{'fraction_non_alphanumeric': 0.027266563347539713, 'fraction_numerical': 0.013463773731111971, 'mean_word_length': 5.105558840922531, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Walnut (Juglans regia L.) Oligopeptide Effects on Enhancing Memory, Cognition and Improving Sleep Quality in Teenagers and Elderly People in a Randomized Double-Blind Controlled Trial

Walnut has been reported to have beneficial effects on improving cognitive performance. This randomized double-blind placebo-controlled clinical trial evaluates the clinical effectiveness and safety of walnut oligopeptide (WO) on memory enhancement, cognition, and sleep quality in teenagers and elderly people. Eighteen teenagers and 18 elderly people were, respectively, randomly allocated to placebo, low dosage (170 mg), and high dosage (340 mg) WO administration groups (n = 6 per group in each population). After 90 days of administration, the Wechsler Adult Intelligence Scale (WAIS) score was significantly increased and the global Pittsburgh Sleep Quality Index (PSQI) score was significantly decreased in the WO administration group. In addition, the average scores for test subjects of Chinese, Mathematics, and English examinations were significantly increased from the baseline for the teenagers in the WO administration group. Our results support the claim that WO has the potential to become a new option for nutritional intervention, to enhance the memory, cognitive ability, and sleep quality of teenagers and elderly people. This study was approved by the Institutional Review Board of the Shanghai Nutrition Society and registered at the Chinese Clinical Trial Registry (http://www.chictr.org.cn) with an ID number of ChiCTR1900028160.


Introduction
The brain is the most important organ in the human body. Its functions include cognition, memory, emotion, and control of physical activity. 1 The brain is also regarded as an energyhungry organ. 2 Despite comprising only 2% of the body's weight, the brain gobbles up more than 20% of the daily energy intake. Because the brain demands such high amounts of energy, the foods we consume greatly affect brain function, including everything from learning and memory to emotions. 1 Perhaps one of the greatest effects of nutrition on brain function is our cognition (thinking). The effects of poor diet on sleeping patterns, energy, and mood, all indirectly affect day to day functioning of the brain at work or school. Cognition is also indirectly affected by the development of other brain functions. For example, nutrition is essential for the development of sensory systems such as hearing and vision and the integration of these processes, the sensorimotor system. The brain is an extremely active organ that demands an extremely high percentage of the overall daily energy requirements supplied by food. Nutrition is an important lifestyle factor that can maintain and protect brain health, such as docosahexaenoic acid, curcumin, nuts, flavonoids, choline vitamin B12, and folate. 1 Walnuts are common nutritious food rich in polyunsaturated fatty acids, proteins, minerals, and bioactive phytochemicals. [3][4][5][6] In traditional Chinese medicine, walnuts are mild-natured and have a sweet and slightly bitter, and astringent taste, which nourish the kidney, strengthen the spleen, replenish blood, relieving constipation, and calming the nerves. A large amount of evidence shows that eating walnuts in the diet can improve cognitive function 7-9 and 1 mood, 10 and reduce the risk of cardiovascular disease and type II diabetes. 8 Wahlut oligopeptide (WO) is a bioactive peptide that is extracted and hydrolyzed from the protein of walnut residues (meal) after removing the lipid content. Compared with walnut protein, oligopeptide, as a small molecule substance, has such properties as easily absorbed by the human body, good solubility, low viscosity and is relatively stable to pH changes. 5,11,12 Peptides have been reported to possess different biological properties, such as antioxidant, antihypertensive, enhance immunity, neuroprotective, enhancing memory, and anti-haze functions. 13 The effects of walnut peptides on boosting brain energy and intelligence have been widely explored. 11 Previous studies showed that peptides from enzymatically hydrolyzed walnut peptides could slow down the aging of mice and improve the learning and memory ability of mice in the water maze experiment by increasing acetylcholine receptors. 14 Insufficient sleep or poor sleep quality can result in neurodegeneration or memory deficit. In 2018, the neuroprotective effects were reported of walnut protein hydrolysates against memory deficits induced by sleep deprivation in rats by alleviating oxidative stress. 15 Furthermore, our previous study indicated that WO protected PC12 cells (rat adrenal medulla pheochromocytoma) against H 2 O 2 -induced oxidative stress. 16 In the zebrafish model, WO showed a neuroprotective effect. 16 Besides, the Morris water maze and Step-Down tests both showed that WO ameliorated memory impairments in scopolamine-, sodium nitrite-or ethanol-induced mice model. 16 With the modern acceleration in the pace of life, increasingly more people are afflicted by insomnia. Insufficient sleep or poor sleep quality can affect physical functions. The glutamic acid-rich walnut peptides are related to the formation of γ-aminobutyric acid in the human body and are helpful to ease nervous tension and mental disorder, which makes it easier for people to fall asleep and secure better sleep quality. 17 There is no reported research on walnut peptides in humans until now, and so we used a limited human study to find out whether walnut peptides can enhance the memory function of healthy people and maintain good sleep quality. This was the first time that the effects were evaluated of WO on enhancing memory, cognitive behaviors, and improving sleep quality in teenagers and elderly people.

