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nutrients
Article Effect of a Hop Extract Standardized in 8-Prenylnaringenin on Bone Health and Gut Microbiome in Postmenopausal Women with Osteopenia: A One-Year Randomized, Double-Blind, Placebo-Controlled Trial Manon Lecomte 1, * , Diego Tomassi 2 , Ren é Rizzoli 3 , Mathieu Tenon 1 , Thierry Berton 1 , Sinead Harney 4 and Pascale Fança-Berthon 1
1 Givaudan France Naturals, 84911 Avignon, France; mathieu.tenon@givaudan.com (M.T.); thierry.berton@givaudan.com (T.B.); pascale.fanca-berthon@givaudan.com (P.F.-B.) 2 Biofortis, 44800 Saint-Herblain, France; diego.tomassi@biofortis.fr 3 Service of Bone Disease, Geneva University Hospitals and Faculty of Medicine, 1211 Geneva, Switzerland; rene.rizzoli@unige.ch 4 Rheumatology Department, Cork University Hospital, T12 DFK4 Cork, Ireland; sinead.harney@hse.ie * Correspondence: manon.lecomte@givaudan.com Abstract: Estrogen deficiency increases the risk of osteoporosis and fracture. The aim of this study was to investigate whether a hop extract standardized in 8-prenylnaringenin (8-PN), a potent phy- toestrogen, could improve bone status of osteopenic women and to explore the gut microbiome roles in this effect. In this double-blind, placebo-controlled, randomized trial, 100 postmenopausal, osteopenic women were supplemented with calcium and vitamin D3 (CaD) tablets and either a hop extract (HE) standardized in 8-PN ( n = 50) or a placebo ( n = 50) for 48 weeks. Bone mineral density (BMD) and bone metabolism were assessed by DXA measurements and plasma bone biomarkers, respectively. Participant’s quality of life (SF-36), gut microbiome composition, and short-chain fatty acid (SCFA) levels were also investigated. In addition to the CaD supplements, 48 weeks of HE supplementation increased total body BMD (1.8 ± 0.4% vs. baseline, p < 0.0001; 1.0 ± 0.6%vs. placebo, p = 0.08), with a higher proportion of women experiencing an increase ≥ 1% compared to placebo (odds ratio: 2.41 ± 1.07, p < 0.05). An increase in the SF-36 physical functioning score was observed with HE versus placebo ( p = 0.05). Gut microbiome α -diversity and SCFA levels did not differ between groups. However, a higher abundance of genera Turicibacter and Shigella was observed in the HE group; both genera have been previously identified as associated with total body BMD. These results suggest that an 8-PN standardized hop extract could beneficially impact bone health of postmenopausal women with osteopenia.
Citation: Lecomte, M.; Tomassi, D.; Rizzoli, R.; Tenon, M.; Berton, T.; Harney, S.; Fança-Berthon, P. Effect of a Hop Extract Standardized in 8-Prenylnaringenin on Bone Health and Gut Microbiome in Postmenopausal Women with Osteopenia: A One-Year Randomized, Double-Blind, Placebo-Controlled Trial. Nutrients 2023 , 15 , 2688. https://doi.org/ 10.3390/nu15122688
Academic Editor: Anna Scotto D’Abusco
Keywords: hop; 8-PN; phytoestrogen; bone; osteoporosis; osteopenia; microbiome; menopause
Received: 3 May 2023 Revised: 21 May 2023 Accepted: 25 May 2023 Published: 9 June 2023
1. Introduction Osteoporosis is a skeletal disorder characterized by reduced bone mass and dete- rioration in bone architecture leading to increased bone fragility and fracture risk [1]. Postmenopausal women are particularly at risk with approximately 30 to 40% affected by osteoporosis in the USA and Europe [2,3]. The decline in endogenous estrogen during and after menopause accelerates the bone remodeling process with an imbalance between bone formation and bone resorption [4]. In this context, phytoestrogens are an interesting non-pharmaceutical intervention to prevent bone loss. Phytoestrogens are polyphenolic plant-derived compounds with a structural similarity to endogenous human estrogen, hence their estrogenic activity. The main dietary sources of phytoestrogens are soy and red clover (isoflavones), flaxseed (lignans) and hops (prenylflavonoids) [5]. Phytoestrogens are already widely used to alleviate menopausal symptoms such as hot flashes and night
Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).
