Abstract
Objectives
We investigated the efficacy and safety of silkworm pupae extract (SWP) consumption for 12 weeks on muscle mass and strength in middle-aged and older individuals with relatively low skeletal muscle mass who do regular low-intensity exercise.
Design
A randomized double-blinded placebo-controlled trial.
Participants
The study was conducted with 54 participants with relatively low skeletal muscle mass (SMM) (64.4 ± 6.1 years; body mass index, 23.8 ± 2.4 kg/m2).
Intervention and Measurements
Participants were randomly assigned to one of two groups: 1000 mg of SWP/day plus regular exercise (SWP group, n=27) or placebo plus regular exercise (placebo group, n=27). All participants were required to engage in 30–60 minutes/day of walking for ≥3 days/week for 12 weeks. The primary outcome was knee extension/flexion strength (Nm), measured at the velocity of 60°/s. Secondary outcomes included body composition, biomarkers (creatine kinase and creatinine), handgrip strength, and quality of life questionnaire.
Results
Both the intention-to-treat (ITT) and per-protocol (PP) analyses revealed no significant impact of SWP on knee strength compared to the placebo group over 12 weeks. On the other hand, the SWP group had significantly greater increases in right-handgrip strength by 1.94 kg (95% CI: 0.08–3.79; p = 0.041) and left-handgrip strength by 1.83 kg (0.25–3.41; p = 0.024) compared to the placebo group in the ITT population, after 12 weeks. Moreover, in the PP population, the SWP group revealed an even greater increase in right-handgrip strength by 2.07 kg (0.15–3. 98; p = 0.035) and left-handgrip strength by 2.21 kg (0.60–3.83; p = 0.008) for the 12-week period. However, this study resulted in a failure to detect significant differences in the body composition, biomarkers, quality of life questionnaire, physical activity, and caloric intake between the groups. None of the participants in the SWP group experienced any significant adverse events. In the placebo group, two participants experienced urticaria and allergic side effects, leading to their withdrawal from the study and two exhibited elevated levels of liver enzyme and increased diastolic blood pressure, respectively at 12 weeks.
Conclusion
SWP, in addition to low-intensity exercise, may enhance handgrip strengths in middle-aged and older adults with relatively lower SMM. Future studies need to use a large sample size over longer periods to validate our findings. This trial was registered at clinicaltrials.gov as NCT04994054.
Key words: Silkworm pupae, muscle, handgrip strength, sarcopenia, middle-aged and older
Introduction
Sarcopenia has emerged as a social issue in a rapidly increasing aging population. Sarcopenia is a disease in which muscle mass, strength, and function are reduced due to aging (1). Muscles perform various functions, such as generating force for motion, maintaining posture and body temperature, blood circulation, and glucose metabolism. Sarcopenia is related to decreased physical function and ability to perform daily activities in older individuals (1, 2). This causes reduced mobility, falls, and fractures, and the resulting physical damage or disability decreases an individual's quality of life (3, 4). Thus, evaluating muscle mass and function is essential for determining the quality of life in older individuals (4). The fundamental strategy for addressing sarcopenia involves ensuring sufficient protein intake and engaging in exercise. A recent previous study that reviewed the effects of exercise on individuals with sarcopenia demonstrated notable benefits of exercise in terms of improving muscle mass, muscle strength, and physical performance even after a three-month intervention period (5).
The mulberry silkworm (Bombyx mori) is a domesticated insect bred to produce silk (6). A silkworm usually goes through five stages over 7 weeks to become a silk moth (7). Silkworm pupae are rich in proteins, amino acids, and unsaturated fatty acids and are traditionally consumed as a delicacy in Asia (6, 7). Recently, several studies have found that the active ingredients in silkworm pupae have pharmacological properties (8, 9, 10, 11, 12, 13). This has generated global interest in silkworm pupae from the food industry (14, 15). Furthermore, given the intense interest in planetary health in the current literature, the silkworm has a huge potential for the environmental benefits of replacing animal sources of protein intake with worms (16).
Proteins and hydrolyzed peptides of silkworm pupae are used to combat fatigue (6, 7, 17, 18). The consumption of silkworm and its by-products, such as pupae and dongchunghacho, has been reported to enhance muscle mass. Administration of silkworm powder as high-quality proteins for two weeks resulted in a notable improvement in the swimming time of mice (17). Also, combining the ingestion of silkworm pupae powder with resistance exercise training for 8 weeks increased the size of the gastrocnemius muscle in mice (18). However, these studies were conducted in animals; human studies are needed to evaluate the potential benefits of silkworm pupae consumption on human muscle strength and mass. To our knowledge, to date, no such human studies have been conducted. Therefore, we aimed to assess the clinical efficacy and safety of silkworm pupae as a functional food for muscle strength and mass in humans.
