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The Role of Prunes in Modulating Inflammatory Pathways to Improve Bone Health in Postmenopausal Women

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The prevalence of osteoporosis among women aged 50 years and older is expected to reach 13.6 million by 2030. Alternative non-pharmaceutical agents for osteoporosis including nutritional interventions are becoming increasingly popular. Prunes (dried plums) (Prunus domestica L.) have been studied as a potential whole food dietary intervention to mitigate bone loss in preclinical models of osteoporosis and in osteopenic postmenopausal women. Sixteen preclinical studies using in vivo rodent models of osteopenia or osteoporosis have established that dietary supplementation with prunes confers osteoprotective effects both by preventing and reversing bone loss. Increasing evidence from ten studies suggests that in addition to anti-resorptive effects, prunes exert anti-inflammatory and antioxidant effects. Ten preclinical studies have found that prunes and/or their polyphenol extracts decrease malondialdehyde and nitric oxide secretion, increase antioxidant enzyme expression, or suppress NF-κB activation and pro-inflammatory cytokine production. Two clinical trials have investigated the impact of dried plum consumption (50–100g/day for 6–12 months) on bone health in postmenopausal women and demonstrate promising effects on bone mineral density and bone biomarkers. However, less is known about the impact of prune consumption on oxidative stress and inflammatory mediators in humans and their possible role in modulating bone outcomes. In this review, the current state of knowledge on the relationship between inflammation and bone health is outlined. Findings from preclinical and clinical studies that have assessed the effect of prunes on oxidative stress, inflammatory mediators, and bone outcomes are summarized, and evidence supporting a potential role of prunes in modulating inflammatory and immune pathways is highlighted. Key future directions to bridge the knowledge gap in the field are proposed.
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REVIEW
The Role of Prunes in Modulating Inammatory
Pathways to Improve Bone Health in
Postmenopausal Women
Janhavi J Damani,1Mary Jane De Souza,2Hannah L VanEvery,3Nicole CA Strock,2and Connie J Rogers3,4
1Intercollege Graduate Degree Program in Integrative and Biomedical Physiology, Huck Institutes of the Life Sciences, The Pennsylvania State University,
University Park, PA, USA; 2Department of Kinesiology, The Pennsylvania State University, University Park, PA, USA; 3Department of Nutritional Sciences, The
Pennsylvania State University, University Park, PA, USA; and 4Center for Molecular Immunology and Infectious Disease, Huck Institutes of the Life Sciences,
The Pennsylvania State University, University Park,PA, USA
ABSTRACT
The prevalence of osteoporosis among women aged 50 y and older is expected to reach 13.6 million by 2030. Alternative nonpharmaceutical
agents for osteoporosis, including nutritional interventions, are becoming increasingly popular. Prunes (dried plums; Prunus domestica L.) have been
studied as a potential whole-food dietary intervention to mitigate bone loss in preclinical models of osteoporosis and in osteopenic postmenopausal
women. Sixteen preclinical studies using in vivo rodent models of osteopenia or osteoporosis have established that dietary supplementation with
prunes confers osteoprotective effects both by preventing and reversing bone loss. Increasing evidence from 10 studies suggests that, in addition to
antiresorptive effects, prunes exert anti-inflammatory and antioxidant effects. Tenpreclinical studies have found that prunes and/or their polyphenol
extracts decrease malondialdehyde and NO secretion, increase antioxidant enzyme expression, or suppress NF-κB activation and proinflammatory
cytokine production. Two clinical trials have investigated the impact of dried plum consumption (50–100 g/d for 6–12 mo) on bone health in
postmenopausal women and demonstrated promising effects on bone mineral density and bone biomarkers. However, less is known about the
impact of prune consumption on oxidative stress and inflammatory mediators in humans and their possible role in modulating bone outcomes.
In this review, the current state of knowledge on the relation between inflammation and bone health is outlined. Findings from preclinical and
clinical studies that have assessed the effect of prunes on oxidative stress, inflammatory mediators, and bone outcomes are summarized, and
evidence supporting a potential role of prunes in modulating inflammatory and immune pathways is highlighted. Key future directions to bridge
the knowledge gap in the field are proposed. Adv Nutr 2022;00:1–17.
Statement of Signicance: Osteoporosis represents a major public health issue and prunes (dried plums) have been extensively studied as
a dietary intervention to mitigate bone loss in preclinical models of osteoporosis. In postmenopausal women, estrogen deficiency triggers
an upregulation of oxidative stress and inflammatory pathways, which promotes bone loss, increasing risk of fracture. In this review, we
summarize the evidence suggesting a potential role of prunes in modulating oxidative and inflammatory pathways, which may contribute
to the protective effect on bone.
Keywords: immunity, dried plums, prunes, bone density, inflammation, gut microbiota, nutritional intervention, osteoporosis, osteopenia
Introduction
Osteoporosis is a debilitating bone disease characterized by
a signicant reduction in bone mineral density (BMD) and
deterioration of bone microstructure, predisposing individ-
uals to increased fracture risk (1). Osteoporosis is estimated
to aect over 200 million women worldwide, causing 8.9
million fractures annually (2), and recent projections indicate
that the prevalence of osteoporosis among women aged 50
y and older will reach 13.6 million by 2030 (3,4). Current
pharmacological therapies include antiresorptive (bisphos-
phonates, denosumab, calcitonin, strontium ranelate), an-
abolic (teriparatide, abaloparatide, romosozumab), or se-
lective estrogen receptor modulators (SERMs) to treat low
BMD in women (5,6). Although these interventions are
eective, high cost, poor compliance, and negative side eects
contribute to declining use and popularity (7,8). Therefore,
osteoporosis represents a major public health issue that
necessitates eective prevention and treatment regimens that
C
The Author(s) 2022. Published by Oxford University Press on behalf of the American Society for Nutrition. All rights reserved. Forpermissions, please e -mail: journals.permissions@oup.com Adv
Nutr 2022;00:1–17; doi: https://doi.org/10.1093/advances/nmab162. 1
are safe, cost-eective, and are associated with fewer adverse
eects than conventional pharmaceutical agents.
Hypoestrogenism manifests as a consequence of ovarian
senescence during menopause and is responsible for the
onset of a 3- to 5-y period of accelerated bone loss, followed
by continued gradual bone loss, which involves loss of
bone strength, density, and poor bone quality, thereby
increasing the risk for osteoporosis and fracture (9). In
addition, hypoestrogenism is a potent stimulus for increased
production of inammatory mediators from immune cells
(10). In particular, estrogen deciency can lead to activation
of macrophages and T cells, which secrete inammatory
cytokines, such as IL-1β,IL-6,andTNF-α,thatstimulate
osteoclast activity and inhibit osteoblast activity, thus col-
lectively promoting bone resorption (11–13). Furthermore,
aging is associated with increased concentrations of cir-
culating proinammatory cytokines, including IL-1β,IL-6,
and TNF-α, which partly contribute to the “inamm-aging”
phenomenon observed in older adults (14,15). Last, changes
in the gut microbiota can modulate inammatory mediators
in healthy individuals (16), and gut dysbiosis is linked to
numerous chronic diseases (17–20), including osteoporosis
(21,22). Commensal bacteria play an important role in
maintaining the integrity of tight junctions within the intesti-
nal epithelium and in secreting metabolites, such as SCFAs,
that exhibit anti-inammatory eects within the intestinal
mucosa (21,23). While the link between the immune system
andboneiswellestablished(24,25), emerging data suggest
that changes in the gut microbiota may be modulating this
relation (26,27). Therefore, the likely factors that contribute
to bone loss during postmenopausal osteoporosis appear to
include increased oxidative stress and inammatory cytokine
production secondary to hypoestrogenism, and the eect of
changesinthegutmicrobiotaoninammatorymediators,all
which contribute to upregulated bone resorption.
There is increasing consumer interest in alternative,
nonpharmacological therapies for osteoporosis, including
nutritional interventions (28,29), which might be used
alone or in combination with pharmacological agents to
reduce their dose or duration. Calcium and vitamin D
supplementation are considered the minimal standard of care
to maintain bone health in postmenopausal women and are
associated with modest reduction in fracture risk among
older adults (30,31). Bone remodeling involves continuous
Supported by the California Prune Board provided funding to MJDS and CJR. Publication funds
came from the Hershey Company endowment, Department of Nutritional Sciences, Penn State
University. California Dried Plum Board (grant no.100804).
Author disclosures: CJR is member of the Nutrition Advisory Panel for the California Dried Plum
Board. The other authors report no conicts of interest.
Address correspondence to CJR (e-mail: cjr102@psu.edu).
