**3. Part 2: synergistic effects of cherry, red raspberry, walnut, and green tea (CRWG)**

The synergistic effects of consuming whole cherry, raspberry, walnut, and green tea in combination were determined in an independent study. The same rodent model as Part 1, C57Bl/6 J mice consuming an obesogenic and diabetogenic high fat diet with added sucrose and cholesterol, was used to evaluate the synergistic impact of these four foods on metabolism and microbiome of the ceca.

Despite consuming similar amounts of energy, the mice supplemented with HF + CRWG had lower final body weight and change in body weight compared to the HF diet alone (**Figure 4A** and **B**). This result is consistent with previous studies on cherry, raspberry, and walnut consumption and reduced weight gain [25–30]. Additionally, walnut and green tea consumption have been demonstrated to increase resting energy expenditure and thermogenesis, which may be a potential mechanism through which decreased weight gain occurred [6, 31]. Elevation of food efficiency in

**Figure 4.**

*Total body weight (A) net change in body weight, (B) average weekly energy intake, (C) energy efficiency, (D) of male C57BL/6 J mice fed either a low fat (LF) diet, a high fat (HF) diet alone, or HF plus cherry, raspberry, walnut, and green tea (CRWG) after 10 weeks. One-way ANOVA indicated significant differences between diet groups (p* ≤ *0.05). Values that do not share a letter differ (p* ≤ *0.05).*

HF-fed mice was also attenuated with the addition of CRWG to the diet (**Figure 4D**). This observation suggests that, although the CRWG group ate similar amounts of energy as the HF control, the CRWG group was less efficient in converting the diet to energy compared to the HF-fed control. Again, this result may be due to increased thermogenesis initiated by the green tea and walnut consumption.

Fasting blood glucose was measured in weeks 9 and 10. Treatment groups fed HF supplemented with CRWG had a slightly reduced fasting blood glucose, but it was not statistically significant from the HF-fed control group (**Figure 5A**). In addition, results from an insulin sensitivity test performed in week 10 showed the HF + CRWG treatment group was not statistically different from the HF-fed control group (data not shown). However, serum collected from the mice at necropsy was used to perform an ELISA to quantify circulating serum insulin, which was indeed reduced with HF + CRWG supplementation compared to HF diet alone such that it was statistically indistinguishable from the LF control mice (**Figure 5B**). Insulin secretion by β-cells increases in response to insulin resistance to moderate elevated blood glucose [32]. Therefore, initial upregulation of β-cell function in response to a HF diet may be regulated with CRWG supplementation as evidenced by reduced serum insulin concentrations compared to HF diet alone. The HOMA-IR and HOMA-%β calculations support this conclusion, as the HF-fed control mice were assessed to have greater development of insulin resistance and heightened β-cell function compared to the HF + CRWG treatment group (**Figure 5C** and **D**). Tart cherry extracts have been shown to increase nitric oxide (NO) production in cell and animal models [33, 34]. Similarly, (−)-epicatechin, found in green tea, may help maintain healthy blood pressure ranges when consumed with HF diet via restoration of NO bioavailability [35]. Increased NO production may improve insulin sensitivity and decrease glucose

*Green Tea as An Ingredient in Food Combinations Provide Metabolic Improvements DOI: http://dx.doi.org/10.5772/intechopen.107347*

#### **Figure 5.**

*Average fasting blood glucose concentration at week 9 and week 10 (A) serum insulin concentration, (B) homeostasis model assessment of insulin resistance (HOMA-IR), (C) and homeostatic model assessment of β-cell function (HOMA-%β), (D) of male C57BL/6 J mice fed either a low fat (LF) diet, a high fat (HF) diet alone, or HF plus cherry, raspberry, walnut, and green tea (CRWG) after 10 weeks. One-way ANOVA indicated significant differences between diet groups (p* ≤ *0.05). Values that do not share a letter differ (p* ≤ *0.05).*


*1 Values are expressed as means ± SEM of each group, values that do not share a letter differ (p* ≤ *0.05). <sup>2</sup> Low fat diet (LF), high fat diet (HF), or HF plus cherry, raspberry, walnut, and green tea (HF + CRWG).*

#### **Table 1.**

*Organ tissue weights and weights as percentage of final body weight of male C57BL/6 J mice1 .*

concentrations via NO-mediated increases in blood flow, resulting in more efficient uptake of glucose by skeletal muscle [36–38]. Therefore, insulin sensitivity and β-cell function may have been improved by CRWG supplementation because of increased NO production from the tart cherry and green tea components of the diet.