Nutrients 2023 , 15 , 2688. https://doi.org/10.3390/nu15122688
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sweats [6]. Moreover, isoflavones from soy and red clover have received considerable atten- tion in the management of postmenopausal bone loss with an overall moderately beneficial effect against bone loss when consumed for at least twelve months [7,8]. There is limited literature regarding the effect of hops ( Humulus lupulus ) on bone metabolism although hops contain one of the most potent phytoestrogens known to date: 8-prenylnaringenin (8-PN). As a novel phytoestrogen, 8-PN is unique in that its receptor specificity and potency is higher than any other phytoestrogens investigated thus far [9,10]. In vitro, 8-PN was shown to enhance differentiation and maturation of osteoblast and inhibit differentiation of osteoclast with intensities of response stronger than that observed with soy isoflavones [11]. Several in vivo studies demonstrated that an oral supplementation with a standardized hop extract was able to prevent estrogen-deficiency-induced bone loss in osteoporotic rodent models [12–15]. Moreover, in ovariectomized rats, supplementation with 68.4 mg/kg of body weight (bw) per day of 8-PN during twelve weeks improved bone biomechanical properties to the same degree as 0.7 mg/kg bw per day of estradiol, while the two other phytoestrogens tested, genistein (60 mg/kg bw per day) and resveratrol (50 mg/kg bw per day), had no significant impact [15]. From a clinical point of view, a bioavailability study per- formed in menopausal women indicates that prenylflavonoids (8-PN, 6-prenylnaringenin, isoxanthohumol, and xanthohumol) from a standardized hop extract are absorbed slowly, undergo enterohepatic circulation, and have long half-lives exceeding 20 h [11]. Moreover, 8-prenylnaringenin seems to be significantly more bioavailable in healthy humans than its isomer 6-prenylnaringenin [12]. In terms of health effect, three clinical studies have demon- strated the efficiency of standardized hop extract in decreasing menopausal symptoms at a doseof 100 µ g of 8-PN per day during a minimum duration of 6 weeks [16–18]. However, to date, the potential impact of hop extract and 8-PN in the prevention of osteoporosis has not been assessed in humans. While the presence of 8-PN in hops is low, other more abundant prenylated phenols such as xanthohumol (X) and isoxanthohumol (IX) can be metabolically converted to 8-PN. The conversion of IX into 8-PN can be accomplished enzymatically by hepatic CYP1A2 or by the gut microbiome [19,20]. However, large inter-individual variability was found for IX conversion capacity by the human gut microbiome, with only about one- third of individuals exhibiting the capability to efficiently execute this transformation [20]. Eubacterium limosum has been identified as one intestinal bacterium capable of facilitating the conversion (O-demethylation) of IX into 8-PN [21,22]. Finally, the gut microbiome impact on host health has been increasingly studied over past decades. There is notably a growing body of evidence indicating that the gut microbiome plays a key role in bone metabolism and osteoporosis pathogenesis even though mechanisms of action have not been clearly elucidated yet [23–25]. Given the role of the gut microbiome in bone health maintenance and its importance in 8-PN generation potency, it is of great interest to investigate the gut microbiome as a potential key factor in the mechanism of action of hop extract. The present clinical trial aimed to determine whether one-year consumption of a hop extract standardized in 8-PN can moderate bone mineral density decreases in post- menopausal women with osteopenia and to explore potential mechanism of action via gut microbiome modulation.
2. Materials and Methods 2.1. Study Design and Participants
This study was a 48-week, parallel-design, placebo-controlled, double-blind, random- ized clinical trial. The clinical aspects of the study were carried out from August 2019 to December 2020 in Cork (Ireland), including screening, recruitment, and follow-up. Partici- pants were recruited through advertisements (local newspaper and social media platforms) or referred by local general practitioners in the area. A total of 221 participants were screened to identify 100 eligible participants, all of whom were postmenopausal women (>1-year post-menopause), between 50 and 85 years of age, with a body mass index (BMI)
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between 18–32 kg/m 2 , and presenting with osteopenia defined as a dual energy X-ray absorptiometry (DXA) T-score between − 1and − 2.5 (based on the lowest T-score at any site). Exclusion criteria included osteoporosis (i.e., T-score ≤− 2.5), currently taking or had taken within the previous three months any drug for osteoporosis (bisphosphonates, parathyroid hormone, strontium ranelate, or denosumab) or any treatment with estrogen or hormone therapy or estrogen agonist/antagonist products (raloxifene or tamoxifene), had taken antibiotics or laxatives during the preceding 2 months, or had experienced gastroenteritis or foodborne illness within 4 weeks prior to the study. Participants had to be healthy, i.e., without uncontrolled hypertension, hypothyroidism, or hyperthyroidism (or must be on stable medication for at least 3 months), without history of cancer within the last five years (except basal cell carcinoma, non-squamous skin carcinoma, or carcinoma in situ with no significant progression over the past 2 years), and without significant cardiovascu- lar, pulmonary, renal, liver, infectious disease, immune disorder, or metabolic/endocrine disorders or other disease that would preclude supplement ingestion and/or assessment of safety and the study objectives. Additionally, participants that were currently taking or had taken any vitamin K or isoflavones supplementation within the previous 4 weeks, were hypersensitive to any of the components of the investigational product (IP), were smokers, were exhibiting excess alcohol consumption, or had been trying to lose weight for the last 3 months were also excluded. Participants who gave written informed consent and were deemed eligible at their screening visit were assigned a randomization number in chronological order. The ran- domization list was generated by an independent biostatistician (Atlanstat, France). A permuted-block, fixed randomization schedule was used with 2 block sizes (the first 14 blocks were size 6 and the next 14 blocks size 4), based on a computer-generated random numbers program (SAS ® Software version 9.4, Cary, NC, USA). All research staff involved in the collection and the analysis of the data remained blinded to the treatment random- ization until all aspects of the study were complete, including the statistical analysis. A total of 50 participants were allocated to the hop extract (HE; 1 capsule per day) group, and 50 participants were allocated to the placebo group (1 capsule per day). Both groups also received calcium and vitamin D (CaD) supplements (2 capsules per day, each capsule consisting