Open access

Short report

AUTHOR PROOF

Improvement of behavioural pattern
and alpha-­synuclein levels in autism
spectrum disorder after consumption ofa beta-­glucan food supplement in arandomised, parallel-­group pilot
clinical study

Kadalraja Raghavan,1,2 Vidyasagar Devaprasad Dedeepiya,3 Nobunao Ikewaki,4,5
Tohru Sonoda,5 Masaru Iwasaki,6 Senthilkumar Preethy,3
Samuel JK Abraham ‍ ‍6,7

To cite: Raghavan K,
Dedeepiya VD, Ikewaki N, et al.
Improvement of behavioural
pattern and alpha-­synuclein
levels in autism spectrum
disorder after consumptionof a beta-­glucan foodsupplement in a randomised,parallel-­group pilot clinicalstudy. BMJ Neurology Open
2021;0:e000203. doi:10.1136/
bmjno-2021-000203
Accepted 19 December 2021

© Author(s) (or theiremployer(s)) 2021. Re-­use
permitted under CC BY-­NC. No
commercial re-­use. See rights
and permissions. Published by
BMJ.
For numbered affiliations see
end of article.
Correspondence to
Dr Samuel JK Abraham;​drsam@​nichimail.​jp

ABSTRACT

imbalance and mutation in synaptic proteins
and receptors have also been reported to be
associated with ASD.1 Synucleins are small
soluble proteins that are present in presynaptic terminals, and they regulate synaptic
plasticity and neurotransmitter release.
Synucleins are important in the context ofbrains and neurons.1 2 Alpha-­
synuclein asa presynaptic neurotransmitter plays a keyrole in the synaptic functions of neurons byregulating CADPS2 mRNA expression.3–5
Alpha-­
synuclein has already been reported
to be associated with several neurodegenerative disorders, collectively called synucleinopathies such as Alzheimer’s disease (AD),Parkinson’s disease (PD), dementia with Lewy
bodies and multiple system atrophy.1 The
levels of alpha-­synuclein have been variedly
reported between ASD and PD wherein in
ASD, levels lower than age-­matched controls
has been reported3–6 while in PD, somehave reported lower than normal levels andothers higher. At present, there is no definitive cure for ASD. Interventions involve
speech and behavioural therapies to improve
the symptoms. According to the research,the microbiota-­gut-­
brain axis is significant
because dysbiosis has been observed in gut-­
INTRODUCTION
related diseases and other generalised disorAutism spectrum disorders (ASDs) are agroup of developmental disabilities that ders, especially of the nervous system, such as
can cause significant impairment in social, AD, multiple sclerosis, PD and ASD.7 Thereemotional and communication skills (​cdc.​ fore, nutritional supplements are consideredgov). Several causes and underlying mecha- potential interventions in alleviating gastrointestinal and behavioural symptoms in ASD.8
nisms have been postulated for the pathogenglucans, especially yeast-­
derived ones,
esis of ASD, including genetic, environmental, Beta-­
have shown a considerable positive outcome
immune dysregulation, neuroinflammation and oxidative stress. Neuronal synaptic as food supplements in modulating gut

Background Autism spectrum disorders (ASDs) are awide range of behavioural disabilities for which there are
no definite interventional modalities available. Remedial
therapies remain the only option but with varying
outcomes. We have evaluated the Childhood Autism
Rating Scale (CARS) and alpha-­synuclein levels in thisparallel-­group, multiple-­arm pilot clinical study after
supplementation with a biological response modifier beta-­
glucan food supplement (Nichi Glucan).
Methods Six subjects with ASD (n=6) Gr. 1 underwent
conventional treatment comprising remedial behaviouraltherapies and L-­carnosine 500 mg per day, and 12
subjects (n=12) Gr. 2 underwent supplementation with
the Nichi Glucan 0.5 g two times per day along with theconventional treatment.
Results There was a significant decrease in the CARS
score in all of the children of the Nichi Glucan Gr.2
compared with the control (p=0.034517). Plasma levelsof alpha-­synuclein were significantly higher in Gr. 2 (Nichi
Glucan) than in the control group Gr. 1 (p=0.091701).
Conclusion Improvement of the behavioural pattern CARS
score and a correlating alpha-­synuclein level, followed
by a safe beta-­glucan food supplement, warrants further
research on other parameters, such as gut-­microbiota
evaluation, and relevant neuronal biomarkers which islikely to cast light on novel solutions.

