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 , René Rizzoli 3 , Mathieu Tenon 1 , Thierry Berton 1 , Sinead Harney 4
and Pascale Fança-Berthon 1

1 Givaudan France Naturals, 84911 Avignon, France; [email protected] (M.T.);

[email protected] (T.B.); [email protected] (P.F.-B.)
2 Biofortis, 44800 Saint-Herblain, France; [email protected]
3 Service of Bone Disease, Geneva University Hospitals and Faculty of Medicine, 1211 Geneva, Switzerland;
[email protected]
4 Rheumatology Department, Cork University Hospital, T12 DFK4 Cork, Ireland; [email protected]
* Correspondence: [email protected]

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
Citation: Lecomte, M.; Tomassi, D.; (BMD) and bone metabolism were assessed by DXA measurements and plasma bone biomarkers,
Rizzoli, R.; Tenon, M.; Berton, T.; respectively. Participant’s quality of life (SF-36), gut microbiome composition, and short-chain fatty
Harney, S.; Fança-Berthon, P. Effect of acid (SCFA) levels were also investigated. In addition to the CaD supplements, 48 weeks of HE
a Hop Extract Standardized in supplementation increased total body BMD (1.8 ± 0.4% vs. baseline, p < 0.0001; 1.0 ± 0.6% vs.
8-Prenylnaringenin on Bone Health
placebo, p = 0.08), with a higher proportion of women experiencing an increase ≥1% compared to
and Gut Microbiome in
placebo (odds ratio: 2.41 ± 1.07, p < 0.05). An increase in the SF-36 physical functioning score was
Postmenopausal Women with
observed with HE versus placebo (p = 0.05). Gut microbiome α-diversity and SCFA levels did not
Osteopenia: A One-Year
differ between groups. However, a higher abundance of genera Turicibacter and Shigella was observed
Randomized, Double-Blind,
Placebo-Controlled Trial. Nutrients
in the HE group; both genera have been previously identified as associated with total body BMD.
2023, 15, 2688. https://doi.org/ These results suggest that an 8-PN standardized hop extract could beneficially impact bone health of
10.3390/nu15122688 postmenopausal women with osteopenia.

Academic Editor: Anna Scotto

Keywords: hop; 8-PN; phytoestrogen; bone; osteoporosis; osteopenia; microbiome; menopause
D’Abusco

Received: 3 May 2023

Revised: 21 May 2023
Accepted: 25 May 2023 1. Introduction
Published: 9 June 2023
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
Copyright: © 2023 by the authors.
by osteoporosis in the USA and Europe [2,3]. The decline in endogenous estrogen during
Licensee MDPI, Basel, Switzerland.
and after menopause accelerates the bone remodeling process with an imbalance between
This article is an open access article bone formation and bone resorption [4]. In this context, phytoestrogens are an interesting
distributed under the terms and non-pharmaceutical intervention to prevent bone loss. Phytoestrogens are polyphenolic
conditions of the Creative Commons plant-derived compounds with a structural similarity to endogenous human estrogen,
Attribution (CC BY) license (https:// hence their estrogenic activity. The main dietary sources of phytoestrogens are soy and red
creativecommons.org/licenses/by/ clover (isoflavones), flaxseed (lignans) and hops (prenylflavonoids) [5]. Phytoestrogens
4.0/). are already widely used to alleviate menopausal symptoms such as hot flashes and night

Nutrients 2023, 15, 2688. https://doi.org/10.3390/nu15122688 https://www.mdpi.com/journal/nutrients

Nutrients 2023, 15, 2688 2 of 20

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
dose of 100 µg 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)
Nutrients 2023, 15, 2688 3 of 20

between 18–32 kg/m2 , and presenting with osteopenia defined as a dual energy X-ray
absorptiometry (DXA) T-score between −1 and −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
Nutrients 2023, 15, 2688 4 of 20

Lifenol® containing 100 µg of 8-PN, 110 µg of 6-PN, 1.25 mg of X, and 2.94 mg of IX

(measured by LC-UV) mixed with maltodextrin (Roquette Frères, 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 ◦ C until
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 µL of plasma or enzymatic hydrolyzed plasma
were loaded on Captiva EMR-lipid plate (Agilent Technologies, Santa Clara, CA, USA).
An amount of 300 µL 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 µL of MeOH/ACN
(50:50) were loaded on the cartridge once again and eluted by positive pressure. For the
urine samples, 300 µL of water were loaded on Captiva ND plate, and 100 µL 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
Nutrients 2023, 15, 2688 5 of 20

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 µL of this solution were
mixed with 60 µL 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 × 2.1 mm, 2.6 µm, 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 µL. 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 R2 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
Nutrients 2023, 15, 2688 6 of 20

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].
Nutrients 2023, 15, 2688 7 of 20

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/cm2 ), and the standard
deviation expected in both groups was 2.2% (corresponding to 0.021 g/cm2 ). 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/m2 ). 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
Nutrients 2023, 15, 2688 8 of 20

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
site of −1.64 ± 0.41 g/cm2 (range: −2.4; −1.0). Two participants (n = 1 HE; 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.
Nutrients 2023, 15, 2688 9 of 20

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 9were
Nutrients 2023, 15, x FOR PEER REVIEW of 21
generally unremarkable (TableCONSORT
S4). There were
2010 no Diagram
Flow notable or clinically meaningful changes
in participants’ anthropometrics or vitals detected during the study.

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) Allocated to placebo (n= 50)
– Received allocated intervention (n= 50) – Received allocated intervention (n= 50)
– Did not receive allocated intervention (n= 0) – Did not receive allocated intervention (n= 0)

Follow-Up
Lost to follow-up (n= 0) Lost to follow-up (n= 0)
Discontinued intervention (n= 3) Discontinued intervention (n= 2)

Analysis
Analysed: Analysed:
– FAS (n=50) – FAS (n=47)
– SS (n=50) – Excluded from analysis: no post baseline
efficacy value (n=3)
– SS (n=50)

Figure1.1.CONSORT
Figure CONSORTflow
flowdiagram.
diagram.FAS:
FAS:full
fullanalysis
analysisset,
set,SS:
SS:safety
safetyset.
set.

3.3. DXA Parameters

Baseline data of the FAS population are presented in Table 1. Both groups were bal-
Foron
anced theallprimary outcome,presented.
the parameters mean changeThe in BMD at L2-L4
participant meanlumbar
age wasspine,
62.2 from baseline
± 6.3 y (range:
after 48 weeks, revealed a slight but not statistically significant increase in the HE group
50; 77 y), and time since menopause was 12.6 ± 7.1 y (range: 1.1–31.8 y) with a slightly
(0.0063 0.0371 g/cm
higher±prevalence of2 )women
compared
> 10toyears
no change in the placeboingroup
post-menopause (0.0002 group,
the placebo ± 0.000270%
g/cm 2 ).
com-
Additionally,
pared to 48%there in thewas
HEno statistically
group significant
(no statistical difference detected
test performed). Mean BMIbetween groups
was 24.9 ± 3.1
(0.0091 0.0089 g/cm 2 and 0.88 ± 0.85 in relative %).
(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/cm2 (range: −2.4; −1.0). Two participants (n = 1 HE;
Nutrients 2023, 15, 2688 10 of 20

Table 1. Baseline characteristics of the HE and placebo group.