Abbreviations used: AGE, advanced glycation end product; ALP, alkaline phosphatase; BMD,
bone mineral density; BMP,bone morphogenic protein; BSAP, bone-specic alkaline
phosphatase; CAT, catalase; CRP, C-reactive protein; CTX, carboxyl-terminal telopeptide of type
1 collagen; GF,germ-free; GPX, glutathione peroxidase; HFD, high-fat-diet; IBD, inammatory
bowel disease; iNOS, inducible NO synthase; MCP, monoc yte chemoattractant protein;NFATc1,
nuclear factor of activated T cells, cytoplasmic 1; OPG, osteoprotegerin; OVX,
ovariectomized/ovariectomy; PBMC, peripheral blood mononuclear cell; RANK, receptor
activator of NF-κB; RANKL, receptor activator of NF-κB ligand; RCT, randomized controlled trial;
ROS, reactive oxygen species; RUNX, runt-related transcription factor; SERM, selective estrogen
receptor modulator; SOD, superoxide dismutase; TRAF, TNF receptor–associated factor.
turnover of the protein matrix and dietary protein is another
nutritional factor required for maintaining bone health (32,
33). Fruits and vegetables rich in bioactive compounds,
such as phenolic acid, avonoids, and carotenoids, have
potential osteoprotective eects in both animal studies and
clinical trials (34–36), and many postmenopausal women use
botanical supplements for osteoporosis management (37).
Of the functional foods and plant-derived compounds
assessed for their eects on bone health, prunes (also known
as dried plums; Prunus domestica L.) have gained increasing
attention and these ndings have been recently summarized
(38,39). Sixteen preclinical studies demonstrate that prune
supplementation not only prevents but also reverses bone
loss in several rodent models of gonadal hormone deciency.
To date, 4 randomized controlled trials (RCTs) (40–43)and
1casestudy(44) assessed the eect of prune consumption
on bone health outcomes in postmenopausal women. These
studies demonstrated promising eects on bone turnover
(40)andBMD(41,42,44), suggesting that supplementation
with prunes may confer signicant benecial eects on bone
outcomes in this at-risk population.
Prunes have been historically consumed for their pur-
ported gastrointestinal health benets (45)andarearich
source of potassium, boron, copper, vitamin K, and phenolic
compounds, such as chlorogenic acids, phenolic acids, and
avonoids (45,46), which have antioxidant properties (47).
Compared with prune juice, prunes have a higher content of
dietary ber and vitamins A and K, and total oxygen radical
absorbance capacity. Furthermore, prunes have a higher
content of total phenolics compared with fresh plums (45).
Overall, prunes are considered a promising functional food
for improving bone health and the bioactive components are
thought to act synergistically within the whole food matrix to
maintain bone health after menopause (39,48). Additionally,
prune consumption may potentially alter gut microbiota (49)
and subsequently aect bone health (49–52). This review
outlines the known associations between oxidative stress,
inammation, and bone health and summarizes ndings
from preclinical and clinical studies that demonstrate a
potential role of prunes in modulating these pathways.
Current Status of Knowledge
Regulation of bone remodeling
Bone formation by osteoblasts and bone resorption by
osteoclasts are tightly coupled processes that constitute bone
remodeling, which maintains homeostasis of bone mass
throughout adult life (53). However, with increasing age,
there is uncoupling of bone turnover. After the age of 40, re-
sorption begins to exceed formation, and this imbalance can
be exaggerated by estrogen deciency, radiation exposure,
or long-term immobility (54). Inammation and oxidative
stress are other factors that can exacerbate bone loss,
particularly in older adults (13,55). Acute inammation is an
immune response that is normally mounted during infection
and tissue repair. However, chronic inammation can favor
bone resorption, thus compromising bone structure and
2 Damani et al.
FIGURE 1 Factors influencing bone remodeling. Bone remodeling is regulated by the coordinated activity and maturation of osteoblasts
and osteoclasts, which are influenced by various factors including estrogen, oxidative stress, and inflammatory mediators. Estrogen 1)
upregulates OPG production by osteoblasts and B cells (66); 2) inhibits proinflammatory cytokine (TNF-α, IL-1, IL-6) secretion from
monocytes and T cells (12,67,68) and RANKL secretion from osteoblasts, T cells, and B cells (69,70); and 3) blocks the production of ROS
(71,72), which upregulates RANKL expression and downregulates OPG expression, thus ultimately preventing bone resorption. HSC,
hematopoietic stem cells; MSC, mesenchymal stem cells; OPG, osteoprotegerin; RANK, receptor activator of NF-κB; RANKL, receptor
activator of NF-κB ligand; ROS, reactive oxygen species.
integrity (12), and contribute to bone loss independently
of hypoestrogenism. Several inammatory diseases, such as
rheumatoid arthritis and inammatory bowel disease (IBD),
are associated with bone loss (13,56) and increased fracture
risk [rheumatoid arthritis—HR: 1.26; 95% CI: 1.15–1.38 (57);
IBD—OR: 1.32; 95% CI: 1.20–1.45 (58)].
Bone remodeling is regulated by the coordinated activity
and maturation of osteoblasts and osteoclasts, which are
inuenced by various factors, including estrogen, oxidative
stress, immune mediators, and growth factors (Figure 1)
(12,59–61). Osteoclasts are derived from hematopoietic stem
cells (HSCs) that dierentiate into granulocyte–macrophage
progenitor cells, and thus, like macrophages, osteoclasts
express innate immune signaling receptors, which, upon
activation, induce the release of proinammatory cytokines
IL-1βand IL-18 (62). Osteoclast dierentiation is regulated
by receptor activator of NF-κBligand(RANKL),whichis
secreted by osteoblasts and various immune cell populations
such as T and B cells (Figure 1), indicating that bone
resorption and the immune response are tightly linked
(63). Upon secretion, RANKL binds its cognate receptor
RANK expressed by osteoclast precursors, triggering the
RANKL/RANK cascade (63). This cascade stimulates down-
stream adaptor proteins called TNF receptor–associated
factors (TRAFs), mainly TRAF6, that activate the transcrip-
tion factor NF-κB, which induces expression of nuclear
factor of activated T cells, cytoplasmic 1 (NFATc1) (54).
NFATc1 is the major transcriptional factor that promotes
expression of osteoclast-specic genes encoding tartrate-
resistant acid phosphate (TRAP) 5b (TRAP-5b), cathepsin
K,andcalcitoninreceptor(62,64). Osteoblasts are derived
from mesenchymal stem cells (MSCs) (Figure 1)andare
regulated by transcriptional factors such as runt-related tran-
scription factor (RUNX)-2, Osterix, and bone morphogenic
proteins (BMPs), which orchestrate osteoblast dierentiation
(65). In addition to expressing RANKL, which promotes
osteoclastogenesis, osteoblasts also express osteoprotegerin
(OPG), a soluble decoy receptor for RANKL, thus preventing
its binding to and activation of its cognate receptor RANK,
inhibiting osteoclast dierentiation, activation, and survival.
Estrogen regulates bone remodeling as an anabolic or
antiresorptive agent that promotes OPG production by
osteoblasts (66) and inhibits proinammatory cytokine pro-
duction by immune cells (12,67,68)andRANKLsecretion
from osteoblasts, T cells, and B cells (69,70)(Figure 1),
thereby inhibiting osteoclast formation and decreasing bone
resorption. Furthermore, oxidative stress alters bone remod-
eling by impacting the activity of osteoclasts and osteoblasts
Prunes, bone health, oxidative stress, and inammation 3
(71,72). Several studies in humans demonstrate that reactive
oxygen species (ROS) and antioxidant systems are involved in
bone loss in aged and/or osteoporotic subjects (73–75). High
levels of ROS inhibit osteoblast activity and dierentiation
(76–78) and thus contribute to reduced bone mineralization.
Increased ROS production also promotes bone loss by
upregulating RANKL and downregulating OPG expression,
thus activating osteoclast dierentiation (78–80). Estrogen
blocks the production of ROS (71), which prevents ROS-
induced bone resorption.
Role of inflammation on bone turnover
The regulatory role of immune and inammatory mediators
in bone remodeling has been well studied (25,81,82). In
addition to osteoclasts and osteoblasts, T cells, B cells, mono-
cytes, macrophages, and dendritic cells can play a role in
inammation and bone loss (83). Macrophages play a major
role in the activation and formation of osteoclasts (84), and
theactivationofmacrophagesmayinducetheproductionof
IFN-γ,IL-1,TNF-α, and numerous inammatory mediators
(85). Osteoclast-mediated bone resorption is activated by
oxidative and inammatory stimuli such as NO, IL-1β,IL-
6, IL-8, IL-18, IL-15, IL-17, IL-32, and TNF-α(62), many
of which induce osteoclastogenesis by upregulating the re-
lease of RANKL (13). Conversely, osteoclast-mediated bone
resorption is downregulated by anti-inammatory cytokines,
such as IL-4, IL-10, IL-13, and IL-3 (86). NO and anti-
inammatory cytokines inuence osteoblasts by increasing
OPG and decreasing RANKL production, thus generating a
highOPGtoRANKLratiothatfavorsinhibitionofosteoclast
dierentiation (86).OPGisalsosecretedbyBcellsand
dendritic cells, demonstrating that osteoclast formation is
also regulated by additional components of the immune
system (25,87). T cells secrete pro-osteoclastic cytokines
such as TNF-αandRANKL,aswellasanti-osteoclastogenic
cytokines such as IFN-γ.IFN-γindirectly inhibits osteoclast
activity by accelerating ubiquitin-proteasomal degradation
of TRAF-6 (88), thus preventing downstream activation
of NF-κB and c-Jun N-terminal kinase and attenuating
osteoclast dierentiation. B cells are also active regulators
oftheRANK/RANKL/OPGsystemandproduceanumber
of regulatory cytokines and chemokines (25). Therefore,
osteoclast maturation appears to be regulated by a balance of
pro- and anti-inammatory cytokines and chemokines (62,
67,68,86,88–91).
