The Efficacy of a Food Supplement Consisting of the Combination of an Olive Extract and a Myrtle Extract for the Improvement of Lipid Metabolism in Subjects With Slightly Altered Cholesterolemia Values

Efficacy Study of the Supplementation of a Food Supplement Consisting of the Combination of an Olive Extract (Olea Europea L., Fruit), and Myrtle Extract (Myrtus Communis L. Leaves) for the Improvement of Lipid Metabolism in Subjects Eith Slightly Altered Cholesterolemia Values: Single-center, Controlled, Randomized, Parallel-arm, Double-blind Clinical Study

Study Objective The aim of the study is to evaluate the efficacy of a dietary supplement based on a mixture of olive (Olea europaea L., fruit) and myrtle (Myrtus communis L., leaves) extracts in maintaining normal plasma cholesterol levels in subjects with mildly altered (borderline) cholesterol levels.

Study Design A single-center, randomized, controlled, two-arm, double-blind, parallel-group clinical trial will be conducted, including a 15-day run-in period.

The treatment period will last 3 months, during which subjects will receive either the dietary supplement or a placebo.

This duration is considered adequate by EFSA to evaluate the clinical efficacy of commonly consumed food components or dietary supplements on hypercholesterolemia.

Before starting the treatment period, enrolled subjects will undergo a 15-day run-in phase during which they will not receive any treatment but will be required to complete a daily food diary.

Experimental Groups

Group 1 (56 subjects): Participants will take one capsule per day of a dietary supplement consisting of a blend of olive (Olea europaea L., fruit) and myrtle (Myrtus communis L., leaves) extracts.

Group 2 (56 subjects): Participants will receive a placebo*.

To control for variables that could bias the interpretation of results, participants will be instructed to:

Follow an isocaloric diet for the entire duration of the study, as specified in the protocol, and record it in a food diary.

Report all pharmacological treatments at each visit.

*Masking methods are described in the dedicated section.

Each subject in both experimental groups will be monitored before, during, and after administration of the dietary supplement or placebo.

Inclusion Criteria

The study will include subjects of both sexes who meet the following criteria:

Age between 18 and 70 years Ability to understand and sign informed consent Negative HIV test Negative pregnancy test Borderline total cholesterol levels (200-239 mg/dL, according to the National Institutes of Health definition) LDL cholesterol levels <159 mg/dL Not taking any medications and willing to avoid medication throughout the study period

Mildly altered LDL cholesterol levels (100-159 mg/dL), according to NCEP guidelines:

Optimal: <100 mg/dL Near optimal/above optimal: 100-129 mg/dL Borderline high: 130-159 mg/dL High: 160-189 mg/dL Very high: ≥190 mg/dL

Exclusion Criteria

Subjects will be excluded if they meet any of the following conditions:

Age <18 or >70 years High cardiovascular risk based on eight risk factors (sex, age, diabetes, smoking status, systolic blood pressure, total cholesterol, HDL cholesterol, antihypertensive treatment) according to the Italian National Institute of Health "Progetto Cuore" parameters Ongoing pharmacological treatment for hypercholesterolemia and/or hyperglycemia, even at low doses Use of supplements for cholesterol, glycemia, or metabolic syndrome within two weeks prior to enrollment Pregnant women, suspected pregnancy, or planning pregnancy Breastfeeding women Blood donors within the three months prior to enrollment Non-self-sufficient individuals Subjects unwilling to cooperate Subjects unable to attend scheduled visits Subjects deemed unsuitable by the investigator due to other conditions requiring pharmacological treatment Subjects with HIV-related immunodeficiency Known allergies to the study product ingredients Alcohol consumption exceeding 30 g/day for men or 20 g/day for women Use of drugs of abuse Subjects currently taking medications

The study plans to recruit 112 subjects (56 subjects per group). As shown in Table 3, power analysis determined a total sample size of 106 subjects, ensuring a power of 95% and a significance level of 0.05, with a medium effect size (f = 0.25). Estimating a dropout rate of approximately 5%, calculated based on an estimate of the subjects who will have to withdraw from the study if they become required to take certain pharmacological treatments, the number of subjects to be enrolled has been increased to 112.

Randomization will be performed upon enrollment according to a randomization table generated by specific software (see the dedicated section).

The study will be double-blind, meaning all enrolled subjects will be unaware of the type of treatment they will receive, and treatment group assignment will be unknown to the investigator, the study sponsor, or any other person involved in conducting the study, except in the event of an emergency. For this reason, the investigator will receive an envelope containing the type of treatment associated with the subject, to be opened in the event of an emergency. Additionally, upon request, the investigator will be able to open the emergency envelope and inform the subject of the treatment to which they have been assigned from the randomization list.

The three treatments will be unrecognizable as the packaging will be identical, and the dosage forms will be identical in color, shape, weight, and flavor.

The study will include subjects of both sexes:

• aged between 18 and 70;

• able to understand and sign the informed consent;

• Negative HIV test

• Negative pregnancy test

• Borderline total cholesterol values, between 200 and 239 mg/dL (National Institute of Health definition)

• LDL cholesterol values < 159 mg/dL (https://www.humanitas-care.it/malattie/colesterolo/)\*

• Who are not taking any medications and will not take any medication throughout the study period * Mildly altered LDL cholesterol between 100 and 159 mg/dL (NCEP Guidelines):

  • Optimal: less than 100 mg/dL
  • Near optimal/above optimal: 100 to 129 mg/dL
  • High limit: 130 to 159 mg/dL
  • High: 160 to 189 mg/dL
  • Very high: greater than 190 mg/dL Education Guidelines National Cholesterol Education Program (NCEP) "Executive summary of the third report of the National Cholesterol Education Program (NCEP) expert panel on the diagnosis, evaluation, and treatment of hypercholesterolemia in adults (Adult Treatment Panel III)" (Revised_2013) https://www.ncbi.nlm.nih.gov/books/NBK542294/#article-19466.r18

Subjects with the following characteristics will be excluded from the study:

• Age < 18 and > 70 years;
• subjects exposed to a high risk of cardiovascular events based on 8 risk factors (gender, age, diabetes, smoking, systolic blood pressure, total cholesterol, HDL cholesterol, and antihypertensive treatment) in accordance with the parameters of the Istituto Superiore di Sanità's Cuore project (http://www.cuore.iss.it/sopra/calc-rischio.asp),
• undergoing pharmacological therapy for cholesterol and/or hyperglycemia, even at low doses,
• taking supplements to control cholesterol, blood sugar, and metabolic syndrome in the two weeks prior to recruitment,
• pregnant women, women who are pregnant or planning to become pregnant,
• breastfeeding women,
• blood donors in the three months prior to recruitment,
• non-self-sufficient subjects,
• subjects who show no willingness to cooperate,
• subjects who have difficulty reaching the clinic reference facility within the required timeframe,
• subjects deemed unsuitable by the investigating physician due to the presence of other conditions deemed incompatible with enrollment and requiring pharmacological treatments,
• subjects with HIV-associated immunodeficiency,
• subjects with known allergies to the ingredients of the study product,
• subjects who consume alcohol in quantities greater than 30g/day for men and 20g/day for women,
• subjects who use drugs of abuse.
• subjects taking medications.