Evaluation of a Dietary Supplementation Combining Protein and a Pomegranate Extract in Older People: A Safety Study

Malnutrition is a highly prevalent condition in older adults. It is associated with low muscle mass and function and increased occurrence of health problems. Maintaining an adequate nutritional status as well as a sufficient nutrient intake in older people is therefore essential to address this public health problem. For this purpose, protein supplementation is known to prevent the loss of muscle mass during aging, and the consumption of various pomegranate extracts induces numerous health benefits, mainly through their antioxidant properties. However, to our knowledge, no study has to date investigated the impact of their combination on the level of malnutrition in older people. The objective of this preliminary study was thus to evaluate the safety of a combination of protein and a pomegranate extract in healthy subjects aged 65 years or more during a 21-day supplementation period. Thirty older participants were randomly assigned to receive protein and a pomegranate extract (Test group) or protein and maltodextrin (Control group) during a 21-day intervention period. The primary outcomes were the safety and tolerability of the supplementation defined as the occurrence of adverse events, and additional secondary outcomes included physical examination and hematological and biochemical parameters. No serious adverse events were reported in any group. Changes in physical, hematological, and biochemical parameters between the initial screening and the end of the study were equivalent in both groups, except for glutamate-pyruvate transaminase (GPT) and prealbumin, for which a decrease was observed only in the Test group. Our initial findings support the safety of the combination of protein and a pomegranate extract in healthy elderly people. Future clinical trials on a larger sample and a longer period are needed to determine the efficacy of this combination.


Introduction
Malnutrition in older adults has been recently identified as a challenging public health problem associated not only with a high mortality and morbidity rate but also with physical, psychological, and functional decline [1,2]. In particular, protein-energy malnutrition (PEM), resulting from a decrease in protein and energy intake, has been associated with a number of health problems such as sarcopenia [3,4]. Sarcopenia is characterized by a gradual loss of muscle mass leading to a decrease in muscle strength and physical performance. This condition can have functional consequences, such as falls and fractures, and worsens the health and the quality of life of the elderly [5].
The treatment of malnutrition requires early identification and efficient intervention, both in hospitalized patients and community-dwelling older adults. Higher protein intake recommendations are made to older adults suffering from malnutrition. Beyond its role in the accretion of muscle mass, protein has been identified as a key nutrient for elderly people. Slightly higher protein intake than usually recommended may improve muscle health

Participants
A total of 39 participants were assessed for eligibility, and 30 participants were randomized between December 2021 and February 2022 into the Test (n = 15) or the Control (n = 15) groups ( Figure 1). One participant from the Test group dropped out of the study for personal reasons. Participants were recruited by posters, mail, social networks, and local newspapers. To be included, the participants had to meet the following criteria: woman or man aged over 65 years; presenting a BMI from 20 to 30; and in good general health as evidenced by medical history and physical examination. The participants were excluded if they presented one of the following exclusion criteria: severe psychiatric disorder or disease within 6 months before inclusion (such as depression, bipolar disorder, severe anxiety, psychosis, schizophrenic disorders, or dementia) or a severe neurologic trouble (such as Alzheimer's, autism, or Parkinson's disease); cancer within less than 3 years before the screening visit (except basal cell skin cancer or squamous cell skin cancer); a severe gastro-intestinal, hepatic, respiratory, kidney, or cardiovascular disorder or severe infection (such as HIV or hepatitis); uncontrolled hormonal disorders (such as thyroid problems or Cushing's syndrome); uncontrolled type 1 or 2 diabetes; known allergy or intolerance to one of the components of the administered products; and participants consuming ONS or protein supplements up to one month before the screening.
Nutrients 2022, 14, x FOR PEER REVIEW 3 of 10 years before the screening visit (except basal cell skin cancer or squamous cell skin cancer); a severe gastro-intestinal, hepatic, respiratory, kidney, or cardiovascular disorder or severe infection (such as HIV or hepatitis); uncontrolled hormonal disorders (such as thyroid problems or Cushing's syndrome); uncontrolled type 1 or 2 diabetes; known allergy or intolerance to one of the components of the administered products; and participants consuming ONS or protein supplements up to one month before the screening. Figure 1. Flow chart of the study samples, including the number of participants who were screened, underwent randomization, completed the study treatment, and were analyzed for the primary and secondary outcomes.
All selected participants provided written informed consent. The trial was approved by the local ethical committee and was carried out in accordance with the Declaration of Helsinki and the Good Clinical Practice guidelines as required by the following regulations: the Belgian law of 7 May 2004 regarding experiments on human beings and the EU Directive 2001/20/EC on Clinical Trials (registration at clinicaltrials.gov as NCT05527249).

