The fallacy of enzymatic hydrolysis for the determination of bioactive curcumin in plasma samples as an indication of bioavailability: a comparative study.

BACKGROUND: Numerous health benefits have been demonstrated for curcumin which is extracted from turmeric (Curcuma longa L). However, due to its poor absorption in the free form in the gastrointestinal tract and rapid biotransformation, various formulations have been developed to enhance its bioavailability. Previous studies indicate that the free form of curcumin is more bioactive than its conjugated counterparts in target tissues. Most curcumin pharmacokinetics studies in humans designed to assess its absorption and bioavailability have measured and reported total (free plus conjugated) curcumin, but not free, bioactive curcumin in the plasma because enzymatic hydrolysis was employed prior to its extraction and analysis. Therefore, the bioavailability of free curcumin cannot be determined. METHODS: Eight human subjects (4 male, 4 female) consumed a single dose of 400 mg curcumin in an enhanced absorption formulation, and blood samples were collected over 6 h. Plasma was treated either with or without glucuronidase/sulfatase prior to extraction. Curcumin and its major metabolites were analyzed using HPLC-tandem mass spectrometry. In addition, the literature was searched for pharmacokinetic studies involving curcumin using PubMed and Google Scholar, and the reported bioavailability data were compared based on whether hydrolysis of plasma samples was used prior to sample analysis. RESULTS: Hydrolysis of blood plasma samples prior to extraction and reporting the results as "curcumin" obscures the amount of free, bioactive curcumin and total curcuminoids as compared to non-hydrolyzed samples. As a consequence, the data and biological effects reported by most pharmacokinetic studies are not a clear indication of enhanced plasma levels of free bioactive curcumin due to product formulations, leading to a misrepresentation of the results of the studies and the products when enzymatic hydrolysis is employed. CONCLUSIONS: When enzymatic hydrolysis is employed as is the case with most studies involving curcumin products, the amount of free bioactive curcumin is unknown and cannot be determined. Therefore, extreme caution is warranted in interpreting published analytical results from biological samples involving ingestion of curcumin-containing products. TRIAL REGISTRATION: ClinicalTrails.gov, trial identifying number NCT04103788 , September 24, 2019. Retrospectively registered.

Beneficial effects of natural eggshell membrane versus placebo in exercise-induced joint pain, stiffness, and cartilage turnover in healthy, postmenopausal women.

PURPOSE: Despite its many health benefits, moderate exercise can induce joint discomfort when done infrequently or too intensely even in individuals with healthy joints. This study was designed to evaluate whether NEM® (natural eggshell membrane) would reduce exercise-induced cartilage turnover or alleviate joint pain or stiffness, either directly following exercise or 12 hours post exercise, versus placebo. PATIENTS AND METHODS: Sixty healthy, postmenopausal women were randomly assigned to receive either oral NEM 500 mg (n=30) or placebo (n=30) once daily for two consecutive weeks while performing an exercise regimen (50-100 steps per leg) on alternating days. The primary endpoint was any statistically significant reduction in exercise-induced cartilage turnover via the biomarker C-terminal cross-linked telopeptide of type-II collagen (CTX-II) versus placebo, evaluated at 1 and 2 weeks of treatment. Secondary endpoints were any reductions in either exercise-induced joint pain or stiffness versus placebo, evaluated daily via participant questionnaire. The clinical assessment was performed on the per protocol population. RESULTS: NEM produced a significant absolute treatment effect (TEabs) versus placebo for CTX-II after both 1 week (TEabs -17.2%, P=0.002) and 2 weeks of exercise (TEabs -9.9%, P=0.042). Immediate pain was not significantly different; however, rapid treatment responses were observed for immediate stiffness (Day 7) and recovery pain (Day 8) and recovery stiffness (Day 4). No serious adverse events occurred and the treatment was reported to be well tolerated by study participants. CONCLUSION: NEM rapidly improved recovery from exercise-induced joint pain (Day 8) and stiffness (Day 4) and reduced discomfort immediately following exercise (stiffness, Day 7). Moreover, a substantial chondroprotective effect was demonstrated via a decrease in the cartilage degradation biomarker CTX-II. Clinical Trial Registration number: NCT02751944.

Beneficial effects of natural eggshell membrane (NEM) on multiple indices of arthritis in collagen-induced arthritic rats.

