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

Abstract

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.

Keywords

walnut oligopeptide clinical trial memory cognition sleep quality WAIS PSQI

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 energy-hungry 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.^3‐6 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 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 H₂O₂-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.

Results and Discussion

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.

Table 1.

Baseline Characteristics (Mean ± SD).

Characteristics	Teenagers (n = 18)				Elderly people (n = 18)
Characteristics	Group A （n = 6）	Group B （n = 6）	Group C （n = 6）	p-value	Group A （n = 6）	Group B （n = 6）	Group C （n = 6）	p-value
Gender
Male	3 (50.0%)	3 (50.0%)	2 (33.3%)	.799	5 (83.3%)	2 (33.3%)	4 (66.7%)	.195
Female	3 (50.0%)	3 (50.0%)	4 (66.7%)	.799	1 (16.7%)	4 (66.7%)	2 (33.3%)	.195
Age	14.2 ± 0.4	14.3 ± 0.5	14.3 ± 0.5	.791	69.7 ± 1.6	71.0 ± 3.2	71.7 ± 3.8	.520
Weight (kg)	51.8 ± 6.5	57.3 ± 5.9	57.7 ± 6.1	.218	63.1 ± 4.3	59.8 ± 5.5	57.3 ± 6.3	.220
Height (cm)	164.3 ± 5.6	168.7 ± 7.4	165.8 ± 4.9	.473	167.0 ± 5.1	162.0 ± 7.0	161.7 ± 7.1	.306
Body mass index (kg/m²)	19.2 ± 2.2	20.1 ± 1.6	21.0 ± 2.6	.367	22.6 ± 0.8	22.8 ± 1.5	21.9 ± 1.0	.369
Sleep quality
Poor	4 (66.7%)	3 (50.0%)	2 (33.3%)	.792	5 (83.3%)	6 (100%)	4 (66.7%)	.301
Good	2 (33.3%)	3 (50.0%)	4 (66.7%)	.792	1 (16.7)	0 (0%)	2 (33.3%)	.301

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.

Table 2.

WAIS Scores (Mean ± SD).