Study Design and Randomization
This was a randomized double-blind placebo-controlled interventional study. A prescreening was proposed with an online questionnaire sent by email. Then, the screening visit, comprising a physical examination (including measurement of body weight, height, heart rate, and blood pressure) and a blood sampling, was organized within 4 weeks before subject inclusion. The participants who met all the criteria were randomly assigned to the Test or the Control groups. Both groups were instructed (1) to consume 20 g protein mixed into vegetable milk, orange juice, or soup and (2) to ingest one capsule with a glass of water every day before lunch for 3 weeks. The participants of both groups received the same protein powder (Fresubin ® ) with a neutral taste and containing 100% whey protein.
The capsule contained 650 mg pomegranate extract (Oxylent GR) in the Test group and 650 mg maltodextrin (Delical, BS Nutrition) in the Control group. Each capsule of pomegranate extract contained at least 65 mg punicalagins (>10%), 260 mg polyphenols (>40%), and more than 13 mg ellagic acid (>2%). The dose of 650 mg was chosen based on the efficacy of this dose to improve muscle strength recovery after eccentric exercise [22]. The capsules containing maltodextrin or pomegranate extract were prepared by a pharmacist. The appearance, i.e., white color, did not distinguish the 2 capsules. All products were

Study Design and Randomization
This was a randomized double-blind placebo-controlled interventional study. A prescreening was proposed with an online questionnaire sent by email. Then, the screening visit, comprising a physical examination (including measurement of body weight, height, heart rate, and blood pressure) and a blood sampling, was organized within 4 weeks before subject inclusion. The participants who met all the criteria were randomly assigned to the Test or the Control groups. Both groups were instructed (1) to consume 20 g protein mixed into vegetable milk, orange juice, or soup and (2) to ingest one capsule with a glass of water every day before lunch for 3 weeks. The participants of both groups received the same protein powder (Fresubin ® ) with a neutral taste and containing 100% whey protein. The capsule contained 650 mg pomegranate extract (Oxylent GR) in the Test group and 650 mg maltodextrin (Delical, BS Nutrition) in the Control group. Each capsule of pomegranate extract contained at least 65 mg punicalagins (>10%), 260 mg polyphenols (>40%), and more than 13 mg ellagic acid (>2%). The dose of 650 mg was chosen based on the efficacy of this dose to improve muscle strength recovery after eccentric exercise [22]. The capsules containing maltodextrin or pomegranate extract were prepared by a pharmacist. The appearance, i.e., white color, did not distinguish the 2 capsules. All products were stored in a closed room at a controlled temperature between 18 and 25 • C and protected from light. Study outcomes were assessed at baseline (V1), in the middle of the intervention (day 10; V2), and at the end of the intervention (day 21; V3). Protocol deviations and subjects' withdrawals were monitored regularly during the study. The database was locked, and the blinding was broken after completion of the whole quality control of the data.

Safety Measurements
The primary outcomes were safety and tolerability of the protein/pomegranate combination defined as occurrence of adverse events. All these events were recorded from the signature of the informed consent to the end of the study.
The secondary outcomes were changes in physical examination, including weight, heart rate, and systolic and diastolic blood pressure; changes in hematological parameters, including hemoglobin and hematocrit levels and white blood cell, red blood cell, and platelet counts; and changes in biochemical parameters (Lims Mbnext group), including uric acid, creatinine, total bilirubin, glutamate oxaloacetic transaminase (GOT), glutamate pyruvate transaminase (GPT), total cholesterol, triglyceride, sodium, potassium, total protein, prealbumin, urea, blood glucose, alkaline phosphatase, gamma-glutamytranspeptidase (GGT), lactate dehydrogenase (LDH), and C-reactive protein (CRP). Compliance to protein and pomegranate/maltodextrin ingestion was assessed at the end of the study by weighing the remaining quantity of powder and capsules and comparing to the quantity given at the beginning of the study.

Statistical Analyses
Given the pilot nature of this study, a sample size of 30 participants was determined based on feasibility considerations. All the continuous variables are presented as mean ± SD. Statistical analyses were performed using the software systems SAS 9.4 (SAS Institute Inc., Cary, NC, USA). Baseline differences in demographic characteristics between the groups were examined using χ 2 tests and independent t tests. Differences in physical, hematological, and biochemical parameters between groups (Control and Test) at each visit (V1, V2, and V3) were analyzed using a linear mixed model for repeated measurements with subjects as the random variable, and groups, visits, and their interaction as fixed independent variables. As variables were not normally distributed, a logarithmic transformation was applied, and when appropriate, post hoc comparison was performed using the Wilcoxon matched-pairs signed-rank test for analyses of within-group differences and the Wilcoxon Mann-Whitney two-sample test for analyses of differences between groups. All analyses were conducted at an alpha level of 0.05.

Participants' Characteristics
The Test group was composed of 5 men and 9 women, and the mean age was 68.4 ± 2.4 years, while the Control group was composed of 9 men and 6 women, and the mean age was 68.7 ± 2.8 years. There were no significant differences in age, gender ratio, weight, BMI, or blood pressure between the two groups at baseline.