Parameter Unit HE (n = 50) Placebo (n = 47)

Age years 60.8 (6.3) 63.6 (6.1)
Time since last menstruation years 11.1 (7.3) 14.1 (6.6)
Anthropometrics
Weight kg 64.4 (9.0) 67.9 (7.5)
BMI kg/m2 24.5 (3.2) 25.3 (3.0)
Waist circumference cm 81.4 (7.8) 84.0 (7.9)
Hip circumference cm 100.1 (6.9) 101.9 (6.8)
Waist-to-hip ratio a.u. 0.8 (0.1) 0.8 (0.1)
Lifestyle
Energy intake Kcal/day 2044.6 (539.5) 1960.5 (570.8)
Calcium intake mg/day 1201.8 (364.1) 1140.4 (432.0)
Vitamin D intake µg/day 4.87 (3.31) 4.71 (2.76)
Physical activity, PASE total score a.u. 165.0 (62.2) 185.9 (67.6)
DXA parameters
BMD at L2-L4 g/cm2 1.02 (0.07) 1.04 (0.10)
BMD at FN g/cm2 0.85 (0.09) 0.84 (0.07)
BMD total body g/cm2 1.06 (0.07) 1.07 (0.08)
T-score at L2-L4 a.u. −1.50 (0.59) −1.30 (0.86)
T-score at FN a.u. −1.09 (0.73) −1.19 (0.59)
Lowest T-score at inclusion a.u. −1.64 (0.41) −1.64 (0.41)
Lean mass kg 38.0 (3.7) 39.7 (3.5)
Fat mass 1 kg 23.5 (6.9) 26.0 (8.9)
Visceral fat kg 1.9 (0.8) 2.2 (0.8)
% Fat % 37.4 (6.6) 38.4 (6.5)
FRAX—Major osteoporotic % 8.14 (5.18) 9.81 (4.98)
FRAX—Hip fracture % 1.68 (3.07) 1.79 (1.40)
Blood parameters
25-OH D3 nmol/L 83.4 (31.2) 74.7 (22.8)
Calcium mmol/L 2.43 (0.10) 2.39 (0.09)
17-β estradiol pmol/L 20.7 (7.5) 18.5 (0.0)
Glucose mmol/L 4.70 (0.48) 4.73 (0.45)
Insulin mIU/L 5.1 (2.3) 5.0 (2.4)
HbA1c mmol/L 35.4 (3.4) 36.2 (2.7)
Total cholesterol mmol/L 5.7 (1.0) 5.7 (1.0)
HDL cholesterol mmol/L 1.9 (0.4) 1.7 (0.3)
LDL cholesterol mmol/L 3.4 (1.2) 3.6 (1.0)
Triglycerides mmol/L 0.92 (0.46) 1.12 (0.65)
HOMA IR a.u. 1.08 (0.58) 1.06 (0.57)
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/cm2 ,
p < 0.0001), while there was no statistically significant increase in the placebo group
(0.0079 ± 0.0026 g/cm2 ). The difference between groups tended to be significant with
a greater increase in the HE group compared to placebo (0.0106 ± 0.0059 g/cm2 , 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/cm2 in the HE group
and 0.0191 ± 0.029 g/cm2 in the placebo group; p < 0.01) without significant difference
between groups.
Nutrients
Nutrients2023,
2023,15,
15,x2688
FOR PEER REVIEW 12 11
ofof2120

Relativechanges
Figure2.2.Relative
Figure changesfrom
from baseline
baseline at
at week
week4848of
ofBMD
BMDatatL2-L4
L2-L4lumbar
lumbarspine and
spine andtotal body
total (A);
body
percentage of women with a total body BMD change from baseline at week 48 ≥ 1% (B); sub-group
(A); percentage of women with a total body BMD change from baseline at week 48 ≥ 1% (B); sub-
analysis
group with relative
analysis changes
with relative from from
changes baseline at week
baseline 48 of48
at week BMD at L2-L4
of BMD lumbar
at L2-L4 spine
lumbar and and
spine total
total
body in vitamin D sufficient vs. insufficient women at baseline (C). All data are representedasas
body in vitamin D sufficient vs. insufficient women at baseline (C). All data are represented
mean
mean±±SEM.SEM.# #Odds
Oddsratio
ratiofor
forrelative
relativechange
changefrom
frombaseline
baseline(probability
(probabilitymodeled
modeledfor forclass
class≥≥1%)
1%)
(HE vs. Placebo) (95% CI): 2.41 (1.01; 5.74), p < 0.05. * p = 0.05 versus placebo.
(HE vs. Placebo) (95% CI): 2.41 (1.01; 5.74), p < 0.05. * p = 0.05 versus placebo.

3.4. Biochemical
Regarding Analysis
body composition, lean mass and fat percentage were not modulated in
anyNo groups, whiledifferences
significant fat mass and werevisceral fat were
observed significantly
between the HE and increased
placeboafter 48 weeks
groups after
48compared
weeks of to baseline in the HE
supplementation for group
any of (median
the plasmachange
bone(Q1; Q3) = 714.0
biomarkers (−123.0;
measured 1285.0)
(Table S5).g
for fat mass
Compared toand 52.5 (−
baseline, 55.0;
CTx 213.0)
level g for visceral
increased after 48fat, p < 0.05),
weeks butgroups
in both no significant difference
(p < 0.001), while
between groups
sclerostin was found.
and TRAP5b decreased in both groups (p < 0.05). uOC decreased significantly
in the Additionally,
HE group after post hoc sub-group
48 weeks (p < 0.01).analysis was performed according to vitamin D
status (sufficient
Similarly, if ≥75 nmol/L
no significant and insufficient
differences between groupsif <75were
nmol/L). In vitamin
observed D suffi-
at 48 weeks for
cient
the women,blood
following there parameters:
was an increase in the HE
triglycerides, group
total compared
cholesterol, to the placebo LDL-
HDL-cholesterol, group
for the BMD
cholesterol, at L2-L4
fasting lumbar
glucose, spine (difference
insulinemia, HbA1c,ofHOMA-IR,
adjusted relative changesD3
serum 25-OH from baseline
concentra-
± SE = 2.29 ± 1.16%; p = 0.051)
tion, and 17-β oestradiol (Table S6). and the total body BMD (difference of adjusted relative
changes from baseline ± SE = 1.44 ± 0.78%, p = 0.066; Figure 2C).
3.5. Antropometrics, Physical Activity, Dietary Intake and Health-Related Quality of Life
3.4. Biochemical Analysis
No significant differences between groups were observed for anthropometric param-
No significant differences were observed between the HE and placebo groups after
eters at any visit (Table S6. Physical activity assessed using the PASE questionnaire
48 weeks of supplementation for any of the plasma bone biomarkers measured (Table S5).
showed similar activity at baseline and throughout the 48 weeks between the HE and pla-
Compared to baseline, CTx level increased after 48 weeks in both groups (p < 0.001), while
cebo groups (Table S7).
sclerostin and TRAP5b decreased in both groups (p < 0.05). uOC decreased significantly in
Dietary analysis using the FFQ showed both groups had similar intake at baseline.
the HE group after 48 weeks (p < 0.01).
After 48 weeks, the HE group showed higher fat, calcium, and vitamin K2 intakes, com-
Similarly, no significant differences between groups were observed at 48 weeks for
pared to the placebo group (p < 0.05; Table S7). For fat, the median change from placebo
the following blood parameters: triglycerides, total cholesterol, HDL-cholesterol, LDL-
at week 48 was + 11 g/d, for calcium +112 mg/d, and for vitamin K2 + 2.3 µg/d in the HE
cholesterol, fasting glucose, insulinemia, HbA1c, HOMA-IR, serum 25-OH D3 concentra-
group compared to the placebo group.
tion, and 17-β oestradiol (Table S6).
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
Nutrients 2023, 15, 2688 12 of 20