of 500 mg of calcium and 400 IU of vitamin D3; manufactured by Pharmavite, Nature Made). Participants were instructed to follow their usual dietary habits and main- tain normal physical activity throughout the study, which was monitored via food and exercise questionnaires (see Section 2.6 below for details). Randomized participants were scheduled to attend 5 visits at the research center at baseline (0), 12, 24, 36, and 48 weeks. Anthropometric parameters (height, weight, BMI, waist circumference, and hip circumference), vitals (blood pressure, heart rate, and temperature), quality of life assessed by the 36-item short form (SF-36), and physical activity assessed by the Physical Activity Scale for the Elderly (PASE) were measured at each visit. DXA measurements (BMD at femoral neck, L2–L4, total hip and total body, T-score at femoral neck and L2–L4, FRAX scores and body composition), fecal samples, and blood samples were collected at baseline, at 24 weeks, and at 48 weeks, while urine samples and dietary intake assessed by the food frequency questionnaire (FFQ) were collected at baseline and at 48 weeks only. Due to the COVID-19 pandemic and Irish government restrictions, it was not possible to conduct interim visits (weeks 12, 24, and 36) at the research center. Notably, DXA, anthropometric, and laboratory assessments were either collected on site later or were unable to be collected at these interim visits. The research was conducted under guidelines stated in the current revision of the Declaration of Helsinki, was approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals, and was registered at ClinicalTrials.gov (NCT04004013). 2.2. Study Product Hop extract (HE) standardized in 8-PN (Lifenol ® , Givaudan France Naturals, Avignon, France) or placebo was administrated orally in capsules. Each HE capsule comprised
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Lifenol ® containing 100 µ g of 8-PN, 110 µ g of 6-PN, 1.25 mg of X, and 2.94 mg of IX (measured by LC-UV) mixed with maltodextrin (Roquette Fr è res, Lestrem, France), and filled in red opaque gelatin capsules size 0. Placebo capsules consisted of only maltodextrin within a similar type of capsules. 2.3. Bone Measurements by Dual-Energy X ray Absorptiometry Body composition was assessed with DXA. DXA examination, performed by the same health care professionals each visit, was conducted using the Lunar iDXA ME +210575 (GE Healthcare, Chicago, IL, USA). DXA was used to determine bone mineral density (BMD) and T score at each body site; DXA was also used to determine body composition (lean mass, fat mass, visceral fat, and fat percentage). Fracture risk assessment was determined using the FRAX tool (www.shef.ac.uk/FRAX, accessed on 7 December 2020) [26]. 2.4. Biomarkers of Bone Turnover and Biochemical Analysis Fasting blood samples collected in EDTA/heparin tubes were centrifuged at 3000 rpm at 4 ◦ C for 10 min within 40 min after collection. Samples were stored at − 80 ◦ Cuntil analysis. Plasma concentrations of osteocalcin and sclerostin were measured using an automated analyzer according to the manufacturer’s instructions (Multiplex Luminex ® As- says, Merck-Milipore, Burlington, MA, USA). Plasma concentrations of undercarboxylated osteocalcin (uOC) (Abbexa, Cambridge, UK), collagen type 1 cross-linked C -telopeptide (CTx) (Abbkine, Wuhan, China), procollagen type I N terminal propeptide (PINP) (Abbexa), human bone alkaline phosphatase (BALP) (MyBiosource, San Diego, CA, USA), tartrate- resistant acid phosphatase isoform 5b (TRAP5b) (MyBiosource), and BALP/TRAP5b ratio were measured using ELISA according to the kit manufacturer’s instructions. The manu- facturer supplied analytic variation coefficients were as follows: PINP and uOC (Abbexa): intra-assay CV < 10% and inter-assay CV < 12%; CTx, (Abbkine): intra-assay CV < 9% and inter-assay CV < 11%; BALP and TRAP5b (MyBiosource): intra-assay CV < 8% and inter-assay CV < 12%. Serum 25- hydroxyvitamin D (25-OH D3), plasma 17- β oestradiol, blood lipids (total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides), glucose homeostasis parameters (blood glucose, insulinaemia, HbA1c, and HOMA IR), and safety parameters were also measured. 2.5. Plasma and Urine Prenylflavonoids and Their Metabolites All prenylflavonoids (X, IX, 6-PN, and 8-PN) were measured both in plasma and urine in their unconjugated, glucuronide, and sulfated forms; each were expressed as a sum of the 3 different forms (i.e., total). Standard of X (purity: 99.6%), IX (purity: 99.6%), 8-PN (purity: 100%), and 6-PN (purity: 97%) were purchased from Phytolab (Vestenbergsgreuth, Germany). β -Glucuronidase enzyme from Escherichia coli , sulfatase enzyme from Helix pomatia , sodium phosphate, acetic acid, and sodium azide used for en- zymatic hydrolysis were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Since the concentrations measured in plasma and urine samples were very low, a calibration curve was prepared at very low concentrations ranging from 0.1 ng/mL to 5 ng/mL in methanol. Urine and plasma samples were stored at − 80 ◦ C until analysis; they were then prepared with an automated workstation (Beckman Coulter, Biomek NX, Brea, CA, USA) in random order. For the plasma samples, 100 µ L of plasma or enzymatic hydrolyzed plasma were loaded on Captiva EMR-lipid plate (Agilent Technologies, Santa Clara, CA, USA). An amount of 300 µ L of MeOH/ACN (50:50) were added and mixed at 700 rpm for 3 min. Samples were then eluted by positive pressure. An amount of 100 µ L of MeOH/ACN (50:50) were loaded on the cartridge once again and eluted by positive pressure. For the urine samples, 300 µ L of water were loaded on Captiva ND plate, and 100 µ L of urine or enzymatic hydrolyzed urine were added and mixed at 700 rpm for 1 min. Samples were then eluted by positive pressure and analyzed by LC-HRMS. Enzymatic hydrolyzed urine and plasma samples were prepared with 10 mg of enzyme diluted in 10 mL sodium
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phosphate buffer (100 mM, pH = 6.8) for β -Glucuronidase and in 10 mL sodium acetate buffer (100 mM, pH = 5) for sulfatase enzyme. An amount of 60 µ L of this solution were mixed with 60 µ L of plasma or urine and incubated during 1 h at 37 ◦ C. Noting the difference between the concentrations measured before and after enzymatic hydrolysis provides the ability to calculate the concentrations of the glucuronide/sulfated forms. Liquid chromatography was performed on a UHPLC Thermo Vanquish (Thermo Scientific, Karlsruhe, Germany) in reverse phase mode with an Accucore RP-MS column (150 × 2.1mm, 2.6 µ m, Thermo Scientific) using solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in ACN). The elution gradient started at 5% B for 1 min, followed by a linear gradient rising to 90% B during 9 min. The mobile phase remained at 90% B for 5 min and then returned to initial condition after 1 min. The column was equilibrated for 4 min in initial conditions (5% B) prior to the next injection, for a total run time of 20 min. The flow rate was 0.5 mL/min, and the injection volume was 2 µ L. The