OBJECTIVES: This study was performed to evaluate the potential efficacy of natural eggshell membrane (NEM) in collagen-induced arthritic rats, a well-established rodent model of inflammation and rheumatoid arthritis. METHODS: Rats with developing type II collagen-induced arthritis (CIA) were treated once daily by oral gavage on study days -14 to 17 with vehicle or NEM (52 mg/kg body weight). Rats were euthanized on study day 17. Efficacy was assessed by daily ankle caliper measurements, ankle diameter expressed as area under the curve (AUCd0-17), and histopathologic evaluation of ankles and knees. Serum biomarkers of cartilage function and inflammation [collagen type II C-telopeptide (CTXII), cartilage oligomeric matrix protein (COMP), and alpha-2-macroglobulin (A2M)] were measured by ELISA. RESULTS: Treatment with NEM resulted in significant beneficial effects on the daily ankle diameter measurements and ankle diameter AUC. Ankle and knee histopathology scores were significantly reduced (36% and 43% reduction of summed individual histopathology scores for ankle and knee, respectively; p < 0.05) toward normal for rats given NEM compared to vehicle controls. The percent reduction of serum CTXII, COMP, and A2M in NEM-treated rats ranged from 30% to 72% (p < 0.05). CONCLUSIONS: NEM significantly improved multiple aspects of inflammatory arthritis including inflammation, pannus, cartilage damage, bone resorption, and periosteal bone formation. This study provides further support for the use of CTXII, COMP, and A2M as relevant biomarkers that were responsive to NEM.

Effectiveness of NEM® brand eggshell membrane in the treatment of suboptimal joint function in dogs: a multicenter, randomized, double-blind, placebo-controlled study.

INTRODUCTION: Sub-optimal joint function is extremely prevalent in dogs. Therefore, a 6-week, prospective, randomized, double-blind, placebo-controlled study was conducted at eight different veterinary clinics to evaluate the efficacy, safety, and tolerability of NEM® brand eggshell membrane (EM), a novel dietary supplement shown in other species to help maintain healthy joints and connective tissues. SUBJECTS AND METHODS: Fifty-one dogs received oral EM ~13.5 mg/kg (6 mg/lb) or placebo (excipients) once daily for 6 weeks. The primary outcome measure of this study was to evaluate the change in mean joint function following 1 week and 6 weeks of supplementation as determined via the Canine Brief Pain Inventory (CBPI) questionnaire (Q#5-10) in the treatment group versus the placebo group. Secondary outcome measures were for changes in mean CBPI pain and CBPI quality of life, and mean joint pain, mobility and lameness via Veterinary Canine Scoring Assessments (VCSA). A final secondary outcome measure was for a change in serum levels of the cartilage degradation biomarker, c-terminal cross-linked telopeptide of type-II collagen (CTX-II). RESULTS: Supplementation with EM produced a significant treatment response versus placebo at 1 week (20.5% improvement, P=0.028), but fell shy of significance at 6 weeks post-treatment (22.5% improvement) for the primary outcome measure (CBPI Function), despite a sizeable treatment effect. Similarly, there was also a significant treatment response versus placebo at 1 week for CBPI Pain (19.4% improvement, P=0.010), but fell just shy of significance at 6 weeks (22.5% improvement), again despite a sizeable treatment effect. Results were not significant versus placebo at 1 week for CBPI quality of life (14.0% improvement), but produced a significant treatment response by the end of the 6-week study (26.8% improvement, P=0.033). Additionally, EM produced a significant treatment response versus placebo at 6 weeks for VCSA pain (23.6% improvement, P=0.012), but fell shy of significance for VCSA mobility and VCSA lameness (walking & trotting). Serum CTX-II levels in EM-supplemented dogs was significantly improved versus placebo at 6 weeks (47.9% improvement, P=0.018). There were no serious adverse events reported during the study and subject dog owners reported that EM was well tolerated by their pets. CONCLUSION: Supplementation with EM, ~13.5 mg/kg (6 mg/lb) taken once daily, significantly reduced joint pain and improved joint function rapidly (CBPI 1 week) and demonstrated a lasting improvement in joint pain (VCSA 6 weeks) leading to an improved quality of life (CBPI 6 weeks). Moreover, a profound chondroprotective effect was demonstrated following 6 weeks of supplementation with EM (CTX-II).

Eggshell membrane hydrolyzates activate NF-κB in vitro: possible implications for in vivo efficacy.