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 K2 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 K2 + 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 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.

Table 2. Health-related quality of life.

SF-36 Scores Statistics HE (n = 50) Placebo (n = 47) p

Physical functioning median (Q1; Q3) 5 (0; 10) 0 (0; 5) 0.049
Role limitations due to physical health median (Q1; Q3) 0 (0; 0) 0 (0; 0) 0.082
Role limitations due to emotional problems median (Q1; Q3) 0 (0; 0) 0 (0; 0) 0.589
Energy/fatigue mean (SD) 2.66 (11.7) 1.6 (12.69) 0.914
Emotional well-being median (Q1; Q3) 0 (−4; 8) 0 (−4; 4) 0.530
Social functioning median (Q1; Q3) 0 (0; 0) 0 (0; 0) 0.555
Pain mean (SD) 1.01 (18.96) 1.06 (23.06) 0.969
General health mean (SD) 2.77 (10.67) −0.43 (14.74) 0.407

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
 Nutrients 2023, 15, 2688 13 of 20
Nutrients 2023, 15, 0 14 of 21

found more abundant in the HE group after 48 weeks. These two genera were largely the
most abundant among the selected most discriminant genera. Two other genera were more
abundant in the HE group, and six others were more abundant in the placebo group with
Coriobacterium being the most relevant. Among enriched species identified in the HE group,
Bifidobacterium saimiriisciurei and Paenibacillus donghaensis had both larger mean abundance
and prevalence in the HE group compared to the placebo group. Akkermansia glycaniphila,
Nutrients 2023, 15, x FOR PEER REVIEW 14 of 21
Hallella seregens, and Ruminiclostridium josui were more abundant and prevalent in the
placebo group.

Figure 3.3. Microbiome

Figure Microbiome components
components differentiating
differentiating between
between HEHE and
and placebo
placebo atat week
week 48.
48. For
For each
each
specific level ((A). Family, (B). Genus and (C). Species), we displayed in the left panel the importance
specific level ((A). Family, (B). Genus and (C). Species), we displayed in the left panel the importance
ofeach
of eachselected
selectedtaxon
taxontotodifferentiate
differentiatebetween
between the
the two
two groups,
groups, in in
thethe middle
middle panel
panel thethe effect
effect size,size,
by
by reporting the log-fold difference between the mean abundance of the taxon in each
reporting the log-fold difference between the mean abundance of the taxon in each group, and finally group, and
finally in the rightmost panel the prevalence of the selected taxa on each compared group. Green =
in the rightmost panel the prevalence of the selected taxa on each compared group. Green = HE;
HE; Black = placebo.
Black = placebo.

Finally,no
Finally, nodifference
differencebetween
betweengroups
groupswaswasobserved
observedregarding
regardingE.E.limosum
limosum abundance
abundance
at baseline and after 48 weeks of supplementation. We further explored
at baseline and after 48 weeks of supplementation. We further explored if treatment if treatment re-
sponsiveness (total body BMD) was correlated with relative abundance of
responsiveness (total body BMD) was correlated with relative abundance of E. limosum atE. limosum at
baseline. There was no specific pattern relating responsiveness to the observed
baseline. There was no specific pattern relating responsiveness to the observed abundance abundance
(FigureS4).
(Figure S4).
Changes
Changes atat week
week 24
24 and
and4848ininthe
theprofile ofof
profile total and
total individual
and SCFA
individual SCFAwere not not
were sig-
nificantly different between groups (Table S8).
significantly different between groups (Table S8).

4.
4. Discussion
Discussion
To
To the best of
the best of our
ourknowledge,
knowledge,this this
is is
thethe
firstfirst randomized
randomized controlled
controlled trialtrial
(RCT) (RCT)
con-
conducted to examine
ducted to examine thethe effect
effect ofofa ahop
hopextract
extracton onbone
bonehealth
health in
in postmenopausal
postmenopausal women women
with
withosteopenia.
osteopenia. In In this
this population,
population, we we demonstrated
demonstrated that that aa daily
daily supplementation
supplementation with with
100 µgof
100 µg of8-PN
8-PNfromfromaastandardized
standardizedhop hopextract
extractfor for4848weeks
weeksincreased
increased total
total body
body BMDBMD
compared
compared to to placebo.
placebo. An An improvement
improvement of of the
the SF-36
SF-36 physical
physical functioning
functioning score
score was
was also
also
observed
observedin in the
the HE
HE group
group suggesting
suggesting aa higher
higher perceived
perceived ability
ability to perform daily
to perform daily activities.
activities.
However,
However, no nosignificant
significant effect
effect was
was found
found inin BMD
BMD at at specific
specific sites
sites (lumbar
(lumbar spine,
spine, femoral
femoral
neck, and total hip), plasma bone biomarkers, and other secondary outcomes, notably
blood lipids and glucose homeostasis parameters.
This beneficial impact on BMD is consistent with previous studies performed in the
ovariectomized rat with standardized hop extract, which showed an increase in BMD fol-
lowing 8 to 12 weeks of supplementation while using a daily dose of 8-PN from 6 to 27
times higher in a human dose equivalent [13,14]. Regarding the effects of other types of
Nutrients 2023, 15, 2688 14 of 20