column was heated at 30 ◦ C to ensure a stable column temperature and a better repeatability between runs, and the autosampler temperature was maintained at 6 ◦ C. The UHPLC system was coupled to an Orbitrap Q-Exactive Focus mass spectrometer (Thermo Scientific, Germany), and analyses were performed using an electrospray interface in negative mode, in full scan with a resolution of 35,000 FWHM in the scan range of m / z 80–1000. ESI parameters were as follows: heater temperature 300 ◦ C, capillary temperature 350 ◦ C, sheath gas 55 (arbitrary units), auxiliary gas 15 (arbitrary units), S-Lens 50 V, spray voltage: 3.5 kV in ESI-. Quan Browser software (Thermo Scientific) was used for quantification and the 4 targeted compounds were extracted with a mass window width of 5 ppm. This analytical method was validated according to internal guidelines and specificity, linearity, repeatability, and limit of quantification (LOQ) are listed in Table S1. Accurate mass used for the four compounds of interest is also listed and specificity was tested by checking that matrices and diluent did not interfere with the analyte masses and retention time. Repeatability was established by analyzing 6 samples of standards solution in diluent and 6 spiked plasmas at 20 ng/mL. The relative standard deviation (RSD) of this analytical method ranged from 3.2 to 4.7% for standards solution in diluent and from 2.5 to 3.8% in spiked plasma according to the compounds (Table S1). Calibration curves also displayed satisfactory linearity with R 2 greater than 0.99 for all compounds, and LOQ were established using a signal-to-noise ratio above 10. 2.6. Dietary Intake, and Physical Activity Level, and Quality of Life Dietary habits, using the EPIC-Norfolk Food Frequency Questionnaire (FFQ; https:// www.epic-norfolk.org.uk/, accessed on 7 December 2020), were evaluated at baseline and after 48 weeks. Data were entered into the Nutritics software version 5.61 (Nutritics, Dublin, Ireland), and average daily intake of total energy, fat, carbohydrate, protein, fiber, calcium, vitamin D, and isoflavones were derived. Physical activity level was registered using the self-reported level of Physical Activity Scale for the Elderly (PASE) [27]. The PASE total score range from 0–400, where higher scores reflect a higher activity level. Health-related quality of life was measured by measuring the means scores of the Short Form 36 (SF-36) [28]. The SF-36 is divided into eight sub-scales (physical function, role limitations-physical, bodily pain, general health, vitality, social function, role limitations- emotional, and mental health). The measurement is scored on a 0–100-point scale for each sub-scale; the higher the score, the more positive the health status.
2.7. Gut Microbiome Analysis 2.7.1. Fecal Samples Collection and DNA Extraction
Fecal samples were collected at baseline, at 24 weeks, and at 48 weeks. Participants were provided stool collection kits and instructed to collect an at-home sample within 48 h of their next research visit. The fecal sample was collected using a collection vial and then placed immediately in the home freezer ( − 20 ◦ C) before being brought to the clinic in a
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provided cooler bag with a cooler block. Samples received at the research center were immediately placed in a freezer at − 80 ◦ C. Genomic DNA was extracted using the ZymoBIOMICS™ 96 MagBead DNA kit (Zymo Research Corp., Irvine, CA, USA) integrating a double lysis (mechanical and chemical) on the Precellys Evolution homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). DNA extraction was performed on the KingFisher Flex automaton (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Once obtained, the DNA solutions were assayed by fluorimetry with the Qubit device (Thermo Fisher Scientific, Waltham, MA, USA). 2.7.2. Libraries Preparation and Shotgun Metagenomic Sequencing Fragmentation of the extracted total DNA was performed using the FS DNA Library Prep Set kit (MGI Tech, Shenzhen, China). After ligation of adapters to each sample, the libraries generated were purified on magnetic beads. Library size was verified by capillary electrophoresis on at least 10% of samples. After quantification by fluorimetry, the libraries were normalized and pooled before denaturation, single-strand circularization, and sequencing using the DNBSEQ-G400 platform (MGI Tech). 2.7.3. Analysis of Overall Association and Taxonomic Profile The MiRKAT family of tests was used to assess overall association between taxonomic compositional profiles and treatment group [29–31]. These are regression-based association tests based on kernels that have been proposed specifically for microbiome data and allow covariate adjustment and repeated measurements. Jaccard and Bray–Curtis beta diversity scores were used to quantify dissimilarity between compositional profiles at several taxonomic ranks. Participant sex, age, and time since menopause were added as covariates. Assessment of microbiota components showing differential abundance between treat- ment groups was evaluated using CoDA-lasso enriched with stability analysis. CoDA-lasso is a multivariate approach that fits a regularized logistic regression model with an addi- tional constraint on the regression coefficient due to the compositional nature of relative abundance data [32]. The set of relevant taxa are those corresponding to non-zero coeffi- cients in the solution of CoDA-lasso. Since the approach can lead to some false positives, a stability analysis was also applied to solutions from CoDA-lasso [33]. It involves refitting the model several times on independent bootstrap samples of the data and picking only those components that are selected almost always, so to delete false positives detected by chance in just a few of the re-samplings. Such stability analysis was performed using 100 bootstrap re-samplings, each comprising 80 percent of the available samples. Only components selected in at least 90% of the replicates were chosen in the final result. The relevance of each selected taxon to discriminate between the treatment groups was investigated assessing variable importance in prediction with random forests. Each random forest comprised 500 non-pruned classification trees. Reported results comprised the selected taxa sorted according to their relative relevance in prediction, a sample estimate of the log-fold difference between the mean abundance of each group, and a heatmap of the taxa prevalence in each compared group. 2.7.4. Quantification of SCFA For the quantification of short-chain fatty acids (SCFA), fecal samples were divided in two aliquots, one for the lyophilization, and the second for a direct measure of the molecules of interest in order to obtain the dry weight-normalized absolute concentration. SCFA (acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, isobutyric acid, isovaleric acid, and isocaproic acid) were measured on the second aliquot of test material via a gas chromatography–flame ionization detector (GC-FID) method as described by De Weirdt et al. [34].