PURPOSE: Eggshell membrane (ESM) has been shown to contain naturally occurring bioactive components, and biological activities such as reducing proinflammatory cytokines, liver fibrosis, and joint pain in osteoarthritis sufferers have also been reported for ESM matrix as a whole. Nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) is a signaling protein found in the cytoplasm of nearly all human and animal cell types and is a primary regulator of immune function. The studies reported herein were designed to investigate the possible role that NF-κB activity might play in the reported biological activities of ESM. METHODS: Three ESM hydrolyzates produced via fermentation, enzymatic, or chemical hydrolysis were evaluated in vitro in either human peripheral blood mononuclear cell or THP-1 (human leukemic monocyte) cell cultures for NF-κB activity following 4-hour exposure. The hydrolyzates were compared with untreated control cells or cells incubated with lipopolysaccharide or ascorbic acid. The source of ESM activity was also evaluated. RESULTS: NF-κB levels were increased above levels found in untreated cells at all three dilutions (1:100, 1:1,000, and 1:10,000) for the fermentation hydrolyzate of ESM (ESM-FH) (P=0.021, P=0.020, P=0.009, respectively) in peripheral blood mononuclear cells. The enzymatic hydrolyzate of ESM (ESM-EH) also produced statistically significant levels of activated NF-κB at the 1:100 and 1:1,000 dilutions (P=0.004, P=0.006, respectively) but fell just shy of significance at the 1:10,000 dilution (P=0.073). Similarly, ESM-FH (P=0.021, P=0.002) and ESM-EH (P=0.007, P=0.007) activated NF-κB in THP-1 cells at 1:1,000 and 1:10,000 dilutions, respectively. The chemical hydrolyzate of ESM (ESM-CH) showed statistically significant levels of activation at the 1:1,000 dilution (P=0.005) but failed to differ from untreated cells at the 1:10,000 dilution (P=0.193) in THP-1 cells. CONCLUSION: Results from our studies provide evidence that ESM hydrolyzates significantly activate NF-κB, and the source of this activity was investigated to confirm that it is inherent to ESM and not derived from bacterial contamination. Based on our findings, we propose a plausible hypothesis as to how increased NF-κB activity might translate into the in vivo efficacy that has been observed with ESM via an "oral tolerance" mechanism.

Safety evaluation of a natural eggshell membrane-derived product.

Natural Eggshell Membrane (NEM®) is a novel dietary ingredient that contains naturally occurring glycosaminoglycans and proteins essential for maintaining healthy joint and connective tissues. NEM® was evaluated for safety via in vitro and in vivo toxicological studies. This included testing for cytotoxicity, genotoxicity, acute oral toxicity, and 90-day repeated-dose oral toxicity. NEM® did not exhibit any cytotoxic effects at a dose of 100 µg in an in vitro human cell viability assay after incubation for up to 20 h. NEM® did not exhibit any genotoxic effects in an in vitro assay of four strains of histidine-dependent Salmonella typhimurium and one strain of tryptophan-dependent Escherichia coli at a dose of up to 5000 µg/plate. NEM® did not exhibit any signs of acute toxicity in rats at a single oral dose of up to 2000 mg/kg body weight, nor signs of toxicity (via urinalysis, hematology, clinical chemistry, or histopathological evaluation) in rats at a repeated oral dose of up to 2000 mg/kg body weight per day for 90 days. The results of these studies suggest that NEM® may be safe for human consumption.

Effects of natural eggshell membrane (NEM) on cytokine production in cultures of peripheral blood mononuclear cells: increased suppression of tumor necrosis factor-α levels after in vitro digestion.

Tumor necrosis factor-α (TNF-α) plays an important role in inflammatory processes. This study examined the effects of natural eggshell membrane (NEM(®)) (ESM Technologies, LLC, Carthage, MO, USA) on interleukin (IL)-2, IL-4, IL-6, IL-10, interferon-γ (IFN-γ), and TNF-α cytokine production by 4-day peripheral blood mononuclear cell (PBMC) cultures exposed to serial dilutions of either an aqueous extract of natural eggshell membrane (NEM-AQ) or NEM subjected to in vitro digestion (NEM-IVD). The effects on cytokine production were also assessed in the presence of phytohemagglutinin (PHA) and pokeweed mitogen (PWM) where exposure to NEM-AQ resulted in reduced levels of proliferation and statistically significant effects on IL-6, IL-10, IFN-γ, and TNF-α cytokine production. NEM-AQ reduced levels of IL-6, IL-10, IFN-γ, and TNF-α in cultures exposed to PHA. In cultures containing PWM, NEM-AQ reduced production of IL-10 and at the highest dose tested increased IL-6 and decreased TNF-α cytokine levels. NEM-IVD, at the two lowest concentrations of product, significantly reduced TNF-α production by PBMC cultures exposed to PWM compared with the in vitro digest control or native NEM. Taken together, these results suggest that NEM-AQ can influence signaling events in response to the T cell-specific mitogen PHA as well as to the mitogen PWM that require cellular cross-talk and that these effects may be partially mediated through a reduction in level of the pro-inflammatory cytokine TNF-α. The suppression of TNF-α production in the presence of NEM-IVD is promising for the use of NEM as a consumable anti-inflammatory product.

