**Regular Consumption of Lipigo**® **Promotes the Reduction of Body Weight and Improves the Rebound E**ff**ect of Obese People Undergo a Comprehensive Weight Loss Program**

### **Marlhyn Valero-Pérez 1, Laura M. Bermejo 2,\*, Bricia López-Plaza 1, Meritxell Aguiló García 3, Samara Palma-Milla <sup>4</sup> and Carmen Gómez-Candela <sup>4</sup>**


Received: 10 June 2020; Accepted: 29 June 2020; Published: 30 June 2020

**Abstract:** Obesity is a global public health problem. Objective: To evaluate the effect of the regular consumption of the product Lipigo® on body weight and rebound effect on overweight/obese subjects undergoing a comprehensive weight loss program. Methods: A randomized, parallel, double-blind, placebo-controlled clinical trial was conducted with male and female subjects presenting a BMI 25–39.9 kg/m2. All subjects underwent a comprehensive weight loss program (WLP) for 12 weeks, which included an individualized hypocaloric diet, physical activity recommendations, nutritional education seminars, and three times a day consumption of the product Lipigo® or Placebo. After-WLP, subjects continued the treatment for 9 months to assess rebound effect. Body weight (BW), BMI, and body composition were measured at the beginning and the end of the WLP, and in the follow-up. Results: A total of 120 subjects (85% women) 49.0 ± 9.5 years old and with a BW of 81.57 ± 13.26 kg (BMI 31.19 <sup>±</sup> 3.44 kg/m2) were randomized and 73 subjects finished the study. At the end of the WLP, there was a tendency toward reduced BW (*p* = 0.093), BMI (*p* = 0.063), and WC (*p* = 0.059) in the treated group. However, subjects with obesity type 1 (OB1) from the treated group significantly reduced body weight (−5.27 <sup>±</sup> 2.75 vs. <sup>−</sup>3.08 <sup>±</sup> 1.73 kg; *<sup>p</sup>* <sup>=</sup> 0.017) and BMI (−1.99 <sup>±</sup> 1.08 vs. <sup>−</sup>1.09 <sup>±</sup> 0.55 kg/m2; *p* = 0.01) compared with placebo. They also presented a minor rebound effect after 9 months with product consumption (−4.19 ± 3.61 vs. −1.44 ± 2.51 kg; *p* = 0.026), minor BMI (−1.61 ± 1.43 vs. <sup>−</sup>0.52 <sup>±</sup> 0.96 kg/m2; *p* = 0.025) and tended to have less fat-mass (−3.44 <sup>±</sup> 2.46 vs. <sup>−</sup>1.44 <sup>±</sup> 3.29 kg; *p* = 0.080) compared with placebo. Conclusions: The regular consumption of the product Lipigo® promotes the reduction of body weight and reduces the rebound effect of obese people after 52 weeks (12 months), mainly in obesity type 1, who undergo a comprehensive weight loss program.

**Keywords:** obesity; overweight; beta-glucans; chitosan; follow up study; weight loss programs; weight gain; weight loss; body weight changes

#### **1. Introduction**

Obesity is a major public health problem that has attained epidemic levels [1]. According to a recent estimate based on population analyses from 195 countries, 603.7 million adults, and 107.7 million children suffered obesity in 2015 worldwide [2]. The mean body mass index (BMI) is calculated to increase by 0.4 and 0.5 points in men and women, respectively, every decade [3]. Metabolic complications of obesity comprise metabolic processes dysfunction such as those controlling blood glucose, lipids, and pressure.

Severe dysregulation of these pathways give rise to a cluster of conditions known as metabolic syndrome [4,5]. As a result, around 3.4 million people die each year due to an overweight or obese condition [6].

Overweight and obesity are partially derived from a dietary energy imbalance stemming from behavioral, biological, and environmental processes [7]. Although not exclusively, obesity is tightly linked with hypercaloric diets, and thus weight reduction strategies are primarily focused on reducing energy consumption (i.e., dieting and reducing fat intake) and enhancing energy expenditure (i.e., regular exercise) [8]. Nevertheless, weight lowering methods based on diet and/or physical activity often fail to ameliorate obesity condition in a long-term period. Furthermore, most studies show weight regain upon medium to long-term follow-up (rebound effect) [9–11]. This phenomenon is likely multifactorial and can be explained by a poor compliance or by behavioral or physiological adaptations and highlights the importance of extending the follow-up period in interventional trials to adequately assess efficacy.

Although a wide range of nutritional interventions pursuing weight loss are nowadays available in the market, evidence of their efficacy is fraught with uncertainty [12], and thus more adequately powered randomized trials with extended follow-up are required. Yeast-derived products as a source of biologically active oral compounds are recently gaining scientific support [13]. In vitro and in vivo data suggest that yeast-derived products, particularly of baker's and brewer's yeast *Saccharomyces cerevisiae*, harbor antioxidant, and free radical-scavenging properties besides the ability to stimulate the immune system [14,15]. Moreover, some clinical trials suggest that yeast hydrolysates may be a useful tool to help manage body weight and fat accumulation [16–18].

Lipigo® is a polysaccharide-rich fraction containing β-glucan, chitin, and chitosan, obtained by a specific hydrolysis procedure of residual *S. cerevisiae* from brewery. A previous randomized, double-blind, placebo-controlled small trial demonstrated a statistically significant benefit of Lipigo® on body weight management in overweight and obese population over 12 weeks [19,20]. In the present study, we sought to replicate the previous findings in a more robust randomized clinical trial, comprising a significantly bigger sample size, a better dietary control, and a longer treatment and follow-up periods. Moreover, we aimed to identify the population group able to obtain the greatest advantage of the oral intake of Lipigo®.

#### **2. Materials and Methods**

The present study was registered at http://clinicaltrials.gov under the number NCT03554525.

#### *2.1. Study Subjects*

Two hundred and twenty-four men and women aged 45–65 years were screened for the present study. Inclusion criteria were as follows: age between 18 and 65 years, BMI of <sup>≥</sup>2 7 and <40 kg/m2, willing to follow a balanced hypocaloric diet to lose weight and perform regulated physical activity, absence of familial or social environment that prevents compliance with dietary treatment, having a suitable understanding of the clinical trial, agreeing to voluntarily participate in the study, and signing the informed consent form. The exclusion criteria were as follows: treatment for CV risk (dyslipidemia, hypertension, diabetes mellitus, and others), mental illness or low cognitive ability, history of severe liver or kidney disease or cancer, pregnancy or lactation, plans to stop smoking or to lose weight, allergy to any of the compounds of Lipigo® as well as subjects who consumed >30 g/day alcohol, subjects were also excluded if they had participated in any program or clinical trials of weight control within the last 6 months.

The trial was approved by the Scientific Research and Ethics Committee of the HULP (Reference number: 4801) in accordance with the International Conference on Harmonization Guidelines on Good Clinical Practice and the ethical standards of the Declaration of Helsinki [21].

#### *2.2. Study Design*

This randomized, double-blinded, placebo-controlled clinical trial with two parallel arms was conducted at the Nutrition Department of La Paz University Hospital (HULP), Madrid (Spain). The total length of the intervention was 52 weeks (12 months). The intervention was divided in two phases: a weight-loss intervention phase (WLP) (12 weeks) in which all subjects were included in a dietary program controlled in 6 visits taking place every two weeks (V0–V6); and a follow-up post-weight lost intervention phase (P–WLP) (40 weeks) controlled in 3 visits taking place every three months (V7–V9). During both phases (WLP and P-WLP) participants were randomized with sex stratification to consume 3 sticks/day of Lipigo® or Placebo (2 sticks just before the lunch and 1 stick just before the dinner).

#### *2.3. Treatments*

#### 2.3.1. Dietary Program

Hypocaloric diets (between 1500 and 3000 Kcal) were prescribed individually for all participants by a dietician expert at the Department of Nutrition of La Paz University Hospital, Madrid. Diets were designed to provide 30% less energy than the total energy expenditure (TEE) at baseline being 1500 kcal the lower limit for caloric restriction. Basal metabolic rate (BMR) was measured by bioelectric impedance Electro Fluid Graph + (EFG) (Akern s.r.l., Florence, Italy). BMR and TEE calculations were corrected according to physical activity and sex as recommended by the World Health Organization (WHO). Proposed hypocaloric diet consisted of 50–55% carbohydrates from total energy intake (added sugars <10%) and 29–34% fat (saturated fatty acids <10%, polyunsaturated fatty acids 5–10% and monounsaturated fatty acids, mainly from virgin olive oil, to complete the lipid profile), according to the recommendations of the Spanish Society of Community Nutrition (SENC, according to its Spanish initials, [22]). Proteins represented 20% of total energy intake (between 0.9–1.8 g/kg of body weight/day), based on body composition benefits observed in a recent meta-analysis [23]. The food intake was distributed in 5 meals: 3 main meals (breakfast, lunch, and dinner) and 2 snacks (mid-morning (11:00 a.m.–11:29 a.m.), and afternoon (5:00–5:29 p.m.)).

