High-fiber basil seed flour reduces insulin resistance and hepatic steatosis in high-fat diet mice

The by-products of the basil seed oil industry have not been explored, and the nutritional composition of the fiber-rich fraction of partially defatted BSF has not been reported. The analysis revealed that BSF has 59.09 ± 2.62 g of dietary fiber per 100 g of flour, a value that is comparable to that previously reported by Capitani et al. ³¹ for chia seeds pressing fiber-rich fraction (51.98 g/100 g), and higher than those fiber-rich fractions from cereals such as wheat (20.93 g/100 g), pearl millet (21.92 g/100 g) and sorghum (23.4 g/100 g)³². According to some authors, the total dietary fiber of whole basil seed ranged between 22.6 to 36.30 g/100 g, which implies that the fraction could possess 1.6–2.6 times more dietary fiber than the seed^28,33,34,35. In this context, less than 50 g of BSF could meet the daily recommendations of European Food Safety Authority (EFSA) and American Dietetic Association (ADA), which recommend an average daily intake > 25 g of dietary fiber^36,37. The protein content (20.24 g/100 g) is also high compared with some pseudo cereals such as quinoa (14.5 g/100 g) and amaranth (16.5 g/100 g). In addition, the amino acid profile illustrates the high nutritional quality of the protein, including all the essential amino acids except S-containing types and tryptophan²⁸. Conversely, despite cold pressing to extract and obtain basil seed oil, the resulting pellet, which is subsequently crushed and sieved to make BSF, still retains 6.35 g/100 g lipids, of which 2.31 g/100 g correspond to ALA and 1.8 g/100 g to linoleic acid (C18:2n-6, LA), amounts that could be enough to provide a health effect²⁸. In general terms, the fiber-rich by products can increase the dietary fiber contents in the food matrix resulting in healthy food products, as previously reported by Elleuch et al. ³⁸.

In the recent decades, the association between dietary fiber intake and health benefits (body weight, lipid regulation and insulin sensitivity), have been extensively studied^15,39,40,41. This association has sparked interest in the search for new sources of fibers with different characteristics that can be used in the development of functional foods. In this context, BSF is presented as a new alternative of dietary fiber source. In our study, after 4 weeks of supplementation with 20% BSF or OAF (as control) in adult male rats with hepatic steatosis without changes in food consumption and energy intake, did present significant changes when comparing the HFD and supplemented CD groups. Fiber supplementation (BSF or OAF) resulted in lower body weight compared to their HFD controls; in particular, the inclusion of BSF, after exhibiting a greater effect on body weight, resulted in a lower body weight compared to the HFD control. Similar behavior was observed by Palou et al. (2015) where pectin supplementation for one month resulted in decreased body-fat content and ameliorate insulin and leptin resistance¹⁶. Additional studies have reported that the intake of dietary fiber can ameliorate adiposity and IR in mice fed a HFD^16,42. In contrast, epididymal adipose tissue weight did not differ in HFD mice with or without supplementation^16,43, presumably because both studies acted from prevention and not from reversion, as in the present investigation.

