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Consumption of Dried Apple Peel Powder Increases Joint Function
and Range of Motion
Gitte S. Jensen, Victoria L. Attridge, Kathleen F. Benson, Joni L. Beaman, Steve G. Carter, and David Ager
NIS Labs, Klamath Falls, Oregon, USA.
ABSTRACT The goal for this study was to evaluate the effects of consumption of dried apple peel powder (DAPP) on joint
function and range of motion (ROM). Additional in vitro and clinical testing was performed to suggest specific mechanisms of
action. An open-label clinical pilot study involved 12 healthy people with moderate loss of joint ROM and associated chronic
pain. The subjects consumed 4.25 g DAPP daily for 12 weeks, with evaluations at baseline, 2, 4, 8, and 12 weeks. ROM was
evaluated at each visit using dual digital inclinometry. Pain scores were collected using Visual Analogue Scales. Blood draws
enabled testing of serum antioxidant protective capacity using the cellular antioxidant protection (CAP-e) bioassay. Additional
in vitro testing involved testing of cyclooxygenase-2 (COX-2) and lipoxygenase inhibition, cellular antioxidant protection by
the CAP-e bioassay, and formation of reactive oxygen species (ROS) by polymorphonuclear (PMN) cells by flow cytometry.
Twelve weeks of consumption of DAPP was associated with improved ROM. DAPP provided antioxidants that were available
to enter into and protect cells from oxidative damage in vitro, and consumption of DAPP for 12 weeks was associated with a
statistically significant improvement in serum antioxidant protective status. DAPP inhibited both COX-2 and lipoxygenase
enzymes, and pretreatment of inflammatory PMN cells with DAPP before inflammatory stimulus resulted in reduced ROS
formation. This suggests multifaceted anti-inflammatory properties of DAPP. Consumption of DAPP was associated with
improved joint function and improved serum antioxidant protection status. The observed pain reduction may be associated
with the improved antioxidant status and linked to the apple polyphenols’ anti-inflammatory effects.
KEY WORDS: antinociceptive antioxidant anti-inflammatory chronic pain digital inclinometry
INTRODUCTION
Joint mobility affects normal activities of daily living
as age-related wear and tear leads to significant struc-
tural, mechanical, and matrix changes.
1
A progressive re-
duction in the ability of chondrocytes to maintain cartilage
homeostasis contributes to overall loss of matrix tensile
strength and stiffness that accompanies aging,
2
resulting in a
restriction of joint movement and loss of mobility.
As interest in natural, nutritional support for overall
health has increased, several studies have shown that poly-
phenol flavonoids, such as anthocyanins, are capable of
neuro-protective, anti-inflammatory, and analgesic func-
tions. These naturally occurring compounds may contribute
to primary prevention measures and provide an alternative
to non-steroidal anti-inflammatory drugs used in the treat-
ment of age-related decrease in joint function.
Contrary to acute inflammation, chronic inflammation
results when the inflammatory response is incomplete, or the
inflammatory initiators persist, rather than being controlled
by the production of anti-inflammatory cytokines.
3
While
inflammation can be beneficial in that it recruits inflamma-
tory cells to the affected tissue and stimulates changes in
local blood vessels, prolonged inflammatory states have
been linked to the development of rheumatoid arthritis and
other inflammatory diseases, which reduce both joint range
of motion (ROM) and activity levels.
Apples, and the apple peel specifically, have been asso-
ciated with multiple health benefits through disease pre-
vention and the maintenance of overall health. Apples have
been shown to display antioxidant and antiproliferative ac-
tivity, which may be responsible for protecting cellular
components from oxidative damage as well as for inhibiting
the growth of tumor cells.
4
Evidence from epidemiological
studies has shown that apples may play a significant role in
reducing the risk of chronic diseases such as cancer, type II
diabetes, cardiovascular disease, pulmonary disease, and
asthma.