Primary Outcomes
The protein/pomegranate combination was well tolerated, with no serious adverse event reported and no difference in the mild and moderate adverse events reported by the Test (n = 5) and the Control (n = 6) groups. None of these mild or moderate adverse effects, including lung infection, COVID-19, and painful tooth extraction, were deemed to be related to the supplementation (Table 1).

Secondary Outcomes
Data related to physical, hematological, and biochemical parameters are presented in Table 2 and Figures 2 and 3.

Secondary Outcomes
Data related to physical, hematological, and biochemical parameters are presented in Table 2 and Figures 2 and 3.

Physical Parameters
The systolic (−4.2%, p = 0.046, Figure 2A) and diastolic (−7.5%, p = 0.003, Figure 2B) blood pressures decreased between V1 and V3 in both the Control and Test groups, with no difference between the two groups. No difference between conditions was observed in the weight, the BMI, or the heart rate ( Table 2).

Hematological Parameters
Plasma hemoglobin levels decreased between V1 and V3 in both groups (−1.8%, p = 0.038), with no difference between groups (Table 2). No difference between conditions was observed in the hematocrit levels or the white blood cell, red blood cell, or platelet counts.

Compliance
A high compliance percentage in both groups was found, with an intake of 100.0 ± 5.7% in the Test group and 100.0 ± 6.8% in the Control group, with no difference between the two groups (Table S1).

Discussion
Malnutrition is a highly prevalent condition in older adults. It is associated with low muscle mass and function and increased occurrence of health problems [1][2][3][4]. Maintaining an adequate nutritional status as well as a sufficient nutrient intake in older people is therefore essential to address this public health problem. For this purpose, protein supplementation is known to prevent the loss of muscle mass during aging [8][9][10], and the consumption of various pomegranate extracts induces numerous health benefits, mainly through their antioxidant properties [13]. Combining protein and a pomegranate extract would be innovative but safety issues may arise as this combination has never been tested in an older population. The aim of this study was therefore to evaluate the safety of a 21-day supplementation combining protein and a pomegranate extract in healthy subjects aged 65 years or more compared to protein alone.
Our results showed that the daily combination of 20 g protein and 650 mg pomegranate extract for 21 days was safe and well tolerated. No serious adverse events were observed, and all mild or moderate adverse events were not related to the dietary supplement consumption. These results reinforce previous studies showing the safety of pomegranate extract supplementation at similar or higher doses in different adult populations, e.g., 1050 mg in hemodialysis patients [23] or 710 and 1420 mg in overweight individuals [24]. In addition, there were no differences between the Test and Control groups in physical and hematological parameters. In line with previous results dealing with protein or pomegranate supplementation [25,26], a decrease in systolic and diastolic blood pressure was observed between the start and the end of the study in both groups. While beyond the primary scope of the present study, these results suggest beneficial actions of protein and protein/pomegranate supplementation on blood pressure. To confirm these preliminary observations, a proper control group with neither protein nor pomegranate supplementation needs to be included in our follow-up study investigating the efficacy of those compounds on physical and hematological parameters in malnourished older people.
Most of the differences in the biochemical parameters observed between baseline screening and the end of the study were present in both groups. Plasma potassium and urea levels increased while uric acid and total cholesterol decreased similarly in both groups, which is consistent with previous reports following protein intake in healthy and pathological populations [27][28][29]. Here, we did not find any additional effect of combined protein/pomegranate supplementation compared to protein alone on the different biochemical parameters studied, possibly excepting GPT, a marker of liver function [30]. The latter decreased in the Test group only. In addition to its already known effects on metabolic risk factors [16], pomegranate could have a potential beneficial effect on parameters related to liver function. Here as well, a proper control group with neither protein nor pomegranate supplementation will be necessary when investigating the efficacy of those compounds on biochemical parameters in malnourished older people. Globally, given the preliminary nature of this safety study, the small number of participants resulted in limited statistical power to demonstrate specific benefits of protein and pomegranate on health-related parameters. That said, the primary aim of the present study was fulfilled, namely, the investigation of the safety and tolerance of combined protein/pomegranate supplementation in a limited sample of older people. Despite some statistical changes, all parameters studied here remained within healthy thresholds. Therefore, our supplementation can be considered safe and well tolerated.
In conclusion, we found that a combined protein/pomegranate supplementation for 21 days was safe and well tolerated in older participants. This first study was required before investigating the efficacy of this combination in a larger sample of older malnourished people for a longer duration to detect changes in muscle mass, and in a more appropriate form such as directly integrated into food to increase ONS compliance.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nu14235182/s1. Table S1: Individual compliance (%) in the Control and Test groups.  Data Availability Statement: Data are available upon request by sending an e-mail to cicn@uclouvain.be.