Randomized, Double-Blind, Four-Arm Pilot Study on the Effects of BRAND' S Chicken Essence and FlexC-II® on Joint, Bone, and Muscle Functions


 BackgroundKnee osteoarthritis (OA) is a leading cause of disability among elderly individuals. Medical and surgical treatments are expensive and have side eﬀects. The current study aimed to investigate the efficacy and safety of FlexC-II®, a type II collagen hydrolysate, and BRAND'S Essence of Chicken with FlexC-II® (BEC-FlexC-II®) on joint, muscle, and bone functions among elderly adults with OA.MethodsPatients (n = 160) with grade 1–3 knee OA based on the Kellgren–Lawrence classification system, joint pain for ≥3 months, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score of >6 were considered eligible in this study. The participants were randomized into four groups (BEC-FlexC-II®, FlexC-II®, glucosamine hydrochloride [HCl], and placebo) and were instructed to perform resistance training for 24 weeks. The outcomes included WOMAC score, visual analogue scale (VAS) score, hand grip strength, fat-free mass (FFM), bone mass, and 36-Item Short-form Survey score. ResultsThe WOMAC scores of all groups improved after 24 weeks. However, the results did not significantly differ. Meanwhile, there was a remarkable difference in the VAS score between groups (P = 0.039). The FlexC-II® group had a greater reduction in pain than the placebo group (mean ± standard error: −1.3 ± 0.45, P = 0.021). In the FlexC-II® group, the VAS score significantly reduced by 0.9 ± 1.89 (P = 0.034) after 14 days. In the adjusted analyses, the BEC-FlexC-II® group had a significantly higher FFM than the glucosamine HCl (P = 0.02) and placebo (P = 0.017) groups and hand grip strength than the glucosamine HCl group (P = 0.002). Further, on the basis of a subgroup analysis, participants with poor training compliance in the BEC-FlexC-II® group had a significantly higher left hip bone mass than those in the placebo (P = 0.01) and glucosamine HCl (P = 0.049) groups.ConclusionsFlexC-II® relieves OA-associated pain within 14 days, and BEC-FlexC-II® increases muscle mass and strength after 24 weeks. Thus, BEC-Flex-CII® is a promising novel, holistic supplement that can improve mobility by promoting joint, muscle, and bone functions among elderly individuals. However, full-scale studies should be conducted in the future to validate these findings.Trial registrationThis clinical trial was retrospectively registered in ClinicalTrials.gov with the ID NCT04483024 on July 20, 2020. URL: https://register.clinicaltrials.gov/prs/app/action/SelectProtocol?sid=S0008FK4&selectaction=Edit&uid=U0004BM2&ts=2&cx=-5y1oh4