5
Although apples contain an abundance of biologically
active compounds, it is well known that the flesh and peel
differ with regard to phenolic distribution. For example,
although both contain compounds such as phloretin gly-
cosides, phloridzin, and chlorogenic acid, the peel con-
tains additional flavonoids that are not found within the
Manuscript received 6 March 2014. Revision accepted 9 September 2014.
Address correspondence to: Gitte S. Jensen, PhD, NIS Labs, 1437 Esplanade, Klamath
Falls, OR 97601, USA, E-mail: gitte@nislabs.com
JOURNAL OF MEDICINAL FOOD
J Med Food 17 (11) 2014, 1204–1213
#Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2014.0037
1204
flesh.
6
In an evaluation of the nutritional quality of apple
peels, the total phenolic and flavonoid contents, antioxi-
dant activities, and inhibition of tumor cell growth were
significantly higher in the peels than in flesh, regardless of
the cultivar.
6
The phytochemical profile of apples has been found to be
affected by their cultivar, conditions of growth and matu-
ration, as well as plant nutritive status and processing.
7
In
addition to the differences in phenolic and flavonoid con-
tents between specific apple cultivars, changes in light ex-
posure during the process of ripening may be significant in
stimulating the production of specific phytochemicals.
These variations in phytochemical concentration have been
positively associated with total antioxidant activity.
6
Al-
though the duration of storage has not been shown to have
significant effects on apple phenolics, the processing of
apples for juice or other apple-related products has been
shown to significantly reduce the concentration of phenolics
and total antioxidant activity compared with fresh apples.
5
In contrast, dried apples and their peel maintain a high level
of polyphenolic antioxidants.
6
Polyphenolic compounds found in fruits have been
documented to display anti-inflammatory and antioxidant
activity both in vitro and in vivo. Apple extracts have been
observed to inhibit the expression of pro-inflammatory
genes, inflammatory enzymes, and transcription factors,
8
thereby modifying signal transduction pathways. Specifi-
cally, quercetin, a polyphenolic flavonoid found in fruits
such as apples, red grapes, and blueberries, has been shown
to have significant anti-inflammatory effects in human and
animal models,
9
suggesting a beneficial effect on chronic
inflammatory diseases and overall joint health. In addition, a
high intake of antioxidant-rich fruits in animal studies has
been positively associated with higher bone mineral density,
mass, thickness, enhanced bone formation, and suppression
of bone resorption, which results in greater overall bone
strength.
10
It is well known that dietary polyphenolic antioxidants
have been associated with improved joint function and anti-
nociceptive effects in both animals and humans. Clinical
evaluation of a beverage containing whole Acai (includ-
ing the skin and pulp) showed improved joint ROM,
11
as
did a clinical study on a complex blend, including the
mushroom-based amino-acid ergothioneine.
12
In addition,
anthocyanin-rich extracts from tart cherries have been
shown to significantly reduce inflammation-induced thermal
and mechanical hyperalgesia in rats.
13
Therefore, due to
their potential for reducing inflammation, improving mo-
bility, and modifying pain perception, interest in a variety of
fruits containing high levels of polyphenols has increased.
Dried apple peel powder (DAPP) is rich in polyphenolic
antioxidants, and it has been demonstrated to inhibit cy-
clooxygenase-2 (COX-2), in addition to other potent
mechanisms of anti-inflammatory action.
14
This study was
performed to explore effects in humans consuming DAPP
over a period of 12 weeks. A detailed evaluation of joint
function was performed, as well as an evaluation of anti-
oxidant status and pain reduction.