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2.8. Compliance and Adverse Events Participants were asked to collect and return empty IP and CaD supplement containers at each visit. Compliance was calculated from the number of IP and CaD supplements returned. Compliance for both IP and CaD was calculated, in percentage, as: (100 × total number of capsules administered)/(theoretical number of capsules per day × extent of exposure in days) at each individual visit and for the entire study period. Participants were considered non-compliant for the IP if they had (1) an overall IP compliance <80% or >120%, or (2) an overall compliance within [80%; 120%], but at least one individual visit IP compliance <70% or >130%, or (3) an overall IP compliance missing and less than three available individual visit IP compliances within [80%; 120%]. Adverse events (AEs) were collected on AE forms throughout the trial. AEs were considered as treatment emergent (TEAE) if they began or worsened from the date of the first IP administration. AEs were recorded in the eCRF at each visit; recordings included a description of the AE, the relationship to the intervention (“not related” or “related or suspected”), whether the AE was serious (i.e., resulted in death, life-threatening, required hospitalization, or resulted in persistent disability) or nonserious, and the intensity of the AE (mild, moderate, severe). 2.9. Power Calculation and Statistical Analysis The sample size was calculated to detect a difference in the change from baseline to 48 weeks in BMD measured by DXA on the L2-L4 lumbar spine region between HE and placebo (primary outcome) with consideration of the findings from previous studies [35–37]. The minimum relevant difference expected in the change in BMD (48 weeks versus baseline) between HE and placebo was 1.45% (corresponding to 0.014 g/cm 2 ), and the standard deviation expected in both groups was 2.2% (corresponding to 0.021 g/cm 2 ). A total of 74 evaluable patients (37 per group) were necessary to ensure an 80% power to detect a sig- nificant difference between treatment groups for two-sided test at the 5% level. Assuming 25% of non-evaluable patients, a total of 100 patients were randomized. Data were analyzed using SAS ® Enterprise Guide software version 8.2 (SAS ® for Windows version 9.4M6). Graphs were created by GraphPad Prism version 9 (GraphPad Software, Inc., Boston, MA, USA). Statistical analyses were performed by an independent biostatistician (Atlantstat, France) on the full analysis set (FAS) according to the analysis group (HE or placebo). FAS is defined as all randomized participants with at least one dose of study treatment (HE or placebo) and with at least one not missing post-baseline value for the efficacy criteria. Due to the COVID-19 pandemic, post-baseline efficacy and safety evaluations were assigned to visits based on time windows around the planned visit dates. Only post-baseline evaluations recorded at the planned day ± 28 days were considered for the statistical analysis. Unfortunately, at week 24, due to the low number of observations for both DXA (8% and 6% for HE and placebo group, respectively) and blood collection (10% and 9% for HE and placebo group, respectively), it was not possible to conduct the statistical analysis for these parameters at this visit. No imputation rule was applied for missing values. The primary outcome, and all other DXA parameters, were analyzed using an analysis of covariance (ANCOVA) including analysis group, baseline values, time since menopause at screening (months), and BMI at baseline (kg/m 2 ). These cofactors were selected based on previous work demonstrating their relevance to bone-related outcomes in postmenopausal women [38,39]. Comparisons within and between groups were studied. The same anal- yses were also conducted on the relative changes from baseline (changes from baseline/ baseline × 100) for all DXA parameters. Moreover, the relative change from baseline in total BMD was categorized in different groupings (<1/ ≥ 1%) and analyzed at week 48 using a logistic regression including analysis group, baseline value, time since menopause at screening, and BMI at baseline. Finally, a subgroup analysis was conducted for BMD L2-L4 and total BMD according to serum 25-OH D3 concentration at baseline (<75/ ≥ 75 nmol/L). The relative changes from baseline at week 48 were analyzed using ANCOVA including
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analysis group, baseline value, time since menopause at screening, BMI at baseline, analysis subgroup, and interaction analysis group × analysis subgroup. For bone biomarkers, the changes from baseline in each parameter were analyzed at week 48 using a mixed model for repeated measures (MMRM), including the following covariates, in addition to analysis group and baseline value: time since menopause at screening and BMI at baseline. Comparisons within and between groups were studied. Regarding the other secondary and exploratory outcomes, depending on planned time points, an ANCOVA at week 48 including analysis group and baseline value or a MMRM including analysis group, visit, baseline values, and group*visit interaction was performed. For all analyses, normality distribution of the residuals was verified by Skewness and Kurtosis. If the adequacy of the model could not be validated, the parameter was derived using the log transformation of the values at each time point and was then modeled with the same model characteristics. If the adequacy of the new model was not able to be validated on the log transformation, a non-parametric analysis of covariance, based on ranks (rank ANCOVA) with the same covariates was performed for the comparison between groups and Wilcoxon signed-rank test for the comparison within group. Regarding safety, all analyses were performed on all participants who received at least one dose of study treatment according to the analysis groups. The incidence of AEs was assessed, and a description according to SOC and PT was tabulated. The number of patients with at least one TEAE was compared between analysis groups using a chi-squared test. Normal data were reported as means ± SD and non-normal data as median (Q1; Q3). All statistical tests were conducted two-sided with a significance level of 5%. No adjustment for multiplicity was considered.