Raghavan K, et al. BMJ Neurol Open 2021;0:e000203. doi:10.1136/bmjno-2021-000203

1

Open access

AUTHOR PROOF

microbiota.9 Nichi Glucan is a black yeast-­derived AFO-­
202 beta-­glucan that has been in consumption for the
past two decades10 and has been shown to have potential
as a nutritional supplement to balance metabolic levelsof glucose, lipids and immunomodulators.11–13 Studies
on children with ASD have indicated there is an underlying neuroinfilammatory process occurring in different
regions of the brain involved in microglial activation,
thus resulting in a loss of connections or underconnectivity of neurons and leading to behavioural manifestations.14 MCP-­1, IL-­6, IL-­10 and TNF-α have been shown
to be expressed in higher levels in children with autism.15
Beta-­glucan has been proven to reduce the expression of
inflammatory and proinflammatory markers, includingIl-­6 and TNF-α,16 apart from having a neuroprotective
effect by attenuating inflammatory cytokine production through microglia.17 In another study, beta-­glucan
reduced induced microglia activation and its phagocytosisof synaptic puncta and upregulation of proinflammatory
cytokine (TNF-α, IL-­1β and IL-­6) mRNA expression apartfrom promoting Tau signalling and improving cognition
and brain function via the gut-­brain axis.18
This study was undertaken to investigate the effectsof Nichi Glucan as a food supplement in) children with
ASD, especially with relevance to the Childhood Autism
Rating Scale (CARS) score and alpha-­synuclein levels.

METHODS
The caregiver of all the subjects gave their informed
consent for inclusion before participation in the study.
The study was conducted in accordance with the Declaration of Helsinki.
Study design
The subjects enrolled in the study had received a diagnosis of ASD by a developmental paediatrician and wereverified by a psychologist using a clinical interview for abehavioural pattern that incorporated CARS.
Eighteen subjects (n=18) with ASD in total wereenrolled in this prospective, open-­label, pilot clinical trial
comprising of two arms. The CONSORT flow diagram ispresented as figure 1.
Arm 1 or Gr. 1: Control: Six subjects with ASD (n=6)
underwent conventional treatment comprising remedial
behavioural therapies and L-­Carnosine 500 mg per day.
Arm 2 or Gr. 2: Treatment arm: 12 subjects (n=12)
underwent supplementation with Nichi Glucan foodsupplement along with conventional treatment (remedial
behavioural therapies and L-­Carnosine 500 mg per day).
Each subject consumed two sachets (0.5 g each) of Nichi
Glucan daily—one sachet with a meal twice daily—for aperiod of 90 days.
Inclusion criteria
1. Age: 3–18 years.
2. Gender: Both male and female.
3. ASD criteria as per CARS score.
2

Figure 1 CONSORT flow diagram for description of theclinical trial.

4. Parents/caretakers willing to provide consent for theirchildren to actively participate in the study.
Exclusion criteria
1. Subjects aged more than 18 years old.
2. Any child with acute general illness or who has been on
any antibiotic, anti-­inflammatory, or antioxidant treatment in the 2 weeks prior to enrolment in the study.
3. Hyperallergic to any of the investigational products.
4. Subjects with long-­standing infections.
Outcome measures
Primary endpoints
1. An improvement on the CARS by at least 4.5 points.19
The CARS was monitored at baseline and after 90 days
between the Gr.1 (control) and Gr.2 (Nichi Glucan). The
CARS score was calculated based on a cumulative scoreobtained on the CARS scale, wherein a score below 30
indicates absence of sufficient signs and symptoms indicative of autism, a score between 30 and 36 indicates mild-­to-­moderately severe autism, and a score from 37 to 60
is correlated with severe autism.19 The psychologist who
performed the assessment and the parents were blind to
the participant’s treatment status.
2. Atleast 30% increase in plasma alpha-­synuclein levels
from baseline.
Human alpha-­synuclein (α-syn) levels in plasma were
measured in peripheral blood at baseline and after the
study’s completion at 90 days. The measurement was
performed using the human α-synuclein (α-syn) ELISA
kit (KINESISDx, USA) as per the manufacturer’s instructions. Normal range of values were 0.50–100 ng/dL.
Raghavan K, et al. BMJ Neurol Open 2021;0:e000203. doi:10.1136/bmjno-2021-000203

Open accessSecondary endpoints
Safety Monitoring: Adverse events frequency and severity;
clinically abnormal safety parameters.

Method of analysis
All data were analysed using Excel software statistics
package analysis software (Microsoft Office Excel);
Student’s paired t-­tests were also calculated using thispackage; and values of p<0.05 were considered significant.