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Background Knee osteoarthritis (OA) is characterized by destruction of knee cartilage and remodeling of the adjacent bone and is a leading cause of disability among elderly adults. 1 The complications of severe knee OA, including loss of mobility and limited daily activities, can lead to economic burden among individuals and societies. The World Health Organization Global Burden of Disease Study was conducted in 21 epidemiological regions worldwide. Results showed that the burden of knee OA increased by 26.6% from 1990 to 2010. 2 The current therapies for OA include different over-the-counter analgesics, non-steroidal antiin ammatory drugs, intra-articular injections of corticosteroids or hyaluronic acid (HA), and tramadol and other opioid analgesics, which can relieve severe pain. 3,4 Although these therapies can alleviate shortterm symptoms, their ultimate impact on the pathophysiologic progression of OA is limited. 5 In addition, there are considerable side effects associated with these drugs. Total joint replacement is the common and only effective treatment for pain and disability.
Individuals with OA typically switch to natural nutraceuticals for pain and discomfort. These products are commonly used because they are well tolerated and considered safe. Nutraceuticals are de ned as functional foods or their components and natural products that have medicinal, therapeutic, or health bene ts, including the prevention or treatment of diseases. Currently, glucosamine hydrochloride (HCl) and chondroitin are the two most commonly used nutraceuticals by humans for alleviating pain associated with arthritis. 6 However, recent randomized controlled trials and meta-analysis of these supplements have shown that they have small-to-moderate e cacy in relieving OA-associated symptoms. 7 Therefore, other disease-modifying osteoarthritic drugs (DMOADs) aiming to prevent the deterioration of joint tissues should be explored. 8 Collagen hydrolysate (CH) is one of the candidate DMOADs that has received considerable attention due to its effects in relieving OA-associated symptoms. CH may reduce OA-associated symptoms by providing building blocks for the collagen brillar network, which promotes tensile strength for the articular cartilage matrix. 9 Oesser et al. have conducted a series of preclinical studies. Results showed that CH passes across the mucosal barrier in the small bowel as a complete peptide that is not subjected to enzymatic cleavage and that accumulates in cartilage tissues and stimulates the production of type II collagen (major protein in the articular cartilage) and proteoglycans in the extracellular matrix of cartilages. 10,11,12 Furthermore, bone changes contribute to the pathogenesis of OA. 13 Preclinical studies on rodents showed that the administration of CH increased bone mineral density 14 and promoted bone metabolism. 15,16 In addition to these preclinical studies, open-label or placebo-controlled randomized clinical trials have been conducted. Several data showed the bene cial effects of CH on joint health in different populations particularly comprising individuals with OA. 17,18,19,20,21,22 The daily administration of CH for 3 months improved joint health or function. That is, there was a decrease in pain 11,12,14 and dependency on pain relievers 14 and improvement in leg strength. 16 When CH is taken together with calcitonin, it inhibited collagen breakdown in postmenopausal women. 23 Considering these ndings, we have developed FlexC-II ® , a type II CH derived from chicken cartilage, and BRAND'S Essence of Chicken (BEC) plus FlexC-II ® (BEC-FlexC-II ® ), a supplement of chicken essence with additional FlexC-II ® . FlexC-II ® is a soluble naturally occurring matrix of hydrolyzed collagen type II, chondroitin sulfate, and HA. Its composition is similar to that of the human articular cartilage lining found in the synovial joints. BEC has been consumed for decades as a health tonic for energy boosting and fatigue recovery. In fact, BEC supplement has been clinically proven to accelerate recovery from exercise by increasing the clearance rate of plasma lactate and ammonia, thereby reducing muscle fatigue. 24 However, all previous data are based on either BEC or CH per se in clinical or in in vitro settings. We hypothesized that FlexC-II ® can relieve joint discomfort and restore joint function and BEC-FlexC-II ® can improve bone and muscle functions. Thus, this randomized, double-blind, four-arm pilot study aimed to investigate the e cacy and tolerability of BEC-FlexC-II ® on joint, bone, and muscle functions.

Study Design
This randomized, double-blind, four-arm, parallel study evaluated the effects of BEC-FlexC-II ® on joint, bone, and muscle functions in patients with knee OA. The current study was approved by the independent Ethics Committee of Linkou Chang Gung Memorial Hospital, Taiwan, prior to commencement. Moreover, it was conducted in accordance with the ethical principles in the latest version of the Declaration of Helsinki and International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use-Good Clinical Practice guidelines. A written informed consent was obtained from each participant before conducting any study procedures.
After enrollment, the participants were randomly allocated in equal proportions to four groups: BEC-FlexC-II ® , FlexC-II ® , glucosamine HCl, and placebo. Then, they were instructed to consume one bottle (68 mL) of the experimental product daily in the morning (after breakfast) for 24 weeks and to perform resistance exercise for 30 min twice a week based on a training manual. Next, they attended four study visits at 0 (baseline), 8, 16, and 24 weeks. A food questionnaire focusing on calcium, vitamin D, and protein intake was used to record weekly dietary status preceding the returning visits.