The hypocaloric dietary program was prescribed at baseline (V0) of the WLP: participants received a 7-day-meal plan as an example of the individualized diet designed for each one. Moreover, each participant received a food exchange list, allowing the personalization of diet plans according to individual preferences, but ensuring that the resulting menu would provide the individual nutritional requirements calculated. Further dietary counseling was given every two weeks (V1-V5) until the end of the WLP (12th week, V6). Dieticians used all these visits to resolve questions and to motivate participants sufficiently to comply with dietary advice. All subjects were given recommended portion sizes and information on possible food swaps. Moreover, nutrition education and motivational sessions were given by the dietician.

#### 2.3.2. Physical Activity Recommendations

In V0 subjects were instructed to perform moderate physical activity for 1 h at least 3 times a week. The subjects began according to their level of physical activity and gradually increased until they achieved 3 sessions per week or more at the end of WLP.

#### 2.3.3. Nutrition and Health Education Sessions

During the WLP, participants attended 5 nutrition and health education sessions (visits 1 to 5) goals to promote healthy eating and physical activity.

#### 2.3.4. Lipigo® or Placebo

During the WLP and P-WLP, subjects consumed 3 sticks/day of Lipigo® or Placebo (2 sticks just before the lunch and 1 stick just before the dinner)

Lipigo® is a fiber combination obtained from *S. cerevisiae* from the brewery industry. Each stick contained a polysaccharide-rich fraction (909 mg β-glucan, 91 mg chitin-chitosan) and 400 mg excipients. The polysaccharide fraction was obtained by a specific hydrolysis procedure of residual *S. cerevisiae* from brewery patented by DAMM S.A (El Prat del Llobregat, Barcelona, Spain). The nutritional composition per 100 g of dry product was: protein, 1.6 g; fat, 3.7 g; carbohydrates 58.7 g; dietary fiber, 29.9 g; and sodium 0.6 g.

Placebo was composed of 1000 mg maltodextrine and 400 mg excipients.

DAMM S.A. prepared the Lipigo® and the Placebo sticks specifically for this study. Both types of sticks were packaged in box packs of 30 sticks. The packs were labeled as either L1 or L2 to maintain blinded conditions. During every visit in the WLP, subjects received all the sticks needed until the next visit every two weeks. At baseline of the P-WLP (V6) and in the V7 and V8, subjects received all the sticks need to complete three months to the next visit. The sticks received by each participant were assigned according to the randomization.

#### *2.4. Endpoints*

The following analyses and measurements were collected:

#### 2.4.1. General Health Status Variables

During both phases (V0–V9), information about special medical conditions and drug consumption was collected. Furthermore, blood pressure and heart rate were measured using a Spot Vital Signs 420 automatic monitor (Welch Allyn, Madrid, Spain; accuracy ±5 mmHg). Three measurements were taken at 5-min intervals, and the means were calculated. Moreover, blood sample was collected to analyze lipid profile including total cholesterol and LDL-cholesterol at the beginning and at the end of each phase (V0, V6, and V9).

#### 2.4.2. Anthropometrics and Body Composition Variables

Anthropometric measurements were performed at the beginning and at the end of each phase (V0, V6, and V9) using standard techniques, adhering to WHO guidelines [24]. All measurements were made by trained personnel in the morning with the subject barefoot and wearing only underwear. Height was determined using a height meter with an accuracy of 1 mm (range, 80–200 cm). Body weight was measured using a TANITA BC-420 MA (Bio Lógica Tecnología Médica S.L, Barcelona, Spain). The BMI was calculated as body weight ((kg)/(height (m)2). According to their BMI, participants were classified in four categories: normal weight (BMI 18.5–24.9 kg/m 2), class I overweight (BMI 25.0–27.9 kg/m 2), class II overweight (BMI 28–29.9 kg/m 2), class I obesity (BMI 30–34.9 kg/m2), and class II obesity (BMI 35–39.9 kg/m2). Waist circumference (WC) was measured using a Seca 201 steel tape (Quirumed, Valencia, Spain). Variables change between V0 and V6 (Dif V0–V6) and between V6 and V9 (Dif V6–V9) was calculated in order to evaluate the anthropometrics and body composition evolution during WLP and P–WLP respectively. Variables change between V0 and V9 (Dif V0–V9) was calculated to evaluate the rebound effect.

Body composition was determined using a specialized tetrapolar bioelectrical impedance analyzer, the EFG ElectroFluidGraph analyzer (Akern s.r.l., Florence, Italy): total fat mass (TFM), fat-free mass (FFM), and muscle mass (MM) were measured. These body composition data were obtained using regression validated equations of the manufacturer (Akern BodyGram Plus 1.0). These validated equations were derived from previous research [25].

All participants were instructed by the researcher to minimize the BIA affecting factors (not having used diuretic medications in the previous seven days, to have been fasting for at least four hours; not having ingested alcohol or caffeinated beverages in the previous 48 h; having abstained from moderate-intense physical activity in the previous 24 h) [26].

The device was calibrated before measurements of each participant. All participants rested (lying on a bed) for at least five minutes prior to the measurement. Electrodes were placed on the dorsal surface of the wrist and the ankle as well as at the base of the second or third metacarpal-phalangeal joints of hand and foot after the skin was cleaned with an alcohol wipe. The lead wires were attached to the appropriate electrodes and participants were instructed to abduct their limbs from the trunk. Triplicate measurements were conducted, and a median value determined.

#### 2.4.3. Dietary Variables

The diet of each subject was recorded during WLP and P-WLP. In V0, V6–V9 participants filled a 24-h recall over 3 consecutive days recording all food and beverages consumed inside and outside the home, including one weekend day [27]. In V2-V5 diet was recorded using a 24 h record questionnaire. Food weight or household measurements (spoonful, cups, etc.) should be self-reported in both food recording questionnaires. Subjects were previously trained by the researchers to obtain accurate data. All food records were thoroughly reviewed by a nutritionist in the presence of the subject during study visits to ensure that the information collected was complete. The energy and nutritional content of the foods consumed were then calculated using DIAL software (Alce Ingeniería, Madrid, Spain).

#### 2.4.4. Physical Activity Variables

Physical activity was assessed using the International Physical Activity Questionnaire-Short Form (IPAQ-SF) for the Spanish population. IPAQ-SF collects the frequency and duration of vigorous-intensity activity, moderate-intensity activity, and walking activity [28]. The questionnaire was filled in V0, V6–V9. Time spent in vigorous, moderate, and walking activity was calculated by the energy spent for these categories of activity, to produce the total Metabolic Equivalent Task (MET)-minutes of physical activity/week.

#### 2.4.5. Compliance and Adverse Events

Subjects received the exact number of sticks required for each period during study visits. Empty and non-empty sticks should be returned to the investigator in each study visit. Compliance was measured by comparing the number of sticks provided and the number of empty sticks returned. A subject was considered compliant when he/she consumed ≥80% of the sticks provided. Adverse events were documented in all study visits (V1-V9). An adverse event was defined as any unfavorable, unintended effect. All such events were recorded along with the symptoms involved (bad breath, nausea, vomiting, diarrhea, constipation, others).

#### *2.5. Statistical Analysis*

The sample size was calculated taking into account the results obtained in a meta-analysis aiming to evaluate the effect of different non-surgical treatments on body weight maintenance in overweight/obese people [29]. Most of the studies included in such meta-analysis have a sample size of around 80 subjects. Moreover, according to these studies, a potential 33% drop-out range should be considered. Given all the above, the total sample size estimated for the present study was of 120 subjects.

The analysis population included all subjects who completed the WLP and P–WLP stages. Data is presented as mean ±standard deviation (SD) or percent (%) and N. The Kolmogorov-Smirnov test was used to check the normal distribution of the data. Outliers (i.e., lying more than two SDs from the mean) in asymmetric distributions were deemed to reflect true results and were included in the analysis. The Levene's test was used to determine whether the variance presented by the measured variables was homogeneous. The Student t test was used to compare the mean values of normally distributed variables for the two treatment groups and the intragroup analysis. Whereas, Mann–Whitney U test

was used when data were not normally distributed. Differences within groups between V0–V6, V6–V9 and V0–V9 were examined using the Student paired t test when the distribution of the results was normal and the Wilcoxon test when it was not. Additionally, Bonferroni's correction for multiple comparisons was performed. All tests were two-tailed.