neck, and total hip), plasma bone biomarkers, and other secondary outcomes, notably
blood lipids and glucose homeostasis parameters.
This beneficial impact on BMD is consistent with previous studies performed in the
ovariectomized rat with standardized hop extract, which showed an increase in BMD fol-
lowing 8 to 12 weeks of supplementation while using a daily dose of 8-PN from 6 to 27 times
higher in a human dose equivalent [13,14]. Regarding the effects of other types of phy-
toestrogens in humans, isoflavones are the most studied in terms of bone health effect in
postmenopausal women. The recent meta-analysis by Sansai et al. included 63 RCTs and
various types of isoflavone interventions [8]. A statistically significant increase in BMD was
found at the lumbar spine, femoral neck, and distal radius with isoflavones. However, these
favorable effects were predominantly associated with the use of genistein pure compounds
and synthetic isoflavones, while the benefits of isoflavone extracts and dietary isoflavone
supplements on BMD remain inconclusive (with up to 300 mg/day of isoflavone aglycone
equivalents). This analysis did not reveal any significant effects on the total hip and the
total body, which might be because no study has investigated the effects of genistein or
synthetic isoflavones on those two sites.
Bone turnover biochemical markers help clinicians to identify patients at high risk for
fracture and to monitor the efficacy of osteoporosis treatments. However, no significant dif-
ference between the HE and placebo groups was observed regarding the bone biochemical
markers concentrations after 48 weeks of supplementation. Among the available biochemi-
cal markers, pro-collagen type I N-terminal propeptide (PINP) and C-terminal telopeptide
(CTX) have been recommended as reference biochemical markers of bone formation and
bone resorption, respectively [41]. Surprisingly, CTX increased compared to baseline in
both groups, while PINP remained stable. The marker of bone resorption TRAP5b and
sclerostin, a marker known to increase in bone diseases, were both decreased in the HE
and placebo groups compared to baseline. Finally, a decrease in serum undercarboxylated
osteocalcin (uOC) was observed only in the HE group compared to baseline, which could
be a sign of a beneficial impact on BMD in the HE group. Indeed, uOC concentration
is a marker of bone turnover, increased serum uOC levels have been associated with an
increased risk of hip fracture [42] and of low BMD of the hip and spine in postmenopausal
women [43].
The magnitude of HE effects on bone density is not comparable to those of osteoporosis
medications, but it could be of interest as a preventive measure for women with low bone
mass that cannot be prescribed medication at this stage. Dawson-Hughes et al. have
reported that after 3 years of vitamin D (700 IU) and calcium (500 mg) supplementation, a
1.1% net increase in total body BMD compared to placebo was associated with a relative
risk reduction of 0.4 of first osteoporotic fracture in men and women [44]. Accordingly, in
our study, the mean total body BMD increase of 1% and the significantly higher proportion
of women with an increase ≥1% following HE consumption compared to placebo might
point toward a similar relative risk reduction. Notably, considering that this 1% net increase
is in addition to a 0.8% increase already observed in both groups in the context of CaD
supplementation. Moreover, several studies have found reduced muscle strength, reduced
physical capacity, and reduced quality of life among patients with low BMD [45–47].
Notably, in a cross-sectional study, 18 postmenopausal women with osteopenia and a healed
wrist fracture had a lower score in the sub-scales on the SF-36 quality of life questionnaire
for physical functioning, role limitation due to physical problem, bodily pain and vitality
compared to a matched, healthy control group with no previous fracture [47]. Therefore,
the increase in the physical functioning SF-36 sub-scale following HE supplementation
compared to placebo may indicate an improved quality of life in women with osteopenia.
Dawnson Hugues et al. have reported in older women without intervention an
annual bone loss of about 1% in the total body and 0.8% in the lumbar spine region [44].
Furthermore, inadequate intakes of vitamin D and calcium lead to increased bone loss,
and CaD supplementation has been demonstrated to be effective in reducing bone loss
in postmenopausal women [48]. In our study, all women were supplemented with CaD,
Nutrients 2023, 15, 2688 15 of 20

which placed favorable conditions of bone loss prevention, therefore potentially limiting
the ability to detect an effect of the HE alone. In the placebo group, supplementation
with 1000 mg of calcium and 800 UI of vitamin D resulted in a mean net increase of
0.8% at the total body, 2.2% at the femoral neck, and no change at the lumbar spine
observed after 48 weeks. Similar total body BMD increase has been previously reported
with CaD supplementation [49–52]. Notably, an open-label, randomized, controlled trial
investigated in 590 postmenopausal women the effect of a similar CaD supplementation
or no intervention over 3 years [50]. A significant increase in total body BMD of 0.8% was
observed in the intervention group compared to control group (0.2%), but no significant
differences were observed at the lumbar spine, femoral neck, trochanter, and total proximal.
In compliant women (those who took at least 80% of their supplementation), greater effect
was observed with a significant increase in BMD at the total body (1.3%, i.e., around
0.4% per year) and all specific sites investigated. Interestingly, these women had a mean
baseline serum 25OHD level of 50 nmol/L, which suggests that they were moderately
vitamin D insufficient. It is known that vitamin insufficient women benefit more from a
higher CaD intake [53]. In our study, following a subgroup analysis, according to vitamin
D status, we observed that vitamin D-insufficient women (defined here as <75 nmol/L)
might have benefited more from the CaD supplements, as in the placebo group, an increase
of 0.8% was observed in these women, while a decrease of 1% was observed in vitamin
D-sufficient women. Moreover, we observed that vitamin D-sufficient women seemed to
have a more beneficial impact of HE supplementation compared to vitamin D-insufficient
women, as an increase of the lumbar spine BMD (+2.3% HE vs. placebo) was observed
primarily in this vitamin D-sufficient population.
The main mechanism of action of HE is the estrogenic activity of 8-PN, which has been
demonstrated in vitro and in vivo and which has been recently extensively reviewed [9–11,54].
Along with its metabolites, 8-PN was only detected in the plasma and urine of women
supplemented with HE, confirming adherence to treatment and an exposition to these
active compounds in this group only. No correlation was found between the levels of total
8-PN and change in total body BMD after 48 weeks. However, the samples were collected
24 h after the last capsule ingestion and therefore are not representative of the acute levels
of metabolites circulating after HE consumption. Another potential mechanism of action
could be the antioxidant properties of the standardized hop extract. Indeed, changes in
reactive oxygen species (ROS) and/or antioxidant systems seem to be involved in the patho-
genesis of bone loss. Additionally, a marked decrease in plasma antioxidants was found in
osteoporotic women, and recent data suggest that diet supplementation with antioxidants
could be an effective strategy to prevent bone loss [55]. Hops prenylflavonoids, and particu-
larly xanthohumol (X), are known to exert diverse antioxidant and free-radical-scavenging
properties and therefore might have beneficially impacted BMD of postmenopausal women
supplemented with HE [56].
Despite the fact that volunteers were asked to maintain a similar diet throughout
the study, significantly higher changes from baseline at week 48 were observed for fat,
vitamin K2 , and calcium in the HE group compared to the placebo group. No difference
was observed between groups in terms of weight, fat mass, and blood lipids, suggesting
that the small difference of fat intake (around 11 g) had no impact on these parameters.
The minimum efficacy dose of vitamin K2 for osteopenia and osteoporosis is known to be
of 45 mg/d; therefore, the differential amount of +2.3 µg/d (20,000 times less) observed
here is unlikely to be responsible for the effect on BMD [57]. Finally, the difference of
calcium between groups does not take into account the calcium supplementation, i.e., an
additional 1000 mg/d of calcium in all women in addition to the initial reported mean
intake of 1170 mg/d at baseline. Therefore, it is also unlikely that the differential amount
of calcium of +112 mg/d could have contributed to the observed effect.
Nutrients 2023, 15, 2688 16 of 20