Outcome	Visit	Teenagers			Elderly people
Outcome	Visit	Group A	Group B	Group C	Group A	Group B	Group C
1. Information	V1	8.17 ± 1.94	8.50 ± 2.07	8.33 ± 2.42	8.67 ± 2.25	7.00 ± 1.55	7.00 ± 1.79
	V2	8.00 ± 2.10	8.83 ± 2.14	8.33 ± 2.42	8.67 ± 1.86	7.00 ± 1.41	8.33 ± 1.03
	V3	9.00 ± 2.71	12.67 ± 1.63**	11.60 ± 1.82	8.40 ± 1.95	10.00 ± 1.79	9.83 ± 1.17
2. Comprehension	V1	6.67 ± 2.80	7.33 ± 2.58	5.83 ± 1.17	5.33 ± 2.16	5.00 ± 1.10	5.17 ± 1.72
	V2	7.00 ± 2.45	8.00 ± 2.28	7.00 ± 2.00	6.00 ± 1.55	5.33 ± 1.03	5.83 ± 0.98
	V3	8.75 ± 2.99	9.67 ± 2.73	11.60 ± 1.52	5.80 ± 1.48	8.83 ± 1.94**	8.83 ± 1.60**
3. Arithmetic	V1	11.17 ± 1.47	11.67 ± 2.07	11.17 ± 1.60	5.83 ± 1.83	5.33 ± 1.75	6.83 ± 2.04
	V2	11.00 ± 1.27	12.33 ± 1.97	11.00 ± 1.26	5.83 ± 1.72	6.17 ± 1.72	8.17 ± 1.94
	V3	10.75 ± 0.96	13.17 ± 1.72	11.40 ± 1.14	6.60 ± 1.82	8.17 ± 1.17	8.17 ± 1.72
4. Similarities	V1	7.83 ± 1.60	7.83 ± 1.33	8.17 ± 1.60	6.17 ± 1.72	6.50 ± 1.05	6.83 ± 1.17
	V2	8.00 ± 1.79	7.83 ± 1.33	8.33 ± 1.37	6.17 ± 1.72	7.17 ± 0.75	7.67 ± 1.75
	V3	8.75 ± 0.96	10.50 ± 1.64	11.00 ± 1.87	6.40 ± 1.95	8.50 ± 1.05	9.33 ± 1.21
5. Digit span	V1	12.67 ± 1.97	11.00 ± 2.76	11.50 ± 1.97	6.83 ± 0.75	7.83 ± 1.33	7.67 ± 2.34
	V2	12.67 ± 1.97	11.50 ± 2.88	11.50 ± 1.97	6.67 ± 0.52	8.17 ± 0.75*	8.17 ± 1.17*
	V3	11.75 ± 1.71	12.00 ± 2.61	11.60 ± 1.95	6.40 ± 0.55	8.00 ± 0.89**	8.00 ± 0.89**
6. Vocabulary	V1	6.67 ± 1.21	6.83 ± 1.17	8.00 ± 0.89	6.00 ± 1.10	5.67 ± 1.63	6.17 ± 1.72
	V2	7.00 ± 1.10	7.50 ± 1.05	8.50 ± 1.22	6.17 ± 0.75	6.17 ± 1.83	6.67 ± 1.37
	V3	6.75 ± 0.96	9.83 ± 1.47**	10.80 ± 1.30***	6.40 ± 1.14	8.83 ± 1.17**	9.50 ± 1.05***
7. VIQ	V1	92.17 ± 3.43	92.17 ± 1.72	92.00 ± 3.95	85.83 ± 3.92	84.33 ± 2.34	86.67 ± 3.88
	V2	92.67 ± 3.61	95.00 ± 3.03	93.67 ± 5.01	86.50 ± 3.67	87.00 ± 2.83	91.83 ± 3.60
	V3	94.75 ± 4.65	107.83 ± 4.49***	108.00 ± 4.42***	87.20 ± 2.39	98.83 ± 2.14***	100.00 ± 3.16***