RESULTS
ITT and PPS subjects
Eighteen patients who fulfilled all the selection criteria
and none of the exclusion criteria were selected to start
the study. These 18 patients were included in the ITT
analysis.
During enrolment, in the treatment group (Gr. 2), oneof them dropped out before the start of the study.
Figure 1 shows the CONSORT diagram of the trial.
During the study, four subjects were lost to follow-­up:
two in Gr. 1 (one dropped out due to social problems in
the family, and the other relocated to another city) and
two in 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 subjects were
included in the PPS.
ITT analysis
Primary end points
CARS score
Among the children in the control group (Gr.1), all four
were in the category of severe autism, and their scoreat baseline ranged from 37 to 52 (mean=42.75 ± 5.76).
Among the nine children in Gr.2, two were in the mild-­to-­moderate category of autism (mean=33.5±2.5), whereas
the remaining seven were in the category of severe autism(mean=43.71±4.80).
ITT analysis
Gr.1 Control: None of the participants achieved theendpoint of improvement in CARS score by 4.5 points.
Gr.2 Nichi Glucan: Only 25% of the subjects achieved
the endpoint of improvement in CARS score by 4.5
points. Proportion of subjects is not greater than 50%
after intervention.
PPS analysis
Gr.1 Control: None of the participants achieved theendpoint of improvement in CARS score by 4.5 points.
Gr.2 Nichi Glucan: Only 33% of the subjects achieved
the endpoint of improvement in CARS score by 4.5
Raghavan K, et al. BMJ Neurol Open 2021;0:e000203. doi:10.1136/bmjno-2021-000203

AUTHOR PROOF

Target population for analysis
Intention-­to-­treat (ITT) analysis included all the subjects
who were enrolled in the study. Per-­
protocol analysis(PPS) was performed on the subjects who completed theentire study without dropping out.

Figure 2 Comparison between Gr.1 and Gr.2 showeda significant decrease in the CARS score, indicating
improvement in autism’s signs and symptoms in Gr.2 (Nichi
Glucan) compared with Gr.1 (control) postintervention(p=0.034). CARS, Childhood Autism Rating Scale.

points. Proportion of subjects is not greater than 50%
after intervention.
Though the proportion of subjects who have achieved
the end point is not greater than 50% in both the groups,
Nichi Glucan is significantly better than Control in terms
of proportion of subjects who have achieved the end
point (p=0.034517).

Absolute values
After the intervention, the mean CARS score in the fourchildren of the control group was 42.5±5.4, while in Gr.2
(Nichi Glucan), the mean of the CARS score in the twochildren with mild-­to-­moderate autism was 32.5±0.5. In
the remaining seven children, the CARS score after Nichi
Glucan intervention had a mean of 40.1±5.96. There wasan average of three points in the improvement of autism’s
signs and symptoms in the Nichi Glucan group Gr.2,
whereas the improvement was very mild or nil in Gr.1
(figure 2). Among the various parameters assessed on the
CARS, there was visible subjective improvement in theemotional response, including reduction in irritability
and anger (88%), sleep improvement (88%), speechcharacteristics with improvement in finger pointing andmonosyllables in 77%, and improved responses to the
caregiver in 77% of the children in Nichi Glucan Gr. 2,
but these improvements were very mild or nil in Gr.1.
Table 1 shows the values of CARS score in all the study
subjects of the PPS analysis.
Plasma levels of alpha-synuclein
ITT analysis
Gr.1 Control: Only 25% of the participants achieved theendpoint of at least 30% improvement in plasma levelsof alpha-­synuclein. Proportion of subjects is not greaterthan 50% after intervention.
Gr.2 Nichi Glucan: Only 31.25% of the participants
achieved the endpoint of at least 30% improvement in

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Table 1 The score on the CARS in Gr.1 (control) and Gr.2
(Treatment—Nichi Glucan) at baseline and postintervention
Patient

Baseline

Postintervention

I

52

50

II

43

45

III

39

39

IV

37

36

I

38

32

II

42

37

III

31

32

IV

37

35

V

45

39

VI

46

45

VII

36

33

VIII

46

42

IX

52

51

AUTHOR PROOF

Gr.1 Control

Gr.2 Treatment

Figure 3 Plasma alpha-­synuclein levels in Nichi Glucan
group GR. 2 showing a significant increase compared with
Gr.1 (p=0.091).

on its own. No adverse effects were found in any of theother children.
DISCUSSION
In this study of 13 subjects, the behavioural pattern evaluated by the CARS score improved in all nine subjectsof Gr.2 (Nichi Glucan) (figure 1), especially on theemotional aspects and sleep-­related parameters, and thealpha-­
synuclein levels increased significantly in thesenine subjects compared with the control (figure 2).
The improvement in behaviour can be attributed to theimprovement in sleep parameters20 as consumption of
Nichi Glucan for 90 days has been able to improve thesleep quality and sleep pattern by a correlating increase inserum melatonin levels as well.21 In a correlating hypothesis of the plasma alpha-­synuclein level, between autism
and neurodegenerative diseases, it has been proposedthat alpha-­synuclein aggregation in the neural synapse