A subgroup of analysis was also conducted based on the presence of overweight or obesity at the baseline (V0). Significance was set at two-sided *p* < 0.05. All calculations were performed using SPSS v.21.0 software (SPSS Inc.).

#### **3. Results**

#### *3.1. Recruitment and Study Population*

The study was performed between April 2017 and August 2018. One hundred and twenty apparently healthy subjects (19 men [15.8%], 101 women [84.2%]) were eligible for their inclusion in the study. Subjects were randomized into either the Placebo or Lipigo® group stratified by sex. Twenty-two subjects dropped-out during the WLP:10 in the Placebo group and 12 in the Lipigo® group. At the end of the study, a total of 47 subjects were lost to follow-up (24 in the Placebo group and 23 in the Lipigo® group) due to personal causes (*n* = 27), failure to follow treatment instructions (*n* = 4), health problems not related to clinical trial procedures (*n* = 6), loss of follow-up (*n* = 9), and low product tolerance (*n* = 1). Thus, 73 subjects (6 men [8.2%], 67 women [91.8%]) completed the 12-months study, and only their results were included in the subsequent analyses (Figure 1).

**Figure 1.** CONSORT diagram.

#### *3.2. Baseline Characteristics*

The mean age of the population was 50.9 <sup>±</sup> 8.9 years old. The mean BMI was 31.2 <sup>±</sup> 3.5 kg/m<sup>2</sup> (Obesity type 1, 39.7%). At baseline, no significant differences existed between subjects assigned to the Lipigo® and Placebo groups in terms of their anthropometric, body composition, blood pressure, and biochemical parameters or other variables such as sex, age, or smoking (Table 1).


**Table 1.** Baseline characteristics of the subjects.

Data is represented as mean ± SD. BMI: Body Mass Index; FM: Fat Mass; MM: Muscle Mass; SBP: Systolic Blood Pressure; DBP: Diastolic Blood Pressure.

Despite existing differences between the number of women and men participating in the study, stratified randomization by sex allowed to obtain 2 homogeneous intervention groups.

#### *3.3. Anthropometric and Body Composition Variables*

Both treatment groups decreased body weight, BMI, and waist circumference at the end of WLP or P-WLP although no significant intra-group differences were detected in any anthropometric or body composition variables. No significant intergroup differences were obtained (Lipigo® vs. Placebo, Table 2).


**Table 2.** Anthropometric and body composition variables throughout the study.

Data is expressed as mean ± SD.V: Visit; WLP: Weight Loss Program; P-WLP: Post-weight Loss Program; OB1: Obesity Type 1; BMI: Body Mass Index.

However, when evaluating the changes in anthropometric and body composition variables at the end of WLP (Dif. V0–V6), subjects included in the Lipigo® group showed a non-significant trend towards reducing body weight (*p* = 0.093), BMI (*p* = 0.063), and waist circumference (*p* = 0.059) when compared to Placebo (Table 3).


**Table 3.** Changes in anthropometric and body composition variables throughout the study.

Data is expressed as mean ± SD. Dif V0–V6: Difference during the WLP; Dif V6–V9: Differences during the P-WLP; Dif V0–V9: Rebound effect. WLP: Weight Loss Program; P-WLP: Post-weight Loss Program; BMI: Body Mass Index.

A significant reduction of the lean mass was observed in subjects included in the Lipigo® group when compared to Placebo during the P-WLP phase (Dif V6–V9, Lipigo®: <sup>−</sup>0.59 <sup>±</sup> 1.57 vs. Placebo: 0.25 ± 1.52 kg; *p* = 0.024).

Next, subgroup analyses based on the presence of overweight type 2 (*n* = 32, 43.8%) or obesity type 1 (*n* = 29, 39.7%) and obesity type 2 (*n* = 12, 16.5%) was performed. Subjects with obesity type 1 and 2 enrolled in the Lipigo® group showed a non-significant higher body weight reduction compared to Placebo at the end of WLP (Dif V0–V6, −5.24 ± 2.53 vs. −3.81 ± 2.2 kg *p* = 0.065). Nevertheless, when analyzing subjects with obesity type 1, Lipigo® group, achieved a significantly higher body weight (−5.27 <sup>±</sup> 2.75 vs. <sup>−</sup>3.08 <sup>±</sup> 1.73 kg; *p* = 0.017) and BMI (−1.99 <sup>±</sup> 1.08 vs. <sup>−</sup>1.09 <sup>±</sup> 0.55 kg/m2; *p* = 0.010) reduction vs. Placebo at the end of WLP (Dif V0–V6). In addition, Lipigo® group seemed to have a higher fat mass reduction than Placebo group at the end of WLP (Dif V0–V6) (−3.44 ± 2.46 vs. −1.44 ± 3.29 kg; *p* = 0.080). Moreover, intragroup difference V0 vs. V6 was observed in BMI only in Lipigo® group (Table 4).

During the P-WLP (Dif v6–v9) both groups gain weight, BMI, and fat mass but the OB1 subjects included in the Placebo group gain more in all the anthropometric and body composition variables during this period (40 weeks) than Lipigo® group, although the differences were not significant.

Moreover, obesity type 1 subjects included in the Lipigo® group presented a significant minor rebound effect (Dif V0–V9) vs. Placebo group when looking at body weight change (−4.19 ± 3.61 vs. <sup>−</sup>1.44 <sup>±</sup> 2.51 kg; *<sup>p</sup>* <sup>=</sup> 0.026) and BMI change (−1.61 <sup>±</sup> 1.43 vs. <sup>−</sup>0.52 <sup>±</sup> 0.96 kg/m2; *<sup>p</sup>* <sup>=</sup> 0.025). Lipigo® group seemed to have a minor rebound effect (Dif V0–V9) in fat mass when compared to Placebo group (−1.62 ± 3.45 vs. 0.62 ± 3.80 kg) although the difference is no significant (*p* = 0.121).


**Table 4.** Changes in body weight, BMI, and Fat mass throughout the study in overweight (*n* = 32) and obesity type 1 (*n* = 29) subjects.

Data are expressed as the means ± SDs. V0–V6: Difference during the WLP; V6–V9: Differences during the P-WLP; V0–V9: Rebound effect. WLP: Weight Loss Program; P-WLP: Post-weight Loss Program; BMI: Body Mass Index; OB1: Obesity type 1. #Intragroup difference V0 vs. V6 (*p* = 0.032).

#### *3.4. Dietary Variables*

At baseline, a mean general caloric intake of 1988.24 ± 437.13 kcal was documented. At the end of WLP both groups significantly reduced energy intake (Dif V0–V6) with no significant (NS) differences intergroup (−278.97 ± 460.7 vs. −288.82 ± 497.34 kcal; NS).

The caloric profile changed in both groups at the end of the WLP (Dif V0–V6) with no significant differences respected to baseline between Lipigo® and Placebo groups: carbohydrates (0.64 <sup>±</sup> 8.08 vs. 2.6± 5.98%; NS), proteins (1.56 ± 3.62 vs. 0.95 ± 2.83%; NS) and lipids (−1.78 ± 7.46 vs. −2.43 ± 5.24%; NS).

At the end of the P-WLP, no significant differences (Dif V6–V9) were observed in the caloric profile between Lipigo® and Placebo groups when compared to baseline: carbohydrates (2.36 <sup>±</sup> 7.89 vs. −0.63 ± 6.73%; NS), proteins (−1.64 ± 3.62 vs. −0.36 ± 3.16%; NS), and lipids (−0.29 ± 6.88 vs. 1.01 ± 6.41%; NS).

#### *3.5. Physical Activity Variables*

Regarding physical activity (total METs), both Lipigo® and Placebo groups showed an increase in the WLP (Dif V0–V6, 530.37 ± 90.47 vs. 372.17 ± 981.91 METs; NS) and a decrease in P-WLP (Dif V6–V9, −186.48 ± 1035.93 vs. −224.89 ± 888.28 METS; NS). No significant differences were found between groups.

Specifically, sitting time was reduced in both Lipigo® and Placebo groups at the end of the WLP (Dif V0–V6, −1.05 ± 2.53 vs. −0.56 ± 2.25 h; NS). On the other hand, walking time was as increased in both groups (12.62 ± 45.05 vs. 21.71 ± 55.13 min; NS). No significant differences were found between groups.