As a critical component, IR strongly promotes hepatic steatosis and there is evidence to support an association between dietary fiber intake and improved IR^2,6,15,42. The present investigation showed that the supplementation with BSF and/or OAF attenuate IR by presenting a reduction in insulin and glycemic parameters compared to HFD. In addition, when comparing blood lipid parameters (TG, total cholesterol, HDL, and LDL), the BSF group showed a significant attenuation in TG, LDL and total cholesterol compared to HFD. Similar results were observed by Jangra et al. ⁴² where the two fermentable dietary fiber (gum acacia and inulin) analyzed resisted the enhancement in serum TG, total cholesterol, LDL and VLDL compared to high fat and sucrose diet used by Villanueva-Suárez et al. ⁴⁴ where the HFD was supplemented with 20% artichoke fiber with a significant reduction of TG, LDL and total cholesterol. Also, it could be assumed that ALA provided by BSF supplementation (33 mg) could partly explain the results whose effect is possibly due to the BSF capacity to increase the excretion of bile salts⁴⁵, and appetite regulation; reported in the group supplemented with BSF, in which a downward trend in food intake was observed, although it was not significant compared to the other groups, since excess calories are determinant in the development of IR^43,46. In this sense, a study by Kim et al, showed that rats fed 10% perilla oil, characterized by a high content of ALA (60%) for 4 weeks, is able to reduce TG and cholesterol levels in plasma⁴⁷. In addition, the mechanism by which dietary fiber intake diminishes TG in rats has been describe as decrease in de novo fatty acid synthesis in the liver through the inhibition of lipogenic enzymes¹⁶. Moreover, the presence of β-glucans, contained in oats and basil seeds, is also linked to lowering total and LDL cholesterol levels by influencing the absorption of carbohydrates and cholesterol in the intestine^26,45. Furthermore, a study identified that basil seeds contained bioactive peptides with antioxidant activity and α-amylase inhibitory activities, by participating as analogs in the hydrogen bonding network in the active site of the amylase/substrate complex, thus inhibiting the hydrolysis process^26,27. In this context, the synergistic effects of dietary fiber, ALA and other bioactives compounds could be attributed to the fact that fiber can trap bile acids, hindering the absorption of cholesterol, increasing the use of circulating cholesterol for its synthesis, thus affecting plasma lipids; reducing the systemic inflammation by lowering the levels of proinflammatory cytokines, improving the SCFAs profile which affect the insulin sensitivity by improving gut barrier function; activating PPARs by PUFAs enhancing insulin sensitivity and promoting fatty acid oxidation, between others^14,48.

As previously reported, high-fat feeding in rodents causes marked hepatic steatosis and insulin resistance due to a mismatch between hepatic lipid uptake and lipid export¹³. In the histology, particularly in those from mice supplemented with BSF and OAF, a decrease in hepatic steatosis was observed, which was corroborated by the steatosis score, the amount of fat in the tissue, hepatic TG, and cholesterol. These results suggest that basil by-products might diminish fat accumulation preventing triglyceridemic and hepatic steatosis. These findings are in agreement with those previously reported by Villanueva-Suárez et al. ⁴⁴ who studied the artichoke dietary fiber composed by a mix of low and large degree of polymerized fructans, which are in synergy with other polysaccharides producing a protective effect against hepatic steatosis.

Besides, the fatty acid profile of the liver tissue was identified and quantified resulting in a higher amount of ALA, EPA and DHA in mice fed with BSF v/s CD. This is presumably due to the amount of ALA contained in the BSF supplementation and because ALA is efficiently converted to DHA in the liver⁴⁷. DHA increases the expression of PPAR-α genes, capable of inhibiting the nuclear transcription factor enhancer of activated NF-κB, restoring β-oxidation of fatty acids, and mediating oxidative, inflammatory (TNF-α, IL-1β, IL-6) and peroxisomal processes^6,49. Proven in this study given that BSF supplementation enhanced the expression of PPAR-α mRNA and increased CPT1α. Consequently, it favors the reversal of NAFLD and hepatocellular damage¹¹. In the process of inflammation and adipocyte dysfunction, M1-type macrophages release IL-6, IL-1β, to enhance phagocytosis and promote an inflammatory response. Additionally, through NLRP3 in the liver stimulated by NF-kβ and oxidative stress, gene expressions of the proinflammatory factors IL-6 and TNF-α have also been demonstrated, which could clarify the origin of the plasma proinflammatory cytokines found in HFD mice^50,51,52. In addition, n-3 PUFAs exert effects on gene expression by regulating two other nuclear lipogenic factors, the carbohydrate response element binding protein ChREBP-1c, and FAS, thereby inhibiting de novo fatty acid synthesis²⁵. Additionally, it has been shown that NAFLD in obese patients may predict a worse long-term prognosis denoted by the higher transaminase levels, relative to non-obese NAFLD¹¹. Concordant with this, animals fed with HFD exhibited significantly higher levels of transaminases (AST, ALT and GGT) compared to those fed with CD, while only the BSF group was able to attenuate liver damage, showing no significant changes compared to CD.