Immune mediators play an important role in post-
menopausal bone loss. Surgical menopause in women is
associated with elevated IL-1 and TNF-αsecretion from
peripheral blood mononuclear cells (PBMCs) with a con-
comitant increase in urinary markers of bone resorption
such as calcium:creatinine ratio (92). Furthermore, estrogen
replacement therapy in these women resulted in decreased
IL-1 and TNF-αsecretion from PBMCs concomitantly with
reduced urinary bone resorption markers (92). Hypoestro-
genism after menopause is also associated with increased
IL-1 activity from circulating human monocytes (93–95),
IL-6 (96)andTNF-α(97) production by mononuclear
cells, and increased secretion of TNF-α(98–100)and
IL-17 by T cells (101). Estrogen deciency also induces
a shift in the T-cell lineage by increasing the ratio of
T-helper (Th)-17 cells to T regulatory (Treg) cells, resulting
in elevated concentrations of proinammatory cytokines,
primarily IL-17 and TNF-α, which stimulate the release of
osteoclast-promoting RANKL (13,87). Moreover, IL-1, IL-6,
and TNF-αsuppress osteoblast activity and formation (25),
demonstrating that bone resorption is favored in the presence
of cytokines that are upregulated during an inammatory
response. PBMCs isolated from women with low BMD
produce higher concentrations of pro-resorptive cytokines,
TNF-α, IL-6, IL-12, and IL-17, and lower concentrations of
antiresorptive cytokines, IL-4, IL-10, and IL-23, compared
with women with normal BMD (102), suggesting that the
cytokine prole during postmenopausal osteoporosis may be
associated with activation of bone resorption.
In addition to estrogen loss, IL-1, IL-6, and TNF-
αincrease with age (14,91), which contributes to the
state of “inamm-aging” observed in older adults (10,
15). Postmenopausal women experience bone loss in 2
phases:anacceleratedtransientphasecausedbyestrogen
loss and a gradual continuous phase attributed, in part,
to inamm-aging associated with increased production of
proinammatory cytokines IL-1, IL-6, and TNF-α(14,
103). Blocking TNF-αwith an inhibitor in postmenopausal
women reduces the concentrations of carboxyl-terminal
telopeptide of type 1 collagen (CTX), a serum marker of bone
resorption, suggesting that elevated TNF-αis key mediator
of bone resorption (104). Estrogen deciency accelerates the
eect of aging on bone by not only elevating inammatory
mediators but also markers of oxidative stress (71). Two
preclinical studies (105,106) provide mechanistic evidence
linking bone loss with estrogen deciency, aging, oxidative
stress, and inammation. In a rat model of postmenopausal
osteoporosis, hepatic expression of lipid peroxide, NO, and
inducible NO synthase (iNOS) increased in aged intact
rats compared with young intact rats, and these eects
were more prominent in ovariectomized (OVX) animals
(105). Furthermore, aging and OVX signicantly increased
liver TNF-α,IL-1β, and IL-6, and downregulated IL-10
(105). In femurs, OVX decreased BMD, impaired bone
micro-architecture parameters, upregulated gene expression
of proinammatory cytokine monocyte chemoattractant
protein (MCP)) and ROS-generating enzymes, and down-
regulated gene expression of anti-inammatory IL-1 receptor
2 gene and antioxidant defense enzymes compared with
sham-operated mice (106). Collectively, these preclinical
data highlight that estrogen deciency enhances markers
of inammation and oxidative stress in the bone mi-
croenvironment. Thus, targeting the age-related increase in
inammatory mediators and/or oxidative stress with novel
treatments may be a strategy to reduce postmenopausal bone
loss.
Thegutmicrobiotaarealsoemergingasanimportant
factor in the pathogenesis of osteoporosis (22). The gut
microbiota produce various immunomodulatory molecules
4 Damani et al.
that promote epithelial barrier integrity, maintain im-
mune tolerance toward commensal bacteria, and confer
anti-inammatory eects on the intestinal mucosa (21).
Dysregulationofthegutmicrobiotaisassociatedwith
various chronic diseases (17–20), including rheumatoid
arthritis (20) and osteoporosis (22), suggesting that microbial
populations in the gut may communicate with distant organs
suchasthebone.Gutdysbiosismayleadtobreakdownof
epithelial barrier integrity, exposing commensal microbiota
to the immune cells in the lamina propria of the intestine
and entry of LPS, a component of the outer membrane
in gram-negative bacteria, and bacterial peptides into the
bloodstream (107). This triggers activation of immune
cells, resulting in increased production of proinammatory
cytokines, including TNF-α,IL-1β,andIL-6(108). These
proinammatory cytokines can promote dierentiation and
activation of osteoclasts, leading to increased bone resorption
(22).
Obesity is a complex, multifactorial disease, involving ge-
netic and environmental factors (including diet and physical
inactivity), wherein increased energy intake and decreased
energy expenditure contribute to pathological expansion of
adiposetissue.Adipocytehypertrophycanresultinhypoxia,
cellular and tissue stress, and ultimately increased production
of proinammatory cytokines, including TNF-α,IL-1β,and
MCP-1 (109), and increased markers of oxidative stress
(110). In both preclinical and clinical studies, increased
adiposity resulted in the accumulation of macrophages in
adipose tissue (111). Macrophages in adipose tissue localize
around adipocytes undergoing hypertrophy-induced cellular
stress and apoptosis (112). Macrophages inltrating adipose
tissue also contributed to the production and secretion of
proinammatory mediators and promoted both local and
systemic proinammatory status (113). In addition, obesity
is associated with an altered composition of the microbiota,
withamarkeddecreaseinmicrobialdiversityandanincrease
in intestinal permeability (114,115), resulting in bacterial
translocation which contributes to the proinammatory
milieu observed in obesity (116). Thus, obesity-induced
inammation and gut dysbiosis represent important factors
that may potentially increase bone resorption through
increased production of proinammatory cytokines (TNF-
α,IL-6),upregulationoftheRANKL-OPGpathway,and
subsequent osteoclast dierentiation (86). Obesity increases
mechanical loading of bone and elevates 17β-estradiol
concentrations, which has been hypothesized to attenuate
bone loss in postmenopausal women (117,118). However,
a recent meta-analysis demonstrated that risk for ankle
fractures increased by 60% (risk ratio =1.60; 95% CI: 1.52,
1.68) in postmenopausal women with obesity compared
with lean postmenopausal women (119). These data suggest
that obesity might not confer osteoprotective eects in
postmenopausal women and may, in fact, have detrimental
eects on bone.
Two preclinical studies provide mechanistic evidence
linking obesity, gut dysbiosis, and inammation to poor bone
outcomes (120,121). High-fat/high-sucrose diet-induced
obese rats developed more severe osteoarthritis compared
with lean rats concurrently with elevated concentrations
of proinammatory cytokines in synovial uid and serum,
and changes in gut microbial populations (Methanobrevibac-
ter and Lactobacillus spp.) (120). Moreover, high-fat-diet
(HFD)-induced obesity in mice impaired osteoblastogenesis
and augmented bone marrow adiposity, resulting in impaired
bone architecture. The aforementioned outcomes were medi-
ated by changes in the gut microbiota as antibiotic treatment
partiallyreversedtheHFD-inducedeectsonbone,andstool
transplanted from HFD-fed mice induced the bone defects in
lean, pellet-fed mice (121). Chronic inammation, oxidative
stress, and dysregulation of the gut microbiota are hallmarks
of obesity (122). Thus, it is postulated that these factors
might contribute to the adverse eects of obesity on bone
outcomes; however, the exact mechanisms underlying the
relation between obesity and bone loss are unknown.
In addition to obesity, diet is an important modulator of
the intestinal microbiota and is a source of phytoestrogens
and prebiotics that are metabolized by the gut microbiota
(26). Numerous dietary factors alter intestinal microbial
composition or metabolites (123,124) and/or immune
responses, which can contribute to systemic inammation.
Thus, accumulating evidence suggests that obesity, alter-
ations in the gut microbiota, dietary factors, estrogen de-
ciency,andagingcontributetoanincreaseininammatory
mediators and markers of oxidative stress, which can enhance
bone resorption and suppress bone formation, resulting
in bone loss (Figure 2). Treatment strategies, including
nutritional interventions that attenuate oxidative stress and
inammation, might potentially improve bone outcomes in
postmenopausal women. While the osteoprotective eects
of prune consumption have been extensively reviewed else-
where (39,48,125), the potential role of prunes in improving
bone outcomes via the modulations of antioxidant and anti-
inammatory mechanisms needs to be further explored.
