A total of 157 community-dwelling individuals aged 65 and up were recruited from senior citizen centers in Bangkok, Thailand. During the initial eligibility assessments, 82 potential participants with pre-existing memory difficulties were enrolled (Petersen et al., 2014). The participants were screened using the following inclusion criteria: being over 65 years old, living independently in the community (i.e., no impairment in activities of daily living), having sufficient hearing and visual acuity to participate in examinations, having Mini-Mental State Examination (MMSE) scores of 23 or lower, meeting the definition for the amnestic type of MCI, and having medical clearance to perform physical activities. Exclusion criteria included a history of major psychiatric illness, as well as other serious neurological or musculoskeletal diagnoses or kidney disease. Sixty participants (50 women and 10 men) aged 74.6±7.3 years old met the inclusion criteria and were randomly assigned to either the intervention or control groups.

The study was approved by the Ethical Review Committee for Research in Human Subjects, Ministry of Public Health, Thailand (Ref. no. 2/2558). Written and verbal informed consent were obtained from all participants; verbal consent was witnessed and formally recorded. This study was registered as a clinical trial with the Thai Clinical Trials Registry (TCTR20180228001). Sample size was based on the ability to detect a moderate to large effect (1.1) according to a previous report by Farhana et al. (2016). As a consequence, it was decided to require 80% power at 0.05 significance. Thus, having at least 11 participants in each group was required to complete the study. The demographic information of the participants in the study is presented in Table 1.

This was a 12-week, single-blind, placebo-controlled randomized trial; measurements were taken at baseline and 12 weeks after the intervention. After baseline measurements, 60 participants were randomly assigned to one of three groups: a multicomponent exercise training group (EXE, n=20); a multicomponent exercise training with Gotu kola supplementation group (EXE+GK, n=20); or a placebo-controlled group (CON, n=20), and 58 participants completed the study. Two subjects in the CON withdrew prior to completion of the intervention, while no subject withdrew in the EXE and EXE+GK. The flowchart illustrates the procedure for selecting participants and carrying out the experiment (Fig. 1). Each participant received one capsule of placebo or 500 mg twice a day of Gotu kola extract. The color, texture, size, and smell of the placebo and Gotu kola extract capsules were identical.

The EXE and EXE+GK participants engaged in three 80-min sessions of multicomponent exercise training each week. Throughout the study period, all participants were instructed to continue their usual levels of physical activity, except for the interventions. They were instructed to maintain their food intake, i.e., the same intake levels as prior to the outset of the study. The participants’ nutritional intake was tracked by monitoring their nutrition diaries. Participants were also notified not to take any other dietary supplements. The review of side-effect compliance was conducted independently by investigators who were also blind to group assignment. At each study visit, adverse effects were examined. During the period of this study, participants were instructed to contact the research center if they experienced any medical complications. To identify any contraindications, fasting blood glucose (FBG), aspartate transaminase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, and creatinine levels were measured and monitored prior to and throughout the trial. This study evaluates cognitive function, which consists of general cognitive function, executive function, and memory domains, oxidative stress, inflammatory markers, and functional mobility at baseline and after 12-week interventions.

The crude extracts were prepared by the Bioresources and Biodiversity Section, Central Laboratory and Greenhouse Complex, Kasetsart University. Gotu kola was collected in Nakhon Pathom, Thailand. Only the leaf portion was used. The fresh leaves were dried at room temperature before being dried in a hot air oven at 70°C for 8 hr. The powdered dried leaves were finely ground. The leaf powder (500 g) was macerated after being extracted with 95% ethanol. To obtain the crude extracts (240 g), ethanol was removed using rotary evaporators under vacuum (Rotavapor R-205, BUCHI, Flawil, Switzerland). The leaf powder (500 g) and fresh leaves (500 g) were boiled in distilled water under reflux for one hour for the water crude extracts. The solution was then filtered and freeze-dried (Telstar Lyoalfa-6, Telstar, Terrassa, Spain) to obtain extract powder of 20 g and fresh leaves of 16 g, respectively. All crude extracts were stored at −20°C until they were used.

The participants exercised for 80 min per day, 3 times per week, for 12 weeks under the supervision of personal trainers. Each supervised session began with a 5-min warm-up period, followed by 30 min of muscle-strengthening exercise, and 40 min of aerobic exercise, postural balance retraining, and dual-task training, followed by a 5-min cool-down. The training program was designed according to the studies reviewed (Suzuki et al., 2012; Suzuki et al., 2013). Strengthening exercises were performed in a progressive sequence from seated to standing positions, with progressive resistance applied to increasing repetitions of each exercise or weight holding. Participants sat in a chair and performed toe raises, heel raises, knee lifts, knee extensions, sit to stand and other exercises. Participants then performed hip flexions, half squats, lateral leg raises, and repetitions of other exercises while standing upright behind the chair and holding the chair’s back for stability. Participants completed circuit training in aerobic exercises and postural balance retraining, which included stair stepping, endurance walking, and postural balance retraining. The average intensity of the aerobic exercises was 65%–75% of the maximum heart rate. Throughout the aerobic exercise session, their heart rate was monitored by taking their pulse. Pulse rates and oxygen saturation were continuously monitored throughout the exercise session using a pulse oximeter (MD300C20 Pulse Oximeter, Beijing Choice Electronic Tech Co., Ltd, Beijing, China). These exercises were also carried out while multitasking. Participants were instructed to memorize a step pattern in consecutive square segments and to step as quickly and accurately as possible while walking. Following that, participants practiced postural balance retraining, which included standing on one leg, multidirectional weight shifts, and walking on balance boards. Throughout the program, researchers and personal trainers provided ongoing safety monitoring to prevent negative accidents such as falls.