3. Results 3.1. Baseline Characteristics of the Study Population
A total of 221 women were screened, among whom 100 were deemed eligible and assigned randomly to HE ( n = 50) or placebo ( n = 50) groups. Five participants were lost to follow up (three in the HE group and two in the placebo group), and ninety-five participants fully completed the 48-week trial. Three participants in the placebo group were excluded from the FAS because they had no post-baseline value for any of the efficacy criteria (Figure 1). Baseline data of the FAS population are presented in Table 1. Both groups were bal- anced on all the parameters presented. The participant mean age was 62.2 ± 6.3 y (range: 50; 77 y), and time since menopause was 12.6 ± 7.1 y (range: 1.1–31.8 y) with a slightly higher prevalence of women > 10 years post-menopause in the placebo group, 70% compared to 48% in the HE group (no statistical test performed). Mean BMI was 24.9 ± 3.1 (range: 18.6; − 31.9), with 54% of the participants within normal range and 46% in an overweight and obese range. Mean serum 25-OH D3 concentration was 79.2 ± 27.7 nmol/L (range: 21; 155). The vitamin D status was considered sufficient if serum 25-OH D3 ≥ 75 nmol/L versus in- sufficient if <75 nmol/L [40]. A slightly lower prevalence of vitamin D insufficient women was observed in the HE group, 42% compared to 52% in the placebo group (no statistical test performed). All participants had osteopenia with an average T-score at the lowest siteof − 1.64 ± 0.41g/cm 2 (range: − 2.4; − 1.0). Two participants ( n =1HE; n = 1 placebo) were enrolled despite that they met the exclusion criteria regarding significant endocrine disorder as they had diabetes. Their data were included in the statistical analysis as they are part of the FAS. 3.2. Safety and Compliance to the Intervention Participant compliance to the IP was good with only 17% and 13% of participants who were non-compliant in the HE and placebo groups respectively. Similarly, compliance of the CaD supplements was good with only 11% and 4% of participants consuming <80% or >120% of the supplements in the HE and placebo group, respectively. Both HE and placebo capsules were well tolerated.
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Analysis of the prenylflavonoids concentrations in urine and plasma confirmed the good adherence to the treatment. Indeed, X, IX, 6-PN, 8-PN, and their metabolites (glu- curonide and sulfated forms) were present after 48 weeks in both urine and plasma of the HE group, while absent or negligible in the placebo group. In urine samples from the HE group, total 8-PN was detected in 94% of the participants with a mean concentration of 13.96 ± 19.11 ng/mL; total 8-PN was detected in 76% of participant plasma samples of the HE group at a mean concentration of 1.09 ± 0.92 ng/mL (Table S2). Regarding safety, there were a total of 127 TEAEs reported during the trial, of which 55 were noted in the HE group and 77 in the placebo group ( p = 0.21; Table S3). Among these, only 15 and 22 were suspected to be related to the IP in the HE and placebo group, re- spectively. Two participants in the HE group versus three in the placebo group discontinued IP and withdrew from trial due to an AE suspected to be related to the IP. Six participants experienced serious TEAEs, two occurred in the HE group and four in the placebo group. These serious TEAEs were unexpected and not related to the IP. Laboratory results were generally unremarkable (Table S4). There were no notable or clinically meaningful changes in participants’ anthropometrics or vitals detected during the study. CONSORT 2010 Flow Diagram
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Assessed for eligibility (n= 221)
Enrollment
Excluded (n=121) – Not meeting inclusion criteria (n=109) – Declined to participate (n=6) – Unable to get a DXA scan due to im- planted jewellery (n= 1) – No longer needed for the study as 100 eligible participants were met (n=5)
Randomized (n= 100)
Allocation
Allocated to intervention (n= 50) – Received allocated intervention (n= 50) – Did not receive allocated intervention (n= 0)
Allocated to placebo (n= 50) – Received allocated intervention (n= 50) – Did not receive allocated intervention (n= 0)
Follow-Up
Lost to follow-up (n= 0) Discontinued intervention (n= 3)
Lost to follow-up (n= 0) Discontinued intervention (n= 2)
Analysis
Analysed: – FAS (n=47) – Excluded from analysis: no post baseline efficacy value (n=3) – SS (n=50)
Analysed: – FAS (n=50) – SS (n=50)
Figure 1. CONSORT flow diagram. FAS: full analysis set, SS: safety set. Figure1. CONSORT flow diagram. FAS: full analysis set, SS: safety set. 3.3. DXA Parameters Baseline data of the FAS population are presented in Table 1. Both groups were bal- anced on all the parameters presented. The participant mean age was 62.2 ± 6.3 y (range: 50; 77 y), and time since menopause was 12.6 ± 7.1 y (range: 1.1–31.8 y) with a slightly higher prevalence of women > 10 years post-menopause in the placebo group, 70% com- pared to 48% in the HE group (no statistical test performed). Mean BMI was 24.9 ± 3.1 (range: 18.6; −31.9), with 54% of the participants within normal range and 46% in an over- weight and obese range. Mean serum 25-OH D3 concentration was 79.2 ± 27.7 nmol/L (range: 21; 155). The vitamin D status was considered sufficient if serum 25-OH D3 ≥ 75 nmol/L versus insufficient if <75 nmol/L [40]. A slightly lower prevalence of vitamin D insufficient women was observed in the HE group, 42% compared to 52% in the placebo group (no statistical test performed). All participants had osteopenia with an average T- score at the lowest site of −1.64 ± 0.41 g/cm 2 (range: −2.4; −1.0). Two participants (n = 1 HE; n = 1 placebo) were enrolled despite that they met the exclusion criteria regarding signifi- For the primary outcome, mean change in BMD at L2-L4 lumbar spine, from baseline after 48 weeks, revealed a slight but not statistically significant increase in the HE group (0.0063 ± 0.0371 g/cm 2 ) compared to no change in the placebo group (0.0002 ± 0.0002 g/cm 2 ). Additionally, there was no statistically significant difference detected between groups (0.0091 ± 0.0089 g/cm 2 and0.88 ± 0.85 in relative %).
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Table1. Baseline characteristics of the HE and placebo group.
Parameter
Unit years years
HE ( n =50)
Placebo ( n =47)
Age
60.8 (6.3) 11.1 (7.3)
63.6 (6.1) 14.1 (6.6)
Time since last menstruation
Anthropometrics Weight
kg
64.4 (9.0) 24.5 (3.2) 81.4 (7.8) 100.1 (6.9)
67.9 (7.5) 25.3 (3.0) 84.0 (7.9) 101.9 (6.8)
kg/m 2
BMI
Waist circumference Hip circumference Waist-to-hip ratio Lifestyle Energy intake Calcium intake Vitamin D intake
cm cm
a.u.
