Cronobióticos Naturales

Natural Chronobiotics: Innovation in the Prevention of Chronic Diseases and Improvement of Biological Balance Background Population aging is a global phenomenon with major public health implications. In Chile, health care spending for older adults is projected to reach about 26% of total health expenditure by 2030, which highlights the need to promote healthy aging. One key contributor to age-related functional decline is disruption of circadian rhythms, the biological processes that regulate homeostasis, metabolism, and the sleep-wake cycle. Circadian rhythms weaken with age, which increases the prevalence of sleep disorders and chronic diseases. Environmental factors such as excessive screen use and exposure to artificial light at night have made circadian disruption (chronodisruption) more common across the population.

An estimated 50 to 70 million adults in developed countries have sleep disorders. In Chile, the use of exogenous melatonin is increasing and is often inappropriate, which has led to cases of intoxication and a need for education on its responsible use. Because endogenous melatonin production declines markedly from about age 25, there is a need for safe, natural alternatives that support the body's own melatonin synthesis and help restore circadian balance.

To address this need, ChronoNaturals (Cronobióticos Chile SpA), a science-based technology company, has developed a proprietary natural chronobiotic formulation made from native berries of southern Chile. The formulation is rich in antioxidant polyphenols and contains no artificial additives. As part of the broader project, the company is also evaluating, in in vitro studies only, whether seaweed-derived nanoparticles can improve the bioaccessibility of the formulation's bioactive compounds. The human pilot study described here uses the original formulation, without nanoparticles.

Hypothesis Daily intake of a natural chronobiotic formulation made from native southern Chilean berries will increase endogenous melatonin levels and improve physiological and metabolic responses in overweight Chilean adults, compared with placebo.

Objectives The general objective of the project is to develop and validate a proprietary chronobiotic formulation that modulates endogenous melatonin levels in adults, with the aim of preventing chronic diseases associated with premature aging.

The specific objectives are:

To validate the incorporation of nanotechnology by determining the bioaccessibility and efficacy of the natural chronobiotic through in vitro experimental studies and composition and quality analyses that confirm the biological properties of the original formulation.

To conduct a pilot study in humans to evaluate the efficacy of the natural chronobiotic and its acceptance by consumers, advancing the product to Technology Readiness Level 5 (TRL5).

To develop an intellectual property strategy to protect the product formula and other intellectual property assets of the company.

To establish key agreements for the production, promotion, and distribution of the natural chronobiotic, and a plan to raise capital and disseminate the company and its product.

This registration corresponds to Specific Objective 2, the human pilot study, which evaluates the original formulation without nanoparticles. The nanotechnology work under Specific Objective 1 is limited to in vitro studies and does not involve human participants.

Study Design

This is a randomized, double-blind, placebo-controlled, two-period crossover pilot trial. Twenty adults will be randomly assigned to one of two sequences:

Sequence 1: Intervention A (4 weeks), washout (4 weeks), Intervention B (4 weeks) Sequence 2: Intervention B (4 weeks), washout (4 weeks), Intervention A (4 weeks) One intervention is the active chronobiotic drink made with berry extracts. The other is a placebo drink without extracts that has similar taste, color, and texture. Total participation lasts about 12 weeks from enrollment.

The allocation sequence will be generated with an online randomization tool (randomization.com) by the Principal Investigator (PI). Only the PI will know which drink (A or B) each participant receives. Participants and the support staff who conduct the visits and collect data will remain blinded. Each participant will receive a unique identification number at screening, and all data will be recorded under this ID.

Study Population Participants will be men and women living in Valdivia, Los Ríos Region, Chile, aged 35 to 55 years, with a body mass index (BMI) of 25 to 29.9 kg/m², a daily intake of fewer than two servings of fruits and/or vegetables, and regular sleep patterns (at least 6 hours per night).