Participants
We enrolled participants who were aged 45-75 years, had a body mass index within the normal range (18.0-30.0 kg/m 2 ), weighed at least 40 kg, experienced knee pain for ≥3 months, had a Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) total pain score of ≥6, presented with mild-tomoderate knee OA (grades 1-3) according to the Kellgren-Lawrence classi cation system, had loss in muscular strength or physical performance for >1 year, and were willing to discontinue hormone therapy and dietary supplements during the study period. The exclusion criteria were as follows: individuals with active viral or bacterial infection based on clinical examinations; those with a history of rheumatoid or other types of arthritis, renal dysfunction, psychiatric disorders, diabetes, stroke or myocardial infarction, mental retardation, schizophrenia, gout, Paget disease of the bone or spinal disc herniation, expected knee arthroscopy or arthroplasty, and life-threatening pathology; those receiving treatments including anti-osteoporotic therapy within the last year, intra-articular injection applied at the target knee joint (most painful knee during screening) within the last 3 months (6 months for HA), and current corticosteroid therapy; those who cannot tolerate protein-based food or supplement; pregnant or lactating women; and those who had alcohol abuse or addiction.

Experimental Products
Both experimental products (i.e., FlexC-II ® and BEC-FlexC-II ® ) and comparators (glucosamine HCl and placebo) were provided in 68-mL bottles by BRAND'S Suntory Asia. FlexC-II ® contained 2.0 g of hydrolyzed type II collagen, thereby providing naturally occurring composition of CH (66.5%), depolymerized chondroitin sulfate (18%), and HA (11%). The uncharacterized components of the sternal cartilage accounted for the remaining 4.5%. Meanwhile, BEC-FlexC-II ® contained 2.0 g of hydrolyzed type II collagen and 5.81 g of BEC with proteins and peptides. BEC was produced via water extraction process from chicken meat for several hours under high temperature, followed by centrifugation to remove fat and cholesterol, vacuum concentration, and sterilization with high temperature and pressure before bottling. Each bottle contained 1.5 g of glucosamine HCl, which is an active comparator. The placebo comprised 6.8 g of maltodextrin, 7 mg of xanthan gum, and yeast extract (9.68%), and its taste was similar to that of BEC-FlexC-II ® . All liquid products in the glass bottles were isocaloric, identical in appearance, and similar in terms of avor and texture.

Study Endpoints and Assessments
The following measurements were used to evaluate the effect of BEC-FlexC-II ® and FlexC-II ® : the WOMAC and visual analogue scale (VAS) scores for joint health; hand grip strength and fat-free mass (FFM) for muscle growth and strength; bone mass for bone health; and 36-Item Short-form Survey (SF-36) score for patient-reported outcomes. The primary endpoint was change in the WOMAC score between the four groups, and the co-primary endpoint was change in the VAS scores. To assess for safety, adverse events (AEs), laboratory test ndings, vital signs, and physical examination results were examined.
The WOMAC is a proprietary set of standardized questionnaire that is widely used to evaluate for OA, including pain, stiffness, and joint function. Higher WOMAC scores indicated worse pain, stiffness, and functional limitations. Pain was measured using the VAS on days 7 and 14 after baseline measurement via patient self-assessment, without scheduled follow-up visits. The VAS is a 100-mm scale used to record pain intensity. Higher VAS scores indicated greater pain. Hand grip strength on both hands was assessed three times using Smedley's DynamoMeter (TTM; Tokyo, Japan), and the maximum value was recorded. The FFM was calculated using the following formula: FFM = body weight − fat mass (FM), where FM was measured via dual-energy X-ray absorptiometry (DXA; HOLOGIC Horizon ® DXA System, USA). Lumbar spine, left hip, and right hip bone masses were also assessed via DXA (HOLOGIC Horizon ® DXA System, USA). The SF-36 is a 36-item, patient-reported measure. 25 It comprises eight scales (vitality, physical functioning, bodily pain, general health perceptions, physical role functioning, emotional role functioning, social role functioning, and mental health). Each scale was directly transformed into a 0-to 100-scale, and a lower score indicated greater disability.