Baseline Characteristics
The baseline characteristics of the teenagers and elderly people are presented in Table 1. No significant difference was observed among the different arms of the same age level.

Wechsler Adult Intelligence Scale
The Wechsler Adult Intelligence Scale (WAIS) subtest scores and composite scores of all 3 visits (time point) by all teenagers and elderly people are listed in Table 2. No significant difference was observed among the groups in each population at visit 1 (the initial time). For the teenagers, the object assembly score of Group C was significantly higher than that of Group A (p = .006). For the aged groups, the Digit Span scores of Groups B and C were significantly higher than that of Group A (p = 0.011). There was no other significant difference on any other scores among the groups at visit 2 (30th day). For both age levels, the verbal intelligence quotient (VIQ), performance intelligence quotient (PIQ), and full-scale intelligence quotient (FSIQ) scores of Groups B and C were all significantly higher than those of Group A, and there was no significant difference between Groups B and C at visit 3 (90th day). For the middle-school student groups, the vocabulary scores and the object assembly scores of Group B (p = .002 and .012, respectively) and Group C (p = .0003 and .002, respectively) were significantly higher than those of Group A. The information score of Group B was also significantly higher than that of Group A (p = .006). For the aged groups, the comprehension, digit span, vocabulary, image completion, block design, and picture sequencing subtest scores for Groups B and C were all significantly higher than those of Group A. The similarity subtest score and object assembly subtest scores for Group C were also significantly higher than those for Group A.

Pittsburgh Sleep Quality Index
The Pittsburgh Sleep Quality Index (PSQI) scores are listed in Table 3 for each group at each visit. No significant difference was observed among the groups at visit 1. At visit 2, for the teenagers, the global PSQI score of Group C was significantly lower than those for Groups A and B (p < .001). This showed that the general sleep quality of Group C subjects was significantly better than that of the other 2 groups. The daytime dysfunction component score of Group C was significantly lower than that for Group A (p = .014). There was no significant difference between Groups A and B on the component scores and the global PSQI score. There was no significant difference in any PSQI component score or global score among the elderly groups at visit 2. At visit 3, for both age levels, the global PSQI scores of Groups B and C were both significantly lower than those for Group A. This shows that the general sleep quality of subjects from these 2 groups was significantly better than Group A subjects. For the teenage groups, the subjective sleep quality component score of Group C was significantly lower than Group A (p = .011). The sleep duration component scores of Group B (p = .005) and Group C (p = .007) were both significantly lower than that for Group A. For both age groups, there was no significant difference in any PSQI component score or global score between Groups B and C at visit 3. No significant between-group difference occurred in the sleep hours at each visit for both age groups.

Academic Performance
The academic performances of the teenage subjects before and after intervention are listed by the group in Table 4. No significant difference was observed among the groups at baseline. After product intervention, the average scores for Groups B and C were significantly higher than those for Group A. There was no significant difference between Groups B and C. The mathematics test score for Group B was also significantly higher than that for Group A (p = .011). The Chinese test score for Group C was also significantly higher than that for Group A (p = .010) after an intervention.
For the correlation between WAIS scores and academic performances, Table 5 shows the correlation analysis between the increase (V3-V1) in the 11 WAIS subtest scores, VIQ score, PIQ score, and FSIQ score, and the increase of school test scores after intervention for middle-school student subjects. The increase in WAIS scores was positively correlated with the improvement in school test scores. The increases in WAIS information, vocabulary, and block design subtest scores, and VIQ, PIQ, and FSIQ scores were all positively and significantly correlated with the increase in Chinese test scores. The correlation between the increases in WAIS information subtest score, VIQ score, and FSIQ score was significantly positive with the increase in mathematics test score. The correlation between the image completion score increase and the English test score increase was also significantly positive. The WAIS score increases in information, comprehension, vocabulary, block design, picture sequencing, and object assembly subtests, and the increases in VIQ, PIQ, and FSIQ scores are all positively correlated with the increase in average scores for Chinese, Mathematics, and English tests, and the positive correlation was statistically significant.