Therefore, the focus of this review is to summarize the
ndings from preclinical and clinical studies that evaluate
antioxidant, anti-inammatory, and other immune outcomes
tooutlinekeyknowledgegapsintheeld.
Prunes (Dried Plums) and Bone Health
In vitro findings
Six in vitro studies (126–131)haveevaluatedtheantiresorp-
tive and/or anabolic eects of prunes using osteoclast (RAW
264.7) and osteoblast (MC3T3-E1) cell lines, and these results
are summarized in Tabl e 1 . One study demonstrates that
concurrent treatment of RAW 264.7 murine macrophages
with prune polyphenol extracts (1000 μg/mL) prevented an
increase in malondialdehyde secretion induced by ferrous
sulfate and hydrogen peroxide (128). A signicant reduction
in LPS-induced NO secretion (126,128)wasreportedin2
studies, and 1 study demonstrated a reduction in iNOS pro-
tein expression following either pretreatment or concurrent
treatment of RAW 264.7 cells with varying concentrations of
prune polyphenol extracts (0.1–1000 μg/mL).
Prunes, bone health, oxidative stress, and inammation 5
FIGURE 2 Factors contributing to an elevation in inflammation and oxidative stress, which may underlie postmenopausal osteoporosis.
The pathogenesis of postmenopausal osteoporosis is attributed to several factors. Ovarian senescence during menopause results in
estrogen deficiency, which is a potent stimulus for increased production of proinflammatory mediators and oxidative stress markers that
promote bone loss. Aging is another factor that is associated with elevated inflammation and oxidative stress. The gut microbiome is
emerging as an important modulator of the bone and immune system and the composition of the intestinal microbiota is affected by the
diet, which is an important source of fiber and phytoestrogens. While these factors directly contribute to increased inflammation and
oxidative stress (indicated by solid black lines), they also modulate one another (indicated by hashed lines). AGE, advanced glycation end
products; ROS, reactive oxygen species.
A signicant reduction in cyclooxygenase 2 (COX-2)
protein expression was reported in 2 studies following
pretreatment or concurrent treatment of RAW 264.7 cells
with varying concentrations of prune polyphenol extracts
(10-1000 μg/mL) (126,128). A reduction in inamma-
tory cytokine production from either RANKL and TNF-
α–stimulated RAW 264.7 cells or TNF-α–stimulated hu-
man synovial broblasts following treatment with prune
polyphenol extracts (10–30 μg/mL) (126)orneochlorogenic
acid (10 μM) (130), respectively, was also reported. One
study found that pretreatment of TNF-α–stimulated bone
marrow–derived macrophages with neochlorogenic acid
(10 mM) reduced NF-κBactivation(130). Another single
study reported a reduction in Toll-like receptor 2 (Tlr2)
gene expression in LPS-stimulated RAW 264.7 cells following
pretreatment with prune phenolic extracts (30 μg/mL)
(126).
Four studies explored the eect of dried plum phenolic
fractions or extracts on bone-related outcomes using either
bone marrow–derived macrophages, osteoclast-osteoblast
co-cultures, RAW 264.7 cells, or the murine pre-osteoblast
cell line MC3T3-E1, which were treated with inammatory
stimuli (RANKL and TNF-αor TNF-αalone; Table 1).
AreductioninNfatc1 gene expression, TRAP+osteoclast
number, and resorptive pit area following treatment with
dried plum phenolic fractions or extracts (126,129)was
reported in 2 studies, suggesting that dried plum phenolic
fractions or extracts can reduce markers associated with
osteoclast dierentiation and function. Two studies using
MC3T3-E1 cells or primary osteoblast cells demonstrated
that dried plum phenolic fractions or extracts increase the
formation of mineralized nodules (127,131), and one of
thesereportedanincreaseingeneexpressionofmodulators
of osteoblastogenesis (Runx2, Osterix, Igf1)(127). Several
polyphenolic fractions upregulate osteoblast activity by en-
hancing BMP signaling, and TNF-α–mediated inhibition of
this cascade results in suppression of the osteogenic response
(131). Furthermore, treatment of MC3T3-E1 cells with hu-
manserumcollectedfromhealthywomen1to2hfollowing
prune consumption increases alkaline phosphatase (ALP)
activity and gene expression of Runx2,connexin 43,andbeta-
catenin (132). Overall, these results suggest that the phenolic
6 Damani et al.
TABLE 1 Eect of phenolic compounds of prunes (dried plum) on antioxidant, anti-inammatory, and bone outcomes in osteoclast and
osteoblast cell lines1
In vitro model Prune product Dose Method
Antioxidant, anti-inflammatory,
or bone outcomes2Reference
RAW 264.7 cells DPPE (Prunus
domestica)
0, 1000 μg/mL Concurrent treatment with DPPE
and FeSO4(100 μg/mL) +H2O2
(1000 μg/mL) for 4 h
MDA secretion (128)
RAW 264.7 cells DPPE (Prunus
domestica)
0, 10, 20, 30 μg/mL Pretreatment with DPPE for 2 h
followed by LPS (10 ng/mL) for
16 h
NO secretion (dose-dependent)
iNOS protein expression
(126)
RAW 264.7 cells DPPE (Prunus
domestica)
0, 0.1, 1, 10, 100,
1000 μg/mL
Concurrent treatment with DPPE
and LPS (1 μg/mL) for 12 h
NO secretion (with 1000 μg/mL
DPPE)
(128)
RAW 264.7 cells DPPE (Prunus
domestica)
0, 10, 20, 30 μg/mL Pretreatment with DPPE for 2 h
followed by LPS (10 ng/mL) for
16 h
COX-2 protein expression (126)
RAW 264.7 cells DPPE (Prunus
domestica)
0, 100, 1000 μg/mL Concurrent treatment with DPPE
and LPS (10 ng/mL) for 6 h
COX-2 protein expression (128)
Bone
marrow-derived
macrophages
(C57BL/6 mice)
Neochlorogenic
acid
10 μM Pretreatment with neochlorogenic
acid for 1 h followed by TNF-α
(10 ng/mL) for 0, 5, 15, 30, and
60 min
NF-κB activation (130)
RAW 264.7 cells DPPE (Prunus
domestica)
0, 10, 20, 30 μg/mL Cells stimulated with RANKL
(30 ng/mL) for 4 d and then
treated with DPPE for 2 h followed
by LPS (10 ng/mL) for 24 h
TNF-αsecretion (dose-dependent) (126)
Human synovial
fibroblasts
Neochlorogenic
acid
10 μM Pretreatment with neochlorogenic
acid for 1 h followed by TNF-α
(10 ng/mL) for 2 d
TNF-α,IL-1β
MCP-1, MIP-1α
MMP-1, MMP-3
(130)
RAW 264.7 cells DPPE (Prunus
domestica)
0, 10, 20, 30 μg/mL Pretreatment with DPPE for 2 h
followed by LPS (10 ng/mL) for 4 h
Tlr2 gene expression (with
30 μg/mL DPPE)
(126)
RAW 264.7 cells DPPE (Prunus
domestica)
0, 10, 20, 30 μg/mL Cells stimulated with RANKL
(30 ng/mL) for 4 d, then treated
with DPPE for 2 h followed by LPS
(10 ng/mL) for 24 h
Nfatc1 gene expression
TRAP+osteoclast number and size
resorptive pit area
(126)
Primary bone
marrow cells
(C57BL/6 mice)
DPPF (Prunus
domestica)
0, 1, or 10 μg/mL Cells stimulated with RANKL
(50 ng/mL) for 4 d, then treated
with DPPF and: TNF-α(1 ng/mL)
for 1 h
Nfatc1,Traf6 ,Sirpb1 gene expression (129)
TNF-α(1 ng/mL) for 30 min–1 h p38, Erk1/2 protein expression
TNF-α(1 ng/mL) for 1–7 d TRAP+osteoclast number
resorptive pit area
Osteoclast-
osteoblast
co-cultures
(C57BL/6 mice)
DPPF (Prunus
domestica)
0or10μg/mL Osteoclast-osteoblast co-cultures
treated with DPPF and: TNF-α
(1 ng/mL) for 6 d; TNF-α(1 ng/mL)
for 10 d
Rankl,Nfatc1,cFos gene expression
TRAP+osteoclast number
(129)
MC3T3-E1 cells DPPE (Prunus
domestica)
0, 2.5, 5, 10, and
20 μg/mL
Pretreatment with DPPE for 24 h
followed by: TNF-α(1 ng/mL) for
18 h; TNF-α(1 ng/mL) for 7–28 d
Runx2, Osterix, Igf1, Lysyl oxidase
gene expression
Rankl gene expression
ALP activity
number and size of mineralized
nodules
(127)
Primary osteoblast
cells (C57BL/6
mice)
DPPF (Prunus
domestica)
10 μg/mL Concurrent treatment with DPPF
and TNF-α(1 ng/mL) for 14 d
mineralized nodule formation (131)
Primary osteoblast
cells (C57BL/6
mice)
DPPF (Prunus
domestica)
10 μg/mL Concurrent treatment with DPPF
and TNF-α(1 ng/mL) for 1 h
Bmp2,Runx2,Tak 1,Smad1,Smad5
gene expression
(131)
1ALP, alk aline phosphatase; COX, cyclooxygenase; DPPE, dried plum polyphenol extracts; DPPF, dried plum polyphenol fractions; Erk, extracellular regulated kinase; Igf,
insulin-like growth factor; iNOS, inducible NO synthase; MCP-1, monocyte chemoattractant protein 1; MDA, malondialdehyde; MIP, macrophage inflammatory
protein; MMP, matrix metalloproteinases; Nfatc1, nuclear factor of activated T cells, cytoplasmic 1; RANKL, receptor activator of NF-κBligand;Runx2, runt-related
transcription factor; Sirpb1, signaling regulatory protein b1; Tak1 , transforming growth factor-beta-activated kinase 1; Tlr, Toll-like receptor; Traf ,TNF
receptor–associated factor; TRAP, tartrate-resistant acid phosphatase; , significant increase; , significant decrease; , no change.
