The Association between Melatonin-Containing Foods Consumption and Students’ Sleep–Wake Rhythm, Psychoemotional, and Anthropometric Characteristics: A Semi-Quantitative Analysis and Hypothetical Application

Food is an important source of melatonin (MT), which belongs to a group known as chronobiotics, a class of substances that affect the circadian system. Currently, no studies have been conducted on how the consumption of foods containing MT (FMT) is associated with indicators characterizing the human circadian system. In this study, we tested the hypothesis that FMT consumption is associated with chronotype and social jetlag. A total of 1277 schoolchildren and university students aged M (SD) 19.9 (4.1) years (range: 16–25 years; girls: 72.8%) participated in a cross-sectional study. Each participant completed an online questionnaire with their personal data (sex, age, height, weight, waist circumference, and academic performance) and a sequence of tests to assess their sleep–wake rhythm (the Munich Chronotype Questionnaire), sleep quality (the Pittsburgh Sleep Quality Index), and depression level (the Zung Self-Rating Depression Scale). Study participants also completed a modified food frequency questionnaire that only included foods containing MT (FMT). They were asked how many foods containing MT (FMT) they had eaten for dinner, constituting their daily serving, in the past month. The consumption of foods containing MT (FMT) during the day (FMTday) and at dinner (FMTdinner) was assessed using this test. Multiple regression analyses were performed to assess the association between the studied indicators. We found that higher FMTday values were associated with early chronotype (β = −0.09) and less social jetlag (β = −0.07), better sleep quality (β = −0.06) and lower levels of depression (β = −0.11), as well as central adiposity (β = −0.08). Higher FMTdinner values were associated with a lower risk of central adiposity (β = −0.08). In conclusion, the data obtained confirm the hypothesis that the consumption of foods containing MT (FMT) is associated with chronotype and social jetlag in adolescents and young adults.


Introduction
The circadian system (CS) in mammals, formed during evolution, is present in all species of organisms living on the Earth's surface [1], including humans [2]. Its main function is maintaining the circadian rhythms of biochemical, physiological, and psychological

Personal Data
The study participants were asked to specify their place of residence, sex, age, academic performance, height, weight, and waist circumference. Weight and height were used to evaluate body mass index (BMI), calculated as weight in kilograms divided by height in meters squared. Sex-and age-adjusted BMI percentiles (BMI%) were determined using BMI growth charts [30]. Four BMI categories (BMIc) were defined according to World Health Organization criteria [30]: (1) underweight (n = 101); (2) normal weight (n = 1003); (3) overweight (n = 122); and (4) obese (n = 51). The waist circumference to height ratio (WHtR) was also calculated [31]. Since the study began near the end of the third wave of the COVID-19 pandemic, some participants (n = 349) completed the questionnaires while remote learning. We adjusted our data analysis to reflect the significant alterations to sleep-wake patterns caused by remote learning [32].

Academic Performance
To assess academic performance, all participants were asked the following question: 'What was your academic performance (GPA) for the quarter or session preceding the study?'. In Russia, a unified, coded grading system for schoolchildren and university students is used. It consists of five grade points. Scores "1" and "2" correspond to unsatisfactory (requiring retakes of exams), "3"-low, "4"-average, and "5"-high academic performance. The mean GPA value among the study participants was M (SD): 4.30 (0.51).

MCTQ
The test questions concerned sleep onset time, awakening time on weekdays and free days, the use of an alarm clock, and the length of the school week. Based on these data, the following indicators were calculated: chronotype (MSF SC ), social jetlag (SJL), average weekly sleep duration (SlD), and sleep efficiency (SlE). The formulas and calculation methods for the indicators listed above were also described in Borisenkov et al. [33]:

PSQI
To assess sleep quality, we used the Russian version of the PSQI [34]. This test consists of 19 questions related to sleep quality, including sleep latency, duration, efficiency, disturbance, use of sleep medication, and daytime sleepiness for a one-month period. Global PSQI scores range from 0 to 21 points. In our sample, the scores ranged from 0 to 16, with an overall group M (SD) of 6.4 (2.8). According to the test authors, a PSQI score of ≤5 indicates good-quality sleep, and a PSQI score of >5 indicates poor-quality sleep [29].

MT-Containing Foods Consumption
FMT intake was assessed using the modified food frequency questionnaire (FFQ). Each study participant was asked to choose from a list of products that, according to previous studies, contain MT. They were also asked to answer the following questions: 1.
How often have you consumed these foods in the past month? Answer options: never, 1-2 times a month, 3-4 times a month, 2-3 times a week, 4-6 times a week, 1-2 times a day, 3-4 times a day, more than 4 times a day.

