**2. Part 1: effect of walnut and synergistic effect of walnut plus green tea consumption**

A rodent model using C57BL/6 J mice fed an obesogenic and diabetogenic high fat diet with added sucrose and cholesterol was designed to evaluate changes in metabolism when supplemented with walnut (W) or walnut plus green tea (W + GT). Walnuts were provided in the diets as ground nuts and the green tea was a dried powder derived from an aqueous extract of dried green tea leaves (Triarco Industries). Diets were formulated to mimic a typical 2000 kcal/day diet with whole walnuts added at an equivalence to 1.5 servings per day and green tea added at 1% (wt:wt), which is consistent with normal human intake levels shown in other rodent studies [13].

The final body weight of the HF diet group provided with walnuts alone did not significantly differ from the HF-fed control. However, the group supplemented with *Green Tea as An Ingredient in Food Combinations Provide Metabolic Improvements DOI: http://dx.doi.org/10.5772/intechopen.107347*

#### **Figure 1.**

*Final body weight (A) and energy intake (B) of obese male mice fed a LF or HF diet alone or one that included walnuts alone or walnut plus green tea for 9 weeks. Values are means ± SEMs, n = 8 mice. One-way ANOVA indicated significant differences between diet groups (p* ≤ *0.05). Values that do not share a letter differ (p* ≤ *0.05).*

additional green tea (W + GT) was significantly lower than the HF-fed control group (*p* ≤ 0.05), despite consuming a similar amount of energy (**Figure 1A** and **B**). This result is consistent with other findings demonstrating that green tea polyphenols, specifically (−)-epigallocatechin-3-gallate, may have protective effects against obesity by increasing thermogenesis [14–17].

Metabolomic analysis comparing 594 different hepatic biochemicals detected in liver samples from the mice in each of the diet groups showed several metabolomic changes, including changes in metabolites related to energetics, inflammation, and redox homeostasis. The W + GT supplemented group showed significant increases in S-methylmethionine, when compared to both HF (*p* ≤ 0.05) and LF (*p* ≤ 0.05) control groups and the W supplemented group (*p* ≤ 0.05). S-methylmethionine is a plant-derived metabolite that can be used as a carbon source for bacterial growth [18] which may imply beneficial effects on the host microbiome with supplementation of green tea. Additionally, increases in xanthine metabolites (e.g., paraxanthine, theobromine, theophylline) in the W + GT supplemented group compared to both the HF (*p* ≤ 0.05) and LF (*p* ≤ 0.05) control groups and the W supplemented group (*p* ≤ 0.05) were observed. This result likely reflects naturally occurring xanthine metabolites in GT as well as liver metabolism of increased caffeine intake from GT extract using the cytochrome P450 system.

The omega-3 polyunsaturated fatty acid linolenate was also elevated in the walnut-supplemented diets, which may indicate potential relevance to inflammation. Both the W and W + GT groups showed significant increases in omega-3 polyunsaturated fatty acids, eicosapentaenoate (EPA), and docosapentaenoate (DPA), compared to LF and HF control groups (**Figure 2**) These results suggest that walnut and green tea supplementation may negatively regulate inflammation. However, increases in docosapentaenoate (DHA) were not significantly altered. These results are consistent with previous studies reporting that increased ALA consumption (herein via walnut)

#### **Figure 2.**

*Box plots demonstrating metabolomic analysis of livers of obese male mice fed a LF or HF diet alone or one that included walnuts alone or walnut plus green tea for 9 weeks.*

results in little to no change in DHA plasma lipid fractions, as large amounts of ALA are required to endogenously convert to significant levels of DHA [19–24].

Supplementation with walnuts alone did not result in significant changes to detected eicosanoids when compared to HF control but did show non-significant trends toward decreased prostaglandin F2alpha (*p* ≤ 0.10) and 12-hydroxyeicosatetraenoic acid (HETE) such that it was statistically indistinguishable from the LF *Green Tea as An Ingredient in Food Combinations Provide Metabolic Improvements DOI: http://dx.doi.org/10.5772/intechopen.107347*

#### **Figure 3.**

*Relative concentration of hepatic proinflammatory fatty acids in obese male mice fed a LF or HF diet alone or one that included walnuts alone or walnut plus green tea for 9 weeks. Values are means ± SEMs, n = 8 mice. One-way ANOVA indicated significant differences between diet groups (p* ≤ *0.05). Values that do not share a letter differ (p* ≤ *0.05). G, walnuts plus green tea; H, high fat, HETE, hydroxyeicosatetraenoic acid; L, low fat; W, walnuts.*

control group (**Figure 3**). However, W + GT, compared to HF control, showed a significant decrease in 12-HETE, suggestive of declining inflammation. These results are consistent with serum cytokine concentrations measured of MCP-1, giving additional insight into the secretion and recruitment of activated immune cells in supplemented mice. MCP-1 concentrations were elevated with consumption of a HF diet compared to LF diet (*p* ≤ 0.05); however, the addition of walnut reduced MCP-1 such that it was intermediate to the LF and HF control groups and statistically indistinguishable from the levels measured in the LF group (data not shown). The addition of green tea to the walnut diet resulted in even further lowering of MCP-1 levels, such that they were statistically equivalent to the LF control group (data not shown). Of note, measured concentrations of TNFα and IL-6 were both measured and saw no significant decreases in HF + W group, but significant elevation of IL-6 was observed in W + GT group (*p* ≤ 0.05) (data not shown).