0.8 (0.1)
0.8 (0.1)
Kcal/day mg/day
2044.6 (539.5) 1201.8 (364.1) 4.87 (3.31) 165.0 (62.2) 1.02 (0.07) 0.85 (0.09) 1.06 (0.07) − 1.50 (0.59) − 1.09 (0.73) − 1.64 (0.41) 38.0 (3.7) 23.5 (6.9) 1.9 (0.8) 37.4 (6.6) 8.14 (5.18) 1.68 (3.07)
1960.5 (570.8) 1140.4 (432.0) 4.71 (2.76) 185.9 (67.6) 1.04 (0.10) 0.84 (0.07) 1.07 (0.08) − 1.30 (0.86) − 1.19 (0.59) − 1.64 (0.41) 39.7 (3.5) 26.0 (8.9) 2.2 (0.8) 38.4 (6.5) 9.81 (4.98) 1.79 (1.40)
µ g/day
Physical activity, PASE total score
a.u.
DXA parameters BMD at L2-L4
g/cm 2 g/cm 2 g/cm 2
BMDat FN
BMD total body T-score at L2-L4
a.u. a.u. a.u.
T-score at FN
Lowest T-score at inclusion
Leanmass Fatmass 1 Visceral fat
kg kg kg
%Fat
% % %
FRAX—Major osteoporotic
FRAX—Hip fracture Blood parameters 25-OHD3
nmol/L mmol/L pmol/L mmol/L mIU/L mmol/L mmol/L mmol/L mmol/L mmol/L
83.4 (31.2) 2.43 (0.10) 20.7 (7.5) 4.70 (0.48) 5.1 (2.3) 35.4 (3.4) 5.7 (1.0) 1.9 (0.4) 0.92 (0.46) 1.08 (0.58) 3.4 (1.2)
74.7 (22.8) 2.39 (0.09) 18.5 (0.0) 4.73 (0.45) 5.0 (2.4) 36.2 (2.7) 5.7 (1.0) 1.7 (0.3) 1.12 (0.65) 1.06 (0.57) 3.6 (1.0)
Calcium
17- β estradiol
Glucose Insulin HbA1c
Total cholesterol HDL cholesterol LDL cholesterol
Triglycerides
HOMAIR
a.u.
1 One aberrant data has been removed after statistical analysis for this parameter. Mean (SD).
Among the other DXA parameters, there was a significant increase in the total body BMD within the HE group at week 48 compared to baseline (0.0180 ± 0.0302 g/cm 2 , p < 0.0001), while there was no statistically significant increase in the placebo group (0.0079 ± 0.0026 g/cm 2 ). The difference between groups tended to be significant with a greater increase in the HE group compared to placebo (0.0106 ± 0.0059 g/cm 2 , p =0.07; 0.99 ± 0.56 relative %, p = 0.08; Figure 2A). Total body BMD increased by at least 1% after 48 weeks in 61% of participants in the HE group compared to 40% in the placebo group, resulting in a significantly higher chance of having a relative change from baseline of ≥ 1% in the HE group versus the placebo group (adjusted odds ratio ± SE=2.41 ± 1.07, p = 0.047; Figure 2B). Compared to baseline, a significant increase in BMD at femoral neck was observed in both groups after 48 weeks (0.0107 ± 0.0289 g/cm 2 in the HE group and 0.0191 ± 0.029g/cm 2 in the placebo group; p < 0.01) without significant difference between groups.
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Figure 2. Relative changes from baseline at week 48 of BMD at L2-L4 lumbar spine and total body ( A ); percentage of women with a total body BMD change from baseline at week 48 ≥ 1% ( B ); sub- group analysis with relative changes from baseline at week 48 of BMD at L2-L4 lumbar spine and total body in vitamin D su ffi cient vs. insu ffi cient women at baseline ( C ). All data are represented as mean ± SEM. # Odds ratio for relative change from baseline (probability modeled for class ≥ 1%) (HE vs. Placebo) (95% CI): 2.41 (1.01; 5.74), p < 0.05. * p = 0.05 versus placebo. 3.4. Biochemical Analysis No signi fi cant di ff erences were observed between the HE and placebo groups after 48 weeks of supplementation for any of the plasma bone biomarkers measured (Table S5). Compared to baseline, CTx level increased after 48 weeks in both groups ( p < 0.001), while sclerostin and TRAP5b decreased in both groups ( p < 0.05). uOC decreased signi fi cantly in the HE group after 48 weeks ( p < 0.01). Similarly, no signi fi cant di ff erences between groups were observed at 48 weeks for the following blood parameters: triglycerides, total cholesterol, HDL-cholesterol, LDL- cholesterol, fasting glucose, insulinemia, HbA1c, HOMA-IR, serum 25-OH D3 concentra- tion, and 17- β oestradiol (Table S6). 3.5. Antropometrics, Physical Activity, Dietary Intake and Health-Related Quality of Life No signi fi cant di ff erences between groups were observed for anthropometric param- eters at any visit (Table S6. Physical activity assessed using the PASE questionnaire showed similar activity at baseline and throughout the 48 weeks between the HE and pla- cebo groups (Table S7). Dietary analysis using the FFQ showed both groups had similar intake at baseline. After 48 weeks, the HE group showed higher fat, calcium, and vitamin K 2 intakes, com- pared to the placebo group ( p < 0.05; Table S7). For fat, the median change from placebo at week 48 was + 11 g/d, for calcium +112 mg/d, and for vitamin K 2 + 2.3 µg/d in the HE group compared to the placebo group. Changes in SF-36 scores after 48 weeks are shown in Table 2. The physical functioning score was signi fi cantly increased in the HE group compared to the placebo group ( p < 0.05), with 25 participants (53%) showing increased scores (>0) in the HE group compared Similarly, no significant differences between groups were observed at 48 weeks for the following blood parameters: triglycerides, total cholesterol, HDL-cholesterol, LDL- cholesterol, fasting glucose, insulinemia, HbA1c, HOMA-IR, serum 25-OH D3 concentra- tion, and 17- β oestradiol (Table S6). Additionally, post hoc sub-group analysis was performed according to vitamin D status (sufficient if ≥ 75 nmol/L and insufficient if <75 nmol/L). In vitamin D suffi- cient women, there was an increase in the HE group compared to the placebo group for the BMD at L2-L4 lumbar spine (difference of adjusted relative changes from baseline ± SE=2.29 ± 1.16%; p = 0.051) and the total body BMD (difference of adjusted relative changes from baseline ± SE=1.44 ± 0.78%, p = 0.066; Figure 2C). 