CARS, Childhood Autism Rating Scale.

plasma levels of alpha-­synuclein. Proportion of subjects is
not greater than 50% after intervention.
PPS analysis
Gr.1 Control: 50% of the participants achieved theendpoint of at least 30% improvement in plasma levelsof alpha-­synuclein. Proportion of subjects is greater than
50% after intervention.
Gr.2 Nichi Glucan: 55% of the subjects achieved theendpoint of at least 30% improvement in plasma levelsof alpha-­synuclein. Proportion of subjects is greater than
50% after intervention.
Nichi Glucan (Gr. 2) is better than Control (Gr. 1) interms of proportion of subjects who have achieved theend point (p=0.091701),

Table 2 Plasma alpha-­synuclein levels in Gr. 1 (control)and Gr.2 (treatment—Nichi Glucan) at baseline andpostintervention
Patient

Absolute values
Plasma levels of alpha-­synuclein ranged between 0.12 and20.41 ng/dL (mean=9.73 ng/dL) in the control groupand between 0.45 and 41.12 ng/dL (mean=9.39 ng/dL)
in the treatment group at baseline. After the intervention,
plasma levels of alpha-­synuclein increased, with a mean
increase in levels of 26.72 ng/dL in the treatment (Nichi
Glucan) Gr.2 group compared with the control group Gr.
1 (mean increase=10.56 ng/dL) (figure 3).
Table 2 shows the values of plasma levels of alpha-­
synuclein in all the study subjects of the PPS analysis.
Secondary endpoint
Adverse effects
Only one child exhibited possible mild adverse effects
related to increased bowel movements in Gr. 2 for 1 week
after supplementation with Nichi Glucan, which settled
4

Baseline(ng/dL)

Postintervention(ng/dL)
Fold increase

1.06

1.75

1.66

Gr.1 Control
I
II

0.12

1.8

15

III

15.99

20.1

1.25

IV

20.41

18.6

0.91

Gr.2 Treatment
I

41.12

56.1

1.36

II

0.9

26.74

29.72

III

3.67

24.1

6.57

IV

0.44

11.12

25.27

V

11.38

9.6

0.84

VI

24.99

28.5

1.14

VII

0.45

70

VIII

2.46

11.98

155.5
4.87

IX

2.18

2.4

1.1

Raghavan K, et al. BMJ Neurol Open 2021;0:e000203. doi:10.1136/bmjno-2021-000203

Open accessRaghavan K, et al. BMJ Neurol Open 2021;0:e000203. doi:10.1136/bmjno-2021-000203

would like to see a 4.5-­point improvement to conclude toa meaningful improvement after an intervention in thecontext of ASD.19 In this study, only a three-­point improvement was observed after Nichi-­Glucan supplementation.
However, this is only a pilot study, and larger randomised,multi-­centric clinical trials of longer duration for validation to attain a clinical meaningful response and to overcome the above-­mentioned limitations are warranted.
Nevertheless, the study is significant as it has identified
a simple nutritional supplemental intervention based ona naturally derived beta-­glucan, the Nichi Glucan, which
could stimulate endogenous alpha-­
synuclein secretion,
promote better synaptic regulation and improve thebehaviour symptoms of children with autism. However,
the results suggest that the benefits will be considerable when evaluated in terms of social and emotional
well-­
being and alleviation of caregiver stress, which isextremely significant.