8. Coding	V1	12.33 ± 0.52	12.00 ± 2.19	12.17 ± 1.47	7.00 ± 2.76	6.50 ± 2.35	5.67 ± 1.97
	V2	12.50 ± 0.84	12.00 ± 2.45	12.67 ± 1.37	7.00 ± 2.53	7.67 ± 1.37	6.83 ± 2.23
	V3	12.50 ± 0.58	12.83 ± 2.04	13.20 ± 1.92	7.00 ± 1.58	8.50 ± 1.05	9.00 ± 1.41
9. Image completion	V1	10.50 ± 1.52	10.50 ± 1.52	10.17 ± 0.98	8.00 ± 1.41	8.50 ± 1.05	8.00 ± 1.67
	V2	11.67 ± 1.63	10.67 ± 1.37	11.00 ± 1.10	8.17 ± 1.47	8.50 ± 1.05	8.17 ± 2.32
	V3	11.25 ± 1.50	12.17 ± 2.14	12.00 ± 1.58	7.80 ± 1.30	10.83 ± 1.33***	9.83 ± 1.17*
10. Block design	V1	11.67 ± 1.63	9.33 ± 1.97	10.17 ± 1.47	5.50 ± 1.22	6.17 ± 1.94	5.00 ± 1.79
	V2	11.83 ± 1.72	9.50 ± 2.07	10.83 ± 1.33	5.67 ± 1.51	7.17 ± 1.83	6.00 ± 1.41
	V3	11.50 ± 1.29	12.83 ± 1.47	13.40 ± 1.14	6.20 ± 1.30	10.33 ± 1.63***	10.00 ± 1.79***
11. Picture sequencing	V1	9.33 ± 2.73	10.33 ± 2.16	10.50 ± 1.52	6.00 ± 2.00	6.83 ± 2.23	7.33 ± 1.51
	V2	9.67 ± 2.07	10.83 ± 1.60	11.33 ± 1.37	6.00 ± 2.00	8.33 ± 1.51	8.33 ± 1.51
	V3	11.25 ± 0.96	13.33 ± 1.51	13.80 ± 0.84	6.60 ± 2.41	11.33 ± 1.03***	10.33 ± 1.21***
12. Object assembly	V1	9.00 ± 1.26	11.00 ± 1.26	10.67 ± 1.51	8.17 ± 1.17	7.33 ± 1.86	7.50 ± 1.87
	V2	9.00 ± 1.10	11.17 ± 1.33	11.67 ± 1.63	8.50 ± 1.22	7.50 ± 1.05	7.50 ± 1.38
	V3	9.75 ± 0.96	12.50 ± 1.38*	13.40 ± 1.14**	8.00 ± 1.41	9.50 ± 1.87	10.83 ± 1.17**
13. PIQ	V1	97.33 ± 7.53	98.00 ± 4.52	98.67 ± 4.27	99.67 ± 6.71	100.67 ± 2.07	98.33 ± 2.42
	V2	100.33 ± 5.24	99.67 ± 4.63	104.50 ± 4.28	100.50 ± 6.75	105.17 ± 3.92	102.50 ± 6.16
	V3	102.50 ± 3.32	114.00 ± 6.69*	117.40 ± 7.16*	101.00 ± 6.60	119.33 ± 4.89***	118.50 ± 5.32***
14. FSIQ	V1	94.00 ± 1.55	94.17 ± 1.33	94.33 ± 1.03	91.17 ± 1.94	90.83 ± 1.17	91.00 ± 2.19
	V2	95.50 ± 1.38	96.67 ± 2.07	98.00 ± 1.79	92.00 ± 2.68	94.50 ± 3.15	95.83 ± 4.07
	V3	98.00 ± 2.16	111.17 ± 3.06***	112.80 ± 3.63***	92.40 ± 2.70	108.33 ± 2.42***	108.67 ± 4.13***