Discussion
This clinical study was the first to evaluate the WO effects on memory and cognitive behavior enhancement and sleep quality improvement in teenagers and elderly people. Cognitive decline is an important global public health issue. Cognitive aging might begin at middle adulthood, the period particularly vulnerable to stress in a human lifespan. The decline in learning and memory is related to nerve cell apoptosis, free radical damage, changes in synaptic plasticity related to learning and memory, the messenger molecule concentration, and the decrease in central cholinergic neurotransmitter synthesis and release. 18,19 In a previous study, walnuts protected against cognitive decline by regulating the N-methyl-D-aspartate receptor and lipid peroxidation levels in the hippocampus. 20 Walnut oil could improve spatial learning and memory ability in rats. The mechanism might be related to the acetyl cholinergic system. 17 Walnut protein with high arginine, aspartic acid, and glutamic acid contents was the active ingredient in walnuts that enhanced learning and memory function. 17 Walnut peptides are rich in glutamate and arginine. The former is linked to neurotransmitters related to signal transduction, and the latter is the precursor substance of messenger nitric oxide. In addition, walnut peptides protected against scopolamine-induced learning and memory impairment, and the mechanism was found to be related to a significant increase in acetylcholine receptors. 14 Walnut peptides can also protect Alzheimer's mice by regulating the antioxidant system and reducing the inflammatory response. 21 Our previous study also demonstrated that WO has enhancing memory effects by antioxidative stress and increases the brain-derived neurotrophic factor level. 16 In this study, we observed that people taking WO for 90 days can increase scores of intelligence quotient (IQ) in teenagers and elderly people. In addition, the students' academic performance was improved. The results suggest WO has beneficial effects for both teenagers and elderly people.
Good sleep quality plays an important role in brain memory function. Several studies showed that sleep improves memory, cognition, and emotion in adults and teenagers. 22,23 It has also recently been suggested that teenagers might represent a second "window of opportunity" in brain development.  Teenagers are at the sensitive period for brain development. 24,25 In recent years, with the increase in life and work pressure from modern society, the incidence of mental and psychologically related diseases has increased significantly, such as insomnia, depression, and anxiety. Domestic research data showed that the incidence of severe insomnia in China is 9.38%, and insomnia seriously affects people's work and quality of life. 26 Table 3 shows that WO has the effect of improving sleep quality after 90 days' administration among teenagers and the elderly. The sleep improvement in mice was related to walnut intake. A daily intake of ≥6.67 g/kg peeled walnut kernels could improve the sleep of mice, and significantly affect their melatonin content. 27 Qualitative analysis of walnuts by highperformance liquid chromatography showed that walnuts contained melatonin (2.5-3.5 ng/g). 28 Radioimmunoassay was used to determine the increase in melatonin content in the serum of mice after walnut consumption. The mechanism by which the walnut diet improved sleep was therefore related to the increase of melatonin in rats. 28 Melatonin has an important protective effect on maintaining normal brain function. 29 Furthermore, the decreased secretion level of melatonin is also closely related to neurodegenerative diseases. 30 Age-related changes in sleep may contribute to cognitive decline among older individuals, yet this detailed mechanism has not been extensively studied. Our unpublished data use the zebrafish model to clarify the mechanism of WO for improving sleep quality.
WO has the effect of stabilizing hyperactive zebrafish, mainly by increasing the expression of gamma-aminobutyric acid (GABA) and melatonin receptors. GABA is an important inhibitory neurotransmitter in the human brain area and is closely related to sleep quality. 31 Previous studies showed that GABA may have an antidepression activity in an animal model and a low GABA level in mood disorder patients. 32 Therefore, WO can promote the expression of GABA receptors and may also have effects on stress relief and emotional regulation.
In this clinical study, 4 teenagers and 8 elderly subjects had symptoms, including upper respiratory tract infections, diarrhea, and colds. It is believed that there is no correlation between these and WO, indicating that WO is safe and has no side effects. In addition, this limited human experiment encounters challenges limited to this research and the need for further in-depth exploration of active single peptides is important for further bioavailability and mechanism studies. We found that WO had beneficial effects in improving cognitive behaviors via 2 major indicators of memory enhancement and sleep quality in both teenagers and elderly people. These findings shed new light on nutrition intervention to prevent cognitive disorders.