Another hypothesis for the important variability in response observed in this trial
could be the inter-individual variation in prenylflavonoids metabolism that was reported
previously [20–22]. The final level of 8-PN absorbed does not only depend on the presence
of 8-PN itself in the HE but also on the transformation of its precursor IX into 8-PN by the
intestinal bacteria E. limosum [21]. However, important inter-individual variability was
observed for conversion efficacy with reported low, moderate, and high 8-PN producers
in humans [19]. E. limosum levels were not statistically different between the HE and
placebo group at baseline and after 48 weeks. Furthermore, subset analysis based on
responsiveness to HE supplementation did not indicate any difference in E. limosum levels
at baseline and after 48 weeks between the women with an increase in their total body
BMD ≥1% vs. <1%. Likewise, the 8-PN levels detected in blood or urine in women who
took the HE supplementation were not correlated with the magnitude of responsiveness.
Altogether, these results suggest that conversion capability of the women included in this
trial was not the primary cause of efficacy. Furthermore, it is likely that a plateau effect
was reached with the dose of 100 µg of 8-PN and that any additional amount brought by
the conversion was not responsible for additional bone health effect. Interestingly, in the
study conducted by Heyerick et al., using the same extract, 100 µg of 8-PN was sufficient to
relieve women from postmenopausal symptoms, and no additional benefit was observed
with the higher dosage of 250 µg of 8-PN [17].
A novel aspect of this trial was the analysis of the participants’ gut microbiome
and its relationship to the responsiveness of treatment. No difference between HE and
placebo was observed for overall association parameters, alpha-diversity indices, and
SCFA levels throughout the study, hence indicating no major shift in the gut microbiome
composition and function. However, the explorative multivariate analysis indicated that
after 48 weeks among the taxa identified as the most relevant to discriminate the two
groups, there was notably a higher abundance of the genera Turicibacter and Escherichia
Shigella in the HE group. In a recent animal study, positive correlations between the
genera Turicibacter and total BMD were observed in ovariectomized mice supplemented
with new antidepressant drug (R)-ketamine [58]. (R)-ketamine significantly attenuated
the reduced abundance of Turicibacter observed after ovariectomy compared to sham
control. Moreover, Escherichia Shigella was found to be more abundant in individuals with
osteopenia compared to those with osteoporosis in a cohort of 181 older adults (including
150 women) [59]. Conversely, with the latter study and our results, Escherichia Shigella was
also found negatively associated with L1-L4 BMD, total BMD, and femur total BMD in the
UK biobank cohort study including middle-aged adults [60]. These results suggest that
there may be an association between Escherichia Shigella and BMD modulation, but causal
relationship needs to be further investigated. Other species identified as discriminating the
two groups have not yet been related to bone health and disease.
Strengths of this study include the sample size calculation, the robust design (random-
ized, placebo-controlled, double-blind), the good adherence rate, the low drop-out rate,
and the integrated analysis, including the impact of treatment on the microbiome and HE
metabolites. The main limitation is the relatively short duration considering bone loss, as
a duration of at least 2 years would incorporate more complete bone remodeling cycles
and further strengthen the evidence provided by DXA in this trial. However, the bone
remodelling cycle is known to last 120–200 days [61]; therefore, a duration of 48 weeks has
allowed 1.5 to 2 bone remodelling cycles to happen.
To conclude, in postmenopausal women with osteopenia, daily consumption of a
standardized hop extract with 100 µg of 8-PN during 48 weeks was found to have a
beneficial increase of 1% of the total body BMD compared to placebo, above and beyond
an increase associated with a calcium and vitamin D supplementation. Furthermore, even
if no major shift of the gut microbiota composition was observed, modulation of some taxa
previously identified as associated with bone loss was noted in the HE group and deserves
further investigation. New clinical trials with notably a longer duration are needed to
confirm the beneficial effect of standardized hop extract on bone health.
Nutrients 2023, 15, 2688 17 of 20

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/nu15122688/s1, Figure S1: β-diversity at baseline (V2), 24 weeks (V4),
and 48 weeks (V6) in the HE and placebo group; Figure S2: Evolution of α-diversity as change from
baseline after 24 weeks (V4) and 48 weeks (V6) in the HE and placebo group; Figure S3: Microbiome
components differentiating between HE and placebo at week 24; Figure S4: Eubacterium limosum
relative abundance between treatment group (A) and between responders and non-responders (B);
Table S1: Targeted compounds and performance of the LC-MS method; Table S2: Plasma and urine
prenylflavonoids and their metabolites; Table S3: Overview of adverse events on all randomized
participants; Table S4: Blood safety parameters at baseline and 48 weeks; Table S5: Plasma bone
biomarkers at 48 weeks; Table S6: Anthropometrics and blood efficacy parameters at 48 weeks;
Table S7: Dietary intake and physical activity at 48 weeks; Table S8: SCFA concentrations at 24 wand
48 weeks.
Author Contributions: Conceptualization, M.L. and P.F.-B.; Formal analysis, D.T.; Investigation, M.T.,
T.B. and S.H.; Methodology, M.L.; D.T., M.T., T.B. and P.F.-B.; Writing—original draft preparation,
M.L., D.T., R.R., T.B. and P.F.-B.; Writing—review and editing, M.L., D.T., R.R., M.T., T.B., S.H. and
P.F.-B. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Givaudan France Naturals, an international group specializing
in plant extraction and production of natural ingredients for food, nutrition, and health, and personal
care sectors.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki and approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals
(on 9 May 2019; Reference number ECM4(e) 07/05/19).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We thank Atlantia Food Clinical trials for the conduct of the clinical tests and all
the volunteers for participating in this study. Ludivine Le Vigouroux, independent biostatistician
(Altanstat, France), is acknowledged for performing the statistical analysis of the study. We thank
Dan Souza and Elizabeth Tilak (Givaudan) for English language proofreading.
Conflicts of Interest: M.L., M.T., T.B. and P.F.-B. are employees of Givaudan France Naturals. D.T.
is an employee of Biofortis and was supported though a service agreement with Givaudan France
Naturals. R.R has received fees for lectures or scientific advisory boards from Abiogen, Givaudan,
Nestlé, ObsEva, and Theramex. S.H. reports no conflict of interest. The funders had no role in study
conduct, analytical, and statistical analysis.