Abbreviations: WAIS, Wechsler Adult Intelligence Scale; VIQ, verbal intelligence quotient; PIQ, performance intelligence quotient; FSIQ, full-scale intelligence quotient.

Multiple comparisons with Bonferroni adjustment *p < .05; **p < .01; ***p < .001.

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.

Table 3.

PSQI Scores (Mean ± SD).

Outcome	Visit	Teenagers			Elderly people
		Group A	Group B	Group C	Group A	Group B	Group C
Subjective sleep quality	V1	2.17 ± 0.41	2.00 ± 0.63	2.50 ± 0.55	2.67 ± 0.52	2.33 ± 0.52	2.83 ± 0.41
	V2	2.17 ± 0.41	2.00 ± 0.63	1.83 ± 0.41	2.83 ± 0.41	2.50 ± 0.55	2.67 ± 0.52
	V3	1.75 ± 0.50	1.17 ± 0.75	0.60 ± 0.55*	2.80 ± 0.45	2.00 ± 0.63	1.67 ± 0.52*
Sleep latency	V1	1.83 ± 0.98	2.17 ± 0.75	2.00 ± 0.63	2.67 ± 0.52	2.67 ± 0.52	2.83 ± 0.41
	V2	1.67 ± 0.82	1.50 ± 0.55	1.33 ± 0.52	2.67 ± 0.52	2.67 ± 0.52	2.83 ± 0.41
	V3	2.00 ± 0.82	1.50 ± 0.55	1.40 ± 0.55	2.80 ± 0.45	2.50 ± 0.55	2.17 ± 0.75
Sleep duration	V1	1.17 ± 0.41	0.83 ± 0.75	0.83 ± 0.41	1.33 ± 1.03	1.50 ± 0.84	1.17 ± 0.41
	V2	1.17 ± 0.41	1.00 ± 0.63	0.67 ± 0.52	1.33 ± 1.03	1.50 ± 0.84	1.17 ± 0.41
	V3	0.50 ± 0.58	0.00 ± 0.00*	0.00 ± 0.00*	1.00 ± 0.71	1.00 ± 0.00	1.00 ± 0.00
Habitual sleep efficiency	V1	0.33 ± 0.52	0.50 ± 0.55	0.67 ± 0.52	1.17 ± 0.75	2.00 ± 1.26	2.17 ± 0.75
	V2	0.33 ± 0.52	0.50 ± 0.55	0.50 ± 0.55	1.17 ± 0.75	2.00 ± 1.26	2.17 ± 0.75
	V3	1.00 ± 0.82	0.50 ± 0.55	0.60 ± 0.55	0.60 ± 0.89	1.17 ± 0.75	1.50 ± 0.55
Sleep disturbances	V1	1.50 ± 0.55	1.50 ± 1.05	1.17 ± 0.75	2.50 ± 0.55	2.50 ± 0.55	2.33 ± 0.52
	V2	1.50 ± 0.55	1.50 ± 1.05	1.00 ± 0.63	2.33 ± 0.52	2.17 ± 0.75	2.00 ± 0.63
	V3	1.00 ± 0.00	0.67 ± 0.52	0.60 ± 0.55	2.40 ± 0.55	1.33 ± 0.82*	1.33 ± 0.52*
Use of sleep medication	V1	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	1.33 ± 1.51	0.83 ± 1.17	0.33 ± 0.52
	V2	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	1.33 ± 1.51	0.83 ± 1.33	0.33 ± 0.52
	V3	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	1.60 ± 1.52	0.50 ± 0.84	0.17 ± 0.41*
Daytime dysfunction	V1	2.33 ± 0.52	2.17 ± 0.41	2.00 ± 1.10	2.67 ± 0.52	2.67 ± 0.52	2.50 ± 0.55
	V2	2.33 ± 0.52	2.00 ± 0.00	1.33 ± 0.82*	2.67 ± 0.52	2.17 ± 0.41	2.33 ± 0.52
	V3	1.25 ± 0.96	0.83 ± 0.41	0.60 ± 0.55	2.60 ± 0.55	1.50 ± 0.55*	1.67 ± 0.82
Global PSQI score	V1	9.33 ± 0.52	9.17 ± 0.41	9.16 ± 0.75	14.33 ± 0.52	14.50 ± 0.55	14.17 ± 0.41
	V2	9.17 ± 0.41	8.50 ± 0.84	6.67 ± 0.82***	14.33 ± 0.52	13.83 ± 0.41	13.50 ± 0.84
	V3	7.50 ± 1.29	4.67 ± 1.03***	3.80 ± 0.84***	13.80 ± 0.84	10.00 ± 0.89***	9.50 ± 0.84***
Sleep hours	V1	6.67 ± 0.61	6.50 ± 1.10	6.67 ± 0.61	5.92 ± 1.28	5.58 ± 1.02	6.08 ± 0.49
	V2	0.67 ± 0.61	6.47 ± 0.67	7.00 ± 0.55	6.08 ± 1.28	5.58 ± 1.02	6.17 ± 0.52
	V3	7.63 ± 0.95	8.42 ± 0.49	8.10 ± 0.22	5.60 ± 1.14	6.33 ± 0.52	6.42 ± 0.49

Abbreviations: PSQI, Pittsburgh Sleep Quality Index; SD, standard deviation.

Multiple comparisons with Bonferroni adjustment *p < .05; **p < .01; ***p < .001.

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.

Table 4.

Academic Performances of the Teenagers (Mean ± SD).

Test subject	Visit	Teenagers
Test subject	Visit	Group A	Group B	Group C
Chinese	V1	1.75 ± 0.50	2.00 ± 0.63	2.17 ± 0.75
Chinese	V3	1.75 ± 0.50	2.67 ± 0.52*	2.80 ± 0.45*
Mathematics	V1	1.50 ± 0.58	1.67 ± 0.52	1.50 ± 0.55
Mathematics	V3	1.50 ± 0.58	2.50 ± 0.55*	2.40 ± 0.55*
English	V1	1.50 ± 0.58	1.67 ± 0.82	1.33 ± 0.52
English	V3	1.50 ± 0.58	2.17 ± 0.41*	2.00 ± 0.00*
Average	V1	1.58 ± 0.32	1.78 ± 0.46	1.67 ± 0.37
Average	V3	1.58 ± 0.32	2.44 ± 0.34***	2.40 ± 0.28**

Multiple comparisons with Bonferroni adjustment *p < .05; **p < .01; ***p < .001.