Methods
Study participants and ethics
The study participants were recruited through bulletin boards and social media advertisements in Yangsan-si, Republic of Korea. The study enrolled participants ≥50 years of age, with body mass index (BMI) ranging from 18.5 to 30.0 kg/m2 and <110% of the skeletal muscle mass (19), measured using the body composition analyzer (InBody 720, Biospace Co. Ltd., Seoul, Republic of Korea). We excluded participants who had any of the following conditions at baseline: vegetarian, central bone fracture within the past year, history of malignancy or severe cerebro-cardiovascular disease within the past 6 months, abnormal liver or kidney function (≥ 2 times the upper limit of normal), uncontrolled diabetes mellitus (fasting blood glucose ≥ 160 mg/dL), uncontrolled hypertension (blood pressure (BP) ≥ 160/100 mmHg), uncontrolled thyroid disease, mental illnesses such as severe affective disorder, schizophrenia, and substance use, alcohol abuse, known allergy to constituent foods, severe gastrointestinal symptoms, pregnancy/lactation or plans to become pregnant during the clinical trial, and participation in other drug trials within the last 3 months. Participants were continuously monitored for new drug use or changes in health status during the study. Participants of the following criteria were discontinued or dropped out of the trial: withdrawal of consent by the participant, occurrence of serious adverse reactions, protocol violations, failure to attend follow-up assessments, and poor adherence. Ethical approval for the involvement of human subjects in this study was granted by the Ethics Committee of the Institutional Review Board of Pusan National University Yangsan Hospital, Reference number 02-2021-023, 07/05/2021. This trial was registered at ClinicalTrials.gov (NCT04994054, 07/30/2021). The study was conducted between July 2021 and February 2022 in accordance with the principles of the Declaration of Helsinki and the Korean Good Clinical Products guidelines. Written informed consent was obtained from all participants.
Study design
This was a randomized, double-blind, placebo-controlled trial. Simple randomization into 2 study groups was performed using a random number table, with the code held by the manufacturer of silkworm pupae extract (SWP) and placebo. Randomization codes were created using the nQuery Advisor 7.0 (Statistical Solutions Ltd., Cork, Ireland). Both the researchers and participants remained blinded to the randomization assignments throughout the study. After the baseline assessment, 54 participants were randomly allocated to one of the 2 intervention groups where SWP or placebo was administered for 12 consecutive weeks. The participants were asked to complete their medication administration records. Adherence to supplementations was assessed using the pill count method. For optimal efficacy, ≥ 80% adherence was necessary.
Every participant was asked to visit the clinic thrice between 9–11 am (visit 1 for screening, visit 2 for randomization and initiation of supplementation, visit 3 for follow-up 6 weeks later, and a final visit (visit 4) at the end of the 12-week treatment period). BP and heart rate were recorded, and blood tests were performed at each visit. All the outcomes were measured at baseline and after 12 weeks of the intervention. In addition, the participants were required to exercise for 30 minutes to 1 hour per day, > 3 times a week, and counseled to maintain their usual diet during the study period. Physical activity was assessed using a self-recorded daily exercise log during the study. Physical activity and nutrition were assessed at baseline and 12 weeks (± 8 days) after treatment (Figure 1).
Figure 1.
Experimental design
Interventions
SWP and placebo were supplied by Day.N BIO Inc. (Jangheung County, Republic of Korea). The SWP capsule contained 1200 mg of SWP (supplied by Day.N BIO Inc., Republic of Korea), 2250 mg of psyllium husk, 1250 mg of oligosaccharide, 250 mg of caramel pigment, and 50 mg of hydroxypropyl methylcellulose (HPMC, total of 5000 mg/ capsule). The placebo capsule contained 2900 mg of psyllium husk, 1800 mg of oligosaccharide, 250 mg of caramel pigment, and 50 mg of HPMC (a total of 5000 mg/capsule). The placebo group was administered an equivalent amount of placebo as the SWP group; however, the placebo did not contain any protein component. Based on the suggested minimum effective dose of 500 mg/kg/day in a previous mouse model (17), the human equivalent dose considering the human body surface would be about 40 mg/kg/day. Then, 2400 mg for individuals weighing 60 kg was selected as the final daily dose. The average weight of the participants was 60.8 ± 8.3 kg. Participants in each group were instructed to take one capsule twice daily, precisely following breakfast and dinner.
Primary outcome measure
The primary outcome measure was the change in the peak torque (TQ) of knee extension/flexion, which represented the muscle function over the 12-week treatment period. The TQ was evaluated in isokinetic condition with the commonly used constant angular velocity of 60°/s (20). Knee extension/ flexion strengths were measured using the Biodex System 3 Pro isokinetic dynamometer (Biodex, Inc., Shirley, NY, USA). The test included a series of 5 extending and flexing movements at the velocity of 60°/s, which is the commonly used constant angular velocity due to the highest intraclass correlation coefficient of knee extension and flexion peak TQ, preceded by three trials with the moderate engagement of the muscles (21, 22).
