Dietary Strategy to Tackle Cognitive and Locomotor Abilities in Early Elderly Subjects

Polyphenols, precisely resveratrol, with red wine as the most substantial source, was associated with improvements in cognitive function. Also, the loss of muscle mass and strength in elderly, that significantly increases dependency of these people, it could be attributed to alterations in gut microbiota through the "gut-muscle axis" and this underline the urgent need to efficiently find out any intervention or preventive approach via modulation of gut microbiota to improve muscle function in elderly. In this context, red wine polyphenols exert their effects through interaction with gut microbiota following the well-known two-way interaction between polyphenols and gut microbiota by promoting the proliferation of beneficial bacteria and increasing their abundance. Similarly, aging cognitive decline can be modulate by microbiota, notably through "gut-brain axis". Additionally, dietary polyphenols can delay inflammation or/and oxidation on the onset of age-related cognitive decline or muscular oxidation or cardiovascular factors risk factors, all of them relevant factors for the onset of physical frailty and dependence in elderly.

Moreover, wine is a singular alcoholic beverage with a high content of phenolic compounds of a very diverse nature on which numerous protective effects on health have been described. In fact, wine, in addition to alcohol, contains a complex mixture of polyphenols, including anthocyanins and non-coloured phenols as proanthocyanidins, flavonols, hydroxycinnamic and hydroxybenzoic acids, stilbenes and lignans. Thus, the bioprotective effects of wine polyphenols could be the consequence of the synergistic effect of this complex mixture of polyphenols from the grape and the winemaking process. That is why it is essential to clarify whether consumption of polyphenols from red wine provided by a nonalcohol red wine within a healthy diet can produce beneficial effects on health, differentiating this pattern from a general consumption of alcohol generally associated with negative effects. Based on the ethical limitations to carry out diet intervention studies with wine in humans, this project proposes the use of a nonalcoholic wine as vehicle of the complex mixture of red wine polyphenols.

The hypothesis of our research is that regular consumption of red wine polyphenols, 150 mg/day, delivered through a nonalcoholic red wine, in the context of a Mediterranean diet (MD pattern), could promote protective mechanisms for a healthy aging, especially through its beneficial effects on cognitive and locomotor abilities and mediated by the modulation of the intestinal microbiota (composition, function and associated metabolome). The main objective of the WinAging project is to add knowledge concerning the diet modulation of molecular mechanisms of the aging process through multi-omic approaches based on the potential health effects of a dietary strategy by a sustained MD supplementation with nonalcoholic red wine rich in polyphenols to tackle cognitive and locomotor abilities in early elderly home-dwelling subjects.

The specific objectives:

* Objective 1. To develop a nonalcoholic red wine with high phenolic content and sensorial acceptability.
* Objective 2. To evaluate the chronic effects of the intake of wine polyphenols (average dose 150 mg/day) delivered through a nonalcoholic red wine in the context of a MD in early elderly home-dwelling subjects, and applying participatory research to increase adherence of subjects in the clinical intervention study.

  * Objective 2.1. To identify selective biological phenolic metabolites in human urine samples to be used as biomarkers of nonalcoholic red wine intake.
  * Objective 2.2. To assess the effects of the diet supplementation with red wine polyphenols on the improvement of cognitive ability.
  * Objective 2.3. To assess the effects of the diet supplementation with red wine polyphenols on the improvement of locomotor ability
  * Objective 2.4. To evaluate the effect of the diet supplementation with red wine polyphenols on the improvement of cardiovascular disease (CVD) risk factors.
* Objective 3. To unravel the underlying mechanisms involved in the potential beneficial effects of red wine polyphenols on aging.

  * Objective 3.1. To evaluate the influence on microbiota composition, function and microbial catabolites.
  * Objective 3.2. To evaluate the impact on inflammation and gut health.
  * Objective 3.3. To evaluate the impact on metabolic pathways related with aging
  * Objective 3.4. To analyze the impact on age-related epigenetic modifications
  * Objective 3.5. To deeply characterize the underlying muscle signalling pathways affected using an animal model of aging.
* Objective 4. To apply integrative computational analyses for the identification of variables (clinical or gut-related) more determinant for a successful prevention of locomotor and cognitive abilities associated with wine polyphenols.

A total of 72 home-dwelling early elderly volunteers (men and women) 60-74 years old will be included in the intervention (36 in each arm of the intervention).