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.

Table 5.

Correlation Between WAIS Scores and Academic Performances (Pearson Correlation).

Changes in WAIS scores（V3-V1）	Pearson r	Changes in teenagers’ test scores (after intervention-baseline)
Changes in WAIS scores（V3-V1）	Pearson r	Chinese	Mathematics	English	Average
1 Information	r	0.647	0.581	0.132	0.683
1 Information	p > \|r\|	0.009**	0.023*	0.640	0.005**
2 Comprehension	r	0.411	0.469	0.329	0.600
2 Comprehension	p > \|r\|	0.129	0.078	0.232	0.018*
3 Arithmetic	r	−0.046	0.262	0.257	0.242
3 Arithmetic	p > \|r\|	0.871	0.346	0.356	0.385
4 Similarities	r	0.353	0.272	−0.266	0.195
4 Similarities	p > \|r\|	0.197	0.326	0.338	0.486
5 Digit span	r	0.236	0.122	−0.152	0.108
5 Digit span	p > \|r\|	0.398	0.666	0.590	0.701
6 Vocabulary	r	0.678	0.377	0.094	0.566
6 Vocabulary	p > \|r\|	0.006**	0.165	0.739	0.028*
7 Coding	r	0.018	0.372	0.218	0.315
7 Coding	p > \|r\|	0.950	0.172	0.435	0.252
8 Image completion	r	0.387	−0.105	0.600	0.389
8 Image completion	p > \|r\|	0.155	0.709	0.018*	0.152
9 Block design	r	0.605	0.404	0.336	0.656
9 Block design	p > \|r\|	0.017*	0.135	0.221	0.008**
10 Picture sequencing	r	0.360	0.501	0.227	0.549
10 Picture sequencing	p > \|r\|	0.188	0.057	0.415	0.034*
11 Object assembly	r	0.447	0.487	0.260	0.596
11 Object assembly	p > \|r\|	0.094	0.066	0.349	0.019*
12 VIQ	r	0.681	0.628	0.169	0.741
12 VIQ	p > \|r\|	0.005**	0.012*	0.548	0.002**
13 PIQ	r	0.543	0.457	0.483	0.724
13 PIQ	p > \|r\|	0.036*	0.087	0.068	0.002**
14 WAIS FSIQ	r	0.634	0.612	0.310	0.774
14 WAIS FSIQ	p > \|r\|	0.011*	0.015*	0.261	0.001*

Abbreviations: WAIS, Wechsler Adult Intelligence Scale; VIQ, verbal intelligence quotient; PIQ, performance intelligence quotient; FSIQ, full-scale intelligence quotient.

Multiple comparisons with Bonferroni adjustment *p < .05; **p < .01; ***p < .001.

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 high-performance 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.

Experimental

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; (2) Group B, WO powder (low dosage, 170 mg); (3) Group C, WO powder (high dosage, 340 mg). The drop-out rate was 16.7%. Two subjects of Group A and 1 subject of Group C left the study. Fifteen subjects in the teenagers’ group completed all the procedures in this study. In addition, 18 eligible subjects were selected from 90 subjects aged over 65, and randomized into 3 groups (Groups A, B, and C), with 6 subjects in each group. The drop-out rate was 5.6%. Seventeen subjects in the elderly people group completed the study. The intervention period was 90 days. 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.

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

WAIS³⁴: 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.

PSQI³⁵: 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.

Footnotes

Acknowledgments

The authors are grateful to Dr. Li Zhang for his assistance in this clinical study.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethical Approval

This study was approved by the Institutional Review Board of the Shanghai Nutrition Society and registered at the Chinese Clinical Trial Registry () with an ID number of ChiCTR1900028160.