Secondary outcome measures
Secondary outcome measures included changes in the appendicular skeletal mass index (ASMI), appendicular skeletal muscle mass index (SMI), skeletal muscle mass index (SMMI), body composition (body weight, SMM, total fat percent, trunk fat percent), handgrip strength, biomarkers [creatine kinase (CK) and creatinine levels], and Euro-QoL-5D-5L (EQ-5D-5L), which is a valid tool for assessing the quality of life in Korean (23), over the 12-week treatment period. Safety was confirmed by measuring the vital signs and performing laboratory tests, including complete blood cell counts, liver enzyme levels, and glucose levels.
Handgrip strengths were measured by conducting three measurements using a Jamar hydraulic dynamometer (Model 5030 J1, Sammons Preston Rolyan, Bolingbrook, IL, USA), and the highest recorded value was obtained. The participant was seated in a comfortable chair, without any arm support, with the elbow in 90° flexion, while ensuring separation between the upper arm and lateral chest. Body composition was assessed using whole-body dual-energy X-ray absorptiometry (Hologic Horizon W, Software: Apex version 5.6.0.5; Hologic Inc., Marlborough, MA, USA) in the supine position according to the manufacturer's guidelines (24, 25). The appendicular skeletal mass (ASM) was defined as the total lean soft-tissue mass in the four limbs. In this study, ASM (kg/m2), SMI (%), and SMMI (kg/m2) were estimated using the following formulas: ASMI = ASM (kg)/[height (m)]2, SMI (%) = ASM (kg)/total body weight (kg) × 100, and SMMI = SMM (kg)/[height (m)]2, respectively (26, 27).
Clinical assessments
BP and heart rate were measured three times in the sitting position after a 10-minute rest using the BP-203 RV II (Colin Corp., Aichi, Japan). Blood samples were collected following a 12-hour overnight fast to assess the effects of SWP and monitor potential side effects. CK concentration was measured by the CK N-acetyl cysteine activation procedure using an AU5800 chemistry analyzer (Beckman Coulter, Brea, CA, USA). Creatinine concentration was measured using a modified Jaffe's kinetic method. Serum glucose concentration was measured using a hexokinase-based test (Glucose HK Gen.3, Roche Diagnostics, Mannheim, Germany), and liver enzyme concentrations were measured according to the recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine on the Cobas c701 module (Roche Diagnostics, Mannheim, Germany).
Physical activity and nutrition assessments
Physical activity was monitored using a self-recorded daily exercise log. Food intake was assessed at baseline and after 12 weeks using a 24-hour dietary recall method, which relied on the recorded meals. Total calories, carbohydrates, lipids, and proteins consumed per day were analyzed using CAN-Pro software ver. 4.0 (The Korean Nutrition Society, Seoul, Republic of Korea).
Statistical analysis
G*Power software (version 3.1.9.7; Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) was used to calculate the sample size, based on our previous study (26 and Supplemental Table 1). The estimated sample size was 21 participants per group for 80% power to detect a mean pre- and post-treatment difference of 4.4 Nm in the knee flexion peak TQ at 60°/s, assuming a standard deviation of 5.0 Nm and an alpha error of 5% (26). The sample size was adjusted to 27 participants per group to allow for a dropout rate of 20%. The intention-to-treat (ITT) analysis encompassed all randomized participants (n = 54), regardless of their compliance or completion of the protocol. In contrast, the per-protocol analysis focused on subjects (n = 47) who successfully completed the study by strictly adhering to the main protocol (28). Intention-to-treat (ITT) analysis (n = 54) was used to compare outcomes between the SWP and placebo groups, with multiple imputations of missing data. Because the percentage of missing values at the 12-week follow-up was 13.0% for all variables, 5 imputed datasets were created, and the results of the analyses from the different imputed datasets were pooled according to Rubin's rules using R software version 3.6.2 (R Foundation for Statistical Computing). Multivariate imputation by the chained equations algorithm was used with the predictive mean matching method. A per-protocol (PP) analysis was also performed (n = 47) to assess the effectiveness of supplementation. The Shapiro-Wilk test was used to test the normality assumption. Baseline characteristics of both groups were compared using the 2-sample t-test or Mann–Whitney's U test for continuous variables and the chi-square or Fisher's exact test for categorical variables. ANCOVA or rank ANCOVA was used for the main analysis, with adjustments for each baseline variable. The model assumptions were checked using histograms, normal probability plots, and residual scatter plots. Safety analyses included all randomized patients who were exposed to at least one dose of the study intervention. A significance level (α) of 5% was adopted for all analyses. Data were analyzed using IBM SPSS Statistics (version 27.0; IBM Inc., Armonk, NY, USA).