People will be excluded if they are unable to give informed consent; have a diagnosed sleep disorder (such as chronic insomnia or sleep apnea); use L-tryptophan, exogenous melatonin, hypnotics, anxiolytics, antidepressants, corticosteroids, vitamin supplements, or supplements containing maqui, cranberry, goji, or other berries; use probiotics in any form; have a BMI below 25 or above 30 kg/m², or a body weight change of ±10% in the previous three months; have blood pressure above 140/90 mmHg; have uncontrolled metabolic or chronic diseases; are pregnant or breastfeeding; are allergic to berries or other components; drink excessive alcohol (more than 40 g per day, or about 3 cans of beer per day) or caffeine (more than 5 cups per day); or take anticoagulants. During the 12-week study, participants must agree not to take supplements high in antioxidants, polyphenols, vitamins, or melatonin.

Recruitment and Consent The study will be advertised through posters, the study's social media, and print and digital press. Interested people will contact the team through an online form or by email and will complete a brief pre-screening questionnaire. Potential participants will then attend an in-person information session at Universidad San Sebastián, Valdivia campus, where the PI will explain the study's objectives, procedures, risks, and benefits, and stress that participation is voluntary and that they may withdraw at any time without consequences. Those who sign the informed consent form will be invited to a screening visit.

Intervention Participants will drink 2 vials per day (one in the morning and one in the afternoon/evening), each about 30 mL, for 4 weeks in each intervention period (8 weeks of product use in total). The active formulation provides approximately 1,000 mg of polyphenols per day. Both the active product and the placebo are produced at the Food Laboratory of Universidad Austral de Chile under quality controls. The formulation was previously evaluated in an animal model in a project approved by the Bioethics Committee of Universidad Austral de Chile, and the preclinical results support its evaluation in humans.

Study Procedures All visits take place at the clinical sample laboratory of the Nutrition program, Universidad San Sebastián, Valdivia campus. Participants must arrive after an 8- to 12-hour fast for all visits that include blood sampling or glucose measurement. A healthy breakfast is offered after sampling.

Screening visit (SCR, about 45 minutes): blood pressure, weight, height, waist and hip circumference, and a health questionnaire administered by a health professional to confirm eligibility. No blood is drawn at screening.

Full visits (Visits 1, 3, 4, and 6; at the start and end of each intervention period):

Blood pressure Capillary blood glucose (Accu-Chek® test strips) Fasting venous blood sample (about 10 mL: 5 mL serum and 5 mL plasma) for biochemical profile, lipid profile, melatonin, and glucose Saliva sample, collected by the participant in a sterile 50 mL tube the night before the visit Body composition: weight, height, waist and hip circumference, and bioelectrical impedance analysis Questionnaires on general health, medical history, eating habits, meal timing, physical activity, and sleep, plus a quantified food frequency questionnaire to characterize baseline diet Intermediate visits (Visits 2 and 5; 2 weeks into each intervention period): blood pressure, capillary blood glucose, and body weight, plus delivery of the remaining vials for the period.

Study vials are delivered at Visits 1, 2, 4, and 5, each time covering 2 weeks of use. No vials are given at Visit 3 (start of washout) or Visit 6 (end of the study). The total blood volume drawn over the study is 40 mL per participant.

A product acceptability survey will assess sensory attributes, consumption habits, and user experience. At the end of the study, each participant will receive a personalized consultation with a nutritionist, including interpretation of their body composition results.

Outcomes The primary outcome is the change in melatonin levels measured in fasting blood samples, from the start to the end of each 4-week intervention period, with the chronobiotic compared with placebo. Secondary outcomes include changes in blood glucose (venous and capillary), lipid and biochemical profile, body weight and body composition, blood pressure, low-grade inflammatory markers (TNF-α and IL-6), reactive oxygen species and antioxidant response, sleep quality, and product acceptability.

Sample Size Sample size was calculated with G*Power 3.1 for a paired-means t test (a priori analysis), assuming melatonin means of 20 and 26 pg/mL, a standard deviation of 10 pg/mL, a within-subject correlation of 0.5, a two-sided alpha of 0.05, and 80% power. This gives an effect size dz of 0.6 and a required sample of 19 participants (achieved power 0.81). To allow for dropouts or incomplete data, 20 participants will be enrolled, which provides adequate power to detect the expected melatonin change of 6 pg/mL.