2Bone outcomes under an inflammatory stimulus (LPS or TNF-α).
Prunes, bone health, oxidative stress, and inammation 7
compounds in prunes may mediate the antioxidant and anti-
inammatory eects observed in osteoclasts and osteoblasts,
and that the osteoprotective eects of prune polyphenols
are mediated both via attenuation of osteoclastogenesis and
augmentation of osteoblastogenesis.
Preclinical findings
The OVX rat model of osteoporosis is established as a suitable
model for assessing bone loss due to ovarian hormone
deciency as it mimics the pattern of rapid bone loss
followed by a period of more gradual bone loss observed
in postmenopausal women (133). Both OVX rats and post-
menopausal women respond similarly to antiresorptive and
anabolic agents such as bisphosphonates, estrogen, SERMs,
parathyroid hormone, calcium, vitamin D, and exercise
(125). These similarities justify the use of the OVX rat model
to evaluate the eects of prune consumption on bone health.
Theeectsofprunesonmeasuresofbonemetabolism
havebeenwidelystudiedinbothratandmousemodelsof
bone loss (134–144) supplemented with varying doses of
prunes, ranging from 5% to 25% wt:wt. To better understand
the relation between prunes and attainment of peak bone
mass, alterations in osteoclast and osteoblast precursor cells
in both growing (1- to 2-mo-old) and skeletally mature
(6-mo-old) C57BL/6 male mice were evaluated following
prune consumption (142). The number of ALP+osteoblast
precursors increased in young growing mice after prune
supplementation (5, 15, or 25% wt:wt). In adult mice, prune
supplementation (25% wt:wt) increased bone volume, which
wasassociatedwithdecreaseinosteoclastsurface,serum
CTX, and multinucleated TRAP+osteoclast precursors.
Although the number of ALP+osteoblast precursors and
serum procollagen type I N propeptide did not change with
prune supplementation in adult mice, surface-based bone
formation rate decreased, suggesting that increased bone
volume in adult mice might be attributed to diminished
bone resorption rather than bone formation. These ndings
demonstrate that prunes increase peak bone mass during
growthandinadulthood(142). Short-term prune supple-
mentation (5, 15, or 25% wt:wt) in an adult osteopenic OVX
rat model not only restored the gene expression of osteoblast
dierentiation factors such as Bmp4 but also suppressed the
gene expression of osteoclast dierentiation factors such as
Nfatc1 (136). Additionally, in comparison to other dried
fruits, prunes (25% wt:wt) exerted a unique eect on bone
by downregulating osteoclast dierentiation in conjunction
with upregulating osteoblast and glutathione activity in an
osteopenic OVX mouse model (141). Combined, these data
suggest that prunes may improve bone metabolism and
biochemical properties in aging and reproductive hormone–
decient animal models.
In addition to improving measures of bone metabolism,
prunes modulate bone structural properties. Overall, pre-
clinical evidence suggests that higher doses of prune sup-
plementation (15% and 25% wt:wt) prevent bone loss by
improving bone strength, indicated by an increase in BMD
of the femur and/or spine. Notably, Smith and colleagues
demonstrated in both female (136)andmale(137) rodent
models of osteopenia that prune supplementation (25%
wt:wt) improves BMD at the whole body, spine, and femur
and confers anabolic eects on the trabecular bone by
improving trabecular volume, number, and thickness at the
lumbar and distal femur. Prunes also showed osteoprotective
eects in a rat model of male osteoporosis, where 25% wt:wt
supplementation increased vertebral and femoral BMD,
vertebral trabecular bone volume, and cortical thickness
(134). In a mouse model of age-related osteoporosis, prune
supplementation (25% wt:wt) improved bone volume and
restored bone loss due to aging in male mice (140). Overall,
animal studies using male and female rodent models of
osteopenia or osteoporosis demonstrate that dietary supple-
mentation with prunes confers osteoprotective eects by not
only preventing bone loss associated with age and estrogen
deciency but also by reversing pre-existing bone loss as a
consequence of these conditions (134–147).
Prunes have also shown promising osteoprotective eects
in other preclinical disease models of bone loss. Cancer
patients undergoing radiotherapy, radiation workers, and
astronauts are examples of populations who are at a higher
risk for bone loss due to exposure to ionizing radiation.
Prune supplementation (25% wt:wt) in mouse models of
radiation-induced bone loss completely prevented cancellous
bone loss, accompanied by reduced gene expression of pro-
osteoclastogenic cytokines Rankl,Mcp1,andTnfa (148).
Prune supplementation ameliorated simulated spaceight-
induced damage in bone micro-architecture and mechan-
ical properties, providing evidence of the use of prune
supplementation as a potential strategy to mitigate bone
loss induced by radiation exposure and microgravity. In
addition, 25% prune supplementation attenuated spinal cord
injury–induced bone loss in a mouse model, suggesting the
benecial eect of prunes on bone outcomes may be widely
applicable.
Several studies have assessed the eect of bioactive
components in prunes on bone outcomes in rat models of
postmenopausal bone loss to determine the mechanisms
underlying the protective eect of prune consumption.
Prunes contain signicant amounts of chlorogenic acids,
which may confer benecial eects on bone (45). However, a
diet supplemented with prunes with a high chlorogenic acid
contentwasnotmoreeectiveatpreventingbonelossthan
a diet supplemented with low chlorogenic acid containing
prunes in an OVX rat model (149). These data suggest that a
dose-dependent eect of chlorogenic acid on bone loss may
not exist (149) and/or other bioactive components in the
whole fruit might contribute to the osteoprotective eects
of prunes. In an aged, osteopenic OVX rat model, prune
polyphenols accounted for 60–80% of the anabolic eect of
prunes on bone. However, when the polyphenolic extract was
combined with vitamin K and potassium, the reversal of bone
loss was equivalent to that observed with consumption of
the whole fruit (150). These ndings suggest that numerous
bioactive compounds in prunes may be contributing their
benecial eect on bone.
8 Damani et al.
TABLE 2 Eect of prunes (dried plum) supplementation on antioxidant and anti-inammatory outcomes in preclinical studies1
Animal model Intervention Study groups
Antioxidant or
anti-inflammatory effect Reference
Aged male BALB/c mice
(12-wk-old, n=35)
5% or 10% wt:wt DP
for 8 wk
5% or 10% wt:wt DP compared
with ND
5% wt:wt DP compared with ND
5% or 10% wt:wt DP compared
with ND
5% or 10% wt:wt DP compared
with ND
5% wt:wt DP compared with ND
Serum MDA
Serum AGE
NRF2 protein expression
KEAP-1 protein expression
CAT, SOD2 protein expression
GPX protein expression
(152)
OVX female C57BL/6J mice
(12-wk-old, n=59)
5%, 15%, or 25%
wt:wt DP for 4 wk
5%, 15%, or 25% wt:wt DP
compared with ND
15% or 25% wt:wt DP compared
with ND
15% wt:wt DP compared with
ND
Peripheral leukocyte count
CD31Ly-6C+granulocytes,
CD115+committed monocytes
Bone marrow lymphoblasts
(151)
Aged male BALB/c mice
(12-wk-old, n=35)
5% or 10% w/w DP
for 8 weeks
5% or 10% wt:wt DP compared
with ND
NF-κB protein expression in the
liver
(152)
OVX female C57BL/6J mice
(12-wk-old, n=59)
5%, 15%, or
25% wt:wt DP for
4wk
Splenocytes stimulated with
Con-A (2.5 μg/mL) for 48 h
15% or 25% wt:wt DP compared
with ND
TNF-αsecretion (151)
Male C57BL/6 mice:
skeletally mature
(6-mo-old, n=15/group)
25% wt:wt DP for
4wk
25% wt:wt DP compared with
ND
Serum IL-1α,IL-1β, IL-10, IL-12
(p70), IL-13, IL-17, TNF-α,MCP-1
(142)
Male C57BL/6J mice with
radiation-induced bone
loss (16-wk-old,
n=5–10/group)
25% wt:wt DP for
7–21 d
25% wt:wt DP compared with
ND
Tnfa , Mcp1 gene expression in
bone marrow cells
(148)
1AGE, advanced glycation end products; CAT, catalase; Con-A, concanavalin A; DP, dried plum; GPX, glutathione peroxidase; KEAP-1, Kelch-like ECH associated protein 1; MCP-1,
monocyte chemoattractant protein 1; MDA, malondialdehyde; ND, normal diet; NRF2, nuclear factor, erythroid 2–like 2; OVX, ovariectomized/ovariectomy; SOD, superoxide
dismutase; , significant increase; , significant decrease.