Results
Baseline characteristics of participants
A total of 65 participants were enrolled, of which 54 were included in this study and 47 (87.0%) completed the study (Figure 2). The average age of the participants was 64.4 ± 6.1 years (range 50–74 years). The supplement adherence exceeded 99% during the 12-week study period. Their general characteristics are reported in Tables 1 and 2. Caloric intake was higher in the SWP group than in the placebo group in the ITT population (n=54), while diastolic BP was higher in the placebo group than in the SWP group in the PP population (n=47). However, there were no differences in the demographic and anthropometric data, smoking history, alcohol consumption, and physical activity parameters at baseline between the 2 groups (Table 1). Additionally, there were no significant changes in calorie intake or physical activity during the study period (Table 2). During the entire study period, a double-blind requirement was maintained.
Figure 2.
CONSORT flow diagram of the study
Table 1.
Baseline characteristics of the study groups1
|
Variables |
Intention-to-treat population |
Per-protocol population |
||||
|---|---|---|---|---|---|---|
| SWP (n=27) | Placebo (n=27) | Pa | SWP (n=23) | Placebo (n=24) | Pa | |
| Age, years | 64.4 ± 6.5 | 64.3 ± 5.8 | 0.930 | 64.4 ± 7.0 | 64.2 ± 5.8 | 0.904 |
| Males, % | 9 (33.3) | 3 (11.1) | 0.050 | 8 (34.8) | 3 (12.5) | 0.071 |
| Body mass index, kg/m2 | 24.05 ± 2.09 | 23.60 ± 2.67 | 0.495 | 24.04 ± 2.13 | 23.73 ± 2.79 | 0.671 |
| Systolic blood pressure, mmHg | 122.89 ± 13.47 | 127.44 ± 13.57 | 0.221 | 121.61 ± 13.78 | 128.33 ± 12.74 | 0.089 |
| Diastolic blood pressure, mmHg | 79.30 ± 7.69 | 83.22 ± 8.91 | 0.089 | 78.96 ± 7.69 | 84.25 ± 8.84 | 0.034 |
| Smoker, % | 2 (7.4) | 0 (0.0) | 0.740b | 2 (8.7) | 0 (0.0) | 0.478b |
| Alcohol, % | 9 (33.3) | 4 (14.8) | 0.111 | 8 (34.8) | 4 (16.7) | 0.154 |
| Co-morbidities | 0.995 | 0.928 | ||||
| Hypertension | 6 (22.2) | 4 (14.8) | 5 (21.7) | 3 (12.5) | ||
| Dyslipidemia | 8 (29.6) | 5 (18.5) | 6 (26.1) | 5 (20.8) | ||
| Diabetes mellitus | 2 (7.4) | 0 (0.0) | 1 (4.3) | 0 (0.0) | ||
| Others | 9 (33.3) | 5 (18.5) | 8 (34.8) | 4 (16.7) | ||
| IPAQ, METs | 990 [1371] | 1386 [2001] | 0.295c | 990 [1371] | 1386 [1952.3] | 0.301c |
| Caloric intake, Kcal/day | 1681.5 ± 417.8 | 1414.7 ± 360.6 | 0.015 | 1621.1 ± 362.5 | 1419.1 ± 382.7 | 0.070 |
| Protein, % | 15.3 ± 2.9 | 16.2 ± 4.1 | 0.357 | 15.4 ± 3.0 | 16.4 ± 3.6 | 0.314 |
| Fat, % | 10.4 ± 3.0 | 10.2 ± 3.4 | 0.850 | 10.4 ± 3.2 | 10.2 ± 3.1 | 0.859 |
| Carbohydrate, % | 62.2 ± 8.9 | 60.6 ± 12.5 | 0.924 | 62.4 ± 9.6 | 60.4 ± 12.1 | 0.932 |
| Adherence of medication, % | 99.4 [1.9] | 99.4 [4.5] | 0.978c | 99.4 [3.0] | 99.7 [4.0] | 0.840c |
| Adherence of physical activity, % | 95.5 [11.0] | 95.2 [18.8] | 0.586c | 93.8 [12.1] | 94.0 [19.1] | 0.965c |
1. Values are presented as mean ± SD or frequency (percentage) or median [IQR]. a. two-sample t-test or chi-square test. b. Fisher's exact test. c. Mann-Whitney U test. IPAQ, International Physical Activity Questionnaire; MET, metabolic equivalent task; SWP, silkworm pupae extract.
Table 2.
