Improvement of sleep and melatonin in children with autism spectrum disorder after β‐1,3/1,6‐glucan consumption: An open‐label prospective pilot clinical study

Abstract Introduction Poor sleep quality is a major problem in patients with autism spectrum disorder (ASD), and is attributed to low melatonin levels. Melatonin supplementation is recommended; however, its effectiveness varies. β‐Glucans have previously been shown to improve melatonin levels in animal studies. Herein, we examined the effectiveness of Aureobasidium pullulans (Nichi Glucan), a species of black yeast that contains beta‐1,3/1,6‐glucan, in a pilot study of children with ASD. Methods Thirteen children (age, 2.5–13 years) with ASD were recruited for the study. The control group consisted of four patients (Gr. 1), while nine patients were classified into the treatment group (Gr. 2). Gr. 2 received 1 g of Nichi Glucan along with conventional therapy, whereas the Gr. 1 (control) patients received conventional therapy alone for 90 days. Serum melatonin levels and sleep patterns, assessed using a subjective questionnaire, were evaluated before and after treatment. Results In Gr. 2, the average serum melatonin level increased from 238.85 ng/L preintervention to 394.72 ng/L postintervention. Eight of nine participants (88%) in Gr. 2 showed improvements in sleep pattern and quality, while no improvement was observed in the participants in Gr. 1. Conclusion The consumption of Nichi Glucan for 90 days resulted in visible improvement in sleep quality, sleep pattern, and serum melatonin levels, which was reported for the first time by our study. A larger multicenter study is required to validate our findings.


INTRODUCTION
Sleep problems are reported in 50%−80% of children with autism spectrum disorder (ASD) , and are most frequent in older children and adolescents. These problems include delayed sleep onset, shorter sleep duration, and daytime sleepiness, whereas bedtime resistance, sleep anxiety, parasomnia, and night waking are predominant in younger children . Sleep problems can exacerbate other features of autism, such as tantrums, aggression, self-injury, inattention, hyperactivity, social interactions, and repetitive behaviors, adding to parental stress and negatively impacting the entire family's well-being (Gagnon & Godbout, 2018;Goldman et al., 2012).
Melatonin, a neurohormone secreted by the pineal gland, regulates circadian rhythms, including sleep patterns, and has been shown to be released at lower levels in individuals with ASD compared to healthy individuals. Melatonin has been shown to exert a positive effect on sleep in individuals with autism by relieving anxiety, improving sensory processing, possessing anti-nociceptive effects, and mitigating gastrointestinal (GIT) dysfunction or gut dysbiosis (Gagnon & Godbout, 2018). Indeed, a significant proportion of children with ASD suffer from chronic GIT problems such as diarrhea, constipation, and irritable bowel syndrome. These GIT symptoms are related to the cortisol response to stress and gut dysbiosis-induced chronic inflammation , which in turn are associated with altered melatonin levels . Thus, melatonin supplementation (Cortesi et al., 2012;Malow et al., 2012;Maras et al., 2018) is one of the main pharmacological approaches used to treat ASD. Clinical studies of melatonin supplementation in children with ASD have shown an improvement in sleep latency and quality (Cortesi et al., 2012;Malow et al., 2012;Maras et al., 2018), although the effectiveness varies (Gagnon & Godbout, 2018). It is also worth noting that although side effects have been reported to be minimal, melatonin has been found to be effective mostly in the short-term treatment of sleep disorders, with clinical studies demonstrating that the positive effects decrease during follow-up (6−12 months) (Russcher et al., 2013).
A previous study showed that when rats were fed an extract consisting of a mix of rice bran and Sarcodon aspratus (mushroom), which contains beta-glucan, blood serum melatonin levels were upregulated (Costa, 2019;Dutta et al., 2021). In addition, clinical research reports have revealed the beneficial effects of Nichi Glucan, a beta-1,3/1,6glucan food supplement derived from the Aureobasidium pullulans strain AFO-202, on metabolic disorders (Dedeepiya et al., 2012;Ganesh et al., 2014) and cancer (Mio, 2014;Mizobuchi et al., 2008), and as a vaccine adjuvant for coronavirus disease 2019 . In a pilot clinical study, we previously explored the effects of Nichi Glucan on sleep patterns and serum melatonin levels in children with ASD (available as preprint; Raghavan et al., 2021).

Study design
The enrolled participants were clinically diagnosed with ASD by developmental pediatricians using standard assessments that involved clinical interviews incorporating the Childhood Autism Rating Scale (CARS). Overall, 18 subjects with ASD were enrolled in this prospective, open-label pilot clinical trial, and were stratified into two groups: Gr. 1: A control group of participants with ASD (n = 6) who underwent conventional treatment comprising remedial behavioral therapy and 500 mg of L-carnosine per day.
Gr. 2: Treatment arm comprising participants (n = 12) who received supplementation with Nichi Glucan along with conventional treatment. Each participant consumed two sachets (0.5 g -21 mg of active ingredient) of Nichi Glucan with meals twice daily for a period of 90 days.
The participants were randomly assigned into the two groups in a 1:2 ratio.
The participants did not take any other psychiatric drugs or supplements that could affect sleep in any way.

Evaluation of serum melatonin:
Melatonin levels in the serum were measured in the peripheral blood collected during the daytime, and analysis was performed using a human melatonin enzyme-linked immunoassay Kit (BT-LAB-Bioassay Technology Laboratory kit, China).
The catalogue number was E1013Hu and the sensitivity was 2.51 ng/L.

Secondary endpoints
Safety monitoring: The frequency and severity of adverse events and any clinically abnormal safety parameters were monitored through telephonic conversation and during the participants' visits on days 28, 56, and 90.