Statistical Analysis Baseline characteristics will be described as proportions for categorical variables, means and standard deviations for approximately normal continuous variables, and medians with interquartile ranges for non-normal continuous variables. Baseline differences between sequences (chronobiotic→placebo vs. placebo→chronobiotic) will be assessed with Fisher's exact test (categorical variables), independent-samples t test (normal continuous variables), or Mann-Whitney U test (non-normal continuous variables).

The intervention effect will be analyzed with linear mixed models for repeated measures. Fixed effects will include treatment (chronobiotic vs. placebo), time (pre vs. post), the treatment × time interaction (the term of interest), period (first vs. second), and sequence, together with prespecified covariates (age, sex, and BMI). A random intercept per participant will account for within-subject correlation in the crossover design. Carryover will be assessed through the period × treatment interaction; if significant, period-stratified analyses will be reported and washout validity will be interpreted with caution.

Sensitivity analyses will include (i) adjustment for the baseline value of each outcome (mixed ANCOVA model), (ii) exclusion of predefined outliers, and (iii) robust models if assumptions are clearly violated. Multiplicity across outcomes will be controlled with the Benjamini-Hochberg false discovery rate procedure (or Bonferroni, as appropriate). A two-sided p < 0.05 will be considered statistically significant. Mean differences (chronobiotic minus placebo) will be reported with 95% confidence intervals and standardized effect sizes. All analyses will be performed in R (version 4.2 or later).

Potential Risks and Safety Possible risks include minor discomfort from blood sampling (mild pain, bruising, dizziness, or rarely fainting); mild allergic reactions in people sensitive to berry pigments or phenolic compounds; gastrointestinal discomfort such as nausea, diarrhea, or abdominal pain from polyphenol intake; possible anticoagulant interactions of anthocyanins and phenolic acids with medications; discomfort answering questionnaires; and time demands of study visits. If any unexpected adverse reaction occurs, participants must stop taking the product and notify the research team, which will evaluate the situation and provide guidance. Risks are minimized by using certified organic raw materials free of pesticides and other chemical agents, and by exclusion criteria that exclude people at risk of interactions with anthocyanin- and polyphenol-rich products. The research team will monitor participants closely throughout the study.

Data Protection Each participant is identified by a unique code. Contact data are stored in a secure, paid Google Workspace cloud account with restricted access. Printed case report forms, laboratory results, and signed consent forms are kept in a locked file cabinet in the PI's office, with access limited to the research team. Digitized documents are stored in the same password-protected cloud. Results will be reported only in aggregated, anonymized form. Data will be kept for 5 years after study closure and then securely destroyed, in accordance with Chilean Law N° 19.628 on the protection of private life. Participants may ask questions before, during, and up to one year after the study, and will receive a plain-language summary of the overall results.

Ethics and Funding The study was approved by the Scientific Ethics Committee of the Los Ríos Health Service (CEC-SSLR; Ord. N° 340, 31 October 2025). An amendment adding capillary blood glucose measurement and saliva collection, together with the updated information and consent document, was approved on 15 April 2026 (Ord. N° 137, 4 May 2026). The project is funded by the Chilean National Agency for Research and Development (ANID) through the Startup Ciencia 2025 program (grant SUC250054) and is carried out by Cronobióticos Chile SpA (ChronoNaturals) in collaboration with Universidad San Sebastián, Valdivia campus.

Expected Results The study is expected to show that 4 weeks of supplementation with the natural chronobiotic, compared with placebo, favorably modulates circadian, metabolic, and inflammatory markers in overweight adults. Expected findings include higher endogenous melatonin levels, lower TNF-α and IL-6, improved metabolic profiles, and a better antioxidant response. These results would support further evaluation of the chronobiotic as a non-pharmacological nutritional strategy to improve circadian regulation and cardiometabolic health in an intermediate-risk population.