Caloric intake and physical activity in the two groups1
|
SWP group |
Placebo group |
Adjusted difference of SWP vs. Placebo | Pa | |||
|---|---|---|---|---|---|---|
| Baseline | 12 week | Baseline | 12 week | |||
| Intention-to-treat (n=54) | ||||||
| Caloric intake, Kcal/d | 1681.49 ± 417.84 | 1471.36 ± 391.05b | 1414.70 ± 360.63 | 1564.40 ± 357.59 | −195.42 (−373.59, 3.33) | 0.054 |
| Protein, % | 15.34 ± 2.85 | 14.78 ± 2.81 | 16.23 ± 4.08 | 16.20 ± 4.22 | −1.45 (−3.45, 0.81) | 0.218 |
| Fat, % | 10.38 ± 2.97 | 8.90 ± 3.46b | 10.21 ± 3.36 | 9.56 ± 3.93 | −0.71 (−2.67, 1.25) | 0.419 |
| Carbohydrate, % | 62.25 ± 8.93 | 66.34 ± 8.56 | 60.61 ± 12.55 | 63.25 ± 10.74 | 2.78 (−2.47, 8.03) | 0.267 |
| IPAQ, METs | 990 [1371] | 1626 [1440]c | 1386 [2001] | 2079 [1930.5] | −6.44 (−623.25, 610.38) | 0.536 |
| Per-protocol (n=47) | ||||||
| Caloric intake, Kcal/d | 1621.14 ± 362.50 | 1471.65 ± 415.27 | 1419.05 ± 382.66 | 1552.73 ± 375.30 | −170.45 (−392.17, 51.27) | 0.128 |
| Protein, % | 15.43 ± 2.95 | 14.71 ± 2.94 | 16.41 ± 3.59 | 15.67 ± 3.28 | −0.92 (−2.80, 0.95) | 0.327 |
| Fat, % | 10.39 ± 3.19 | 8.62 ± 3.53b | 10.23 ± 3.10 | 9.26 ± 4.03 | −0.7 (−2.84, 1.44) | 0.641 |
| Carbohydrate, % | 62.45 ± 9.64 | 67.31 ± 8.63b | 60.38 ± 12.12 | 63.13 ± 10.94 | 3.64 (−2.01, 9.29) | 0.656 |
| IPAQ, METs | 990 [1371] | 1386 [1482]b | 1386 [1952.3] | 1963 [2212.9] | −98.41 (−674.15, 477.33) | 0.658 |
1. Values are presented as mean ± SD or median [IQR] or mean (95% CI). a. ANCOVA or rank ANCOVA with adjusted for each baseline as covariate. b. P < 0.05, c. P < 0.005 by paired t-test or Wilcoxon sign-rank test within each group. IPAQ, International Physical Activity Questionnaire; MET, metabolic equivalent task; SWP, silkworm pupae extract.
Primary outcome measure
Based on the within-group differences, it was found that SWP increased the extensor strength performance of the right knee after 12 weeks. However, as Table 3 shows, there were no treatment-related effects of SWP on knee extension/flexion strength compared to placebo in both the ITT and PP analyses, based on between-group differences.
Table 3.
Primary outcome measures in the two groups1
| SWP group | Placebo group | Adjusted difference of SWP vs Placebo 12 week | Pa | |||
|---|---|---|---|---|---|---|
| Baseline | 12 week | Baseline | 12 week | |||
| Intention-to-treat (n=54) | ||||||
| 60°/s knee extension peak TQ (right), Nm | 54.92 ± 28.98 | 62.12 ± 25.49b | 56.73 ± 21.99 | 59.12 ± 19.88 | 4.69 (−3.10, 12.48) | 0.232 |
| 60°/s knee extension peak TQ (left), Nm | 56.07 ± 24.66 | 60.05 ± 25.64 | 56.51 ± 23.98 | 66.17 ± 22.52b | −3.63 (−12.91, 5.65) | 0.436 |
| 60°/s knee flexion peak TQ (right), Nm | 19.98 ± 11.70 | 20.87 ± 12.71 | 20.16 ± 8.91 | 21.51 ± 9.45 | −1.11 (−6.16, 3.95) | 0.662 |
| 60°/s knee flexion peak TQ (left), Nm | 18.91 ± 12.06 | 19.44 ± 10.57 | 20.05 ± 7.77 | 21.80 ± 8.79 | −0.497 (−4.40, 3.41) | 0.799 |
| Per-protocol (n=47) | ||||||
| 60°/s knee extension peak TQ (right), Nm | 56.85 ± 30.43 | 63.57 ± 26.34b | 56.25 ± 23.16 | 59.82 ± 20.82 | 3.31 (−4.60, 11.22) | 0.274 |
| 60°/s knee extension peak TQ (left), Nm | 55.87 ± 25.49 | 62.17 ± 26.76 | 56.86 ± 25.22 | 66.68 ± 23.75b | −3.75 (−13.43, 5.92) | 0.438 |
| 60°/s knee flexion peak TQ (right), Nm | 19.41 ± 11.82 | 20.50 ± 13.06 | 20.15 ± 9.47 | 21.76 ± 9.93 | −0.75 (−6.01, 4.52) | 0.793 |
| 60°/s knee flexion peak TQ (left), Nm | 18.35 ± 12.27 | 19.80 ± 11.02 | 20.52 ± 8.11 | 22.11 ± 9.24 | −0.75 (−4.91, 3.40) | 0.494 |
1. Values are presented mean ± SD or mean (95% CI). a. ANCOVA or rank ANCOVA with adjusted for baseline caloric intake and each baseline value as covariates. b. P < 0.05 by paired t-test within each group. SWP, silkworm pupae extract; TQ, torque.