#### *3.6. Compliance and Adverse Events*

When analyzing compliance, 88.2 ± 8.04% of the participants showed proper adherence to treatment. No serious adverse events were observed with Lipigo® consumption.

Bad breath, diarrhoea, constipation, bloating, nausea, and heartburn were documented as adverse events throughout the study. No differences were observed between treatment groups during all the study (V0–V9), except for Bloating having a significantly higher incidence in Lipigo® vs. Placebo group in V2 only (24.3 vs. 2.9%; *p* = 0.008).

#### **4. Discussion**

This is the first randomized clinical trial conducted during 52 weeks (12 months) to study the effect of fibers combination supplement obtained from *S. cerevisiae* (Lipigo®) on weight loss after a 12 weeks Weight Loss Program (based in hypocaloric diet, physical activity recommendations, and nutritional education) and on rebound effect during 40 weeks follow-up.

The study demonstrates that three sticks/day of a polysaccharide-rich fraction (909 mg β-glucan, 91 mg chitin-chitosan) decrease significantly body weight and BMI after WLP only in OB1 subjects. Other BMI classifications (overweight or obese type II) reduce body weight and BMI classification but not significantly. Moreover, body weight, BMI, and waist circumference showed a slight reduction in Lipigo® group compared to Placebo after WLP, independently of BMI classification.

In a previous double-blinded, randomized study, Santas et al. showed that Lipigo® was beneficial in OB1 and overweight subjects. In that study, body weight and waist circumference were also reduced when compared to Placebo after 3 months of treatment. Importantly, participants were not enrolled in a body weight loss program [19]. Then, the present study results on body weight and waist circumference reduction support the efficacy of Lipigo® even when combined with a controlled diet.

Furthermore, a possible role of fiber combinations including β-glucan and chitin-chitosan on body weight loss in obese and overweight populations has been widely studied by others. Pittler et al., was the first group to perform a randomized, double-blinded, Placebo-controlled trial providing data on BMI and body weight from overweight subjects after fiber intake [30]. However, their results showed a non-significant effect for 1g/day of chitin-chitosan fibers on body weight or BMI reduction after 4 weeks of treatment. Despite that, other groups have evidenced short-and mid-term effects on body weight and BMI loss after longer administration periods of fibers combination including chitin-chitosan [31]. Two independent groups have published significant body weight and BMI reduction in obese population concomitantly following a calorie restriction and physical activity program [32,33]. The study design followed on both studies is the most similar to the present study. Nevertheless, subjects enrolled in previous studies underwent longer dietary and physical activity programs when compared to the present study (12 or 6 months vs 3 months).

The present study also showed a lean mass decrease after WLP. This result could be associated with a good adherence to the WLP and Lipigo® intake, instead of physical activity recommendations. Significant body weight loss was also achieved in other studies which provided a controlled diet but not physical activity advice [34–36]. However, the treatment period in these studies ranged from 2 to

6 months. In addition, the daily dosage of chitosan fibers (3 g) was supplemented with β-glucan and oat fibers more than betaine hydrochloride and aloe saponins in the Kaats et al. study [36]. Moreover, other studies have shown positive effects of a chitosan compound on the depletion of excess body fat under free-living conditions with minimal loss of fat-free or lean body mass [37,38]. Together, these findings suggest that β-glucan and/or chitosan combination fibers can impact body weight independently of physical activity.

To our knowledge, no previous studies have assessed the effect of β-glucan and chitosan treatments over rebound effect during a follow-up period after a weight loss program. The most highlighted result of this study is that OB1 subjects get the most benefit from consuming the product, not only during a 12 weeks weight loss program, but also decreasing the rebound effect especially in weight and BMI after 52 weeks (12 months) of starting the study including a 9 months follow-up period. This result could be due to the OB1 subjects included in the Lipigo group getting lower gain in anthropometric and body composition variables than the placebo groups during the 9 months follow-up period (P-WLP), although the differences were not significant.

OB1 subjects being the most benefited population from Lipigo® treatment may have several explanations. On the one hand, overweight non-obese participants have been previously associated with less interest in body weight reduction than the obese population [39,40]. This fact may lead to decreased motivation to strictly follow a calorie restriction diet. On the other hand, greater amounts of fat may increase the difficulty of losing weight [41]. This particularity might be the reason OB2 subjects are not getting a benefit from product consumption. However, the small OB2 sample size does not allow any firm conclusions to be drawn in this subgroup.

One limitation of the present study was that body composition was measured by bioimpedance electrical. This method is not a gold standard but is a method relatively simple, inexpensive, and non-invasive technique suitable in field studies. In fact, the European Society of Parenteral and Enteral Nutrition (ESPEN) suggests that BIA works well to evaluate body composition in healthy and ill subjects (including overweight and obese people), using validated BIA equation appropriate to age, sex and race [42]. This aspect has been considered in the present study.

In summary, these results support the efficacy of Lipigo® on body weight reduction as a concomitant and follow-up treatment to a controlled diet. Furthermore, we could for the first time demonstrate an important rebound effect reduction after 52 weeks (12 months) follow up period. Future studies with Lipigo® aim to investigate the ability of the product to improve serum lipid profile and other biochemical and cardiovascular risk factors.

#### **5. Conclusions**

Regular consumption of Lipigo® presented an adjuvant effect on body weight loss in the context of an individualized intervention, with a hypocaloric diet, physical activity practice, and nutritional education sessions. In this sense, the action of Lipigo® is more evident in OB1 participants undergoing a weight control program. Moreover, in OB1 subjects, Lipigo® intake reduced the rebound effect on body weight and BMI after 52 weeks (12 months) of starting the study including a 9 months follow-up period.

Based on the results obtained, the use of fiber mix from *S. cerevisiae* could be a complementary product to be included in weight control programs as well as in follow-up phases in obesity subjects.

**Author Contributions:** Conceptualization, L.M.B., B.L.-P. and C.G.-C.; Methodology, L.M.B.; Formal Analysis, M.V.-P. and B.L.-P.; Investigation, B.L.-P., L.M.B., C.G.-C. and M.V.-P.; Resources, S.P.-M.; Data Curation, M.V.-P.; Writing-Original Draft Preparation, M.V.-P., M.A.G and L.M.B.; Writing-Review and Editing, B.L.-P., L.M.B., C.G.-C., M.V.-P. and M.A.G.; Visualization, S.P.-M., M.A.G.; Supervision, C.G.-C. and L.M.B., B.L.-P.; Project Administration, L.M.B., B.L.-P., and C.G.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the DAMM S.A. group through the project RTC-2016-5317-1 from the RETOS COLABORACIÓN 2016 program of Economy and Competitiveness Ministry of Spain (MINECO).

**Acknowledgments:** We thank DAMM S.A. for the manufacture and supply of the experimental product (Lipigo®) and placebo. Also, thanks to the Biostatistics Department at the HULP for their continuous support.

**Conflicts of Interest:** M.A.G is a full-time employee of AB-Biotics SA, a company doing contract research work for DAMM SA. Other authors declare no conflicts of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Raspberry Ketone [4-(4-Hydroxyphenyl)-2-Butanone] Di**ff**erentially E**ff**ects Meal Patterns and Cardiovascular Parameters in Mice**

### **Dushyant Kshatriya 1,2, Lihong Hao 2, Xinyi Li 1,2 and Nicholas T. Bello 1,2,\***


Received: 1 May 2020; Accepted: 9 June 2020; Published: 11 June 2020

**Abstract:** Raspberry ketone (RK; [4-(4-hydroxyphenyl)-2-butanone]) is a popular nutraceutical used for weight management and appetite control. We sought to determine the physiological benefits of RK on the meal patterns and cardiovascular changes associated with an obesogenic diet. In addition, we explored whether the physiological benefits of RK promoted anxiety-related behaviors. Male and female C57BL/6J mice were administered a daily oral gavage of RK 200 mg/kg, RK 400 mg/kg, or vehicle for 14 days. Commencing with dosing, mice were placed on a high-fat diet (45% fat) or low-fat diet (10% fat). Our results indicated that RK 200 mg/kg had a differential influence on meal patterns in males and females. In contrast, RK 400 mg/kg reduced body weight gain, open-field total distance travelled, hemodynamic measures (i.e., reduced systolic blood pressure (BP), diastolic BP and mean BP), and increased nocturnal satiety ratios in males and females. In addition, RK 400 mg/kg increased neural activation in the nucleus of the solitary tract, compared with vehicle. RK actions were not influenced by diet, nor resulted in an anxiety-like phenotype. Our findings suggest that RK has dose-differential feeding and cardiovascular actions, which needs consideration as it is used as a nutraceutical for weight control for obesity.