ORCID iD

Chien-Liang Chao

Statement of Human and Animal Rights

The study has been conducted in accordance with ICH GCP and the best interests of study subjects would have been protected well in the study at any time.

Statement of Informed Consent

All subjects enrolled in this study have signed the informed consent form.

References

Gómez-Pinilla

. Brain foods: the effects of nutrients on brain function. Nat Rev Neurosci. 2008;9(7):568‐578. doi:10.1038/nrn2421

Mergenthaler

Lindauer

Dienel

Meisel

. Sugar for the brain: the role of glucose in physiological and pathological brain function. Trends Neurosci. 2013;36(10):587‐597. doi:10.1016/j.tins.2013.07.001

Fukuda

Ito

Yoshida

. Antioxidative polyphenols from walnuts (Juglans regia L.). Phytochemistry. 2003;63(7):795‐801. doi:10.1016/S0031-9422(03)00333-9

Ruijun

Shi

Yijun

, et al. Antitumor effects and immune regulation activities of a purified polysaccharide extracted from Juglan regia. Int J Biol Macromol. 2015;72: 771‐775. doi:10.1016/j.ijbiomac.2014.09.026

Martínez

Labuckas

Lamarque

Maestri

. Walnut (Juglans regia L.): genetic resources, chemistry, by-products. J Sci Food Agric. 2010;90(12):1959‐1967.

Cosmulescu

Achim

Botu

Trandafir

. Mineral composition of fruits in different walnut (Juglans regia L.) cultivars. Not Bot Hort Agrobot Cluj. 2009;37(2):156‐160.

Poulose

Miller

Shukitt-Hale

. Role of walnuts in maintaining brain health with age. J Nutr. 2014;144(4 Suppl):561s‐5566 s. doi:10.3945/jn.113.184838

Chauhan

. Beneficial effects of walnuts on cognition and brain health. Nutrients 2020;12(2):550‐560. doi:10.3390/nu12020550

Esselun

Dilberger

Silaidos

, et al. Walnut diet in combination with enriched environment improves cognitive function and affects lipid metabolites in brain and liver of aged NMRI mice. Neuromol Med. 2021;23(1):140‐160. doi:10.1007/s12017-020-08639-7

10.

Pribis

. Effects of walnut consumption on mood in young adults-a randomized controlled trial. Nutrients 2016;8(11):668‐677. doi:10.3390/nu8110668

11.

Joseph

Shukitt-Hale

Willis

. Grape juice, berries, and walnuts affect brain aging and behavior. J Nutr. 2009;139(9):1813s‐1817s. doi:10.3945/jn.109.108266

12.

Mao

Hua

. Chemical composition, molecular weight distribution, secondary structure and effect of NaCl on functional properties of walnut (Juglans regia L) protein isolates and concentrates. J Food Sci Technol. 2014;51(8):1473‐1482. doi:10.1007/s13197-012-0674-3

13.

Zhang

Liu

Fan

, et al. Walnut protein isolates attenuate particulate matter-induced lung and cardiac injury in mice and zebra fish. RSC Adv. 2019;9:40736‐40744. doi:10.1039/C9RA06002B

14.

Zhao

Zhang

, et al. Effect of walnut protein hydrolysate on scopolamine-induced learning and memory deficits in mice. J Food Sci Technol. 2017;54(10):3102‐3110. doi:10.1007/s13197-017-2746-x

15.

Wang

Zhang

, et al. Walnut (Juglans regia) peptides reverse sleep deprivation-induced memory impairment in rat via alleviating oxidative stress. J Agric Food Chem. 2018;66(40):10617‐10627. doi:10.1021/acs.jafc.8b03884

16.

Liu

Yang

, et al. Neuroprotective and memory-enhancing effects of antioxidant peptide from walnut (Juglans regia L.) protein hydrolysates. Nat Prod Commun. 2019;14:1‐13.

17.

Zhu

. Research progress of biological activity of walnut peptide. Chin Food sci. 2018;24(12):58‐62.