Liver weight to body weight ratios were measured postmortem (**Table 1**). Liver weight and liver weight to body weight ratio of HF-fed control group was significantly higher than the LF-fed mice, while liver weight and liver weight to body weight ratio of HF + CRWG treatment group was reduced such that it was statistically indistinguishable from the LF-fed control group. Additionally, stained cross sections of the liver tissue were used to identify liver lipid accumulation in each group. A representative image for each group is shown in **Figure 6**. White globules are indicative of lipid accumulation, red staining indicates cytosol, and

#### **Figure 6.**

*Hematoxylin–eosin stained liver cross sections of male C57BL/6 J mice fed either a low fat (LF) diet, a high fat (HF) diet alone, or HF plus cherry, raspberry, walnut, and green tea (CRWG) after 10 weeks. Slides were observed under 400x magnification (40x objective) using Nikon Eclipse 50i microscope (Nikon Corporation, Tokyo, Japan) equipped with an Infinity 1-3C camera (Lumenera Corporation, Ottawa, ON, Canada).*

#### **Figure 7.**

*Liver lipid percentage of hematoxylin–eosin-stained liver cross sections of male C57BL/6 J mice fed either a low fat (LF) diet, a high fat (HF) diet alone, or HF plus cherry, raspberry, walnut, and green tea (CRWG) after 10 weeks. One-way ANOVA indicated significant differences between diet groups (p* ≤ *0.05). Values that do not share a letter differ (p* ≤ *0.05).*

dark red circles are the nuclei. Analysis of lipid percentage within the hepatocytes of each group is shown in **Figure 7**. Although not significant, HF + CRWG diet did appear to reduce liver lipid accumulation by approximately 30% compared to HF diet alone. Typically, liver weight increases proportionally to body weight gain, thus these results may suggest potential improvement in liver lipid accumulation in the CRWG supplemented mice.


*Green Tea as An Ingredient in Food Combinations Provide Metabolic Improvements DOI: http://dx.doi.org/10.5772/intechopen.107347*

*1 Values are expressed as means ± SEM of each group, values that do not share a letter differ (p* ≤ *0.05). <sup>2</sup> Some populations sequenced were not complete down to the lowest taxonomic level due to incomplete sequences for these bacteria within the Greengenes database in QIIME2, and thus cannot be differentiated. 3 Low fat diet (LF), high fat diet (HF), or HF plus cherry, raspberry, walnut, and green tea (HF + CRWG).*

#### **Table 2.**

*Relative frequency of the top seven bacteria in the ceca1 .*

The microbiome was quantified from the cecum of each mouse, and variation in the top seven most prolific gut bacteria of each group can be seen in **Table 2**. Among these bacteria, one group was from the phylum Bacteroidetes and family S24–7. This group of bacteria has previously been demonstrated to triple in number in HF diet fed mice that developed diabetes versus HF diet fed mice that were resistant to diabetes development [39]. Our findings showed that LF-fed animals had significantly higher

number of S24–7 bacteria than any of the HF-fed groups, suggesting that these bacteria may be beneficial for maintaining a healthy metabolism. Another study found increased abundance of S24–7 bacteria following remission of colitis, again inferring a positive relationship within the host [40]. This variability between studies may be due to differences in diet formulation, animal model, and microbiome composition analysis and demonstrates the difficulty in reaching a scientific consensus on the beneficial and harmful effects of certain intestinal bacterial on metabolism. The second group of bacteria were from the genus Bacteroides, which have been reported to act commensally in the gut [41]. We observed a marked increase in Bacteroides in the HF + CRWG group such that it was significantly higher than both the LF and HF controls. Added fiber, especially from the raspberry component of the diet, may be a contributing factor to the increased frequency.

One of the bacteria analyzed was from the phylum Verrucomicrobia, specifically Akkermansia muciniphila (A. muciniphila). In the current study, we observed the highest degree of A. muciniphila proliferation in the HF control group, which is inconsistent with previous publications demonstrating its beneficial effect on weight maintenance and metabolism [42, 43]. Furthermore, other reports have showed that polyphenol intake promotes A. muciniphila growth; [44, 45] whereas, in the present study, CRWG fed mice had lower relative abundance than the HF-fed controls. The variability of our result with other literature could have been due to differences in diet administration and/or microbiome analysis.

Four of the analyzed bacteria belonged to the order Clostridiales from the phylum, Firmicutes. Members of this order have been previously associated with obesity; however, there is still vast variability within the lower taxonomic levels [43, 46, 47]. In our findings, we observed an increased shift in the frequency of two of the four Clostridiales bacteria in HF-fed mice. These two bacterial populations belonged to the family Ruminococcaceae. Supplementation with HF + CRWG resulted in reduced frequency of both Ruminococcaceae populations identified when compared to the HF diet alone. Alternatively, we observed a decrease in Clostridiales bacteria belonging to the family Lachnospiraceae in HF-fed mice. Supplementation with HF + CRWG resulted in increased frequency of this population. Increased proliferation of Lachnospiraceae with LF diet as well as polyphenol intake has been previously demonstrated [48]. Thus, our findings may suggest a positive intestinal shift in bacterial strains associated with both lean and obese phenotypes of Clostridiales bacteria with supplementation of CRWG.