**Keywords:** frambinone; meal frequency; open-field test; elevated plus maze; sensory motor gating; pre-pulse inhibition; c-Fos

#### **1. Introduction**

Raspberry ketone (RK; 4-(4-hydroxyphenyl)-2-butanone) is a naturally occurring phenolic compound responsible for the aroma and flavor of raspberries (*Rubus idaeus*) [1,2]. Naturally derived RK is costly to produce, therefore synthetic sources of RK are readily available [3,4]. In the United States, RK is designated as a generally recognized as safe (GRAS) food additive and is listed by the Food and Drug Administration (FDA) as a synthetic flavoring substance [5]. Recently, RK has been marketed as a weight loss agent and an appetite suppressant, and used as single- or multi-ingredient supplement [6–9].

A few studies reveal the efficacy of RK in the prevention of fat accumulation. RK has been shown to reduce lipid accumulation and alter expression of lipolytic and adipogenic genes in 3T3-L1 adipocytes [10–14]. Further, it can increase fat oxidation in vitro and the effect may be mediated by heme oxygenase-1 and brown-like adipocyte formation [13,14]. RK mitigated ovariectomy-induced weight gain [12,14], and reduced high-fat diet induced nonalcoholic steatohepatitis in rats [15]. Adulteration of diet with RK has shown to prevent weight gain [16,17], however, the strong sensory profile of RK could

have potentially affected food intake. Previous work from our lab supports the preventative actions of oral gavage administration of RK against weight gain and fat accumulation in a high-fat diet induced obesogenic environment [18]. Along with management of weight gain, RK can serve cardio protective roles. RK pretreatment reduced isoproterenol-induced cardiac tissue damage and dyslipidemia [19,20]. Because obesity therapeutic agents often are associated with adverse cardiovascular events [21], the RK doses that prevent weight gain should also be examined for their hemodynamic effects. Indeed, weight loss nutraceuticals are typically consumed on a long-term basis for several months, therefore, understanding the cardiovascular risks and benefits are critical for their assessment.

Diet-induced obesity often involves hyperphagia and changes in feeding patterns such as increased meal frequency and meal size [22,23]. Therefore, it is valuable to understand the influence of RK, as a preventative weight loss agent, on meal patterns. The present study was designed to characterize the dose-dependent effect of RK on meal microstructure in male and female mice. In addition, we assessed the interaction of diet and RK dose on hemodynamic parameters. We hypothesized that there would be a dose-dependent preventative effect of RK on the high-fat diet- induced alterations in meal patterns and cardiovascular outcomes. Because weight changes have been associated with anxiogenic or antipsychotic agents [24,25], we also conducted a series of behavioral tests to determine whether the effects were secondary to an anxiogenic effect of RK or impairments in sensory-motor gating.

Previous bioavailability studies from our laboratory have shown that oral RK is rapidly absorbed and a 200 mg/kg dose can peak in the plasma after 15 min in male and female mice [26]. In addition, RK and metabolites were detected in the brain and white adipose tissue of normal weight and diet-induced obese mice [27]. While there is accumulating evidence that RK has direct actions in adipose tissue [10,11,13], the ability of RK to activate brain areas involved in feeding and cardiovascular control has not been explored. One central site that overlaps to regulate these two physiological systems, is the nucleus of the solitary tract (NTS) in the caudal hindbrain [28–30]. Therefore, we examined whether RK, at doses that influence meal patterns and hemodynamic parameters, also activates the NTS and associated area postrema (AP).

#### **2. Materials and Methods**

#### *2.1. Mice*

A total of 270 male and female C57BL/6J mice (7 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were fed ad libitum standard chow (Purina Mouse Diet 5015, St. Louis, MO, USA; 25.34% fat, 19.81% protein, 54.86% carbohydrate, 3.7 kcal/g) upon arrival. One week later, mice (8 weeks old) were equally divided by body weight and were switched to a high-fat diet (HFD; D12451, Research Diets, Inc., New Brunswick, NJ; 45% fat, 20% protein, 35% carbohydrate; 4.73 kcal/g) or sucrose-matched control diet (LFD; D12450H; Research Diets, Inc., New Brunswick, NJ; 10% fat, 20% protein, 70% carbohydrate; 3.85 kcal/g) and fed ad libitum. Water was available at all times. Mice were single-housed, unless otherwise noted, and maintained on a 12-h light and 12-h dark cycle with lights on from 07:00 h to 19:00 h. The animal care protocol was approved by the Institutional animal Care and Use Committee of Rutgers University (OLAW #A3262-01). Coincident with the diet switch, daily oral dosing was initiated in mice with the following treatments: vehicle (VEH; 50% propylene glycol, 40% water, and 10% dimethyl sulfoxide (DMSO) or raspberry ketone (RK, 200 mg/kg or 400 mg/kg; (4[4-hydroxyphenyl]-2-butanone; 99%; cat#178519; Sigma Aldrich). A reference sample from each lot number of raspberry ketone batch was deposited in a secure, climate-controlled repository [31]. Oral dosing of the mice was performed using single-use, sterile plastic feeding tubes (20 ga × 30 mm; cat# FTP-20-30, Instech Laboratories, Plymouth Meeting, PA, USA). Mice were fed the individual diets and oral dosed for 2 weeks, unless otherwise noted. Daily body weight and cumulative food intake were measured throughout the entire dosing period. Daily dosing was performed between 10:00h and 12:00h. Mice were divided into six groups based on diet and

dose; HFD-Vehicle, LFD-Vehicle, HFD-RK (200 mg/kg), LFD-RK (200 mg/kg), HFD-RK (400 mg/kg), and LFD-RK (400 mg/kg), see Supplementary Materials Figure S1.

#### *2.2. Meal Patterns and Meal Microstructures*

Meal microstructures were analyzed in male and female C57BL/6J mice (n = 4 – 16 per group/sex) using the Biological Data Acquisition System (BioDAQ; Research Diets, New Brunswick, NJ, USA). This system utilizes standard shoe-box style cages with a gated front-mounted food hopper. The gated hopper sits upon a sensor that detects net changes in food weight per second. A feeding bout was defined as a change in stable weight of the hopper. Bouts were clustered into meals, defined by an inter-meal interval of 300 s and a minimum of 0.02 g consumed. Meal patterns were measured over the entire two-week period. Data were analyzed and averaged over the 14-day period and presented separately for the dark-cycle (nocturnal) and light-cycle (diurnal). Data recordings in the BioDAQ computer were paused when the mice were handled for measurement of body weight and dosing. Daily data were used to calculate, meal frequency (meals/day), meal size (kcal), meal duration (min/meal), eating rate (kcal/min), and satiety ratio (Inter meal interval (min)/kcal). Mice that demonstrated food shredding were not included in the analyses [18].

#### *2.3. Anxiety-Related and Sensorimotor Gating Behavioral Testing*

After the BioDAQ data collection, mice were kept in their respective diet and daily dosing groups for an additional 8 days and exposed to three behavioral tests. Mice were exposed to the following tests, one teste per day in the following order: open field test (Days 18–20), elevated plus maze (Days 19–21), and pre-pulse inhibition (Days 20–22). Daily dosing and diet regimen continued until completion of all behavioral tests. Daily testing times were staggered and were performed >1 h after mice received their respective daily dose.

#### 2.3.1. Open Field

The open field tests took place in a brightly lit box (40 × 40 × 38 cm) with white floors and luminescent walls. Mice were placed in the center of the apparatus for a ten-minute test period. Measurements of locomotion, exploration, and anxiety were video recorded and scored offline by an individual blinded to the grouping of the study [32].

#### 2.3.2. Elevated Plus Maze

Mice were exposed to the elevated plus maze apparatus for 5 min. The elevated plus maze apparatus has two open arms (25 × 5 × 0.5 cm) across from each other and perpendicular to two closed arms (25 × 5 × 16 cm) with a center platform (5 × 5 × 0.5 cm) and is 50 cm above the floor. At the commencement of the test, each mouse was placed at the junction of the open and closed arms and faced the open arm. The number of entries into open and closed arms, risk assessment, number of feces, and self-grooming behavior were video recorded for five minutes and scored offline by an individual blinded to the study [33].