18.

Massaad

Klann

. Reactive oxygen species in the regulation of synaptic plasticity and memory. Antioxid Redox Signal. 2011;14(10):2013‐2054. doi:10.1089/ars.2010.3208

19.

Lynch

Rex

Gall

. Synaptic plasticity in early aging. Ageing Res Rev. 2006;5(3):255‐280. doi:10.1016/j.arr.2006.03.008

20.

Hicyilmaz

Vural

Delibas

, et al. The effects of walnut supplementation on hippocampal NMDA receptor subunits NR2A and NR2B of rats. Nutr Neurosci. 2017;20(3):203‐208. doi:10.1179/1476830514Y.0000000166

21.

Zou

Cai

Xiong

Ruan

. Neuroprotective effect of peptides extracted from walnut (Juglans sigilata dode) proteins on Aβ25-35-induced memory impairment in mice. J Huazhong Univ Sci Technol. 2016;36(1):21‐30. doi:10.1007/s11596-016-1536-4

22.

Walker

. The role of sleep in cognition and emotion. Ann N Y Acad Sci. 2009;1156:168‐197. doi:10.1111/j.1749-6632.2009.04416.x

23.

Potkin

Bunney

. Sleep improves memory: the effect of sleep on long term memory in early adolescence. PLoS One 2012;7(8):e42191‐e42191. doi:10.1371/journal.pone.0042191

24.

Fuhrmann

Knoll

Blakemore

. Adolescence as a sensitive period of brain development. Trends Cogn Sci. 2015;19(10):558‐566. doi:10.1016/j.tics.2015.07.008

25.

Dorn

Hostinar

Susman

Pervanidou

. Conceptualizing puberty as a window of opportunity for impacting health and well-being across the life span. J Res Adolesc. 2019;29(1):155‐176. doi:10.1111/jora.12431

26.

Chen

. Study and treatment of insomnia. Chin J Rehabil Med. 2006;22:151‐153.

27.

Wang

Dai

, et al. Effects of walnuts on sleep improvement in mice and its mechanism. Acta Nutr Sin. 2017;39:189‐193.

28.

Reiter

Manchester

Tan

. Melatonin in walnuts: influence on levels of melatonin and total antioxidant capacity of blood. Nutrition 2005;21(9):920‐924. doi:10.1016/j.nut.2005.02.005

29.

Acufla-Castroviejo

Escames

Macks

, et al. Minireview: cell protective role of melatonin in the brain. J Pineal Res. 1995;19(2):57‐63. doi:10.1111/j.1600-079X.1995.tb00171.x

30.

Chen

Zhang

Lee

T.H

. Cellular mechanisms of melatonin: insight from neurodegenerative diseases. Biomolecules. 2020;10(8):1158‐1184. doi:10.3390/biom10081158

31.

Boonstra

de Kleijn

Colzato

, et al. Neurotransmitters as food supplements: the effects of GABA on brain and behavior. Front Psychol. 2015;6:1520‐1525. doi:10.3389/fpsyg.2015.01520

32.

Brambilla

Perez

Barale

Schettini

Soares

. GABAergic dysfunction in mood disorders. Mol. Psych. 2003;8(8):721‐737. doi:10.1038/sj.mp.4001362

33.

Liu

Yang

Hong

, et al. A simple and convenient method for the preparation of antioxidant peptides from walnut (Juglans regia L.) protein hydrolysates. Chem Cent J 2016;10:39‐50. doi:10.1186/s13065-016-0184-x

34.

Estevis

Basso

Combs

. Effects of practice on the Wechsler Adult Intelligence Scale-IV across 3- and 6-month intervals. Clin Neuropsychol. 2012;26(2):239‐254. doi:10.1080/13854046.2012.659219

35.

Nicassio

Ormseth

Custodio

, et al. Confirmatory factor analysis of the Pittsburgh sleep quality index in rheumatoid arthritis patients. Behav Sleep Med. 2014;12(1):1‐12. doi:10.1080/15402002.2012.720315