Body weight was measured via an electronic scale (Filizzola PL 150, Filizzola Ltda, São Paulo, Brazil). Height was measured minus shoes using a standard stadiometer (Health o Meter Professional, Sunbeam Products Inc., Boca Raton, FL, USA). Body mass index (BMI) was calculated as weight divided by height squared. Waist circumference was measured at the end of inspiration from the midpoint between the lower rib margin and the iliac crest with a flexible and inextensible measuring tape (Hoechstmass Balzer GmbH, Sulzbach, Hessen, Germany). Hip circumference was also measured at the level of the trochanter major, with a waist-to-hip circumference (W/H) ratio also calculated. Body composition, including % body fat, fat mass, and fat free mass, was measured using a bioimpedance analysis device (Inbody 720, Biospace Inc., Seoul, Korea) with light clothing and without shoes.

Blood samples for inflammatory markers were collected in standardized fasting conditions (12 hr) in the mornings between 7:00 and 9:00 a.m. at baseline and 7 days after the last training session to minimize any acute effects of exercise. Blood was collected in a vacuum test tube and centrifuged at 1,500 g for 15 min at 4°C. The plasma aliquots were then frozen at −80°C until they were analyzed.

Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) concentrations (mg/dL) were determined using an automated colorimetric assay (BS-200, Shenzhen Mindray Bio-medical Electronics Co., Nanjing, China). Low-density lipoprotein cholesterol (LDL-C) concentration was calculated via the Friedewald equation (Friedwald, 1972). Atherogenic index of plasma (AIP) was computed by transforming the ratio of TG to HDL-C by a logarithmic scale. The non-HDL-C was computed by subtracting HDL-C from TC, and the atherogenic index (AI) was computed as the ratio of non-HDL-C to HDL-C. FBG levels were also measured using standard methods at Nakhon Pathom Hospital in Nakhon Pathom, Thailand.

Plasma concentrations of IL-6 and TNF-α were measured using an enzyme-linked immunosorbent assay (ELISA) (Cayman Chemical Company, Ann Arbor, MI, USA) in accordance with the manufacturer’s instructions. The plasma 8-iso-PGF2α concentration was analyzed by ELISA using commercial kits (Cayman Chemicals, Ann Arbor, MI, USA) in accordance with the manufacturer’s instructions. The blood samples were run in duplicate. Researchers conducting ELISAs were blinded to all patient information.

Cognitive function, including general cognitive function, executive function, and memory domains, was assessed at baseline and 12 weeks following the intervention. Occupational therapists conducted cognitive evaluations using standardized methods, including the MMSE, Trail Making test (TMT), and Digit Span test (DST). The assessors introduced and demonstrated the assessments to facilitate understanding of the tests by the participants before they conducted the tests. The MMSE was used to assess general cognitive function. The TMT part A (TMT-A) and part B (TMT-B) were used to assess executive function. TMT-A requires participants to connect numbers ranging from 1 to 25 on a paper sheet. The TMT-B is similar to the TMT-A, except that participant connect numbers and letters alternately. Participants were asked to complete the tests as quickly and accurately as possible. The total time to complete the task is the score for each section. The DST, which consists of two subsections, the Digit Span Forward (DSF) test and the Digit Span Backward (DSB) test, was used to assess memory function. The DSF, which measures short-term memory, requires participants to memorize and repeat the numbers shown on a computer screen in the same order. In the DSB, measuring working memory, participants have to repeat the numbers in reverse order. The score for each test is determined by the number of correctly repeated sequences until participants are unable to reproduce two sequences of equal length.

Functional mobility and balance were assessed before and after the intervention using the 4-m timed walk test (4-MWT) and Timed Up and Go test (TUG). Participants were instructed to walk a straight 4-m distance at their normal ambulation pace for the 4-MWT. The mean duration of two trials was converted to walking speed (m/sec) (Miller et al., 2012). For TUG, participants were instructed to stand up from a chair with armrests without using their hands, walk for 3 m, and then return to their starting position (seated on the chair). The time required to complete the test was recorded. Assistive walking devices were only used at the participant’s request or if the investigators observed a risk of falling.

The results were presented as the mean and standard deviation. The Shapiro–Wilk test was used to determine normality. A one-way analysis of variance or Pearson chi-square were used to compare baselines between groups. Covariates were used as baseline values. Prior to the analysis of covariance (ANCOVA), the data were transformed logarithmically to meet the normal distribution criterion. Aside from that, the measurement results were examined to see if they met the assumptions of normality, linearity, and variance homogeneity. ANCOVA was used to compare outcome variables between groups. The Bonferroni test was used for pairwise comparisons. The dependent t-test was used for within-group analysis. An effect size analysis was performed using eta-squared (η²) for the ANCOVA, and interpreted 0.2, 0.5, and 0.8 as small, medium, and large, respectively. The Spearman correlation was used to examine correlations between changes (Δ; 12-week value - baseline value) in cognitive domains and oxidative stress and inflammatory markers. The level of statistical significance was set at P<0.05. Statistical analyses were conducted by employing IBM SPSS Statistics ver. 26.0 (IBM Co., Armonk, NY, USA).