#### 2.3.3. Pre-Pulse Inhibition

Each pre-pulse inhibition session was conducted in a ventilated soundproof startle chamber (Med Associates Inc., Fairfax, VT, USA) and proceeds with a 5-min acclimation period with 70 dB background noise followed by five successive 110 dB trials for habituation. Six different trial types were then presented: startle pulse (ST110, 110 dB/40 ms), low prepulse stimulus given alone (P74, 74 dB/20 ms), high prepulse stimulus given alone (P90, 90 dB/20 ms), P74 or P90 given 100 ms before the onset of the startle pulse (PP74 and PP90, respectively), and finally a trial where only the background noise was presented in order to measure the baseline movement in the cylinders. All trials were applied

ten times and presented in random order (P74 and P90 were only given five times) and the average inter-trial interval was 15 s (10–20 s) [34].

#### *2.4. Hemodynamic Measures*

In a separate set of male and female C57BL/6J mice (n = 11–12 per group/sex) measurements of hemodynamic parameters were performed using the noninvasive blood pressure CODA system (Kent Scientific, Torrington, CT, USA) system. This computerized system measures systolic and diastolic blood pressure, mean blood pressure, and heart rate via tail volume pressure recordings. Animals were acclimated to the holder and cuff of the CODA system for 5 min before each recording trial. CODA trials were performed 1–2 h after daily dosing of either RK or vehicle. Animals were exposed to 5 consecutive days of recording trials, data from the last 2 days were used for data analysis (i.e., days 13 and 14 of the 2-week dosing period), see Supplementary Materials Figure S2.

#### *2.5. Immunohistochemistry of Area Postrema (AP) and Nucleus of The Solitary Tract (NTS)*

A separate set of male C57BL/6J mice (n = 5–6 per group) were dosed with RK (400 mg/kg) or vehicle for 14 days. On day 14, all mice were returned to their cage without food or water and left undisturbed for 120 min. Mice were then deeply anesthetized with 0.1% euthasol (pentobarbital sodium and phenytoin sodium) solution intraperitoneal (IP), exsanguinated with 0.9% saline, and perfused with 4% paraformaldehyde in phosphate buffered saline (PBS). Brains were extracted and post-fixed for 24 h in 4% paraformaldehyde in PBS, then switched to 20% sucrose in 4% paraformaldehyde until sectioning. Free-floating sections (40 μm) of the forebrain were obtained by using a Leica cryostat (Leica Microsystems, Rijswijk, The Netherlands). Sections were stored in cryoprotectant until immunohistochemistry was performed. Sections were transferred to a new clean plate containing PBS (10 mM phosphate, 150 mM NaCl, pH 7.5). Initial PBS was removed, sections were then washed 3 × 10 min in PBS. Endogenous peroxidases were neutralized with 0.3% H2O2 in dH2O. After a 3 × 10 min PBS wash, sections were incubated in normal goat serum (PK-4001, Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA) with 0.3% Triton-X-100 in PBS for 30 min. c-Fos immunolabeling was performed with a polyclonal rabbit IgG anti c-Fos antibody (ab190289, Abcam, Cambridge, MA, USA), diluted 1:100 in PBS. Tissue sections were incubated overnight (~20 h). Sections were transferred to a new clean plate, washed 3 × 10 min in 0.1% Triton X-100 in PBS, then incubated for 30 min in biotinylated secondary antibody (goat IgG anti-rabbit, PK-4001, Vectastain ABC kit, Vector Laboratories) with 0.3% Triton X-100 in PBS. After 3 × 10 min wash in PBS, sections were incubated in an avidin-peroxidase complex (PK-4001, Vectastain ABC kit, Vector Laboratories) for 45 min. Sections were washed 3 × 10 min in PBS. Staining was performed using nickel diaminobenzidine tetrahydrochloride (Ni-DAB) Chromagen (SK-4100, DAB Peroxidase Substrate Kit, 3,3 -diaminobenzidine, Vector Laboratories) for approximately 30 s to stain Fos-like products black. PBS was added immediately after desired stain was reached and sections were washed in 3 × 10 min in PBS to halt the Ni-DAB reaction. Sections were mounted on gelatin-coated slides (Fisherbrand Double Frosted Microscope Slides, Thermo Fisher Scientific Inc., Bridgewater, NJ, USA) and dehydrated with ethanol and xylenes prior to coverslipping with permount [35], see Supplementary Materials Figure S3.

#### *2.6. Imaging and Quantification of C-Fos Positive Nuclei*

Coronal sections from area postrema (AP) and four rostrocaudal levels of the nucleus of the solitary tract (NTS) were analyzed per animal. The anterior-posterior levels were determined by coordinates from Bregma [36]. The NTS areas consisted of anatomically matched sections from caudal (cNTS; −7.92 mm), at the level of the obex, corresponding to the posterior edge of the AP; medial (mNTS; −7.48 mm) at the maximal extent of the AP; intermediate (iNTS; −7.08 mm), anterior to the AP, corresponding to the maximal extent of the gelatinous subnucleus of the NTS; rostral (rNTS; −6.84 mm) consisting of the area rostral to the gelatinous nucleus and the caudal aspect of the medial vestibular nucleus on the dorsal boundary [36]. This analysis provided a view for the rostral-caudal extent

of c-Fos activation [29]. Imaging was performed using an Olympus FSX-BSW imaging scope and FSX100 software (Olympus videoscope, Tokyo, Japan). Quantification was performed by identifying c-Fos positive black nuclei using Image J software system (NIH, Bethesda, MD, USA) image analysis software. Three anatomically matched tissue slices of each region (unilateral) of each mouse were used in data analysis. Cells were counted by two observers blinded to the experimental conditions [35].

#### *2.7. Statistical Analyses*

Data are presented as means ± standard error of the mean (SEM). Separate two-way analysis of variance (ANOVA) or two-way ANOVA with repeated measures were performed to determine the effects of treatment conditions on individual measured parameters. When justified, Newman-Keuls post-hoc tests were performed unless otherwise specified. All statistical and power analyses were performed using Statistica 7.1 software (StatSoft; Dell Inc, Round Rock, TX, USA) and significance was set at α = 0.05.

#### **3. Results**

#### *3.1. Bodyweights Over 14-Day Dosing and Diet Acesss*

At the start of the experiments and group assignment, males baseline body weights were 23.2 ± 0.3 g for HFD-Vehicle, 23.3 ± 0.5 g for HFD-RK (200 mg/kg), 23.3 ± 0.5 g for HFD-RK (400 mg/kg), 23.1 ± 0.4 g for LFD-Vehicle, 23.6 ± 0.4 g for LFD-RK (200 mg/kg), and 23.1 ± 0.6 g for LFD-RK (400 mg/kg). For body weight gain over the 14-day diet and dosing regimen, there were effects for diet (F (1, 58) = 9.6, *p* < 0.005) and dose (F (2, 58) = 6.9, *p* < 0.005), days (F (12, 696) = 37.9, *p* < 0.005) and dose x days (F (24, 696) = 2.2, *p* < 0.001). There was increased body weight gain in the HFD fed male mice (*p* < 0.05). The 400 mg/kg dose produced an overall reduction in body weight gain over the 14-days for males (*p* < 0.05) compared with vehicle dose. There were body weight reductions with 400 mg/kg, compared with vehicle, for days 2–5 of dosing (*p* < 0.05 for all days), see Figure 1A. For females, baseline body weights were 18.3 ± 0.5 g for HFD-Vehicle, 18.1 ± 0.3 g for HFD-RK (200 mg/kg), 18.2 ± 0.4 g for HFD-RK (400 mg/kg), 18.4 ± 0.4 g for LFD-Vehicle, 17.8 ± 0.5 g for LFD-RK (200 mg/kg), and 17.9 ± 0.4 g for LFD-RK (400 mg/kg). For body weight gain there were effects for diet (F (1, 41) = 4.2, *p* < 0.05), dose (F (2, 41) = 3.2, *p* < 0.05), and days (F (12, 492) = 43.0, *p* < 0.0005). There was an increase in body weight gain in the HFD fed female mice (*p* < 0.05). The 400 mg/kg dose produced a reduction in body weight gain, compared with 200 mg/kg, over the 14-days for females (*p* < 0.05), see Figure 1B.