3.4. Biochemical Analysis No significant differences were observed between the HE and placebo groups after 48 weeks of supplementation for any of the plasma bone biomarkers measured (Table S5). Compared to baseline, CTx level increased after 48 weeks in both groups ( p < 0.001), while sclerostin and TRAP5b decreased in both groups ( p < 0.05). uOC decreased significantly in the HE group after 48 weeks ( p <0.01). Figure2. Relative changes from baseline at week 48 of BMD at L2-L4 lumbar spine and total body ( A ); percentage of women with a total body BMD change from baseline at week 48 ≥ 1%( B ); sub-group analysis with relative changes from baseline at week 48 of BMD at L2-L4 lumbar spine and total body in vitamin D sufficient vs. insufficient women at baseline ( C ). All data are represented as mean ± SEM. # Odds ratio for relative change from baseline (probability modeled for class ≥ 1%) (HE vs. Placebo) (95% CI): 2.41 (1.01; 5.74), p <0.05. * p = 0.05 versus placebo. Regarding body composition, lean mass and fat percentage were not modulated in any groups, while fat mass and visceral fat were significantly increased after 48 weeks compared to baseline in the HE group (median change (Q1; Q3) = 714.0 ( − 123.0; 1285.0) g for fat mass and 52.5 ( − 55.0; 213.0) g for visceral fat, p < 0.05), but no significant difference between groups was found.
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3.5. Antropometrics, Physical Activity, Dietary Intake and Health-Related Quality of Life No significant differences between groups were observed for anthropometric parame- ters at any visit (Table S6). Physical activity assessed using the PASE questionnaire showed similar activity at baseline and throughout the 48 weeks between the HE and placebo groups (Table S7). Dietary analysis using the FFQ showed both groups had similar intake at baseline. After 48 weeks, the HE group showed higher fat, calcium, and vitamin K 2 intakes, compared to the placebo group ( p < 0.05; Table S7). For fat, the median change from placebo at week 48 was + 11 g/d, for calcium +112 mg/d, and for vitamin K 2 +2.3 µ g/d in the HE group compared to the placebo group. Changes in SF-36 scores after 48 weeks are shown in Table 2. The physical functioning score was significantly increased in the HE group compared to the placebo group ( p <0.05), with 25 participants (53%) showing increased scores (>0) in the HE group compared to 14 (30%) in the placebo group. The role limitations due to physical health score trended toward a greater increase in the HE group compared to placebo ( p = 0.08); however, at least 50% of the participants had no change in both groups (Q1; Q3 changes from baseline = 0; 0), and only eight and five participants had increased scores in the HE and placebo group, respectively, indicating a weak effect. No difference between groups was observed for the other scores.
Table2. Health-related quality of life.
SF-36 Scores
Statistics
HE ( n =50)
Placebo ( n =47)
p
Physical functioning
median (Q1; Q3) median (Q1; Q3)
5 (0; 10) 0 (0; 0) 0 (0; 0)
0 (0; 5) 0 (0; 0) 0 (0; 0)
0.049 0.082 0.589 0.914 0.530 0.555 0.969 0.407
Role limitations due to physical health
Role limitations due to emotional problems median (Q1; Q3)
Energy/fatigue
mean (SD)
2.66 (11.7) 0 ( − 4; 8) 0 (0; 0) 1.01 (18.96) 2.77 (10.67)
1.6 (12.69) 0 ( − 4; 4) 0 (0; 0) 1.06 (23.06) − 0.43 (14.74)
Emotional well-being Social functioning
median (Q1; Q3) median (Q1; Q3)
Pain
mean (SD) mean (SD)
General health
3.6. Gut Microbiome Modulation As the gut microbiota is a key player in prenylflavonoid metabolism and bone home- ostasis, potential differences in the microbiome composition between the HE and placebo groups were explored. Low-dimensional representations of the taxonomic profiles com- puted using non-metric multidimensional scaling (MDS) on Bray–Curtis and Jaccard β -diversity scores suggested that there were no differences between the groups at any visit. These exploratory results were also confirmed with MiRKAT overall association tests based on β -diversity (Figure S1). There was also no significant difference between groups in terms of α -diversity assessed as change from baseline by the inverse Simpson index, Shannon index, and observed number of species (Figure S2). Despite this lack of significant differences between the overall compositions of samples between the HE and placebo groups, an exploratory multivariate analysis was run to identify the taxa that seem more relevant to distinguish between the two groups. Results for each taxonomic rank (family, genus, specie) are displayed in Figure 3. Results shown in Figure 3A highlight five families enriched in the HE group and seven enriched in the placebo group. The most discriminant family to differentiate between the two groups was Barnesiellaceae, which was more abundant in the HE group. Observed prevalence, however, was high and roughly the same in both groups. Turicibacteraceae was also enriched in the HE group, showing larger mean abundance and prevalence than in the placebo group. This finding was consistent with the identification of Turicibacter as a differentiating genus more abundant and prevalent in the HE group. To be noted, Turicibacter was also identified as one of the most discriminant genus to differentiate between the two groups after 24 weeks, being more abundant and prevalent in the HE group compared to the placebo group such as after 48 weeks (Figure S3). Shigella was also
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