Eggshell membrane: a possible new natural therapeutic for joint and connective tissue disorders. Results from two open-label human clinical studies.

BACKGROUND: Natural Eggshell Membrane (NEM) is a novel dietary supplement that contains naturally occurring glycosaminoglycans and proteins essential for maintaining healthy joint and connective tissues. Two single center, open-label human clinical studies were conducted to evaluate the efficacy and safety of NEM as a treatment for pain and inflexibility associated with joint and connective tissue disorders. METHODS: Eleven (single-arm trial) and 28 (double-arm trial) patients received oral NEM 500 mg once daily for four weeks. The primary outcome measure was to evaluate the change in general pain associated with the treatment joints/areas (both studies). In the single-arm trial, range of motion (ROM) and related ROM-associated pain was also evaluated. The primary treatment response endpoints were at seven and 30 days. Both clinical assessments were performed on the intent-to-treat (ITT) population within each study. RESULTS: Single-arm trial: Supplementation with NEM produced a significant treatment response at seven days for flexibility (27.8% increase; P = 0.038) and at 30 days for general pain (72.5% reduction; P = 0.007), flexibility (43.7% increase; P = 0.006), and ROM-associated pain (75.9% reduction; P = 0.021). Double-arm trial: Supplementation with NEM produced a significant treatment response for pain at seven days for both treatment arms (X: 18.4% reduction; P = 0.021. Y: 31.3% reduction; P = 0.014). There was no clinically meaningful difference between treatment arms at seven days, so the Y arm crossed over to the X formulation for the remainder of the study. The significant treatment response continued through 30 days for pain (30.2% reduction; P = 0.0001). There were no adverse events reported during either study and the treatment was reported to be well tolerated by study participants. CONCLUSIONS: Natural Eggshell Membrane (NEM) is a possible new effective and safe therapeutic option for the treatment of pain and inflexibility associated with joint and connective tissue (JCT) disorders. Supplementation with NEM, 500 mg taken once daily, significantly reduced pain, both rapidly (seven days) and continuously (30 days). It also showed clinically meaningful results from a brief responder analysis, demonstrating that significant proportions of treated patients may be helped considerably from NEM supplementation. The Clinical Trial Registration numbers for these trials are: NCT00750230 and NCT00750854.

Eggshell membrane in the treatment of pain and stiffness from osteoarthritis of the knee: a randomized, multicenter, double-blind, placebo-controlled clinical study.

Natural Eggshell Membrane (NEM(R)) is a new novel dietary supplement that contains naturally occurring glycosaminoglycans and proteins essential for maintaining healthy articular cartilage and the surrounding synovium. The randomized, multicenter, double-blind, placebo-controlled Osteoarthritis Pain Treatment Incorporating NEM(R) clinical study was conducted to evaluate the efficacy and safety of NEM(R) as a treatment for pain and stiffness associated with osteoarthritis of the knee. Sixty-seven patients were randomly assigned to receive either oral NEM(R) 500 mg (n = 34) or placebo (n = 33) daily for 8 weeks. The primary endpoint was the change in overall Western Ontario and McMasters Universities (WOMAC) Osteoarthritis Index as well as pain, stiffness, and function WOMAC subscales measured at 10, 30, and 60 days. The clinical assessment was performed on the intent-to-treat population. Supplementation with NEM(R) produced an absolute rate of response that was statistically significant (up to 26.6%) versus placebo at all time points for both pain and stiffness, but was not significantly improved for function and overall WOMAC scores, although trending toward improvement. Rapid responses were seen for mean pain subscores (15.9% reduction, P = 0.036) and mean stiffness subscores (12.8% reduction, P = 0.024) occurring after only 10 days of supplementation. There were no serious adverse events reported during the study and the treatment was reported to be well tolerated by study participants. Natural Eggshell Membrane (NEM(R)) is an effective and safe option for the treatment of pain and stiffness associated with knee osteoarthritis. Supplementation with NEM(R), 500 mg taken once daily, significantly reduced both joint pain and stiffness compared to placebo at 10, 30, and 60 days.