Secondary outcome measures
Based on the within-group differences, SWP significantly increased the handgrip strengths. Handgrip strengths were higher in the SWP group than in the placebo group in the ITT (right, p = 0.041, left, p = 0.024) and PP analysis (right, p = 0.035, left, p = 0.008), based on between-group differences (Table 4 and Supplemental Table 2). The median percentage difference (interquartile range) of the right and left handgrip strengths were 6.7% (26.6) and 4.8% (22.4), respectively, in the ITT analysis, and 5.6% (25.0) and 4.8% (21.7), respectively, in the PP analysis. Based on the within-group differences, SWP showed improvements in total fat percent and EQ-5D-5L score while also reducing SMMI; however, no significant difference was observed when comparing the two groups (Table 4 and Supplemental Table 2).
Table 4.
Secondary outcome measures in the two groups (Intention-to-treat analysis)1
| SWP group (n = 27) | Placebo group (n = 27) | Adjusted difference of SWP vs Placebo 12 week | Pa | |||
|---|---|---|---|---|---|---|
| Baseline | 12 week | Baseline | 12 week | |||
| Handgrip (right), kg | 25.96 ± 7.48 | 28.17 ± 8.23b | 25.12 ± 4.88 | 25.20 ± 4.86 | 1.94 (0.08, 3.79) | 0.041 |
| Handgrip (left), kg | 25.12 ± 7.03 | 26.66 ± 7.32b | 24.13 ± 4.50 | 23.80 ± 4.35 | 1.83 (0.25, 3.41) | 0.024 |
| ASMI, kg/m2 | 5.69 [1.36] | 5.60 [1.38] | 5.42 [0.88] | 5.50 [0.82] | −0.11 (−0.29, 0.06) | 0.159 |
| SMI, % | 23.04 [4.66] | 22.78 [4.28] | 23.15 [2.37] | 22.96 [2.7] | −0.13 (−0.67, 0.42) | 0.478 |
| Total fat percent, % | 40.90 [9.7] | 41.20 [8.3]b | 40.2 [5.3] | 40.5 [6.6] | 0.33 (−0.55, 1.20) | 0.553 |
| Trunk fat percent, % | 41.47 ± 5.98 | 42.02 ± 6.28 | 41.57 ± 4.56 | 42.20 ± 5.02b | 0.07 (−0.75, 0.88) | 0.867 |
| Creatinine, mg/dl | 0.70 ± 0.13 | 0.70 ± 0.15 | 0.68 ± 0.12 | 0.68 ± 0.11 | −0.01 (−0.05, 0.04) | 0.816 |
| Skeletal muscle mass, kg | 35.84 ± 6.33 | 35.71 ± 6.29 | 33.48 ± 5.61 | 33.63 ± 5.38 | −0.22 (−0.85, 0.42) | 0.495 |
| SMMI, kg/m2 | 9.02 [1.85] | 8.99 [1.62]b | 9.33 [1.12] | 9.49 [0.94] | −0.18 (−0.38, 0.02) | 0.139 |
| Body weight, kg | 62.38 ± 7.54 | 62.63 ± 7.37 | 58.57 ± 8.49 | 59.24 ± 8.47 | −0.16 (−1.24, 0.92) | 0.770 |
| Creatine kinase, U/L | 101.0 [78.0] | 88.0 [30.0] | 112.0 [111.0] | 92.0 [43.0] | −6.22 (−36.85, 24.41) | 0.798 |
| EQ-5D-5L, score | 0.90 [0.11] | 0.93 [0.06]b | 0.96 [0.10] | 0.96 [0.10] | 0.02 (−0.02, 0.05) | 0.346 |
| EQ-5D-5L VAS, score | 77.21 ± 13.09 | 80.40 ± 14.31 | 76.12 ± 17.90 | 83.23 ± 11.20c | −3.38 (−9.91, 3.15) | 0.303 |
1. Values are presented as mean ± SD or median [IQR] or mean (95% CI). a. ANCOVA or rank ANCOVA with adjusted for baseline caloric intake and each baseline value as covariates. b. P < 0.05, c. P < 0.005 by paired t-test or Wilcoxon signed rank test within each group. SWP, silkworm pupae extract; ASMI, appendicular skeletal muscle index; SMI, appendicular skeletal muscle mass index; SMMI, skeletal muscle mass index; EQ-5D-5L, European quality of life 5 dimensions 5 level version; VAS, visual analogue scale.