Target population for analysis
The intention-to-treat (ITT) analysis included all participants enrolled in the study. Per-protocol analysis (PPS) was performed on participants who completed the entire study without dropping out.

Method of analysis
All data were analyzed using statistics package of Excel software (Microsoft Office Excel®). Paired Student's t-tests were also calculated using this package, and p values < .05 were considered significant.

ITT and PPS participants
Eighteen patients who met the inclusion and exclusion criteria were included in the study and the ITT analysis. One participant in the treatment group (Gr. 2) dropped out before the start of the study. Figure S1 shows a CONSORT diagram of the trial.
During the study, four subjects were lost to follow-up: two each in Gr. 1 (one dropped out due to social problems in the family, and the other relocated to another city) and Gr. 2 (one dropped out due to social problems in the family and the other relocated to another city).
After excluding these four subjects, 13 were finally included in the PPS.
Their ages ranged from 2.5 to 14 years (average, 6.8 years). Though one subject was only 2.5 years, which falls out of the inclusion criteria, the investigator felt that the subject would benefit from the study, he applied for a waiver from the sponsor and notified the Independent Ethics Committee (IEC) about enrolling this subject who did not meet the inclusion criteria.

Improvement in sleep pattern
In the CSHQ, a reduction in the total score was observed in Gr. 2 compared with that of Gr. 1 (Figure 1), particularly in terms of bedtime resistance and time of sleep onset ( When the groups were compared, the total sleep score was found to be lower in Gr. 1 both pre-and postintervention than in Gr. 2, although the difference was not significant (p = .105).

Adverse effects
Only one patient in Gr. 2 exhibited mild adverse effects. One week after initiating Nichi Glucan supplementation, the patient showed an increase in the number of bowel movements, which resolved without treatment. No adverse effects were observed in any of the other participants.

DISCUSSION
In this open-label clinical trial, we found that the majority of children treated with Nichi Glucan (8/9; 88%) experienced an improvement in indicated that melatonin is more effective in the short term than in the long term, although these have mostly been conducted in cohorts of individuals without ASD (Russcher et al., 2013).
Nutritional supplements that are easy to administer and have minimal-to-no adverse effects are an ideal alternative to melatonin.
In the current study, Nichi Glucan, which has been consumed as a food supplement for several decades (Braam et al., 2018) and has been proven to be beneficial for the treatment of diseases such as metabolic disorders and cancer (Dedeepiya et al., 2012;Ganesh et al., 2014;Mio, 2014;Mizobuchi et al., 2008), was shown to be a promising alternative to melatonin. This supplement functions to improve sleep quality and increase daytime serum melatonin levels.
It has been postulated that the etiology of low melatonin levels in children with ASD arises from melatonin deficiency in the mothers, and that melatonin exerts its effects on the embryo during neurodevelopment (Li et al., 2019). Another study reported a clear correlation between the gut microbiome profiles of children with ASD and their mothers, suggesting the importance of assessing the microbiome during the early stage of a mother's pregnancy, as well as the importance of planning personalized treatment and prevention of ASD via microbiota modulation (Li et al., 2019). Beta-glucans have also been shown to reduce the underlying chronic inflammation associated with gut dysbiosis, as well as to help foster a healthy microbiome. This is advantageous for individuals with ASD as chronic inflammation has been shown to be associated with symptom severity (Jyonouchidoi, 2019). Thus, as the current study showed that β-glucans can enhance melatonin and sleep quality in children with ASD, it is necessary to further investigate the ability of β-glucans to modulate gut microbiota and reverse gut dysbiosis, which are the possible mechanisms behind altered melatonin levels (Cheng et al., 2020;Russcher et al., 2013;Shi et al., 2020), and thereby improve sleep.
This study has several limitations, which should be noted. First, the Autism Diagnostic Interview-Revised (ADI-R) and Autism Diagnostic Observation Schedule (ADOS) are the standard measures used to diagnose ASD. However, we used CARS for ASD diagnosis because it is the most widely used tool in low-and middle-income countries (LMICs) (Samms-Vaughan et al., 2017). The administration requirements of the ADOS and the ADI-R, which are widely used in high-income countries (HICs), make them less feasible for the diagnosis of ASD in LMICs.

CONCLUSIONS
In this open-label pilot clinical study, patients with ASD showed improved sleep quality and improved levels of serum melatonin following nutritional supplementation with a beta-1,3/1,6-glucan (Nichi Glucan) derived from the AFO-202 strain of black yeast Aureobasidium pullulans. Given that Nichi Glucan was found to be effective for improvement of sleep and other parameters in this pilot study of children with ASD, it may be worth recommending it as a supplement in such children, given that results from larger studies with longer-term follow-up are consistent with our findings. Further in-depth evaluation of these mechanisms and their correlation with other neurological parameters is recommended. These findings may shed light on the development of novel solutions and drug candidates for ASD.

ACKNOWLEDGMENTS
The authors would like to dedicate this paper to the memory of Mr.
Takashi Onaka, who passed away on June 1, 2022 at the age of 90 years, who played an instrumental role in successfully culturing an industrial scale up of AFO-202 strain of Aureobasidium pullulans after their isolation by Prof. Noboru Fujii, producing the novel beta-glucan described in this study.

FUNDING
No external funding was received for the study.

CONFLICT OF INTEREST
Author Samuel Abraham is a shareholder in GN Corporation, Japan, which holds shares of Sophy Inc., Japan, the manufacturers of novel beta-glucans using different strains of Aureobasidium pullulans; a board member in both the companies; and also an applicant to several patents of relevance to these beta-glucans.

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in the article (and its supplementary information files).

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/brb3.2750