During the study there will be 11 visits in total. Visits will be every 4 weeks form the V1. The study visits will be the following:

* Screening visit (V0).
* Basal visit (V1).
* Visits during intervention (V2, V3, V4, V5, V6, V7, V8, V9).
* 3-month study visit (V4)
* 6-month study visit (V7)
* Telephone visits (V2, V3, V5, V6, V8, V9)
* Final study visit (V10). In visits V0, V1, V4, V7 and V10 volunteers must present themselves in fasting conditions of 8 hours to obtain fasting blood samples.

  * In addition, future candidates will be provided with the nonalcoholic wine, selected for study and allowed to taste it for one week to check its tolerability and sensory acceptance and thus ensure that candidates can commit to drinking the study wine daily for 9 months and reduce follow-up losses during the intervention.

Basal visit (V1; week 0):

* Nuclear Magnetic Resonance (NMR) and muscle biopsy (±5 days V1)\*.
* Isokinetic assessment (±5 days V1)\*
* Revision of study clinical history.
* Vital signs (blood pressure/resting heart rate).
* Checking the concomitant medication.
* Anthropometry waist circumference; hip; body weight and composition; height).
* Checking the physical activity and sedentary habits (IPAQ-E).
* Checking the quality of life (SF-36 Health survey questionnaire)
* Checking depression symptoms (Geriatric Depression Scale Questionnaire)
* Checking MD adherence
* Checking the cognitive and locomotor abilities
* Spanish validated of the Mini Mental State Examination (MMSE), Alzheimer disease questionnaire (AD8), Memorey Alteration tets (TAM test), Fototest
* Sarcopenic parameters (muscle strength based on grip dynamometry; skeletal muscle mass index (SMI) based on bioimpedance analysis (BIA), kg/m2 and appendicular skeletal muscle mass index (ASMI) based on bioimpedance analysis (BIA), low physical performance or physical function based on 4 m gait speed, m/s.
* Blood sample extraction.
* Ultrasound (muscle mass and abdominal fat).
* Checking the 3-day dietary record and Food Frequency Questionnaire (FFQ).
* Collection of feaces samples.
* Collection of saliva samples.
* Collection of urine samples.
* MD guidelines explanation.
* Schedule the next visit and instructions.
* Administration of red nonalcoholic wine through opaque bottles to protect from light.

  * In a subsample of n= 15 subjects/group, 5 days before or after the visit V1, volunteers must have a:

    * NMR, in order to measure changes in hippocampal volume, and volume of white matter hyperintense lesions,
    * Muscle biopsys, in order to measure changes in signalling pathways in skeletal muscle,
    * Isokinetics analysis in order to measure changes in muscle function through variables such as torque, work and power.

Telephone visits: (V2 and V3; week 4 and 8):

* Checking the FFQ
* Checking changes in concomitant medication
* Product intake control
* Record adverse effects
* Schedule the next visit and instructions.

  3-month study visit (V4; week 12):
  * Revision of study clinical history.
  * Vital signs (blood pressure/resting heart rate).
  * Checking the concomitant medication.
  * Anthropometry waist circumference; hip; body weight and composition; height).
  * Checking the physical activity and sedentary habits (IPAQ-E).
  * Checking the quality of life (SF-36 Health survey questionnaire)
  * Checking depression symptoms (Geriatric Depression Scale Questionnaire)
  * Checking MD adherence
  * Checking the cognitive and locomotor abilities
  * Spanish validated of the MMSE, AD8, TAM test, Fototest
  * Sarcopenic parameters (muscle strength based on grip dynamometry; skeletal muscle mass index (SMI) based on bioimpedance analysis (BIA), kg/m2 and appendicular skeletal muscle mass index (ASMI) based on bioimpedance analysis (BIA), kg/m2; low physical performance or physical function based on 4 m gait speed, m/s.
  * Blood sample extraction.
  * Ultrasound (quadricep muscle mass and abdominal fat).
  * Checking the Food Frequency Questionnaire, Mediterranean adherence (FFQ).
  * Collection of urine samples.
  * Product intake control
  * Record adverse effects
  * MD guidelines explanation.
  * Schedule the next visit and instructions.
  * Administration of red nonalcoholic wine through opaque bottles to protect from light.