Safety
When comparing the two groups after 12 weeks, no difference was observed in the changes in fasting blood glucose, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) concentrations or BP (Table 5). However, at the end of the 12-week period, it was observed that 1 participant in the placebo group had elevated ALT concentration, and 1 participant had higher diastolic blood pressure (≥100 mmHg) (Supplemental Table 3). Additionally, 2 (3.7%) of the 54 participants reported two adverse reactions (urticaria and allergy). These 2 participants were assigned to the placebo group.
Table 5.
Laboratory findings evaluating the adverse effects1
| SWP group | Placebo | group | Adjusted difference of SWP vs Placebo 12 week | Pa | ||
|---|---|---|---|---|---|---|
| Baseline | 12 week | Baseline | 12 week | |||
| Intention-to-treat (n=54) | ||||||
| Glucose, mg/dl | 97.70 ± 17.94 | 101.26 ± 16.71b | 96.93 ± 12.70 | 102.52 ± 15.97 | −1.86 (−8.01, 4.30) | 0.547 |
| AST, IU/L | 27.0 [9.0] | 25.0 [11.0] | 27.0 [10.0] | 24.0 [10.0] | 3.07 (0.18, 5.95) | 0.158 |
| ALT, IU/L | 18.0 [18.0] | 24.0 [22.0] | 21.0 [13.0] | 21.0 [11.0] | 3.81 (−2.27, 9.90) | 0.227 |
| Systolic BP mmHg | 122.89 ± 13.47 | 125.67 ± 9.38 | 127.44 ± 13.57 | 127.15 ± 12.37 | 0.61 (−4.45, 5.68) | 0.809 |
| Diastolic BP mmHg | 79.30 ± 7.69 | 78.96 ± 6.35 | 83.22 ± 8.91 | 81.89 ± 9.71 | −0.84 (−4.76, 3.08) | 0.669 |
| Per-protocol (n=47) | ||||||
| Glucose, mg/dl | 99.13 ± 19.09 | 101.09 ± 17.74 | 96.50 ± 12.46 | 101.46 ± 16.09 | −2.42 (−9.23, 4.39) | 0.595 |
| AST, IU/L | 27.0 [10.0] | 25.0 [12.0] | 26.0 [9.75] | 24.0 [9.75] | 3.24 (−0.02, 6.49) | 0.194 |
| ALT, IU/L | 18.0 [18.0] | 22.0 [28.0] | 21.5 [13.5] | 21.0 [11.75] | 4.47 (−2.50, 11.43) | 0.206 |
| Systolic BP, mmHg | 121.61 ± 13.78 | 124.91 ± 9.74 | 128.33 ± 12.74 | 126.46 ± 12.74 | 2.10 (−3.32, 7.52) | 0.840 |
| Diastolic BP, mmHg | 78.96 ± 7.69 | 79.26 ± 6.80 | 84.25 ± 8.84 | 82.38 ± 10.16 | 0.02 (−4.45, 4.50) | 0.991 |
1. Values are presented as mean ± SD or median [IQR] or mean (95% CI). a. ANCOVA or rank ANCOVA with adjusted for each baseline as covariate. b. P < 0.05 by paired t-test within each group. SWP, silkworm pupae extract. AST, aspartate aminotransferase; ALT, alanine aminotransferase; BP, blood pressure.
Discussion
Herein, we assessed the potential benefits and safety of a 12-week course of SWP (with regular exercise) on muscle strength, body composition, and biomarkers of muscle metabolism in middle-aged and older people with relatively low muscle mass. The results confirmed that SWP may enhance muscle strength but not mass in middle-aged and older individuals who perform a regular low-intensity exercise. SWP consumption for 12 weeks increased both hand grip strengths (right and left, median 6.7% and 4.4%, ITT; 5.6% and 4.8%, PP) compared to the baseline. As participants in this study, individuals who met the Asian Working Group for Sarcopenia (AWGS) criteria for sarcopenia (29) were also included, although the number of such individuals was limited. In the SWP group, 5 participants were initially diagnosed with sarcopenia according to the AWGS criteria. However, this number decreased to 2 after 12 weeks of the trial. On the other hand, in the placebo group, only 1 participant was diagnosed with sarcopenia at baseline, and this number remained unchanged at 1 after 12 weeks. This finding shows that SWP can be an alternative source of protein to animal-derived protein for improving muscle health. However, the change in knee strength did not differ between the SWP and placebo groups after 12 weeks of the intervention. In addition, no significant change was observed in the ASMI, SMI, SMMI, weight, body composition, creatinine and CK concentrations, and EQ-5D-5L and its EQ-VAS scores between 2 groups after 12 weeks. In safety of SWP, ITT analysis revealed that the AST concentration increased in the SWP group, compared with those in the placebo group after 12 weeks, despite being within the reference range. Thus, further studies are required to confirm this finding.