References
1. Compston, J.E.; McClung, M.R.; Leslie, W.D. Osteoporosis. Lancet 2019, 393, 364–376. [CrossRef] [PubMed]
2. Melton, L.J.; Chrischilles, E.A.; Cooper, C.; Lane, A.W.; Riggs, B.L. Perspective how many women have osteoporosis? J. Bone
Miner. Res. 2009, 7, 1005–1010. [CrossRef] [PubMed]
3. Kanis, J.A.; World Health Organization Scientific Group. WHO Scientific Group Technical Report. In Assessment of Osteoporosis at
the Primary Health Care Level; WHO Collaborating Centre for Metabolic Bone Diseases, University of Sheffield Medical School:
Sheffield, UK, 2008.
4. Tella, S.H.; Gallagher, J.C. Prevention and treatment of postmenopausal osteoporosis. J. Steroid. Biochem. Mol. Biol. 2014, 142,
155–170. [CrossRef] [PubMed]
5. Canivenc-Lavier, M.-C.; Bennetau-Pelissero, C. Phytoestrogens and Health Effects. Nutrients 2023, 15, 317. [CrossRef]
6. Franco, O.H.; Chowdhury, R.; Troup, J.; Voortman, T.; Kunutsor, S.; Kavousi, M.; Oliver-Williams, C.; Muka, T. Use of Plant-Based
Therapies and Menopausal Symptoms. JAMA 2016, 315, 2554. [CrossRef]
7. Akhlaghi, M.; Ghasemi Nasab, M.; Riasatian, M.; Sadeghi, F. Soy isoflavones prevent bone resorption and loss, a systematic
review and meta-analysis of randomized controlled trials. Crit. Rev. Food Sci. Nutr. 2019, 60, 2327–2341. [CrossRef]
8. Sansai, K.; Na Takuathung, M.; Khatsri, R.; Teekachunhatean, S.; Hanprasertpong, N.; Koonrungsesomboon, N. Effects of
isoflavone interventions on bone mineral density in postmenopausal women: A systematic review and meta-analysis of
randomized controlled trials. Osteoporos. Int. 2020, 31, 1853–1864. [CrossRef]
9. Milligan, S.R.; Kalita, J.C.; Heyerick, A.; Rong, H.; De Cooman, L.; De Keukeleire, D. Identification of a Potent Phytoestrogen in
Hops (Humulus lupulus L.) and Beer. J. Clin. Endocrinol. Metab. 1999, 84, 2249. [CrossRef]
Nutrients 2023, 15, 2688 18 of 20