Statistical Analysis
In a previous study, the data of each group had a normal distribution, with a standard deviation (SD) of 13.81. 26 The target sample size was 36 per study group based on an estimated difference, with a mean of 10.26, two-sided alpha value of 0.05, statistical power of 80%, and anticipated dropout rate of 25%. A total of 144 participants was considered su cient for this study.
The study population comprised participants who had a supplement compliance rate of ≥70% and who did not meet any protocol deviations leading to study withdrawal. To investigate the effects of supplements when minimal resistance training was performed, we also carried out a subgroup analysis on participants with poor resistance training compliance (de ned as the average minutes of training below the 10 th percentile). Analysis of variance (ANOVA) or analysis of covariance using a mixed effect model repeated measurement with supplement-by-visit interaction included as xed effect factors was used to compare the difference in mean/changes in continuous e cacy endpoints between groups, with the use of baseline value, sex, visit, and supplement serving as covariates. The covariates were selected a priori by identifying key physiological factors, such as differences in FFM and bone mass loss in terms of sex, and baseline values re ecting different disease severities, which may in uence the measured outcome. Variables between groups were compared using the Kruskal-Wallis test for continuous data and the chi-squared test for categorical data. Changes in continuous variables from baseline to the study endpoint were analyzed using the paired t-test or Wilcoxon signed-rank test. Safety data were summarized descriptively using the Medical Dictionary for Regulatory Activities 22.1. No imputation method was used to estimate missing values. Statistical analysis was performed using the Statistical Analysis Software (SAS) version 9.4 (SAS Institute, Cary, NC, USA). A P-value of 0.05 was considered statistically signi cant.

Results
The participants were recruited from November 2018 to March 2019 from a single center. In total, there were 160 eligible participants, and they were equally allocated to four groups (n = 40 for each). The study dropout rate was low (n = 9, 5.6%), with three, two, two, and two from the BEC-FlexC-II ® , FlexC-II ® , glucosamine HCl, and placebo groups, respectively. All dropouts withdrew their consent and were excluded from the analysis (Figure 1).

Baseline Characteristics of the Participants
The baseline characteristics of participants are presented in Table 1. There was no statistically signi cant difference between the study groups at baseline. The mean age ranged from 56.4 to 59.1 years.
Approximately 84.8% of participants were women, and they were in the menopausal stage (87.9%, 73.3%, 75.0%, and 60.6% in the BEC-FlexC-II ® , FlexC-II ® , glucosamine HCl, and placebo groups, respectively). Almost all participants were neither smokers nor alcoholics. There was no signi cant difference in baseline knee OA severity, and over half of the participants presented with grade 1 OA based on the Kellgren-Lawrence classi cation system.   The WOMAC score signi cantly decreased in all groups after 24 weeks (Figure 2A). However, no signi cant difference was detected between groups (P = 0.848). The VAS score signi cantly differed between baseline and day 14 between the treatment groups (P = 0.039) ( Figure 2B). The FlexC-II ® group experienced a signi cant reduction (0.9 ± 1.89; P = 0.034) in VAS score after 14 days. Meanwhile, in the placebo group, the VAS pain score increased by 1.0 ± 2.24 from baseline to day 7 (P = 0.012). On the basis of the repeated-measure ANOVA with supplement-visit interaction term, the FlexC-II ® group had a greater decrease in pain than the placebo group (P = 0.021) ( Table 2). The changes in FFM and hand grip strength after 24 weeks did not signi cantly differ between groups (data not shown). The initial ANOVA in the adjusted analyses revealed that the supplement had signi cant effects on FFM and hand grip strength (FFM, P = 0.006; hand grip strength, P < 0.001) and the relationship between supplementation and sex (FFM, P = 0.006; hand grip strength, P = 0.007). ANOVA adjusted for signi cant factors showed a signi cant increase in FFM after 24 weeks in the BEC-FlexC-II ® group compared with that in the glucosamine HCl (P = 0.02) and placebo (P = 0.017) groups (Supplementary Table 1). Similarly, the BEC-FlexC-II ® group had a signi cantly higher adjusted hand grip strength at week 24 than the glucosamine HCl group (P = 0.002).

Effects on Bone Mass
After 24 weeks of supplementation, all participants presented with sustained bone mass in the lumbar spine, left hip, and right hip. However, there were no signi cant differences between groups (data not shown).

