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Gut health immunomodulatory and anti-inflammatory functions of gut enzyme digested high protein micro-nutrient dietary supplement-Enprocal.

Kanwar JR, Kanwar RK - BMC Immunol. (2009)

Bottom Line: Our results indicate that Enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies.Furthermore, through downregulation of proinflammatory cytokines, Enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease (IBD).Stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioDeakin, Institute for Technology & Research Innovation, Deakin University, Geelong Technology Precinct, Geelong, Victoria, Australia. jagat.kanwar@deakin.edu.au

ABSTRACT

Background: Enprocal is a high-protein micro-nutrient rich formulated supplementary food designed to meet the nutritional needs of the frail elderly and be delivered to them in every day foods. We studied the potential of Enprocal to improve gut and immune health using simple and robust bioassays for gut cell proliferation, intestinal integrity/permeability, immunomodulatory, anti-inflammatory and anti-oxidative activities. Effects of Enprocal were compared with whey protein concentrate 80 (WPC), heat treated skim milk powder, and other commercially available milk derived products.

Results: Enprocal (undigested) and digested (Enprocal D) selectively enhanced cell proliferation in normal human intestinal epithelial cells (FHs74-Int) and showed no cytotoxicity. In a dose dependent manner Enprocal induced cell death in Caco-2 cells (human colon adencarcinoma epithelial cells). Digested Enprocal (Enprocal D: gut enzyme cocktail treated) maintained the intestinal integrity in transepithelial resistance (TEER) assay, increased the permeability of horseradish peroxidase (HRP) and did not induce oxidative stress to the gut epithelial cells. Enprocal D upregulated the surface expression of co-stimulatory (CD40, CD86, CD80), MHC I and MHC II molecules on PMA differentiated THP-1 macrophages in coculture transwell model, and inhibited the monocyte/lymphocyte (THP-1/Jurkat E6-1 cells)-epithelial cell adhesion. In cytokine secretion analyses, Enprocal D down-regulated the secretion of proinflammatory cytokines (IL-1beta and TNF-alpha) and up-regulated IFN-gamma, IL-2 and IL-10.

Conclusion: Our results indicate that Enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies. Furthermore, through downregulation of proinflammatory cytokines, Enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease (IBD). Stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies.

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Effect of Enprocal D and other digested milk product controls on cell proliferation of normal human intestinal cells (FHs74-Int) determined by MTT assay. All treatments were performed in triplicate and assay was repeated three times independently with similar results. Each bar presented in the histogram was mean ± SD values of all experiments in triplicates. **P < 0.001 is the highly significant value from the control with media only or enzyme cocktail. *P < 0.05 is the significance value from the control with media only or enzyme cocktail.
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Figure 4: Effect of Enprocal D and other digested milk product controls on cell proliferation of normal human intestinal cells (FHs74-Int) determined by MTT assay. All treatments were performed in triplicate and assay was repeated three times independently with similar results. Each bar presented in the histogram was mean ± SD values of all experiments in triplicates. **P < 0.001 is the highly significant value from the control with media only or enzyme cocktail. *P < 0.05 is the significance value from the control with media only or enzyme cocktail.