A growing body of evidence suggests that prunes and
their polyphenols also modulate inammatory pathways
and oxidative stress. Four preclinical studies (142,148,151,
152)haveinvestigatedtheeectofprunesonmodulating
inammatory and oxidative stress markers, ndings of which
have been summarized in Tab l e 2. One study demonstrated
that prune supplementation (5% or 10% wt:wt) for 8 wk
signicantly decreased markers of oxidative damage [serum
malondialdehyde and advanced glycation end product
(AGE)] and increased protein expression of nuclear factor,
erythroid 2–like 2 (NRF2) and downstream antioxidant en-
zymes [catalase (CAT), superoxide dismutase (SOD)-2, and
glutathione peroxidase (GPX)] in 12-wk-old male BALB/c
mice (152). Prunes also exhibited immunomodulatory ef-
fects in an ovarian hormone deciency–induced model of
osteoporosis. Prune supplementation (15% or 25% wt:wt)
for 4 wk increased peripheral blood leukocytes, CD31Ly-
6C+granulocytes, and CD115+committed monocytes but
decreased bone marrow lymphoblasts in 12-wk-old OVX fe-
male C57BL/6J mice (151). Four preclinical studies in mouse
models of aging and bone loss (148,151–153)reportedthat
prune supplementation (5–25% wt:wt for 1–8 wk) suppressed
NF-κB expression and production of inammatory cytokines
(IL-1α,IL-1β, IL-10, IL-12-p70, IL-13, IL-17, TNF-α,and
MCP-1). Prune supplementation (20% wt:wt) also restored
bone loss and is associated with fewer TRAP +cells,
indicating downregulation of osteoclastogenesis in a TNF-α-
dependent rodent model of arthritis (130). Combined, these
ndings suggest prune supplementation may be modulating
inammatory and oxidative pathways.
A relation between the changes in gut microbiota and
overt alterations in bone mass has been demonstrated
in numerous preclinical studies (154–159). However, the
mechanisms underlying this relation are unknown. In young,
female germ-free (GF) mice, femoral bone volume fraction
was 39% greater and was associated with a lower number
of osteoclasts and reduced gene expression of Il6 and Tnf a
in bone, likely contributing to reduced bone resorption
compared with conventionally raised mice (154). Greater
femoral cortical volume and cortical thickness were also
observed in female GF mice compared with conventionally
raised mice (156). Short-term colonization of 2-mo-old male
andfemaleGFmicewithcommensalbacteriareducedbone
mass; however, long-term colonization of GF mice (i.e., 8 mo)
resulted in increased femur length and bone mass compared
with GF counterparts (155). Yan et al. (155) demonstrated
that microbiota-regulated bone growth may be mediated
by SCFA-induced modulation of IGF-1, a bone trophic
hormone. Broad-spectrum antibiotic treatment depleted the
microbiota and reduced SCFAs, as well as serum IGF-1, to
Prunes, bone health, oxidative stress, and inammation 9
approximately half of the concentrations observed in the
vehicle-treated control. However, when SCFA-supplemented
waterwasprovided,serumIGF-1increasedbyapproximately
50% and bone mass was comparable to control mice (155).
Furthermore, OVX in GF mice did not result in increased
osteoclastogenic cytokine production, bone resorption, and
trabecular bone loss, demonstrating that the gut microbiota
plays a key role in OVX-induced bone loss (156). Together,
these preclinical studies provide evidence that the gut
microbiota inuences bone outcomes.
In summary, there is sucient preclinical evidence to
suggest that the osteoprotective eects of prune consump-
tion are due to a decrease in the rate of bone turnover,
where resorption is downregulated more than formation.
The dietary levels of prunes in some in vivo models are
high—for example, 25% by weight, which is equivalent
to approximately 20 prunes daily for a 2000-kcal diet in
humans (142). Although the high doses of prunes used
in animal models may not represent ideal quantities for
human consumption, these preclinical ndings suggest that
the osteoprotective eects of prunes in animal models of
osteopeniamaybepartlymediatedbyanti-inammatoryand
antioxidative pathways.
Clinical Findings
Postmenopausal women represent a clinically relevant popu-
lation in which bone health is a primary concern. As such,
several investigators have administered prunes to women
to test their osteoprotective eects by evaluating if prune
consumption alters bone biomarkers and prevents bone loss
over time. To date, the results from 4 RCTs (40–43)and1case
study (44) investigating the eects of prune consumption on
bone health outcomes in postmenopausal women indicate
promising eects on bone turnover (40)andBMD(41,42,
44). Prune consumption at a dose of 100 g/d for 3 mo
signicantly increased the serum concentrations of bone-
specic alkaline phosphatase (BSAP) by 5.8% and IGF-1
by 17% in postmenopausal women who were not receiving
any hormone replacement therapy (40). Additionally, prune
consumption at the same dose of 100 g/d for 1 year improved
BMDoftheulnaandlumbarspineincomparisontothe
control (75 g/d dried apple) (41,43), and decreased serum
markers of bone turnover, BSAP, TRAP-5b, and osteocalcin
(41). Prune consumption at 50 g/d and 100 g/d for 6 mo
prevented loss of total BMD, but not ulnar BMD (reported
as change from baseline), and decreased TRAP-5b compared
with women consuming 0 g/d of prunes (n=16/group)
(42). In a recent case study describing changes in BMD over
thecourseof28mo,apostmenopausalosteopenicwoman
participated in an ongoing RCT as a control participant
(1200 mg calcium carbonate and 800 IU vitamin D3daily
for12mo)andthenvolitionallyconsumed50gprunesdaily
in addition to calcium and vitamin D3for an additional 16
mo (44). During participation in the RCT, the participant
experienced a 7.6% decrease in lumbar spine BMD; however,
from month 12 to 28, which included voluntary prune
consumption, there was a notable improvement in lumbar
spine BMD (7.8%) while preventing further decline in total
body and total hip BMD. Together, these results suggest that
50 g/d of prunes may be an eective dose to improve bone
health in postmenopausal women, with longer duration of
consumption (42,44). However, data from the 6-mo RCT and
case study should be interpreted with caution until replicated
due to the limited sample size. Overall, the clinical ndings
to date suggest that prune consumption may be eective
in improving bone outcomes in postmenopausal women,
possibly by enhancing bone formation and reducing bone
resorption.
In total, 3 clinical studies (41,42,160)haveassessed
the eect of prune consumption on markers of oxidative
stress or inammation in postmenopausal women, and
ndings are summarized in Tab l e 3 .Onestudy(160)
reported a signicant increase in plasma total antioxidant
capacity and plasma antioxidant enzyme activity of SOD
in postmenopausal women after 6 mo of consuming 50 or
100 g/d prunes compared with baseline; however, no eects
were observed on plasma CAT and GPX activities. Two
studies (41,42) investigated the eect of prune consumption
in postmenopausal women with osteopenia on serum C-
reactive protein (CRP), a widely used serum marker of
systemic inammation. One study reported a signicant
reduction in CRP in postmenopausal women after 3 mo of
consuming 100 g/d of prunes compared with the control
group consuming 75 g/d of dried apple; however, no
dierence in CRP between the prune- versus dried apple–
consuming group was observed after 6 and 12 mo on the trial
(41). The lack of dierence in CRP concentrations at 6 and 12
mo in the prune group and dried apple control group was due
to a reduction in CRP in the dried apple–consuming control
group, which likely resulted because apples also contain
phenolic compounds (161). The second study measuring
CRP reported no eect of 50 g/d or 100 g/d of prune
consumption on serum CRP at 3 and 6 mo compared with
of 0 g/d of prune consumption (42). Although serum CRP
isawidelyusedmarkerofinammation,itmaynotbe
modulated by prune consumption. One study (160)inves-
tigated the eect of 50 g/d or 100 g/d prune consumption
for 6 mo on additional markers of inammation in the
same study population as reported in Hooshmand et al.
(42). Low-dose (50 g/d) prune consumption signicantly
decreased plasma proinammatory cytokines IL-6 and TNF-
αcompared with baseline. Overall, these 3 clinical trials
provide preliminary evidence suggesting potential antioxi-
dant and anti-inammatory eects of prune consumption
in postmenopausal women with bone loss, but results vary
depending on what oxidative or inammatory marker is
assessed.