MATERIALS AND METHODS
Reagents
The following buffers and reagents were obtained from
Sigma-Aldrich (St. Louis, MO, USA): Histopaque 1077 and
1119, phosphate-buffered saline (PBS), RPMI-1640 culture
medium, fetal calf serum, L-glutamine 200 mM, penicillin-
streptomycin 100 ·solution, gallic acid, dimethylsulfate
(DMSO), fibronectin, and bovine serum albumin. The pre-
cursor dye [5-(and-6)-chloromethyl-20,70-dichlorodihydro-
fluorescein diacetate, acetyl ester] (DCF-DA) was obtained
from Molecular Probes (Eugene, OR, USA). 2,20-Azobis (2-
amidinopropane) dihydrochloride (AAPH) was obtained
from Wako Chemical USA (Richmond, VA, USA); sodium
azide (NaN
3
) was obtained from LabChem, Inc. (Pittsburgh,
PA, USA). Leukotriene B4 was obtained from Cayman
Chemical (Ann Arbor, MI, USA).
Dried apple peel powder
Leahy dried apple peel powder (Leahy DAPP), com-
mercially available as AppleActivand AppleBoost, was
obtained from Leahy Orchards, Inc. (Quebec, Canada). For
the in vitro testing, DAPP was prepared as follows: 500 mg
DAPP was added to 5 mL saline, incubated on a rocker for
1 h, after which solids were removed by centrifugation fol-
lowed by filtration through a sterile 0.22 lm cellulose ace-
tate filter. The dose range chosen for the bioassays was
based on an initial cellular viability assay, to establish the
dose range to be used in cellular assays. For some tests, a
low-molecular-weight (LMW) fraction was prepared by
applying this sterile saline solution, containing the water-
soluble portion of DAPP, to an ultrafiltration device and
centrifuging according to the manufacturer’s instructions to
isolate compounds smaller than the 3 kDa cut-off for the
ultrafiltration device. For the clinical pilot study, DAPP was
encapsulated in veggie capsules. Participants consumed
three capsules thrice daily, for a daily dose of 4.25 g, which
is the upper daily dose recommended by the manufacturer.
Antioxidant capacity using the oxygen radical
absorbance capacity assay
The antioxidant capacity was evaluated by a panel of
chemical oxygen radical absorbance capacity (ORAC) tests,
where each test measures quenching of specific oxidative
reactions. Total ORAC provides a measure of the total an-
tioxidant capacity against these five predominant reactive
species: peroxyl radicals, hydroxyl radicals, peroxynitrite,
super oxide anion, and singlet oxygen. Testing was per-
formed at Brunswick Laboratories (Southborough, MA,
USA).
Antioxidant capacity using the Folin–Ciocalteu assay
The antioxidant capacity was also tested using the Folin–
Ciocalteu assay. The Folin–Ciocalteu’s phenol reagent was
added to serial dilutions of extract, thoroughly mixed, and
incubated for 5 min. To start the chemical reaction, sodium
DRIED APPLE PEEL IMPROVES JOINT RANGE OF MOTION 1205
carbonate was added to produce color. The microplate was
incubated for 30 min at 37C, and the optical absorbance at
765 nm was read in a colorimetric plate reader (BioTek
PowerWave, Winooski, VT, USA).
COX-2 inhibition
The effects of DAPP on COX-2 was evaluated using the
COX Inhibitor Screening Assay Kit (Cayman Chemical).
Arachidonic acid was used as a substrate for human re-
combinant COX-2 enzyme. The assay measures PGF2a
produced by SnCl2 reduction of COX-derived PGH2. The
prostanoid product is quantified via enzyme immunoassay
using a broadly specific antibody that binds to all major
prostaglandin compounds.
Lipoxygenase inhibition
DAPP’s effect on the enzymatic activity of lipoxygenase
was tested using lipoxygenase Inhibitor Screening Assay Kit
(Cayman Chemical). Purified soybean lipoxygenase was
provided with the substrate arachidonic acid in the absence
versus presence of DAPP. The hydroxyperoxides produced
as a result of the lipoxygenase enzymatic reaction were
measured in a colorimetric assay. Chromogen was used to
stop enzyme catalysis and develop the reaction. The 96-well
plate was read in a microplate reader (BioTek PowerWave)
at 490–500 nm absorbance.