Mentions: Near complete digestion of Enprocal and other digested milk product controls was achieved after 4 hours incubation with gut enzyme cocktail mixture (Fig 1). In our preliminary experiments we treated Enprocal with the gut enzyme cocktail at different enzyme cocktail: protein ratios at pH 7.0–7.4. A 1:50 ratio of enzyme cocktail: protein at pH 7.2 was found to be most optimal and complete digestion was achieved after 6 hours. Further it was deemed necessary to find a concentration range which would not kill/disrupt the cell monolayers needed to test in bioassays on permeability, immunomodulation, cell adhesion, and oxidative stress. We treated Caco-2 and FHs 74 Int cells with digested and undigested samples of Enprocal at different concentrations (100–8000 μg/ml) to determine the effect on cell viability and growth determined by trypan blue exclusion assay (Fig 2A). With both Enprocal undigested and digested samples, the Caco-2 cell viability as compared to that of FHs 74 Int was significantly reduced in (P < 0.001) at concentrations 4000 and 8000 μg/ml. There was visible reduction in Caco-2 viability with Enprocal D compared to undigested Enprocal. However, these levels did not reach any significance. There was no effect of all concentrations tested in the range of 100–8000 μg/ml on the cell viability of normal intestinal FHs 74 Int cells. This was further confirmed in the LDH released cytotoxicity assay (Fig 2B). There was no significant increase (P > 0.05) in the cytotoxicity as compared to control (media only) when FHs 74 Int cells were treated with digested and undigested samples of Enprocal from 100–8000 μg/ml. The enzyme cocktail showed no effect of its own on cell viability and cytotoxicity in both the cell lines. Although there was no significant difference between the cell viability and cytotoxicity effects of digested and undigested Enprocal samples but to mimic the in vivo system we selected the digested form for bioassays. From the cell viability and cell cytotoxicity results 500–2000 μg/ml was selected as the optimal concentration range for the bioassays. The other control milk product digests at these concentrations were tested for their effects on cell viability in trypan blue assay (Fig 2C). When compared to media only control (no treatment) and Enprocal D, heat SMP digest samples reduced significantly (P < 0.001) the Caco-2 cell viability at 2000 μg/ml. The control milk products were further tested in LDH release assay (Fig 3A), and cytotoxicity levels were below 10% and not significant (P > 0.05) at 2000 μg/ml concentration when compared with Enprocal D and media only control. In TUNEL and Annexin-V-fluos assays, Enprocal D did not significantly (P > 0.05) increase the apoptotic index (AI) and necrotic index (NI), however heat SMP induced significant (P < 0.01) AI as compared to media only control in Caco-2 cells (Fig 3B). In a dose dependent manner (500–4000 μg/ml) Enprocal D increased significantly (P < 0.001) the proliferation of FHs 74 Int cells (Figure 4), determined by MTT assay, as compared to untreated and positive control (EGF 50 ng/ml) cells. When compared with other control products this effect was significant (p < 0.001) except for P2 (500 μg/ml) where the difference was insignificant between Enprocal and P2. However the cell proliferation of Caco-2 cells was decreased significantly following treatment with Enprocal D (data not shown).


Gut health immunomodulatory and anti-inflammatory functions of gut enzyme digested high protein micro-nutrient dietary supplement-Enprocal.

Kanwar JR, Kanwar RK - BMC Immunol. (2009)

Effect of Enprocal D and other digested milk product controls on cell proliferation of normal human intestinal cells (FHs74-Int) determined by MTT assay. All treatments were performed in triplicate and assay was repeated three times independently with similar results. Each bar presented in the histogram was mean ± SD values of all experiments in triplicates. **P < 0.001 is the highly significant value from the control with media only or enzyme cocktail. *P < 0.05 is the significance value from the control with media only or enzyme cocktail.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667481&req=5