#### *3.2. Meal Pattern Analysis Over 14 Days Dosing and Diet Access*

For nocturnal meal frequency, in males there were diet (F (1, 54) = 22.9, *p* < 0.005) and dose (F (1, 54) = 13.5, *p* < 0.005) effects. More meals were consumed in male mice with HFD than LFD (*p* < 0.001). Fewer meals were consumed by RK 200 mg/kg (*p* < 0.001) and RK 400 mg/kg (*p* < 0.05) dosed mice, compared with vehicle. In females, there were diet (F (1, 34) = 46.9, *p* < 0.001) and dose (F (2, 34) = 11.9, *p* < 0.05) effects. More meals were consumed in female mice with HFD than LFD (*p* < 0.05). More meals were consumed with RK 200 mg/kg compared with RK 400 mg/kg (*p* < 0.001) and vehicle (*p* < 0.05), see Figure 2A. For nocturnal meal size, in males there was a dose (F (2, 54) = 7.6, *p* < 0.05) effect. Meal sizes were increased by RK 200 mg/kg (*p* < 0.05). In females, there were diet (F (1, 34) = 9.6, *p* < 0.001) and dose (F (2, 34) = 4.3, *p* < 0.05) effects. Meal sizes were increased by LFD (*p* < 0.05) and decreased by RK 200 mg/kg compared with RK 400 mg/kg and vehicle (*p* < 0.05 for both). For nocturnal meal duration, in males there were diet (F (1, 54) = 56.5, *p* < 0.001) and dose (F (2, 54) = 11.3, *p* < 0.001) effects. Meal duration was shorter with HFD compared with LFD (*p* < 0.005) and RK 200 mg/kg dose increased meal duration (*p* < 0.001). In females, there was a diet effect (F (1, 34) = 30.7, *p* < 0.005) for meal duration. Meal duration was shorter with HFD (*p* < 0.05), see Figure 2C. For nocturnal eating rate, in males there were diet (F (1, 54) = 18.9, *p* < 0.001) and

dose (F (2, 54) = 4.9, *p* < 0.05) effects. There was an increase in eating rate with HFD (*p* < 0.001), and RK 400 mg/kg had a higher eating rate compared with VEH and RK 200 mg/kg (*p* < 0.05 for both). In females, there were diet (F (1, 32) = 43.6, *p* < 0.005) and diet X dose (F (2, 32) = 4.4, *p* < 0.05) effect for eating rate. In HFD, there was increase in eating rate compared with LFD (*p* < 0.005) and 200 mg/kg had the highest eating rate of all groups (*p* < 0.05), see Figure 2D. For nocturnal satiety ratio, in males, there were diet (F (1, 54) = 5.2, *p* < 0.05) and dose (F (2, 54) = 6.2, *p* < 0.005) effects. Satiety ratio was higher in male mice fed HFD than LFD (*p* < 0.05). RK 200 mg/kg and 400 mg/kg increased satiety ratio compared with vehicle (*p* < 0.05 for both). For satiety ratio in females, there was a dose effect (F (2, 32) = 5.9, *p* < 0.05), with higher satiety ratio in female mice receiving RK 400 mg/kg (*p* < 0.05), see Figure 2E.

**Figure 1.** Body weight change in grams over the 14 days of diet access and oral RK dosing compared with baseline. Data are represented as means ± standard error of the mean (SEM). High-fat diet (45% fat; HFD, solid symbols) and low-fat diet (10% fat; LFD, open symbols) and oral gavage with raspberry ketone (RK) or vehicle (50% propylene glycol, 40% water, and 10% dimethyl sulfoxide; DMSO) for 14 days. Comparisons are separate within each sex. (**A**): Males, (**B**): Females. \* indicates overall diet difference from LFD (*p* < 0.05), # indicates overall difference from all other doses (*p* < 0.05), + indicates overall daily dose difference from VEH dose (*p* < 0.05), & indicates overall dose difference from 200 mg/kg dose (*p* < 0.05). HFD-Vehicle (males: n = 16, females n = 8), HFD-RK (200 mg/kg) (males: n = 8, females: n = 8), HFD-RK (400 mg/kg) (males: n = 8, females: n = 7), LFD-Vehicle (males: n = 16, females: n = 8) LFD-RK (200 mg/kg)(males: n = 8, females: n = 8), and LFD-RK (400 mg/kg)(males: n = 8, females: n = 8).

**Figure 2.** Average meal patterns over the 14 days of daily oral RK and diet access. Meal patterns are from the nocturnal period. Data are represented as means ± standard error of the mean (SEM). High-fat diet (45% fat; HFD, solid lines) and low-fat diet (10% fat; LFD, stripped line) and oral gavage with raspberry ketone (RK) or vehicle (50% propylene glycol, 40% water, and 10% dimethyl sulfoxide; DMSO) for 14 days. Comparisons are separate within each sex. Meal patterns were averaged over fourteen days of dosing and diet exposure. (**A**): Meal frequency; average number of meals each day, (**B**): meal size (kcal), (**C**): meal duration (min), (**D**): meal eating rate (kcal/min), and (**E**): satiety ratio (inter meal interval in min/kcal). \* indicates overall diet difference from LFD (*p* < 0.05), \*\* indicates overall diet difference from LFD (*p* < 0.005), # indicates overall dose difference from vehicle (*p* < 0.05), ## indicates overall dose difference from vehicle (*p* < 0.01), && indicates overall dose difference from RK200 dose (*p* < 0.001), & indicates overall dose difference from RK200 dose (*p* < 0.05). HFD-Vehicle (males: n = 14, females n = 4, HFD-RK (200 mg/kg) (males: n = 8, females: n = 6), HFD-RK (400 mg/kg) (males: n = 7, females: n = 6), LFD-Vehicle (males: n = 16, females: n = 8) LFD-RK (200 mg/kg)(males: n = 7, females: n = 8), and LFD-RK (400 mg/kg)(males: n = 8, females: n = 8).

There were no effects of treatment on diurnal meal size, meal duration, and satiety ratio, in male mice. For meal frequency, there was diet X dose (F (2, 54) = 3.5, *p* < 0.05) effect, with an increased meal number in male mice receiving HFD and RK 200 mg/kg. There was a diet effect on eating rate (F (1, 54) = 11.6, *p* < 0.05), with a higher rate in HFD fed mice. There were no effects of treatment on diurnal meal frequency, satiety ratio and eating rate in female mice. There was an effect of dose (F (2, 34) = 6.9, *p* < 0.05) on diurnal meal size, with an increase in RK 400 mg/kg compared with RK 200 mg/kg and vehicle (*p* < 0.05 for both). For diurnal meal duration in female, there was an effect of diet (F (1, 34) = 18.7, *p* < 0.005), with shorter meals in HFD compared with LFD fed mice.

At the end of the 14-day meal pattern analysis the male cumulative food intake was 147.3 ± 2.8 kcal for HFD-Vehicle, 137.3 ± 2.2 kcal for HFD-RK (200 mg/kg), 136.2 ± 4.3 kcal for HFD-RK (400 mg/kg), 130.9 ± 3.2 kcal for LFD-Vehicle, 135.5 ± 2.3 kcal for LFD-RK (200 mg/kg), and 132.1 ± 13.6 kcal for LFD-RK (400 mg/kg). The female cumulative food intake was 177.5 ± 12.3 kcal for HFD-Vehicle, 175.3 ± 4.7 kcal for HFD-RK (200 mg/kg), 156.1 ± 7.1 kcal for HFD-RK (400 mg/kg), 152.2 ± 9.4 kcal for LFD-Vehicle, 149.5 ± 3.1 kcal for LFD-RK (200 mg/kg), and 133.1 ± 6.6 kcal for LFD-RK (400 mg/kg). There was no effect of treatment on cumulative food intake.

#### *3.3. Open-Field After 14 Days of Dosing and Diet Access*

For time spent in center, in males there was an effect of dose (F (2, 55) = 5.6, *p* < 0.01). Male RK 200 mg/kg mice spent more time in the center of the open field than VEH (*p* < 0.05) and RK 400 mg/kg (*p* < 0.01), see Figure 3A. There was a significant effect of dose (F (2, 55) = 5.8, *p* < 0.01) on the number of entries into the outer zone in male mice, with RK 400 mg/kg mice entering the outer zone fewer times than VEH (*p* < 0.01). Similarly, dose (F (2, 41) = 5.2, *p* < 0.05) had a significant effect on number of entries of female mice into outer zone, with RK 400 mg/kg mice entering the outer zone fewer times than VEH (*p* < 0.05) and RK 200 mg/kg (*p* < 0.05) mice, see Figure 3B. Whereas, in male mice both dose (F (2, 55) = 6.7, *p* < 0.01) and diet (F (1, 55) = 10.0, *p* < 0.01) had an effect on total distance travelled. RK 400 mg/kg treated male mice travelled less than VEH (*p* < 0.01), and HFD mice overall travelled less than LFD mice (*p* < 0.05). Dose (F (2, 41) = 3.4, *p* < 0.05) had a significant effect on total distance travelled by female mice, with RK 400 mg/kg mice travelling less than RK 200 mg/kg, see Figure 3C.

