Subjects

A total of 46 healthy male and female subjects aged between 35 and 65 years were recruited either as walk-in or referred from their general practitioners for counseling for life-style related issues. They were not suffering from any medical condition and did not require any medication. Their psychological well-being was assessed using General Health Questionnaire, Beck Anxiety Inventory, Beck Depression Inventory, and Sheehan Disability Scale and confirmed that they did not suffer from other psychiatric disorders or serious medical illnesses. The study adhered to the strict standard Good Clinical Practice (GCP) of Malaysia and also Guidelines of Declaration of Helsinki in dealing with human subjects. Compliance of subjects was monitored by requesting them to bring the unfinished or empty bottles to exchange for new ones. Data from subjects non-compliant to the supplementation instructions was not included for statistical analyses. At the end of the study, a total of 26 subjects were excluded from the data analyses due to either non-compliance of supplementation or withdrawal from the study. The remaining fully compliant subjects consisted of 10 male and 10 female subjects respectively. Each supplementation group consisted of 5 male and 5 female subjects. Subjects were also required to provide a dietary diary throughout the study to determine if there were any substantial variations among the subjects, as well as any changes in the dietary profiles throughout the study. All the subjects were on mixed diets that included protein intake from sources such as eggs and meats from their normal diets.

CMI-168

Chicken meat ingredient-168 (CMI-168) was a hydrolyzed chicken extract prepared from chicken meat that had been processed by a proprietary technology, involving bio-processing and aqueous extraction. It was produced through the advancement and optimization of the process used for production of essence of chicken (EOC).

Test sample

Tablets containing the chicken meat ingredient-168 (CMI-168) (335 mg per tablet) were prepared. Subjects were supplemented with 2 tablets per day (i.e. 670 mg per day).

Placebo

Tablets containing, instead of CMI-168, 335 mg of microcrystalline cellulose were prepared. Subjects were supplemented with 2 tablets per day as the CMI-168-supplemented group.

Both test sample and placebo were supplied by Cerebos Pacific Limited, Singapore.

Study design

A randomized, double-blind, placebo-controlled study was conducted to evaluate the effect of CMI-168 on cognitive performance. Subjects were randomly divided into 2 groups, namely the placebo group and CMI-168 group. Subjects were supplemented orally with either the placebo or test sample daily for 6 weeks. The subjects and the investigator who conducted the tests were blinded to the information about the group allocation and samples provided. Instead, an independent investigator, who has no information about the cognitive assessment and psychological well-being, maintained the record of all the samples and group allocation. During the 6 weeks of supplementation that subjects self-administered the supplements (either placebo or CMI-168), the subjects were required to return the unfinished supplements or empty bottle before they were issued with new bottles of supplements. There were about 20 subjects who did not fully comply with the supplementation regime and their data were excluded from the final statistical analyses.

Measurements

The subjects were examined using a battery of psychological assessments as well as cognitive performance tasks at the beginning of study (day 0) and day 42 (week 6). They were also tested on day 56 (week 8) to continue monitoring the effects of CMI-168 on cognitive performance after termination of supplementation. This post-supplementation assessment of cognition was intended to provide information on the sustainability of the cognition-enhancing benefits of CMI-168. The psychological assessments were Beck Anxiety Inventory, Beck Depression Inventory, General Health Questionnaire, Sheehan Disability Scale; the cognitive performance tasks were Digit Span, Letter Number Sequencing and Rey Auditory Verbal Learning Test (RAVLT). These pre- and post-supplementation psychological assessments were helpful to ensure that the subjects are free from any psychological or psychiatric disorders at beginning of the study and there was no negative psychological effect on the subjects as a result of the supplementation.

Beck Anxiety Inventory and Beck Depression Inventory

Beck Anxiety Inventory (BAI) is a 21 item self report scale designed to measure the severity of anxious symptoms [12]. The score ranges from 0–63. The test-retest reliability is 0.75 and concurrent validity is 0.65 with Hamilton rating score. Beck Depression Inventory (BDI) is another self report scale that measures the severity of depression symptoms [13]. The score ranges from 0–63. Any score above 30 would indicate severe depression. The normal range is 0–9. The test-retest reliability is 0.8 and it has a high validity index.

General Health Questionnaire

General Health Questionnaire (GHQ) is a structured questionnaire validated for use in Malaysia in 1996 [14]. It has become a commonly used instrument in multicentre, international trials designed to detect psychiatric disorders, particularly, states of depression, anxiety and psychiatric morbidity.

Sheehan Disability Scale

The Sheehan Disability Scale (SDS) was developed to assess functional impairment in three inter-related domains; work/school, social and family life [15]. It is a brief self-report tool with a 10 point visual analogue scale. Scores above 5 are associated with significant functional impairment.

Digit Span Backwards

The Digit Span assessment typically consists of Digit Span Forwards and Digit Span Backwards. The Digit Span Forwards is generally simpler task and is a measure of short-term memory storage capacity. In contrast, as the Digit Span Backwards [16] requires the subject to recall and repeat auditory information in the reverse sequence, it not only requires the short-term memory storage capacity, but also involves the manipulation of information within the phonological buffer. As such it is also a measure of working memory. As one of our key objectives of the current study is to understand how CMI-168 could be beneficial to the cognitive resource optimization for effective information processing and manipulation, Digit Span Backwards task was employed in this study to more selectively examine the working memory. The subjects were presented with a series of digits and were asked to immediately repeat them verbally in reverse order. The length of the longest list a subject can remember is that subject’s digit span.

Letter-Number Sequencing

Letter-Number Sequencing (LNS) requires the subject to attend to a series of letters and numbers that have been read to him or her, hold them in memory, manipulate them into a new order, and repeat the sequence. It was indicated while much of the variance on the LNS task was explained by performance on the more traditional Digit Span, additional unique prediction of LNS performance are provided by measures of processing speed and visual spatial working memory [17].

Rey Auditory Verbal Learning Test

The Rey Auditory Verbal Learning Test (RAVLT) is an efficient neuropsychological instrument for evaluating verbal memory and learning. It provides scores for assessing immediate memory, new verbal learning, susceptibility to interference, retention of information after a period of time, and memory recognition [18, 19]. Briefly, it consists of 3 lists. The first list consists of 15 words and participants are allowed 3 attempts to remember as many words as possible (termed ‘Immediate Memory’ , ‘Best Learning’ and ‘Total Learning’ respectively). Participants are then presented with a distractor list (List B) followed by a free-recall test from List B (termed ‘Proactive interference’). Immediately after, participants are asked to recall List A (termed ‘Retroactive interference’). After a 20 min interval, participants are asked again to recall the words from List A (termed ‘Delayed Recall’). Finally, the last task presents a 3rd list (List C) which includes words from List A. The participants need to identify the words that are part of List A (termed ‘Recall’).

Statistics

Requirement of the sample size to provide statistical significance was computed using Epi Info™ 7 software with the anticipated effect size was 0.8, power of 0.8 and confidence level of 95%. Statistical Package for Graphpad Prism was used to analyze the data collected in this study. All values are expressed as Score for each test. All values were expressed as mean ± standard error of mean (SEM). For all comparisons, the probability level of p < 0.05 is considered to be statistically significant using 2-way repeated measures ANOVA followed by Bonferroni post-hoc test.