Subgroup Analysis on Poor Training Compliance
To understand the effect of supplementation in participants with minimal resistance exercise training, a subgroup analysis was performed (Supplementary Table 2). Repeated-measure ANOVA with supplement term showed that the three experimental groups had greater reduction in VAS score than the placebo group after 14 days (BEC-FlexC-II ® vs. placebo, P = 0.047; FlexC-II ® vs. placebo, P = 0.021; and glucosamine HCl vs. placebo group, P = 0.006).
Moreover, the BEC-FlexC-II ® group had higher hand grip strength than the glucosamine HCl group (P = 0.025; with supplement and sex as signi cant factors) and left hip bone mass than the glucosamine HCl and placebo groups after 24 weeks (P = 0.049 and P = 0.01, respectively; with supplement as a signi cant factor).

Effects on Patient-reported Outcomes
The participants in all four groups experienced improvement in the physical component scores of SF-36.
Moreover, the FlexC-II ® and placebo groups had signi cant improvement in the mental component scores of SF-36 after 24 weeks. However, no statistically signi cant differences were observed between groups (data not shown).

Safety
Approximately 98.8% of patients in the four groups presented with mild AEs. Both BEC-FlexC-II ® and FlexC-II ® were considered safe and well tolerated, and there were no clinically signi cant abnormalities or changes correlated with BEC-FlexC-II ® and FlexC-II ® . Only one patient in the glucosamine HCl group and one in the placebo group presented with serious AEs (urinary incontinence and adnexa uteri cyst, respectively) leading to hospitalization. However, these AEs were not associated with the supplements.