Telephone visits: (V5 and V6; week 16 and 20):

* Checking the FFQ
* Checking changes in concomitant medication
* Product intake control
* Record adverse effects
* Schedule the next visit and instructions. 6-month study visit (V7; week 24):

  * Revision of study clinical history.
  * Vital signs (blood pressure/resting heart rate).
  * Checking the concomitant medication.
  * Anthropometry waist circumference; hip; body weight and composition; height).
  * Checking the physical activity and sedentary habits (IPAQ-E).
  * Checking the quality of life (SF-36 Health survey questionnaire)
  * Checking depression symptoms (Geriatric Depression Scale Questionnaire)
  * Checking MD adherence
  * Checking the cognitive and locomotor abilities
  * Spanish validated of the MMSE, AD8, TAM test, Fototest
  * Sarcopenic parameters (muscle strength based on grip dynamometry; skeletal muscle mass index (SMI) based on bioimpedance analysis (BIA), kg/m2 and appendicular skeletal muscle mass index (ASMI) based on bioimpedance analysis (BIA), kg/m2; low physical performance or physical function based on 4 m gait speed, m/s.
  * Blood sample extraction.
  * Ultrasound (quadricep muscle mass and abdominal fat).
  * Checking the 3-day dietary record and Food Frequency Questionnaire (FFQ).
  * Collection of feaces samples.
  * Collection of saliva samples.
  * Collection of urine samples.
  * MD guidelines explanation.
  * Schedule the next visit and instructions.
  * Administration of red nonalcoholic wine through opaque bottles to protect from light.

Telephone visits: (V8 and V9; week 28, and 32):

* Checking the FFQ
* Checking changes in concomitant medication
* Product intake control
* Record adverse effects
* Schedule the next visit and instructions.

Final visit (V10; week 36):

* NMR and muscle biopsy (±5 days V1)\*.
* Isokinetic assessment (±5 days V1)\*
* Revision of study clinical history.
* Vital signs (blood pressure/resting heart rate).
* Checking the concomitant medication.
* Anthropometry waist circumference; hip; body weight and composition; height).
* Checking the physical activity and sedentary habits (IPAQ-E).
* Checking the quality of life (SF-36 Health survey questionnaire)
* Checking depression symptoms (Geriatric Depression Scale Questionnaire)
* Checking MD adherence
* Checking the cognitive and locomotor abilities
* Spanish validated of the MMSE, AD8, TAM test, Fototest
* Sarcopenic parameters (muscle strength based on grip dynamometry; skeletal muscle mass index (SMI) based on bioimpedance analysis (BIA), kg/m2 and appendicular skeletal muscle mass index (ASMI) based on bioimpedance analysis (BIA), kg/m2; low physical performance or physical function based on 4 m gait speed, m/s.
* Blood sample extraction.
* Ultrasound (quadricep muscle mass and abdominal fat).
* Checking the 3-day dietary record and Food Frequency Questionnaire (FFQ).
* Collection of feaces samples.
* Collection of saliva samples.
* Collection of urine samples. \*In a subsample of n= 15 subjects/group, 5 days before or after the visit V10.

  * Nuclear Magnetic Resonance (NMR), in order to measure changes in hippocampal volume, and volume of white matter hyperintense lesions,
  * Muscle biopsys, in order to measure changes in signalling pathways in skeletal muscle,
  * Isokinetics analysis in order to measure changes in muscle function through variables such as torque, work and power.

Eligibility Criteria:
Inclusion Criteria:

1. Men or women between 60-74 years old; Sensory tolerance to red wine; Written informed consent provided before the initial screening visit.

Exclusion Criteria:

1. Men or women \>75 years old,
2. Hypoglucaemiant treatment or type 1 and type 2 diabetes mellitus diagnosed
3. Anaemia (hemoglobin ≤13 g/dL in men and ≤12 g/dL in women)
4. Subjects diagnosed of intestinal disorders such as chron disease, colitis ulcerous, and irritable bowel syndrome
5. To present a clinical active chronic disease
6. To present severe sarcopenia
7. To present cognitive impairment (MMSE ≤ 24 or clinical diagnosis of mild cognitive impairment or dementia)
8. Dietary allergies to: Mediterranean foods (eg, nuts), sulphytes or nitrates
9. Use of antioxidants supplements
10. Regular consumers of red wine who do not agree to change the consumption of red wine with alcohol to nonalcholized wine during the intervention
11. Chronic alcoholism
12. Current or past participation in a clinical trial or consumption of a research product in the 30 days prior to inclusion in the study
13. Failure to follow the study guidelines. For participation in the muscle biopsy, additional exclusion criteria included: Use of prescription anti-platelet medication; Prescription anticoagulant use (or antiaggregant, or acenocoumarol; Conditions which would reduce healing; or known allergy to lidocaine.