Conclusions
This randomized double-blind clinical study first observed that WO can enhance memory, cognition, and improve the sleep quality of teenagers and elderly people. After 90 days of administration, the WAIS score was significantly increased and the PSQI score was significantly decreased in the WO administered group. In addition, the students' academic performance was improved. This is also the first time that actual subject examination scores were used to evaluate the efficacy of WO. Brain health and sleep quality are inextricably related to each other. This work provides new ideas for nutritional intervention in brain health and sleep quality.

Walnut Oligopeptide
The walnut oligopeptide (WO) is developed and manufactured by Sinphar TianLi Pharmaceutical Co., Ltd. The ingredients and the production process met the standards of the food safety law in China. WO was obtained from walnuts (Juglans regia L.) using a continuous countercurrent extraction process and includes 90% oligopeptide with most in the range <1000 Da. 33

Study Population and Dosing Method
Eighteen eligible subjects were selected from 100 teenagers aged from 12 to 16, and randomized into 3 groups (6 subjects for each group): (1) Group A, placebo containing maltodextrin;

Study Design
Two study populations were enrolled: teenagers aged 12-16 years, and aged people aged above 65 years. Exclusion criteria included: allergic to dairy products; serious intolerance to milk and other dairy products; unable to give written informed consent; have been taking antibiotics during the 2 weeks before screening or at screening phase; have been on a diet, increasing exercise, or taking any drugs that could affect appetite or help to control weight in the last 3 months; have participated in other similar nutrition studies using dairy or probiotic products in the last 3 months; have been taking drugs for cardiovascular or metabolic diseases; have a medical history of or have been diagnosed with any of the following diseases: obvious gastrointestinal dysfunction, liver, kidney, endocrine, blood, respiratory and cardiovascular diseases, which may interfere with the study evaluation; suffering from a gastrointestinal disorder or any other gastrointestinal disease, including, but not limited to, irritable bowel syndrome, enteritis, ulcerative colitis and celiac disease; have a history of hospitalization during the 3 months before screening; subjects who are frequently taking drugs that the investigator believes may affect the gastrointestinal function or the immune system.

Efficacy Evaluation Methods
WAIS 34 : verbal (6 subtests) and nonverbal (5 subtests) components. The composite verbal IQ (VIQ), performance IQ (PIQ; nonverbal), and FSIQ scores were calculated by adding the corresponding subtests' scores scaled from raw scores. PSQI 35 : The PSQI is a self-rated questionnaire that assesses sleep quality and disturbances over a 30-day interval. Nineteen individual items generated 7 "component" scores: subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication, and daytime dysfunction. The sum of scores for these 7 components yielded 1 global score.
Test score collection form (teenagers only): a 5-score scale was used in this study to analyze the academic performance of teenager subjects: 1 = fail the exam (test score range of 0-71); 2 = just above the passing score (test score range of 72-83); 3 = middle rank (test score range of 84-95); 4 = good performance (test score range of 96-107); 5 = excellent performance (test score range of 108-120).

Statistical Analysis
The mean and standard deviation were calculated for continuous variables. The frequency and percentage were calculated for discrete variables. The test indicators comparison was carried out separately for teenagers and elderly people. For continuous variables, the comparison among groups was performed with the F-test. The pairwise comparison results among groups were modified with the Bonferroni method. The significance level should be divided by the number of pairwise comparisons, which was 3 in this study for 3 groups, to get the significance level after the data correction, ie, 0.05/3≈0.017. For discrete variables, the chi-square test was used for comparison among groups. The significance level of the 2-sided test in this study was 0.05.