There are limited data in humans on prune consumption
andchangesinthegutmicrobiota,aswellasimmuneand
bone health alterations. To date, 3 clinical studies (162–164)
have investigated the eect of the gut microbiota on bone
health in postmenopausal women. These studies demon-
strate that postmenopausal women with low BMD have
altered diversity and composition of the gut microbiota, with
10 Damani et al.
TABLE 3 Eect of prunes (dried plum) supplementation on antioxidant and anti-inammatory outcomes in clinical studies1
Study
design Study population Intervention2Control2
Duration of
intervention
Antioxidant or
anti-inflammatory effect Reference
RCT, par-
allel
arm
Postmenopausal
women (n=40,
completed)
50 g/d DP
consumption
(n=14, age =
68.5 y)
100 g/d
DP consumption
(n=13, age =
70.4 y)
0g/dDP
consumption
(n=13,
age =71 y)
6mo Plasma TBARS in 50-g/d and
100-g/d DP groups
PlasmaTACin50-g/dDP
group
Plasma SOD activity in 50-g/d
and 100-g/d DP groups
Plasma GST activity in 50-g/d
DP group
Plasma CAT and GPX
activities
(160)
RCT, par-
allel
arm
Postmenopausal
women with
osteopenia
(n=100,
completed)
100 g/d DP
consumption
(n=55, age =
57.5 y)
75 g/d dried apple
consumption
(n=45,
age =55.6 y)
3, 6, and 12
mo
Serum CRP at 3 mo in DP
group
Serum CRP at 6 or 12 mo
(41)
RCT, par-
allel
arm
Postmenopausal
women with
osteopenia
(n=42,
completed)
50 g/d DP
consumption
(n=16, age =
68.5 y)
100 g/d
DP consumption
(n=13, age =
70.4 y)
0g/dDP
consumption
(n=13,
age =71 y)
6mo Serum CRP (42)
RCT, par-
allel
arm
Postmenopausal
women (n=40,
completed)
50 g/d DP
consumption
(n=14, age =
68.5 y)
100 g/d
DP consumption
(n=13, age =
70.4 y)
0g/dDP
consumption
(n=13,
age =71 y)
6mo Plasma IL-6, TNF-αin 50-g/d
DP group
(160)
1CAT, catalase; CRP, C-reactive protein; DP, dried plum; GPX, glutathione peroxidase; GST, glutathione Stransferase; RCT, randomized controlled trial; SOD, superoxide dismutase;
TAC, total antioxidant capacity; TBARS, thiobarbituric acid reactive substances; , significant increase; , significant decrease; , no change.
2Includes 500 mg calcium +400 IU (10 mg) vitamin D3. Ages are means.
ahigherabundanceofBacteroides (164). Plasma markers
of gut barrier dysfunction, including LPS-binding protein
and soluble CD14, increase during the menopausal transition
and are associated with lower BMD. These data suggest that
greater gut permeability may associated with elevated inam-
matory mediators, which may contribute to postmenopausal
bone loss (162). Additionally, reduced BMD in older adults
with osteopenia and osteoporosis is associated with an
altered microbiota (165–167). In the only study published
to date examining prune consumption on gut microbiota,
120 healthy males and females were randomly assigned to
1 of 3 arms—control (no prunes), 80 g/d of prunes, or
120 g/d of prunes (n=40/group)—for 9 wk (168). Changes
in stool characteristics (i.e., stool weight and frequency) were
reported, but no eect of prune consumption on gut bacterial
composition, SCFA concentration, or stool pH were observed
(168).
Overall, evidence from in vitro, preclinical studies, and
limited clinical studies suggests the potential role of prunes
in ameliorating bone loss. These ndings may be attributed
to altered bone turnover and by inhibiting inammation-
induced NF-κB and NFATc1 signaling and their downstream
expression of cytokines in osteoclast precursors, attenuating
TNF-αsecretion from monocytes and T cells, and suppress-
ing markers of oxidative stress.
Potential mechanisms and future directions
Potential mechanisms underlying the protective eects of
prunes on bone health have been described by Arjmandi et
al. (39), suggesting that the components of prunes, including
minerals, vitamin K, phenolic compounds, and dietary ber,
might work additively or synergistically to mediate benecial
eects. Polyphenol-rich extracts have been shown to promote
the growth of commensal bacteria (52). Dietary ber and
phenolic compounds may contribute to the potential anti-
inammatory properties of prunes by modulating the gut
microbiota (169). These changes may include shifts in the
microbiota composition and increased SCFA production
upon microbial fermentation of dietary ber. SCFAs have
the potential to modulate host immune response and
promote gut barrier integrity (52,170). Reduction in chronic
inammation can potentially reduce the dierentiation and
activation of osteoclasts and prevent bone loss. Therefore,
any potential anti-inammatory eect of prune consumption
Prunes, bone health, oxidative stress, and inammation 11
FIGURE 3 Proposed mechanisms linking prune consumption to improved bone health via changes in inflammatory mediators, oxidative
stress, and the gut microbiome. Prunes are rich in minerals, vitamin K, phenolic compounds, and dietary fiber, all of which might work
additively or synergistically to mediate beneficial effects on bone (39). Dietary fiber and polyphenolic compounds found in prunes may
alter the composition of the gut microbiota (169), increasing SCFA production and promoting increased gut barrier integrity. These
changes in the gut may be associated with decreased secretion of proinflammatory cytokines (IL-1, IL-6, TNF-α) and oxidative damage
markers from immune cells in the lamina propria and in the periphery (169). Preclinical evidence supports the role of prune polyphenols
in enhancing osteoblast function and suppressing osteoclast function. It is hypothesized that reductions in markers of inflammation and
oxidative stress are linked to improvements in bone outcomes. OPG, osteoprotegerin; RANK, receptor activator of NF-κB; RANKL, receptor
activator of NF-κB ligand; ROS, reactive oxygen species.
may mediate, at least in part, the benecial eect of prune
on bone outcomes. Proposed mechanisms underlying the
osteoprotective eects of prunes via changes in inammatory
and oxidative stress mediators are outlined in Figure 3.Prune
consumption may lead to changes in the gut microbiota
due to the ber and phenolic content, promoting increased
SCFA production, which decreases colonic inammation
and improves gut integrity (39,170). This may lower
secretion of proinammatory cytokines (IL-1, IL-6, TNF-
α) and oxidative damage markers from immune cells
in the lamina propria and in circulation (169). Overall,
reduction in mediators of inammation (IL-1, IL-6, TNF-
α) and oxidative stress (ROS) contributes to enhanced bone
formation and reduced bone resorption, ameliorating bone
loss. A substantial body of literature demonstrates the role
of the gut–bone axis in the pathogenesis of osteoporosis,
suggesting that dietary phytochemicals with osteoprotective
properties improve the gut barrier integrity and favorably
modulate the gut microbiota and mucosal immune cells
(171). For example, prune consumption in healthy individ-
uals signicantly increases the growth of Bidobacteria, a
genus of bacteria generally thought to provide benecial
eects to the host (168). However, further studies are needed
to determine whether prunes exert prebiotic potential to
positively modulate the host gut microbiota and reduce
inammation, and whether these changes occur following
prune interventions of longer duration. In addition to prunes,
there is emerging evidence on the osteoprotective eects
of other polyphenol-rich fruits such as tart cherry (172–
175), blueberry (176–178), and watermelon (179), which
may mitigate bone loss via similar biological mechanisms.
The biological mechanisms underlying the potential eect
of prunes on inammatory and immune mediators require
further investigation.
12 Damani et al.
Clinical trials assessing the eect of dietary interventions
on inammatory responses frequently measure plasma or
serum cytokines; however, measurement of these circulating
inammatory markers alone might not adequately capture
the immunomodulatory eects of nutritional interventions.
We have demonstrated that in vitro stimulation of PBMCs
with LPS, which mimics in vivo activation, may be an
additional endpoint to include in human intervention studies
to assess the inammatory response following a dietary
intervention (180,181). Therefore, additional measurements
of proinammatory mediators, including serum cytokines
and/or cytokines secreted from stimulated PBMCs collected
from postmenopausal women following prune consumption,
might provide a more comprehensive understanding of the
eectofprunesoninammatoryoutcomesinhumans.
In addition to exploring the mechanistic role of prunes
on bone health, it is important to determine the clini-
cal feasibility of prunes as a nutritional intervention by
addressing questions such as whether the osteoprotective
eects of prunes are long-lasting and if they are associated
with clinical endpoints such as reduction in fractures.
Further studies are needed to determine the optimal dosage
and duration associated with minimal adverse eects and
to develop strategies for obtaining maximum compliance.
Additional large-scale RCTs are needed to enhance the
generalizability of these ndings among postmenopausal
women, as well as other target populations such as osteopenic
men. Furthermore, investigating whether the osteoprotective
eects of prunes are mediated through anti-inammatory,
antioxidant, and/or immunomodulatory mechanisms and
measuring phenolic compounds in the plasma or urine after
long-term prune consumption in conjunction with inam-
matory mediators and fecal microbial populations are critical
next steps. There is an emerging but poorly understood
link between gut microbiota, inammatory mediators, and
bone health. Future mechanistic studies are warranted to
advance our understanding of the complexities of these
relations.