Furthermore, in interventional mice, plasma levels of the inflammatory cytokines TNF-α, IL-6 and IL-1β were significantly increased after 14 weeks with HFD, whereas in mice supplemented with BSF or with OAF these values were attenuated compared to HFD. HFD increases the risk of metabolic complications, and because in humans, excess lipid tissue would be susceptible to mononuclear cell infiltration and secretion of proinflammatory cytokines such as TNF-α, IL1-β, IL-6 and resistin^6,53,54. In this context, it was proposed that these cytokines can induce IR by activating several serine-threonine kinase pathways (such as the transcription factor NF-κB) which, in turn, phosphorylate insulin receptor substrate 1 (IRS-1) at serine or threonine leading to IRS degradation, affecting the downstream signaling cascade of insulin and consequently suppresses GLUT4 localization in cell membranes, favoring a reduced capacity for glucose uptake and utilization by cells^6,53,54.

Another key event in the pathogenesis of NALFD is the formation of reactive oxygen species (ROS) during inflammation, which, in addition to mitochondrial damage, perpetuates the accumulation of metabolic intermediates, with induction of endoplasmic reticulum stress, that over time, triggers exacerbated ROS production¹¹. As expected, the HFD group exhibited an increase (p < 0.05) in TBARS and hepatic isoprostanes, markers of lipid peroxidation; curiously, even though the BSF group was associated with the highest amount of DHA, a fatty acid sensitive to oxidation due to the elevated number of double bonds, it was the group with the lowest levels of TBARS, presenting significant differences compared to HFD. This protection could be attributed to the action of PPAR-α activity and the peptides with antioxidant activity previously mentioned²⁶. In addition, lipid peroxidation by-products have been shown to be highly reactive and capable of modifying nucleophilic lysine, cysteine, and histidine residues in proteins, predominantly in later stages of NALFD⁵⁵. In this sense, BSF group presented a significantly lower amount of protein carbonyls in contrast to the HFD group, indicating that supplementation attenuated ROS production and therefore lipid and protein oxidation, making lipid/protein peroxidation a validated marker of oxidative stress.

In contrast to this feature, in all living organisms, ROS levels are controlled by a complex network of antioxidant defenses, which reduce (but do not completely prevent) oxidative damage to biomolecules⁵⁶ Glutathione peroxidase (GSH-Px) is one of the main cellular antioxidant enzymes, which reduces complex hydroperoxides into their respective alcohols using GSH as a reducing agent⁵⁷. This may explain why, in this study, it was observed that although HFD mice showed a significant increase in GSH and GSSG levels (possibly as a compensatory mechanism) compared to CD and supplemented mice, their oxidative stress parameters were exacerbated.

There are limited studies addressing the impact of the amount of dietary ALA on its own accumulation and conversion to longer chain n-3 PUFAs, an aspect being relevant to study because (i) its storage (adipose tissue) may represent a reservoir of slow-release ALA that other tissues use over time (e.g., brain)²³; and (ii) the generation of lipid mediators that can be produced in the conversion of ALA to DHA, namely, the specific pro-resolving mediators (SPMs) with dual anti-inflammatory and proresolving bioactivity, including resolvins, protectins and maresins, which modulate the function of endothelial cells and the immune system⁵⁸. Along with this, it is essential to counteract the harmful effects of high intakes of carbohydrates and palmitic acid (C:16:0), given that they induce vascular inflammation by activating the immune system (Toll-like receptors (TLR) 2 and 4). Consequently, enhancement in the formation of precursors of lipid intermediates such as ceramides, IL-6, and NF-κB activation, represents a mechanism by which NAFLD promotes the development of vascular damage and atherosclerosis^12,59,60. In this context, hamsters fed different amounts of ALA for 5 weeks (between 1 and 40 g ALA/100 g total fatty acids), showed a dramatic ALA content increase in epididymal adipose tissue, but to a much lesser extent than in red blood cells, although DHA content did not change⁴⁷. The results of the present investigation corroborated that the ALA (2.6 g/100 g) contained in the supplementation with BSF, increased the levels of this fatty acid in epidemic adipose tissue and in erythrocytes. However, in these mice there was unexpectedly a significant increase of DHA in erythrocyte phospholipids compared to the HFD group. In addition, HFD diet has the ability to alter the fatty acid composition of tissues which can be corrected with dietary intervention, as they contribute to preserve the antioxidant capacity⁶¹, as demonstrated in our study. Regarding stool composition, supplementation reduced the absorption of saturated fats, leading to a higher amount of palmitic acid in the feces compared to the HFD group, as well as increased levels of n-6 and n-3 polyunsaturated fatty acids. This effect may be attributed to the fiber content mentioned earlier ⁽⁶⁴⁾. These findings may partially explain the observed changes in the fatty acid profiles of various tissues and the overall lipid profile.