Figure 4: Effect of Enprocal D and other digested milk product controls on cell proliferation of normal human intestinal cells (FHs74-Int) determined by MTT assay. All treatments were performed in triplicate and assay was repeated three times independently with similar results. Each bar presented in the histogram was mean ± SD values of all experiments in triplicates. **P < 0.001 is the highly significant value from the control with media only or enzyme cocktail. *P < 0.05 is the significance value from the control with media only or enzyme cocktail.
Mentions: Near complete digestion of Enprocal and other digested milk product controls was achieved after 4 hours incubation with gut enzyme cocktail mixture (Fig 1). In our preliminary experiments we treated Enprocal with the gut enzyme cocktail at different enzyme cocktail: protein ratios at pH 7.0–7.4. A 1:50 ratio of enzyme cocktail: protein at pH 7.2 was found to be most optimal and complete digestion was achieved after 6 hours. Further it was deemed necessary to find a concentration range which would not kill/disrupt the cell monolayers needed to test in bioassays on permeability, immunomodulation, cell adhesion, and oxidative stress. We treated Caco-2 and FHs 74 Int cells with digested and undigested samples of Enprocal at different concentrations (100–8000 μg/ml) to determine the effect on cell viability and growth determined by trypan blue exclusion assay (Fig 2A). With both Enprocal undigested and digested samples, the Caco-2 cell viability as compared to that of FHs 74 Int was significantly reduced in (P < 0.001) at concentrations 4000 and 8000 μg/ml. There was visible reduction in Caco-2 viability with Enprocal D compared to undigested Enprocal. However, these levels did not reach any significance. There was no effect of all concentrations tested in the range of 100–8000 μg/ml on the cell viability of normal intestinal FHs 74 Int cells. This was further confirmed in the LDH released cytotoxicity assay (Fig 2B). There was no significant increase (P > 0.05) in the cytotoxicity as compared to control (media only) when FHs 74 Int cells were treated with digested and undigested samples of Enprocal from 100–8000 μg/ml. The enzyme cocktail showed no effect of its own on cell viability and cytotoxicity in both the cell lines. Although there was no significant difference between the cell viability and cytotoxicity effects of digested and undigested Enprocal samples but to mimic the in vivo system we selected the digested form for bioassays. From the cell viability and cell cytotoxicity results 500–2000 μg/ml was selected as the optimal concentration range for the bioassays. The other control milk product digests at these concentrations were tested for their effects on cell viability in trypan blue assay (Fig 2C). When compared to media only control (no treatment) and Enprocal D, heat SMP digest samples reduced significantly (P < 0.001) the Caco-2 cell viability at 2000 μg/ml. The control milk products were further tested in LDH release assay (Fig 3A), and cytotoxicity levels were below 10% and not significant (P > 0.05) at 2000 μg/ml concentration when compared with Enprocal D and media only control. In TUNEL and Annexin-V-fluos assays, Enprocal D did not significantly (P > 0.05) increase the apoptotic index (AI) and necrotic index (NI), however heat SMP induced significant (P < 0.01) AI as compared to media only control in Caco-2 cells (Fig 3B). In a dose dependent manner (500–4000 μg/ml) Enprocal D increased significantly (P < 0.001) the proliferation of FHs 74 Int cells (Figure 4), determined by MTT assay, as compared to untreated and positive control (EGF 50 ng/ml) cells. When compared with other control products this effect was significant (p < 0.001) except for P2 (500 μg/ml) where the difference was insignificant between Enprocal and P2. However the cell proliferation of Caco-2 cells was decreased significantly following treatment with Enprocal D (data not shown).

Bottom Line: Our results indicate that Enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies.Furthermore, through downregulation of proinflammatory cytokines, Enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease (IBD).Stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioDeakin, Institute for Technology & Research Innovation, Deakin University, Geelong Technology Precinct, Geelong, Victoria, Australia. jagat.kanwar@deakin.edu.au

ABSTRACT

Background: Enprocal is a high-protein micro-nutrient rich formulated supplementary food designed to meet the nutritional needs of the frail elderly and be delivered to them in every day foods. We studied the potential of Enprocal to improve gut and immune health using simple and robust bioassays for gut cell proliferation, intestinal integrity/permeability, immunomodulatory, anti-inflammatory and anti-oxidative activities. Effects of Enprocal were compared with whey protein concentrate 80 (WPC), heat treated skim milk powder, and other commercially available milk derived products.

Results: Enprocal (undigested) and digested (Enprocal D) selectively enhanced cell proliferation in normal human intestinal epithelial cells (FHs74-Int) and showed no cytotoxicity. In a dose dependent manner Enprocal induced cell death in Caco-2 cells (human colon adencarcinoma epithelial cells). Digested Enprocal (Enprocal D: gut enzyme cocktail treated) maintained the intestinal integrity in transepithelial resistance (TEER) assay, increased the permeability of horseradish peroxidase (HRP) and did not induce oxidative stress to the gut epithelial cells. Enprocal D upregulated the surface expression of co-stimulatory (CD40, CD86, CD80), MHC I and MHC II molecules on PMA differentiated THP-1 macrophages in coculture transwell model, and inhibited the monocyte/lymphocyte (THP-1/Jurkat E6-1 cells)-epithelial cell adhesion. In cytokine secretion analyses, Enprocal D down-regulated the secretion of proinflammatory cytokines (IL-1beta and TNF-alpha) and up-regulated IFN-gamma, IL-2 and IL-10.

Conclusion: Our results indicate that Enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies. Furthermore, through downregulation of proinflammatory cytokines, Enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease (IBD). Stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies.

Show MeSH
Related in: MedlinePlus