#### *3.4. Elevated Plus Maze After 14 Days of Dosing and Diet Access*

Dose (F (2, 56) = 4.1, *p* < 0.05) had a significant effect on time spent in the open arms by male mice, with RK400 mice spending less time than VEH (*p* < 0.05) in open arms, see Figure 4A. There was a significant effect of dosing (F (2, 56) = 6.5, *p* < 0.01) on number of entries into open arms, with RK400 mice entering the open arms less frequently than VEH (*p* < 0.01), see Figure 4C. There were no significant effects of dosing or diet on elevated plus maze parameters of female mice, Figure 4A–D.

**Figure 3.** Open-field test during daily oral RK and diet access. Open field tests were performed during days 18–20 of daily dosing and diet access. Data are represented as means ± standard error of the mean (SEM). High-fat diet (45% fat; HFD, solid lines) and low-fat diet (10% fat; LFD, stripped line) and oral gavage with raspberry ketone (RK) or vehicle (50% propylene glycol, 40% water, and 10% dimethyl sulfoxide; DMSO). Comparisons are separate within each sex. (**A**): Time spent in center of open field (s), (**B**): number of entries into outer zone, (**C**): total distance travelled (m). \* indicates overall diet difference from LFD (*p* < 0.05), # indicates overall dose difference from all other doses (*p* < 0.05), + indicates overall dose difference from VEH dose (*p* < 0.05), ++ indicates overall dose difference from VEH dose (*p* < 0.01), & indicates overall difference from RK200 dose (*p* < 0.05). HFD-Vehicle (males: n = 15, females n = 8), HFD-RK (200 mg/kg) (males: n = 7, females: n = 8), HFD-RK (400 mg/kg) (males: n = 8, females: n = 7), LFD-Vehicle (males: n = 16, females: n = 8) LFD-RK (200 mg/kg) (males: n = 7, females: n = 8), and LFD-RK (400 mg/kg)(males: n = 8, females: n = 8).

**Figure 4.** Elevated plus maze behavior test during daily oral RK and diet access. Elevated plus maze tests were performed during days 19–21 of daily dosing and diet access. Data are represented as means ± standard error of the mean (SEM). High-fat diet (45% fat; HFD, solid lines) and low-fat diet (10% fat; LFD, stripped line) and oral dosed with raspberry ketone (RK) or vehicle (50% propylene glycol, 40% water, and 10% dimethyl sulfoxide; DMSO). Comparisons are separate within each sex. (**A**): Time spent in open arms (s), (**B**): time spent in closed arms (s), (**C**): number of entries into open arms, (**D**): number of entries into closed arms. + indicates overall dose difference from VEH dose (*p* < 0.05), ++ indicates overall dose difference from VEH dose (*p* < 0.01). HFD-Vehicle (males: n = 15, females n = 8), HFD-RK (200 mg/kg) (males: n = 7, females: n = 8), HFD-RK (400 mg/kg) (males: n = 8, females: n = 7), LFD-Vehicle (males: n = 16, females: n = 8) LFD-RK (200 mg/kg)(males: n = 8, females: n = 8), and LFD-RK (400 mg/kg)(males: n = 8, females: n = 8).

#### *3.5. Pre-Pulse Inhibition After 14 Days of Dosing and Diet Access*

One day after completing the open field test, the mice underwent testing for the pre-pulse inhibition of an acoustic startle. This was measured in mice after three weeks of their respective daily treatment. Data were analyzed separately for each sex using a two-way ANOVA, with diet and dose as variables. There were no significant effects of diet or dose on parameters of startle response in both sexes, see Figure 5A–B.

**Figure 5.** Pre-pulse inhibition of an acoustic startle response behavior test. Pre-pulse inhibition tests were performed during days 21–22 of daily dosing and diet access. Data are represented as means ± standard error of the mean (SEM). High-fat diet (45% fat; HFD, solid lines) and low-fat diet (10% fat; LFD, stripped line) and oral dosed with raspberry ketone (RK) or vehicle (50% propylene glycol, 40% water, and 10% dimethyl sulfoxide; DMSO). Comparisons are separate within each sex. (**A**): Startle amplitude, (**B**)**:** pre-pulse inhibition. HFD-Vehicle (males: n = 15, females n = 8), HFD-RK (200 mg/kg) (males: n = 7, females: n = 8), HFD-RK (400 mg/kg) (males: n = 8, females: n = 7), LFD-Vehicle (males: n = 16, females: n = 8) LFD-RK (200 mg/kg)(males: n = 8, females: n = 8), and LFD-RK (400 mg/kg)(males: n = 8, females: n = 8).

#### *3.6. Hemodynamics Di*ff*erences After 14 Days of Dosing and Diet Access*

For systolic blood pressure (SBP), in males, there was a dose effect (F (2, 62) = 6.2, *p* < 0.005) with a reduction in SBP with 400 mg/kg (*p* < 0.05). In females, there was a dose effect (F (2, 62) = 5.4, *p* < 0.01) for SBP. In females, the 400 mg/kg also reduced SBP (*p* < 0.05), see Figure 6A. For diastolic blood pressure (DBP), in males there was a dose effect (F (2, 62) = 5.2, *p* < 0.01) with a reduction in DBP with 400 mg/kg (p < 0.05). In females, there was a dose effect (F (2, 62) = 4.0, *p* < 0.05) with a reduction in DBP with 400 mg/kg (*p* < 0.05), see Figure 6B. For mean blood pressure (MBP), in males, there was a dose effect (F (2, 62) = 5.7, *p* < 0.05) with a reduction in 400 mg/kg (*p* < 0.05). Similar effects were observed with dose (F (2, 62) = 4.4 *p* < 0.05) in females with a reduction in MBP (*p* < 0.05), see Figure 6C. For heart rate, in males, there was a dose effect (F (2, 61) = 3.4, *p* < 0.05) with 400 mg/kg only different from 200 mg/kg (*p* < 0.05). In females, there was also a dose effect (F (2, 61) = 14.9, *p* < 0.0005) with 400 mg/kg reducing heart rate (*p* < 0.005), see Figure 6D.

**Figure 6.** Hemodynamic measurements during the last two days of the 14-day daily oral RK and diet access. Blood pressure and heart rate were measured by noninvasive tail-cuff method. Data are represented as means ± standard error of the mean (SEM). High-fat diet (45% fat; HFD, solid lines) and low-fat diet (10% fat; LFD, stripped line) and oral dosed with raspberry ketone (RK) or vehicle (50% propylene glycol, 40% water, and 10% dimethyl sulfoxide; DMSO) for 14 days. Comparisons are separate within each sex. Hemodynamic measurements were averages over the last 2 days of dosing and diet exposure. (**A**): Systolic blood pressure (mm Hg), (**B**): diastolic blood pressure (mm Hg), (**C**): mean blood pressure (mm Hg), (**D**): heart rate (beats per minute; BPM). # indicates overall dose difference from vehicle and RK 200 mg/kg (*p* < 0.05), ## indicates overall dose difference from vehicle and RK 200 mg/kg (*p* < 0.005) & indicates overall dose difference from RK200 dose (*p* < 0.05). HFD-Vehicle (males: n = 12, females n = 12), HFD-RK (200 mg/kg) (males: n = 12, females: n = 11), HFD-RK (400 mg/kg) (males: n = 11, females: n = 11), LFD-Vehicle (males: n = 11, females: n = 11) LFD-RK (200 mg/kg)(males: n = 11, females: n = 11), and LFD-RK (400 mg/kg) (males: n = 11, females: n = 12).

#### *3.7. c-Fos Immunopositive Cells of The Caudal Hindbrain in Mice Receiving Vehicle or Raspberry Ketone (400 mg*/*kg) and Diet Access*

In the AP, there were no significant effects for dose, diet, or dose x diet, see Table 1. In the NTS, there was only a significant effect for dose (F (1, 19) = 4.7, *p* < 0.05) with mice receiving RK having a greater number of immunopositive cells (*p* < 0.05), see Table 1. The regions with the highest number of immunopositive cells were the mNTS and iNTS, see Figure 7.

**Table 1.** Average immunoreactive c-Fos counts in the area postrema (AP) and nucleus of the solitary tract (NTS) from mice orally dosed with vehicle or raspberry ketone (400 mg/kg). Mice were fed high-fat diet (HFD; 45% fat) or low-fat diet (LFD: 10% fat) and orally dosed vehicle (VEH) or raspberry ketone (400 mg/kg) for 14 days. Mice were euthanized on day 14, 120 min after respective dosing. Immunopositive cell counts are means ± SEM.

