However, it is important to consider the limitations of our study. We solely employed histochemical techniques to elucidate our findings, and the bioavailability of silymarin was not directly measured in the experimental group. Despite the limitation posed by a modest sample size, our group's prior investigations have demonstrated compelling outcomes of this nutraceutical in preclinical models of obesity and type 2 diabetes, utilizing an identical sample size.²³⁴ These earlier studies yielded statistically significant results, thus providing a strong foundation for the current study's rationale and potential implications. However, it is crucial to acknowledge the necessity for larger sample sizes in future research to further validate and consolidate these findings, ensuring greater generalizability and robustness of the conclusion drawn. Notwithstanding these limitations, our study boasts several noteworthy strengths. We introduced a novel nutraceutical supplementation that potentially modulates small intestine morphology, thereby increasing absorption capacity. Future investigations utilizing immunohistochemical and molecular techniques may further elucidate the underlying pathways involved in villus genesis and the effects of each individual compound within this nutraceutical on the gastrointestinal system.

In conclusion, the combination of silymarin, prebiotics, yeast β-glucan, and minerals in a dietary supplementation regimen may play a crucial role in shaping small intestine morphology and enhancing absorption capacity. Thus, silymarin's role in shaping small intestine morphology and its combination with other compounds for improved bioavailability are noteworthy, offering implications for the nutraceutical industry.