AUTHOR PROOF

may lead to lower plasma levels.5 Whether the increase
in alpha-­synuclein levels in plasma in the ASD patients
after Nichi Glucan supplementation is due to regulation/prevention of alpha-­synuclein’s aggregation in theneural synapse must be investigated because an earlier
study on beta-­
glucan from yeast showed reduction inalpha-­synuclein expression on the brain substantia nigra
in Parkinson’s rat model.22 Another postulated mechanism is association of the gut microbiota. Gram-­negative
enteric bacteria such as the Enterobacter and Escherichia colisecrete the amyloid curli which causes misfolding23 andaccumulation of the neuronal protein alpha-­synuclein in
the form of insoluble amyloid aggregations, which has
also been shown to propagate in a prion-­like fashion fromthe gut to the brain via the vagus nerve and/or spinal
cord, thus culminating in the neurological disorders such
as ASD and PD.24 In a follow-­up analysis of this study, we
have reported a significant decrease in Enterobacter andE. coli25 which logically will result in lesser production ofalpha-­synuclein. In spite of such decreased production,
the increase in plasma alpha-­synuclein levels can be probably due to the disintegration of the amyloid deposits
by natural killer cells leading to these alpha-­synuclein
entering the blood stream.26 27 These postulated mechanisms while need further in-­depth research, no single
intervention, or therapy has proven its ability to regulatealpha-­synuclein levels, especially in children with ASD.
This study, thus, gains significance as the first of its kindwhich demonstrates a significant increase in the plasmaalpha-­
synuclein levels after Nichi Glucan supplementation and the levels being in line with those that werereported for children without ASD,28 probably implies aregulation of the alpha-­synuclein but this requires further
validation.
The mechanism by which the beta-­glucan promoted
behavioural improvement in this study and correlatedwith the regulation of alpha-­
synuclein levels needs
further in-­depth research, not only for ASD but also forneurodegenerative diseases such as AD, PD, and so on,
especially with regard to its effects on the gut-­microbial
ecosystem. The evolving data on the gut-­brain axis and gut
microbiota indicate there are two major approaches tobalancing gut microbiota: probiotic and prebiotic. Probiotic approaches, such as nutritional probiotics, faecal
transplantation, and so on, involve direct administrationof the beneficial microorganisms that have to colonise
the gut.23 However, the gut environment must be conducive for such probiotic supplementation. This is whereprebiotic approaches come in, such as Nichi Glucan,
which help in regulating the gut-­microbial ecosystem andpreventing chronic inflammatory status28 ; this must be
validated by future studies in terms of the effects of Nichi
Glucan and gut microbiota in their relevance to ASD.
The limitation of the study is the limited number ofparticipants, the unequal distribution of genders, andthe number of participants between the groups. Also,
the drop-­out rate from the study is very high. Further, a
recent study has suggested that clinicians on an average

CONCLUSION
Patients with ASD showed improvement in behaviouralsymptoms and improved levels of plasma alpha-­synuclein;
thus, this pilot clinical study of nutritional supplementation with an AFO-­202 strain of black yeast Aureobasidium
pullulans produced the biological response modifier beta-­
glucan (Nichi Glucan). Evaluation as per the CARS scorehas also shown significant beneficial effects. Althoughfurther validations need to be performed, the study definitely confirms the potential of Nichi Glucan as a simple
but effective food supplement to be considered as aroutine in children with ASD. Further research on themechanisms of its action in improving alpha-­synuclein
levels and balancing the immune system in the context
of managing chronic inflammation and gut-­microbiota
regulation as a prebiotic is likely to improve understanding of other diseases caused by neuroinflammationsuch as PD and AD.
Author affiliations
1
Kenmax Medical Services Pvt. Ltd, Madurai, India
2
Department of Paediatric Neurology, Sarvee Integra Private Limited, Madurai, India
3
Nichi-­In Centre for Regenerative Medicine, Chennai, India
4
Kyushu University of Health and Welfare, Nobeoka, Miyazaki, Japan
5
Junsei Educational Institute, Nobeoka, Japan
6
University of Yamanashi Faculty of Medicine Graduate School of Medicine, Chuo,
Yamanashi, Japan
7
GN Corporation Co Ltd, Kofu, Yamanashi Prefecture, Japan
Contributors KR, NI and SJKA contributed to conception and design of thestudy. KR and NI helped in data collection and analysis. SJKA and SP drafted themanuscript. VDD, TS and MI performed critical revision of the manuscript. All theauthors read, and approved the submitted version.

Funding The authors have not declared a specific grant for this research from any
funding agency in the public, commercial or not-­for-­profit sectors.
Competing interests SJKA is a shareholder in GN Corporation, Japan which inturn is a shareholder in the manufacturing company of the AFO 202 Beta Glucan.
Patient consent for publication Consent obtained directly from patient(s)Ethics approval The study was approved by the Institutional Ethics Committee(IEC) of Saravana Multispeciality Hospital, Madurai, India on 24th August, 2019 (Ref
ID: GLU/2020/01).

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Open access
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in thearticle or uploaded as online supplemental information. Not applicable.
Open access This is an open access article distributed in accordance with theCreative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which
permits others to distribute, remix, adapt, build upon this work non-­commercially,and license their derivative works on different terms, provided the original work is
properly cited, appropriate credit is given, any changes made indicated, and the use
is non-­commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

AUTHOR PROOF

ORCID iD
Samuel JK Abraham http://orcid.org/0000-0003-2646-2687

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