2.
How many servings of these foods did you consume in one meal (this question was accompanied by a picture indicating the size of one serving and the product's weight in grams)? Answer options: 0.5, 1, 2, 3, 4, or 5 servings.

3.
What percentage of the foods above was eaten during dinner? The response options were 0, 25, 50, 75, or 100%.
These data were used to calculate FMT consumption per day (FMT day ) and per dinner (FMT dinner ) by multiplying the average number of MT-containing foods consumed per day and at dinner by the average MT content in those products (Table S1). The calculations are presented in Supplementary Materials.

Data and Statistical Analyses
We used SPSS version 20 (SPSS, Inc., Chicago, IL, USA) for statistical data analyses. Table 1 presents the continuous variables (mean, standard deviations, and estimates of normality of distribution) used in this study. The distribution of six variables (Age, BMI, WHtR, SlE, FMT day , and FMT dinner ) differed from normal. Therefore, in further analyses, transformed indicators (Agec, BMI%, WHtRc, SlEc, FMT1 day , and FMT1 dinner ) with a normal distribution were used (Table 1). Table 2 presents the categorical variables and codes.
Analysis of covariance showed a significant association between FMT day and central adiposity, chronotype, social jetlag, sleep quality, and depression (Table 4, Figure 1A,B). A significant association between FMT dinner and central adiposity was noted ( Table 5). The indices characterizing central adiposity, chronotype, social jetlag, and depression were significantly lower in people consuming FMT day more than 1651.1 ng/day ( Table 3). The central adiposity index was significantly lower in people consuming FMT dinner more than 577.3 ng/dinner (Table 3).
Students who consumed more FMT day and FMT dinner had lower central adiposity index values (Models 1 and 2, Table 6). Students who consumed more FMT day had an earlier chronotype (Model 3, Table 6), less social jetlag (Model 4, Table 6), better sleep quality (Model 5, Table 6), and fewer depression symptoms (Model 6, Table 6).
The logistic regression analysis indicated that schoolchildren and students with higher FMT day consumption did not show moderate/severe depression symptoms (
Analysis of covariance showed a significant association between FMTday and central adiposity, chronotype, social jetlag, sleep quality, and depression (Table 4, Figure 1A,B). A significant association between FMTdinner and central adiposity was noted ( Table 5). The indices characterizing central adiposity, chronotype, social jetlag, and depression were significantly lower in people consuming FMTday more than 1651.1 ng/day ( Table 3). The central adiposity index was significantly lower in people consuming FMTdinner more than 577.3 ng/dinner (Table 3).