10. Milligan, S.; Kalita, J.; Pocock, V.; Heyerick, A.; De Cooman, L.; Rong, H.; De Keukeleire, D. Oestrogenic activity of the hop
phyto-oestrogen, 8-prenylnaringenin. Reprod. Camb. 2002, 123, 235–242. [CrossRef]
11. Luo, D.; Kang, L.; Ma, Y.; Chen, H.; Kuang, H.; Huang, Q.; He, M.; Peng, W. Effects and mechanisms of 8-prenylnaringenin on
osteoblast MC3T3-E1 and osteoclast-like cells RAW264.7. Food Sci. Nutr. 2014, 2, 341–350. [CrossRef]
12. Xia, T.-s.; Lin, L.-y.; Zhang, Q.-y.; Jiang, Y.-p.; Li, C.-h.; Liu, X.-y.; Qin, L.-p.; Xin, H.-l. Humulus lupulus L. Extract Prevents
Ovariectomy-Induced Osteoporosis in Mice and Regulates Activities of Osteoblasts and Osteoclasts. Chin. J. Integr. Med. 2019, 27,
31–38. [CrossRef] [PubMed]
13. Ban, Y.-H.; Yon, J.-M.; Cha, Y.; Choi, J.; An, E.S.; Guo, H.; Seo, D.W.; Kim, T.-S.; Lee, S.-P.; Kim, J.-C.; et al. A Hop Extract Lifenol®
Improves Postmenopausal Overweight, Osteoporosis, and Hot Flash in Ovariectomized Rats. Evid.-Based Complement. Altern.
Med. 2018, 2018, 2929107. [CrossRef]
14. Keiler, A.M.; Helle, J.; Bader, M.I.; Ehrhardt, T.; Nestler, K.; Kretzschmar, G.; Bernhardt, R.; Vollmer, G.; Nikolić, D.; Bolton, J.L.;
et al. A standardized (Humulus lupulus L.) ethanol extract partially prevents ovariectomy-induced bone loss in the rat without
induction of adverse effects in the uterus. Phytomedicine 2017, 34, 50–58. [CrossRef] [PubMed]
15. Sehmisch, S.; Hammer, F.; Christoffel, J.; Seidlova-Wuttke, D.; Tezval, M.; Wuttke, W.; Stuermer, K.; Stuermer, E. Comparison of
the Phytohormones Genistein, Resveratrol and 8-Prenylnaringenin as Agents for Preventing Osteoporosis. Planta Med. 2008, 74,
794–801. [CrossRef]
16. Erkkola, R.; Vervarcke, S.; Vansteelandt, S.; Rompotti, P.; De Keukeleire, D.; Heyerick, A. A randomized, double-blind, placebo-
controlled, cross-over pilot study on the use of a standardized hop extract to alleviate menopausal discomforts. Phytomedicine
2010, 17, 389–396. [CrossRef] [PubMed]
17. Heyerick, A.; Vervarcke, S.; Depypere, H.; Bracke, M.; Keukeleire, D.D. A first prospective, randomized, double-blind, placebo-
controlled study on the use of a standardized hop extract to alleviate menopausal discomforts. Maturitas 2006, 54, 164–175.
[CrossRef]
18. Aghamiri, V.; Mirghafourvand, M.; Mohammad-Alizadeh-Charandabi, S.; Nazemiyeh, H. The effect of Hop (Humulus lupulus L.)
on early menopausal symptoms and hot flashes: A randomized placebo-controlled trial. Complement. Ther. Clin. Pract. 2016, 23,
130–135. [CrossRef]
19. Bolca, S.; Possemiers, S.; Maervoet, V.; Huybrechts, I.; Heyerick, A.; Vervarcke, S.; Depypere, H.; De Keukeleire, D.; Bracke, M.;
De Henauw, S.; et al. Microbial and dietary factors associated with the 8-prenylnaringenin producer phenotype: A dietary
intervention trial with fifty healthy post-menopausal Caucasian women. Br. J. Nutr. 2007, 98, 950–959. [CrossRef]
20. Possemiers, S.; Bolca, S.; Grootaert, C.; Heyerick, A.; Decroos, K.; Dhooge, W.; De Keukeleire, D.; Rabot, S.; Verstraete, W.;
Van de Wiele, T. The Prenylflavonoid Isoxanthohumol from Hops (Humulus lupulus L.) Is Activated into the Potent Phytoestrogen
8-Prenylnaringenin In Vitro and in the Human Intestine. J. Nutr. 2006, 136, 1862–1867. [CrossRef]
21. Possemiers, S.; Rabot, S.; Espín, J.C.; Bruneau, A.; Philippe, C.; González-Sarrías, A.; Heyerick, A.; Tomás-Barberán, F.A.; De
Keukeleire, D.; Verstraete, W. Eubacterium limosum Activates Isoxanthohumol from Hops (Humulus lupulus L.) into the Potent
Phytoestrogen 8-Prenylnaringenin In Vitro and in Rat Intestine. J. Nutr. 2008, 138, 1310–1316. [CrossRef]
22. Possemiers, S.; Heyerick, A.; Robbens, V.; De Keukeleire, D.; Verstraete, W. Activation of Proestrogens from Hops (Humulus lupulus
L.) by Intestinal Microbiota; Conversion of Isoxanthohumol into 8-Prenylnaringenin. J. Agric. Food Chem. 2005, 53, 6281–6288.
[CrossRef]
23. Li, S.; Mao, Y.; Zhou, F.; Yang, H.; Shi, Q.; Meng, B. Gut microbiome and osteoporosis. Bone Jt. Res. 2020, 9, 524–530. [CrossRef]
24. Seely, K.D.; Kotelko, C.A.; Douglas, H.; Bealer, B.; Brooks, A.E. The Human Gut Microbiota: A Key Mediator of Osteoporosis and
Osteogenesis. Int. J. Mol. Sci. 2021, 22, 9452. [CrossRef] [PubMed]
25. Rizzoli, R. Nutritional influence on bone: Role of gut microbiota. Aging Clin. Exp. Res. 2019, 31, 743–751. [CrossRef] [PubMed]
26. Kanis, J.A.; McCloskey, E.V.; Johansson, H.; Oden, A.; Ström, O.; Borgström, F. Development and use of FRAX® in osteoporosis.
Osteoporos. Int. 2010, 21, 407–413. [CrossRef] [PubMed]
27. Washburn, R.A.; Smith, K.W.; Jette, A.M.; Janney, C.A. The physical activity scale for the elderly (PASE): Development and
evaluation. J. Clin. Epidemiol. 1993, 46, 153–162. [CrossRef]
28. Ware, J.E., Jr.; Sherbourne, C.D. The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection.
Med. Care 1992, 30, 473–483. [CrossRef]
29. Koh, H.; Li, Y.; Zhan, X.; Chen, J.; Zhao, N. A Distance-Based Kernel Association Test Based on the Generalized Linear Mixed
Model for Correlated Microbiome Studies. Front. Genet. 2019, 10, 458. [CrossRef]
30. Chen, J.; Chen, W.; Zhao, N.; Wu, M.C.; Schaid, D.J. Small Sample Kernel Association Tests for Human Genetic and Microbiome
Association Studies. Genet. Epidemiol. 2016, 40, 5–19. [CrossRef]
31. Zhao, N.; Chen, J.; Carroll, I.M.; Ringel-Kulka, T.; Epstein, M.P.; Zhou, H.; Zhou, J.J.; Ringel, Y.; Li, H.; Wu, M.C. Testing in
Microbiome-Profiling Studies with MiRKAT, the Microbiome Regression-Based Kernel Association Test. Am. J. Hum. Genet. 2015,
96, 797–807. [CrossRef]
32. Susin, A.; Wang, Y.; Lê Cao, K.-A.; Calle, M.L. Variable selection in microbiome compositional data analysis. NAR Genom.
Bioinform. 2020, 2, lqaa029. [CrossRef] [PubMed]
33. Meinshausen, N.; Bühlmann, P. Stability Selection. J. R. Stat. Soc. Ser. B Stat. Methodol. 2010, 72, 417–473. [CrossRef]
34. De Weirdt, R.; Possemiers, S.; Vermeulen, G.; Moerdijk-Poortvliet, T.C.W.; Boschker, H.T.S.; Verstraete, W.; Van de Wiele, T. Human
faecal microbiota display variable patterns of glycerol metabolism. FEMS Microbiol. Ecol. 2010, 74, 601–611. [CrossRef]
Nutrients 2023, 15, 2688 19 of 20