Silkworm pupae, a popular food in Asia, including China, Korea, India, and Japan, are rich in proteins, fats, minerals, and vitamins (6). In the dry powder form, proteins and fats account for 55.6% and 32.2% of the total content, respectively (30). While meat contains essential amino acids, it also has a high saturated fatty acid content; hence, there is a risk of metabolic diseases if excessive meat is consumed (18). However, silkworm pupae have high unsaturated fatty acids and essential amino acids necessary for humans (30). Thus, silkworm pupae have been proposed as an economical and eco-friendly alternative to animal proteins (18, 31).
Previous in vitro and in vivo studies have established that silkworm pupae consumption has several pharmacological functions, including anticancer (11, 32), antioxidant (33, 34), antibacterial (10, 35), antihypertensive (36), antilipemic (37), hypoglycemic (12), hepatoprotective (9), and anti-fatigue (17, 18) effects. Most studies consistently show that silkworm pupae consumption increases muscle mass. One previous animal study established that silkworm pupae powder consumption tended to increase muscle mass after resistance exercise training; however, the difference was not significant (18). However, one recent animal study found that silkworm powder consumption produced a dual effect; it increased muscle (114% compared to control; p < 0.01) and reduced fatigue (plasma creatinine and D-lactate concentrations: 63%, and 78%, respectively, compared to control; p < 0.05), with increasing forced swimming time (17). Another animal study reported that silkworm pupae consumption increased the total amino acids content of the muscle (111.5% compared to control; p < 0.05) and muscle mass (38). Muscle synthesis is increased by the C-Myb expression, which promotes myoblast differentiation via inhibition of glycogen synthase kinase-3β (GSK3β) expression (38, 39). Protein kinase B (Akt), which is related to the Akt-mammalian target of rapamycin (mTOR) signal, tends to increase with silkworm pupae consumption, which is greater when combined with exercise. The Akt-mTOR signaling pathway is related to muscle wasting and affects the rise in resistance exercise-induced muscle synthesis (18, 40).
The current study had some limitations. The study duration was limited to 12 weeks. Changes in handgrip strength were observed within 12 weeks; however, changes in knee strength, body composition, and biomarkers after SWP consumption were not observed. These results need to be verified after the administration of SWP for a longer period. Second, as this is the first human study of muscle health in SWP, the sample size estimate was based on a prior study that examined changes in knee flexion strength. Third, this study did not measure the expression levels of GSK3β, Akt, Akt-mTOR, etc., which are associated with the increase in SW-related muscle mass in animal experiments. Therefore, it remains unknown whether changes in the expression of GSK3β, Akt, and Akt-mTOR also occur in the human body; further studies are needed that measure their expression levels. Finally, elevated concentrations of AST were minimal and not much clinically meaningful. However, the effect may be substantive if the dose level increases or if the supplement use lasts longer.
Despite these limitations, to the best of our knowledge, this is the first well-designed clinical study to evaluate the safety and efficacy of SWP supplementation in increasing muscle strength and mass, with regular low-intensity exercise, in middle-aged and older people with relatively low muscle mass. None of the participants who consumed SWP experienced any adverse events.
In conclusion, daily consumption of 2400 mg of SWP for 12 weeks could enhance handgrip strength in middle-aged and older individuals with relatively low muscle mass who engage in regular low-intensity exercise. However, there was no change in muscle mass after 12-week SWP administration. SWP has the potential to provide an alternative source of protein to animal-derived protein for enhancing muscle health. Further studies with large sample sizes over longer periods are needed to determine the mechanism of action of SWP on muscle in humans, the effective dose and optimal duration of SWP supplementation for muscle strength and mass, and the potential interaction between SWP and exercise.
Funding
None.
Author Contributions
The authors' responsibilities were as follows—YLL and SYL: designed and conducted the research; HYK, JHL, and SYL: conceived the study idea and the analysis design; JIC, YLL, and SYL: carried out the statistical analysis and wrote the initial drafts. SYL had primary responsibility for the final content. All authors read and approved the final manuscript.
Conflicts of Interest
All authors have no conflicts of interest.
Ethical standards
This study was granted by the Ethics Committee of the Institutional Review Board of Pusan National University Yangsan Hospital (Reference number 02-2021-023).
ClinicalTrials.gov Identifier
NCT04994054. Web site: https://clinicaltrials.gov/ct2/show/NCT04994054.
Electronic supplementary material
Supplementary material is available for this article at https://doi.org/10.1007/s12603-023-1942-9 and is accessible for authorized users.
Efficacy of Silkworm pupae extract on muscle strength and mass in middle-aged and older individuals: a randomized, double-blind, placebo-controlled trial
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Supplementary Materials
Efficacy of Silkworm pupae extract on muscle strength and mass in middle-aged and older individuals: a randomized, double-blind, placebo-controlled trial