Discussion
Our study is the first to show that total FMT intake is associated with sleep-wake rhythm and social jetlag in adolescents and young adults. Adolescents and young adults who consume more FMT-containing foods throughout the day have a less pronounced delay in sleep-wake rhythm phase and circadian misalignment. We found a direct association between FMT day and MCTQ-derived "weekend mid-sleep phase adjusted for school-day sleep debt (MSFsc)" [7]. This indicator is a quantitative measure of an individual's chronotype. Previously, MSFsc and dim light MT onset (DLMO) have been closely associated [38,39], representing a reliable marker of the endogenous rhythm in human CS [5]. In addition, we noted an inverse association between FMT day and SJL, a quantitative measure of circadian misalignment [6].
The data obtained indicate that FMT consumption is one of the ways to prevent SJL and, consequently, circadian-misalignment-related problems in adolescents, such as low academic performance [10], depression [12], and obesity [13]. This concept is supported by the association between FMT and anthropometric measures (central adiposity) as well as psychoemotional indicators (depression). This conclusion has practical importance since circadian misalignment has become widespread among students. In different countries, social jetlag detection rates widely vary, from 40.1% in Japan [40] to 86.4% in Russia [8].
Our study also showed that schoolchildren and university students who consume more FMT during the day have higher sleep quality. These data are consistent with previously published data on FMT's positive effect on sleep function [19][20][21][22][23]. In particular, MT-rich foods for dinner and breakfast have previously been shown to increase sleep duration and efficiency [19,21,22] as well as reduce sleep latency [19] in adults and the elderly. One study [22] found that drinking cherry juice concentrate with high MT content in the morning and evening for a week increases the amplitude and mesor, but not the phase of the 24-h rhythm of MT metabolite excretion in the urine. The authors also noted an increase in sleep duration and efficiency, as assessed by actimetry. Cherry juice concentrate similarly affected the sleep quality of 65-year-olds in another study [20]. In young adults with low self-reported sleep quality, consuming two kiwifruit an hour before bedtime for four weeks increased PSQI-derived total sleep duration and efficiency [21].
It should be noted that not all study results can be logically explained within our hypothesis regarding FMT's chronobiotic effects. The close relationship between FMT day and the studied indicators, compared with FMT dinner , does not fit with the framework of this concept. The weak association of FMT dinner with sleep-wake rhythm characteristics may be due to the presence of substances in food that prevent FMT's action. However, analysis of such substances was not performed in the present study. It can be speculated that food containing an effective FMT dose also contains an excess amount of carbohydrates, fats, etc., which can interfere with FMT's positive effects. Sleep quality is adversely affected by excessive food intake before bedtime [41], and excessive fat content in one's daily diet negatively affects CS function [42]. Eating a high-calorie meal for dinner may also delay the sleep-wake rhythm phase [43].
A more pronounced chronobiotic effect of FMT day , compared with FMT dinner , could be explained by the fact that adolescents and young adults consume the bulk of their daily diet in the afternoon. Circadian misalignment causes significant changes in eating behavior, such as skipping breakfast [44] and refusing a full lunch during classes [45]. As previously demonstrated, the sleep-wake rhythm phase shifted earlier when exogenous MT was administered in the afternoon, even 11 h before the sleep midpoint [46].
At a dose of 1300 ng/day of FMT day , we observed a significant change in most of the parameters studied. This dosage is significantly less than the minimum dose of exogenous MT (0.3-0.5 mg), at which chronobiotic effects were previously noted [46,47]. Ingesting 0.3 mg of MT in the second half of the day has been found to shift the sleep-wake rhythm phase to an earlier time of day [46]. In this regard, we should note that our methodology can only be characterized as semi-quantitative, providing a rough estimate of FMT consumption. We did not evaluate the dietary intake of tryptophan, a precursor to serotonin and MT biosynthesis. The consumption of cereals enriched with tryptophan (60 mg) for dinner and breakfast leads to an increase in urinary excretion of MT metabolites, positively affecting sleep function and reducing depression risk in the elderly [48]. Furthermore, it cannot be ruled out that the FMT dose affecting CS function during chronic consumption is significantly lower than with short-term exogenous MT administration. Nagata et al. [27], who carefully estimated total dietary FMT intake, found even lower FMT day values of 29.8-32.3 ng/day. At the same time, the authors noted a significant inverse relationship between FMT day and total mortality risk in a large sample of Japanese residents > 35 years old (about 30,000 people).
Our work has several strengths and perspectives. An inverse relationship was first noted between FMT and social jetlag and indicators closely related to circadian misalignments, such as obesity and depression. We suggested that FMT day and FMT dinner can be used as integrated indicators of MT-containing foods consumption to help develop regimens and diets to prevent the negative consequences of circadian desynchrony. At the same time, this work has limitations. It should be noted that FFQ validation is required to determine food MT intake. The FMT day and FMT dinner indicators were calculated based on the literature data. MT content in individual products, according to different authors, widely varies depending on many factors. We used a semi-quantitative scale to reduce the effects of a wide range of factors on MT content in food. The study did not take into account the influence of lifestyle factors, such as the level of physical activity, caloric intake, coffee, and alcohol and nicotine consumption. This appeared to have reduced the accuracy of the analysis of the relationship between FMT and indicators characterizing human CS.
Respondents were asked to fill out a questionnaire in which they indicated the frequency of consumption of melatonin-containing foods over the past month. It should be taken into account that this approach reduced the reliability of the collected data due to possible inaccuracies in the respondents' recollection of their diet. Most of the study participants (72.8%) were women. In this study, we did not take into account the influence of another factor, the state of the reproductive function. It is known that there are significant changes in sleep function and psychoemotional state in women during the menstrual cycle, associated with changes in the production of sex hormones [49] and melatonin [50]. In the future, it will be necessary to conduct a special study to take into account the influence of the state of reproductive function on the association among studied indicators. The cross-sectional design of our study did not allow us to judge causal relationships between the studied indicators.

Conclusions
As a result of the study, it was found that higher consumption of melatonin-containing foods per day is associated with early chronotype and less social jetlag, better sleep quality and lower levels of depression, and central adiposity. Higher consumption of melatonincontaining foods for dinner is associated with a lower risk of central adiposity. The findings indicate that the potential chronobiotic effect of the diet may be partly due to dietary melatonin.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/nu15153302/s1, Figure S1: Picture indicating portions size; Table S1: FMT concentration in food products, ng/g fresh weight; Table S2: Rounded values of FMT content in 5 product categories; Table S3: Frequency of product consumption; Table S4: the sizes of food portions in grams; Table S5: Examples of a list of products containing threshold amounts of FMT corresponding to the lower threshold of the 3rd tertile of FMT day and FMT dinner . References [15][16][17]20,22, are cited in the supplementary materials.  Informed Consent Statement: Verbal informed consent was obtained from all study participants. Additionally, the schoolchildren's parents provided written informed consent.

Data Availability Statement:
The data of this study are available on request from the corresponding author.