35. Lambert, M.N.T.; Thybo, C.B.; Lykkeboe, S.; Rasmussen, L.M.; Frette, X.; Christensen, L.P.; Jeppesen, P.B. Combined bioavailable
isoflavones and probiotics improve bone status and estrogen metabolism in postmenopausal osteopenic women: A randomized
controlled trial. Am. J. Clin. Nutr. 2017, 106, 909–920. [CrossRef]
36. Bolton, K.L.; Egerton, T.; Wark, J.; Wee, E.; Matthews, B.; Kelly, A.; Craven, R.; Kantor, S.; Bennell, K.L. Effects of exercise on bone
density and falls risk factors in post-menopausal women with osteopenia: A randomised controlled trial. J. Sci. Med. Sport 2012,
15, 102–109. [CrossRef] [PubMed]
37. Atkinson, C.; Compston, J.E.; Day, N.E.; Dowsett, M.; Bingham, S.A. The effects of phytoestrogen isoflavones on bone density in
women: A double-blind, randomized, placebo-controlled trial. Am. J. Clin. Nutr. 2004, 79, 326–333. [CrossRef] [PubMed]
38. Kim, S.J.; Yang, W.-G.; Cho, E.; Park, E.-C. Relationship between Weight, Body Mass Index and Bone Mineral Density of Lumbar
Spine in Women. J. Bone Metab. 2012, 19, 95. [CrossRef]
39. Fistarol, M.; Rezende, C.R.; Figueiredo Campos, A.L.; Kakehasi, A.M.; Geber, S. Time since menopause, but not age, is associated
with increased risk of osteoporosis. Climacteric 2019, 22, 523–526. [CrossRef]
40. American Geriatrics Society Workgroup on Vitamin D Supplementation for Older Adults. Recommendations Abstracted from the
American Geriatrics Society Consensus Statement on Vitamin D for Prevention of Falls and Their Consequences. J. Am. Geriatr.
Soc. 2014, 62, 147–152. [CrossRef]
41. Garnero, P. The Utility of Biomarkers in Osteoporosis Management. Mol. Diagn. Ther. 2017, 21, 401–418. [CrossRef]
42. Vergnaud, P.; Garnero, P.; Meunier, P.J.; Bréart, G.; Kamihagi, K.; Delmas, P.D. Undercarboxylated Osteocalcin Measured with a
Specific Immunoassay Predicts Hip Fracture in Elderly Women: The EPIDOS Study1. J. Clin. Endocrinol. Metab. 1997, 82, 719–724.
[CrossRef] [PubMed]
43. Booth, S.L.; Broe, K.E.; Gagnon, D.R.; Tucker, K.L.; Hannan, M.T.; McLean, R.R.; Dawson-Hughes, B.; Wilson, P.W.; Cupples,
L.A.; Kiel, D.P. Vitamin K intake and bone mineral density in women and men. Am. J. Clin. Nutr. 2003, 77, 512–516. [CrossRef]
[PubMed]
44. Dawson-Hughes, B.; Harris, S.S.; Krall, E.A.; Dallal, G.E. Effect of Calcium and Vitamin D Supplementation on Bone Density in
Men and Women 65 Years of Age or Older. N. Engl. J. Med. 1997, 337, 670–676. [CrossRef] [PubMed]
45. Hallberg, I.; Rosenqvist, A.M.; Kartous, L.; Löfman, O.; Wahlström, O.; Toss, G. Health-related quality of life after osteoporotic
fractures. Osteoporos. Int. 2004, 15, 834–841. [CrossRef] [PubMed]
46. Pasco, J.A.; Sanders, K.M.; Hoekstra, F.M.; Henry, M.J.; Nicholson, G.C.; Kotowicz, M.A. The human cost of fracture. Osteoporos.
Int. 2005, 16, 2046–2052. [CrossRef]
47. Hakestad, K.A.; Nordsletten, L.; Torstveit, M.K.; Risberg, M.A. Postmenopausal women with osteopenia and a healed wrist
fracture have reduced physical function and quality of life compared to a matched, healthy control group with no fracture. BMC.
Womens Health 2014, 14, 92. [CrossRef]
48. Tang, B.M.; Eslick, G.D.; Nowson, C.; Smith, C.; Bensoussan, A. Use of calcium or calcium in combination with vitamin D
supplementation to prevent fractures and bone loss in people aged 50 years and older: A meta-analysis. Lancet. 2007, 370, 657–666.
[CrossRef]
49. Liu, C.; Kuang, X.; Li, K.; Guo, X.; Deng, Q.; Li, D. Effects of combined calcium and vitamin D supplementation on osteoporosis
in postmenopausal women: A systematic review and meta-analysis of randomized controlled trials. Food Funct. 2020, 11,
10817–10827. [CrossRef]
50. Kärkkäinen, M.; Tuppurainen, M.; Salovaara, K.; Sandini, L.; Rikkonen, T.; Sirola, J.; Honkanen, R.; Jurvelin, J.; Alhava, E.;
Kröger, H. Effect of calcium and vitamin D supplementation on bone mineral density in women aged 65–71 years: A 3-year
randomized population-based trial (OSTPRE-FPS). Osteoporos. Int. 2010, 21, 2047–2055. [CrossRef]
51. Jackson, R.D.; LaCroix, A.Z.; Gass, M.; Wallace, R.B.; Robbins, J.; Lewis, C.E.; Bassford, T.; Beresford, S.A.A.; Black, H.R.;
Blanchette, P.; et al. Calcium plus Vitamin D Supplementation and the Risk of Fractures. N. Engl. J. Med. 2006, 354, 669–683.
[CrossRef]
52. Daniele, N.; Carbonelli, M.; Candeloro, N.; Iacopino, L.; Delorenzo, A.; Andreoli, A. Effect of supplementation of calcium and
Vitamin D on bone mineral density and bone mineral content in peri- and post-menopause womenA double-blind, randomized,
controlled trial. Pharmacol. Res. 2004, 50, 637–641. [CrossRef] [PubMed]
53. Bischoff-Ferrari, H.A.; Kiel, D.P.; Dawson-Hughes, B.; Orav, J.E.; Li, R.; Spiegelman, D.; Dietrich, T.; Willett, W.C. Dietary Calcium
and Serum 25-Hydroxyvitamin D Status in Relation to BMD Among U.S. Adults. J. Bone Miner. Res. 2009, 24, 935–942. [CrossRef]
[PubMed]
54. Pohjanvirta, R.; Nasri, A. The Potent Phytoestrogen 8-Prenylnaringenin: A Friend or a Foe? Int. J. Mol. Sci. 2022, 23, 3168.
[CrossRef] [PubMed]
55. Domazetovic, V. Oxidative stress in bone remodeling: Role of antioxidants. Clin. Cases Miner. Bone Metab. 2017, 14, 209. [CrossRef]
[PubMed]
56. Zugravu, C.-A.; Bohiltea, R.-E.; Salmen, T.; Pogurschi, E.; Otelea, M.R. Antioxidants in Hops: Bioavailability, Health Effects and
Perspectives for New Products. Antioxidants 2022, 11, 241. [CrossRef]
57. Ma, M.-l.; Ma, Z.-j.; He, Y.-l.; Sun, H.; Yang, B.; Ruan, B.-j.; Zhan, W.-d.; Li, S.-x.; Dong, H.; Wang, Y.-x. Efficacy of vitamin K2 in
the prevention and treatment of postmenopausal osteoporosis: A systematic review and meta-analysis of randomized controlled
trials. Front. Public Health 2022, 10, 2654. [CrossRef]
Nutrients 2023, 15, 2688 20 of 20

58. Wan, X.; Eguchi, A.; Fujita, Y.; Ma, L.; Wang, X.; Yang, Y.; Qu, Y.; Chang, L.; Zhang, J.; Mori, C.; et al. Effects of (R)-ketamine on
reduced bone mineral density in ovariectomized mice: A role of gut microbiota. Neuropharmacology 2022, 213, 109139. [CrossRef]
59. Das, M.; Cronin, O.; Keohane, D.M.; Cormac, E.M.; Nugent, H.; Nugent, M.; Molloy, C.; O’Toole, P.W.; Shanahan, F.; Molloy, M.G.;
et al. Gut microbiota alterations associated with reduced bone mineral density in older adults. Rheumatology 2019, 58, 2295–2304.
[CrossRef]
60. Cheng, B.; Wen, Y.; Yang, X.; Cheng, S.; Liu, L.; Chu, X.; Ye, J.; Liang, C.; Yao, Y.; Jia, Y.; et al. Gut microbiota is associated with
bone mineral density. Bone Jt. Res. 2021, 10, 734–741. [CrossRef]
61. Kenkre, J.; Bassett, J. The bone remodelling cycle. Ann. Clin. Biochem. 2018, 55, 308–327. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.