Acknowledgments
Images in the review were created with BioRender.com. The
authors’ responsibilities were as follows—MJDS and CJR:
designed the research; JJD, HLVE, and NCAS: conducted
the research and collected data; JJD, HLVE, NCAS, MJDS,
and CJR: participated in data analysis and interpretation and
wrote the manuscript; CJR: had primary responsibility for
thenalcontent;andallauthors:readandapprovedthenal
manuscript.
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Prunes, bone health, oxidative stress, and inammation 17
... Previous studies have shown GM and VM regulates inflammation (9,23,(33)(34)(35), which is related to osteoporosis, so we hypothesized that osteoporosis may be associated with an increase in systemic inflammation by GM and VM. The results in this study showed that the level of the anti-inflammatory factor IL-10 was significantly lower in the osteoporosis group than in the control and osteopenia groups ( Figure 5A). ...
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Background: Postmenopausal osteoporosis (PMO) is influenced by estrogen metabolism and immune response, which are modulated by several factors including the microbiome and inflammation. Therefore, there is increasing interest in understanding the role of microbiota in PMO. Objectives: To investigate variations in gut microbiota (GM) and vaginal microbiota (VM) in postmenopausal women with osteoporosis. Methods: A total of 132 postmenopausal women were recruited for the study and divided into osteoporosis (n = 34), osteopenia (n = 47), and control (n = 51) groups based on their T score. The serum levels of interleukin (IL)-10, tumor necrosis factor (TNF)-α, and lipopolysaccharide-binding protein were determined via enzyme-linked immunosorbent assay. Additionally, 16S rRNA gene V3-V4 region sequencing was performed to investigate the GM and VM of the participants. Results: Significant differences were observed in the microbial compositions of fecal and vaginal samples between groups (p < 0.05). It was noted that for GM, Romboutsia, unclassified_Mollicutes, and Weissella spp. were enriched in the control group, whereas the abundances of Fusicatenibacter, Lachnoclostridium, and Megamonas spp. were higher in the osteoporosis group than in the other groups. Additionally, for VM, Lactobacillus was enriched in the control group, whereas the abundances of Peptoniphilus, Propionimicrobium, and Gallicola spp. were higher in the osteoporosis group than in the other groups. The predicted functional capacities of GM and VM were different in the various groups. We also found that the serum level of IL-10 in the osteoporosis group was significantly lower than that in the control group and osteopenia group, while TNF-α was significantly higher in the osteoporosis group than that in the control group (p < 0.05). Conclusion: The results show that changes in BMD in postmenopausal women are associated with the changes in GM and VM; however, changes in GM are more closely correlated with PMO than VM.
... In addition to prunes, other polyphenol-rich foods exhibit similar biological activity in the prevention of bone loss (Lucas et al., 2020). Future research should aim to investigate whether this protective effect is associated with alterations in gut microbes (Damani et al., 2022). Foods rich in anthocyanins, calcium, unsaturated fatty acids, bioactive peptides, and polyphenols regulate the diversity and composition of the intestinal microbiota to a certain extent and thus have a beneficial effect on the bone. ...
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The gut microbiota has been shown to play an important role in the pathogenesis of various diseases, including metabolic diseases, cardiovascular diseases, and cancer. Recent studies suggest that the gut microbiota is also closely associated with bone metabolism. However, given the high diversity of the gut microbiota, the effects of different taxa and compositions on bone are poorly understood. Previous studies demonstrated that the mechanisms underlying the effects of the gut microbiota on bone mainly include its modulation of nutrient absorption, intestinal permeability, metabolites (such as short-chain amino acids), immune responses, and hormones or neurotransmitters (such as 5-hydroxytryptamine). Several studies found that external interventions, such as dietary changes, improved bone health and altered the composition of the gut microbiota. This review summarises the beneficial gut bacteria and explores how dietary, natural, and physical factors alter the diversity and composition of the gut microbiota to improve bone health, thereby providing potential new insight into the prevention of osteoporosis.
... Flow cytometric analysis: Phenolic compounds in prunes are reported to have numerous anti-inflammatory and anti-oxidative effects [35]. We chose to assess the effect of prunes on the number and activation status of monocytes as they are one of the main producers of inflammatory cytokines. ...
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The use of non-pharmacological alternatives to pharmacological interventions, e.g., nutritional therapy, to improve or maintain bone mineral density (BMD) in postmenopausal women has gained traction over the past decade, but limited data exist regarding its efficacy. This paper describes the design of the Prune Study, a randomized controlled trial (RCT) that explored the effectiveness of a 12-month intervention of daily prune consumption on bone density, bone structure and strength estimates, bone turnover, various biomarkers of immune function, inflammation, and cardiovascular health, as well as phenolic and gut microbiota analyses. Postmenopausal women between the ages of 55–75 years were randomized into either control group (no prune consumption; n = 78), 50g prune (50g prune/day; n = 79), or 100g prune (100g prune/day; n = 78). All participants received 1200mg calcium +800 IU vitamin D3 daily as standard of care. The Dried Plum study is the largest and most comprehensive investigation of a dose response of prune consumption on bone health, biomarkers of immune function, inflammation, and cardiovascular health, as well as detailed phenolic and gut microbiota analyses in postmenopausal women. 235 women were randomized, and 183 women completed the entire study. The findings of this study will help expand our current understanding of clinical implications and mechanisms underlying the resultant health effects of prune as a functional food therapy.
Article
Background Dietary consumption of prunes has favorable impacts on bone health, however, more research is necessary to improve upon study designs and refine our understandings. Objectives We evaluated the effects of prunes (50 g or 100 g/day) on bone mineral density (BMD) in postmenopausal females during a 12-month dietary intervention. Secondary outcomes include effects on bone biomarkers. Study Design The single center, parallel arm 12-month randomized controlled trial (RCT; NCT02822378) tested the effects of 50 g and 100 g prunes vs. a Control group on BMD (every 6 months) and bone biomarkers in postmenopausal females. Results 235 females (age 62.1 ±5.0 yr) were randomized into Control (n = 78), 50 g Prune (n = 79), or 100 g Prune (n = 78) groups. Compliance was 90.2 ± 1.8% and 87.1 ± 2.1% in the 50 g and 100 g Prune groups. Dropout was 22%; however, the dropout rate was 41% for the 100 g Prune group (compared to other groups 10% Control; 15% 50 g Prune; (p < 0.001)). A group × time interaction for total hip BMD was observed in Control vs 50 g Prune groups (p < 0.05), but not in Control vs 100 g Prune groups (p > 0.05). Total hip BMD decreased -1.1 ± 0.2% in the Control group at 12 months, while the 50 g Prune group preserved BMD (-0.3 ± 0.2%) at 12 months (p < 0.05). While hip fracture risk (FRAX) worsened in the Control group at 6 months compared to baseline (10.3 ± 0.5% vs 9.8 ± 0.5%, p < 0.05), FRAX score was maintained in the pooled (50g + 100 g) Prune groups. Conclusions A 50 g daily dose of prunes can prevent loss of total hip BMD in postmenopausal females after six months, which persisted for 12-months. Given that there was high compliance and retention at the 50 g dosage over 12 months, we propose that the 50 g dose represents a valuable non-pharmacological treatment strategy that can be used to preserve hip BMD in postmenopausal females and possibly reduce hip fracture risk. Clinical Trials Registry Number: NCT02822378
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Osteoporosis or porous bone disorder is the result of an imbalance in an otherwise highly balanced physiological process known as ‘bone remodeling’. The immune system is intricately involved in bone physiology as well as pathologies. Inflammatory diseases are often correlated with osteoporosis. Inflammatory mediators such as reactive oxygen species (ROS), and pro-inflammatory cytokines and chemokines directly or indirectly act on the bone cells and play a role in the pathogenesis of osteoporosis. Recently, Srivastava et al. (Srivastava RK, Dar HY, Mishra PK. Immunoporosis: Immunology of Osteoporosis-Role of T Cells. Frontiers in immunology. 2018;9:657) have coined the term “immunoporosis” to emphasize the role of immune cells in the pathology of osteoporosis. Accumulated pieces of evidence suggest both innate and adaptive immune cells contribute to osteoporosis. However, innate cells are the major effectors of inflammation. They sense various triggers to inflammation such as pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), cellular stress, etc., thus producing pro-inflammatory mediators that play a critical role in the pathogenesis of osteoporosis. In this review, we have discussed the role of the innate immune cells in great detail and divided these cells into different sections in a systemic manner. In the beginning, we talked about cells of the myeloid lineage, including macrophages, monocytes, and dendritic cells. This group of cells explicitly influences the skeletal system by the action of production of pro-inflammatory cytokines and can transdifferentiate into osteoclast. Other cells of the myeloid lineage, such as neutrophils, eosinophils, and mast cells, largely impact osteoporosis via the production of pro-inflammatory cytokines. Further, we talked about the cells of the lymphoid lineage, including natural killer cells and innate lymphoid cells, which share innate-like properties and play a role in osteoporosis. In addition to various innate immune cells, we also discussed the impact of classical pro-inflammatory cytokines on osteoporosis. We also highlighted the studies regarding the impact of physiological and metabolic changes in the body, which results in chronic inflammatory conditions such as ageing, ultimately triggering osteoporosis.
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