Discussion
This pilot, randomized, placebo-controlled study is the rst clinical trial that evaluated the e cacy of FlexC-II ® , a newly developed chicken-derived CH ingredient, alone and in combination with chicken essence, a food supplement that has long been consumed in Asia. The main nding was that FlexC-II ® had a fast-acting effect in reducing pain, as measured using the VAS after 14 days. Such an effect was found to be superior to that of the placebo. The total WOMAC score improved in all four groups after 24 weeks. However, the between-group differences were not signi cant. After 24 weeks, the BEC-FlexC-II ® group had a higher FFM than the glucosamine HCl and placebo groups and had higher hand grip strength than the glucosamine HCl group.
Previous trials investigating the role of collagen in treating OA had contrasting results. Compared with treatment with glucosamine HCl 27 and placebo, 28 treatment with type II collagen (10 g/day) for 13 weeks was found to be more effective in reducing WOMAC scores in patients with knee OA. A recent study used magnetic resonance imaging to examine changes in the cartilage. 29 Results showed that collagen peptides increased the proteoglycan content in the knee cartilage after 6 months of treatment. This nding is consistent with in vitro data showing a stimulation of extracellular matrix synthesis by collagen peptides. 30,31 In contrast, no differences were found in patients with OA when type II collagen was either added to glucosamine plus chondroitin sulfate supplementation or administered as a standard analgesic therapy. 32,33 A key nding in this trial is the signi cant improvement in WOMAC scores in all four-arm groups. This result could be attributed to the following: (1) psychological effects from supplement and trial regimens (exercise, consultations, and counseling) and placebo effects and (2) physiological bene ts of some regimens, including resistance training and lifestyle modi cations. To identify the effect of treatment with exercise as the least factor, a subgroup analysis of patients with low exercise compliance was performed.
Indeed, all treatment arms (BEC-Flex-CII ® , Flex-CII ® , and glucosamine HCl) had better outcomes than the placebo arm even though the size of each subgroup was small (data not shown). Thus, resistance training had a bene cial (yet confounding) role in improving WOMAC scores among participants who comply with training. Systematic reviews have concluded that resistance training is effective in managing the symptoms of OA by improving joint function and reducing pain, 34,35,36 possibly via the enhancement of muscle strength and rebalancing leg muscle activation patterns. 37 As the overall resistance training compliance was high in this study (>100% for all groups), this trial likely underestimated the e cacy of supplements, and the actual effects were more pronounced in the absence of exercise.
BEC-FlexC-II ® differs from other type II collagen supplements in terms of ingredient (chicken essence), amount of collagen, and liquid format of supplements. Potential confounders such as food, exercise, and environmental factors (e.g., amount of sunlight and physical activities) could also explain the difference between BEC-FlexC-II ® and other type II collagen supplements. The overall nutritional status of individuals in Taiwan is good, and there is su cient exposure to sunlight in this tropical country. However, one limitation of this study is that the daily diet and physical activities of participants were not monitored closely. This might have affected physical functions and, hence, the study results. Second, as the study was powered for the WOMAC score, the small sample size could have been inadequate for other endpoints, as the heterogeneity of small cohorts might cause biases or variations in results. Indeed, post hoc analyses adjusted for the characteristics of participants within a more de ned cohort showed that CH-containing products can decrease OA-derived pain and can increase muscle mass and strength. Third, a study period of 6 months could have been too short to detect a clinically signi cant change in joint and bone conditions in patients with knee OA. Thus, future long-term studies with a larger sample size should be conducted to validate the effect of BEC-FlexC-II ® and FlexC-II ® supplements on locomotor functions.
Finally, female predominance (84.8% of patients) might have limited the generalizability of our results to the general population. Nevertheless, the difference in sex re ects the higher prevalence of knee OA in women than in men. 38,39,40 OA in women is commonly more advanced and aggressive, 41 thereby leading to a higher level of pain and disability. 42,43 In a 15-year retrospective study utilizing data from the Taiwanese Nationwide National Health Insurance database, the prevalence of OA among women was three times higher than that among men. 44 Further, women had higher incidence rates of total knee replacement (2.5-3 times) than men, and this value was higher than that of other countries. 45,46 The immediate and rapid effect of FlexC-II ® in reducing pain based on the VAS is a key nding in this trial.
Synovial in ammation has been associated with joint pain in patients with OA. 47  Moreover, BEC-FlexC-II ® could also increase muscle mass and hand grip strength after 24 weeks. In accordance with this result, BEC has an ergogenic effect by improving exercise performance, endurance capacity, and grip strength. 55 Another plausible mechanism is the prevention of in ammation-induced muscle atrophy, as suggested by preliminary in vitro data. 56 Furthermore, the effects of collagen peptides on muscle recovery 57 and preservation of lean body mass 58 have been clinically proven in few studies on elderly participants. The aforementioned information supported the notion that the combination of chicken essence and CH could contribute to muscle growth and strength particularly in elderly individuals.
In addition, the BEC-FlexC-II ® group had better left hip bone mass than the placebo and glucosamine HCl groups who had minimal resistance training. In accordance with this result, BEC-FlexC-II ® containing GPEGAPGKD was found to reduce bone resorption in an in vitro model for the regulation of osteoclastic differentiation and bone resorption by the receptor activator of nuclear factor kappa-Β ligand system. This could be the underlying mechanism associated with increased bone mass (unpublished data). To the best of our knowledge, only two clinical studies have shown that collagen peptides relative to the use of calcitonin and calcium plus vitamin D have an additive effect on the inhibition of bone collagen breakdown 59 and loss of bone mineral density, respectively. 60 Hence, the e cacy of BEC-FlexC-II ® in the management of osteopenia or osteoporosis should be further assessed.
The BEC-FlexC-II ® and FlexC-II ® were safe and well tolerated by participants, and no safety issues were detected. Most AEs were mild and were not correlated to BEC-FlexC-II ® and FlexC-II ® . The AEs differed between groups. However, in general, infections and gastrointestinal disorders were common in all groups. In addition, the incidence of musculoskeletal and connective tissue disorders was higher in the placebo group than in the treatment groups. Musculoskeletal and connective tissue disorders re ected joint, muscle, and bone health. Thus, BEC-FlexC-II ® and FlexC-II ® may have protective effects on the bone, muscle, and joint. Nevertheless, further studies should be conducted to validate these ndings.

Conclusions
Flex-CII ® and BEC-FlexC-II ® are promising ingredients and combination supplements. The study was reviewed and approved by the independent Ethics Committee of Linkou Chang Gung Memorial Hospital, Taiwan, prior to commencement. Moreover, it was conducted in accordance with the ethical principles of the Declaration of Helsinki and ICH-GCP guidelines. A written informed consent was obtained from each participant before conducting any study procedures.

Consent for publication
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Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.