In this study, the analysis of the SCFAs composition in the stool indicates that mice fed with BSF had a significantly higher amount of total SCFAs than the CD and HFD groups. This aspect has been little studied in the literature so it would be important to further assess the SCFA profile in the future.

In addition, the stool of the BSF mice had higher moisture and fat compared to the control groups (CD and HFD) due to the water absorption/retention capacity produced mainly by the soluble fiber and the action of SCFAs that regulate the absorption of water, minerals and fat^62,14. Furthermore, SCFAs also contribute to decrease the pH which inhibit potential pathogens and promote growth of beneficial bacteria¹⁴. Conversely, the pH analysis reflected a slight decrease in the supplemented groups compared to CD and HFD, presumably due to the production of SCFAs, since, being weak acids, they can decrease the colonic pH and, as a result the pH of stool. This could be a potential advantage because it has been observed that a low pH can inhibit the growth of sensitive pathogenic bacteria and favor the growth of beneficial bacteria¹⁴. This latter aspect is relevant in the pathogenesis of NALFD because intestinal dysbiosis could alter the production of bacteria and metabolites such as tryptophan, bacterial lipopolysaccharides (LPS) and thus deregulate the inflammatory activation of Kupffer cells, change the enterohepatic circulation of bile acids, causing inflammation and finally hepatic steatosis². Evidence further highlighted the beneficial role of gut microbiota-derived SCFAs in managing energy homeostasis by regulating PPAR-α and enzymes involved in mitochondrial lipid oxidation such as acetyl-CoA carboxylase and carnitine-palmitoyl transferase 1¹³.

The analysis of the SCFA profile in mice stool, identified that the BSF group contributed significantly to the production of SCFAs followed by those fed with OAF, acetic acid > propionic acid > butyric acid. Similar findings were reported by several authors, which detected significant increase of these SCFAs, both in vitro, mice and human models^14,43,63.

On the other hand, the dietary fiber contained in supplements could be considered as potential prebiotic, since it is known that bacteria belonging to the phylum Bacteroidetes make up a large proportion of the intestinal microbiota and mainly produce propionate together with acetate¹⁴. In addition, Wongputtisin and Khanongnuch⁶⁴ investigated the prebiotic properties crude basil oligosaccharide. The results revealed an increase in total lactic acid bacteria and decrease in Salmonella-Shigella spp group, suggesting the prebiotic potential of this fiber.

Also, the specific effect of oat on the previously mentioned results could be based on what was recently published by Gao et al. (2022), who reported that oat fiber supplementation is able to block the TLR4 signaling pathway and decrease the expression of NF-κB p65 in intestinal tissues of male mice. In addition, they indicated that oat fiber increases the expression of tight junction proteins, including zonula occludens-1 (ZO-1) and occludin, thus contributing to maintaining the integrity of the intestinal barrier⁶⁵. The mechanisms by which BSF and OAF can reduce IR and steatosis, mentioned in the discussion, are summarized in Fig. 5.

In conclusion, this research aimed to determine the effects of fiber-rich fraction of partially defatted basil seeds against IR and hepatic steatosis induced by a high-fat diet and its contribution on the tissue content of n-3 PUFAs and SCFAs in mice. The HFD diet induced IR, hepatic steatosis, proinflammatory status, oxidative status and a significant decrease in SCFA production. In contrast, the HFD diet supplemented with BSF and to a lesser extent with OAF achieved protective effects by exhibiting an improvement in IR-related parameters, attenuation of steatosis, liver damage and oxidative stress, as well as, decreased inflammatory status, increased n-3 PUFAs in liver, adipocytes and erythrocyte and increased SCFAs. Taking this into account, supplementation with BSF exerted protection, so this product could be considered as a potential therapeutic line for the management of IR and reversal of hepatic steatosis in humans. In addition, this study allowed the revaluation of industrial residues from the production of basil seed oil emerging in the market, by proving that the expeller obtained can become an excellent source of dietary fiber, insoluble fiber and ALA. It remains for future research to determine and quantify the polyphenols that may remain in the product since there is a possibility that they could contribute to or explain the decrease in the metabolic complications studied.