Purification of polymorphonuclear cells and erythrocytes
Healthy human volunteers between the ages of 18 and 65
years served as blood donors after written informed consent
was obtained, as approved by the Sky Lakes Medical Center
Institutional Review Board (FWA2603). Isolation of poly-
morphonuclear (PMN) cells was performed as previously
described.
15,16
The PMN cells were used for evaluation of
anti-inflammatory activity in assays for production of re-
active oxygen species (ROS) and migratory response to the
inflammatory mediator leukotriene B4. The erythrocytes
were stored at 4C in aliquots for later use in the cellular
antioxidant protection (CAP-e) bioassay.
CAP-e bioassay
The CAP-e assay was performed according to the method
published by Honzel et al.,
15
but using an accelerated
and more sensitive microplate-based protocol, and the im-
plementation of ex vivo storage of the erythrocytes to
achieve improved interassay performance. Briefly, a sus-
pension of erythrocytes was prepared for the CAP-e bioas-
say by adding 0.1 mL packed red blood cells to 10 mL
physiological saline (pH 7.4) in a V-bottom 96-well mi-
croplate. Six wells served as negative controls (no induced
oxidative damage), and six wells served as positive controls
(maximum oxidative damage in the absence of any antiox-
idants). Gallic acid was used as a standard reference com-
pound. For assessment of the cellular antioxidant protection
provided by the DAPP in vitro, serial dilutions of the
aqueous extract were tested in duplicate. For assessment of
the changes in serum antioxidant protection capacity, serum
samples were tested in quadruplicate. The cells were incu-
bated for 20 min with aqueous extract or serum, to enable
available antioxidants to penetrate into the cells. After this
incubation, antioxidant compounds that were not absorbed
into the erythrocyte cells were removed from the assay plate
by two washes in physiological saline. Cell pellets were re-
suspended and lysed in water to accelerate the final steps,
and the precursor dye DCF-DA was added to the wells for
15 min. Subsequently, oxidative damage was induced by the
addition of AAPH and incubation for 1 h. The oxidation
leads to the transformation of the precursor dye to a green
fluorescent marker, where the fluorescence intensity is a
measure of the level of oxidative damage. The green fluo-
rescence intensity was recorded at 488 nm using a Tecan
Spectrafluor plate reader (Durham, NC, USA). Cellular
antioxidant protection was calculated as the inhibition of
oxidative damage reflected by the reduced fluorescence in-
tensity in the wells where cells were pretreated with test
products, compared with the baseline (negative controls)
and maximum oxidative damage (positive controls).
Evaluation of ROS formation in PMN cells
PMN cells were used for the evaluation of ROS. They
were incubated at 37C, in 5% CO
2
for 20 min as either
untreated PMN cells or serial dilutions of DAPP aqueous
extract treated PMN cells. The precursor dye DCF-DA was
prepared by adding 0.18 mL DMSO to a 50 lg aliquot of
DCF-DA (stock solution). By adding 0.01 mL stock to
10 mL PBS, the working solution was prepared. PBS was
used to wash the PMN cells twice to remove any unabsorbed
and unbound compounds. The DCF-DA working solution
was used to resuspend the cells. They were incubated for 1 h
at 37C so the precursor dye could be absorbed into the
PMN cells. All samples were then exposed to 167 mM H
2
O
2
for 45 min to induce severe oxidative stress, except for the
three negative controls. The peroxide was removed from the
samples by washing twice in PBS, transferred to cold RPMI
1640 medium, and stored in the dark on ice. Flow cytometry
(Becton Dickinson FACSCalibur, San Jose, CA, USA) was
used to immediately analyze the DCF-DA fluorescence in-
tensity. Data for controls and each dilution of the DAPP
extract were collected in triplicate. Untreated, H
2
O
2
-treated,
and extract-pretreated cells were compared using the mean
fluorescence intensity of PMN cells.
Migratory response to the inflammatory mediator
leukotriene B4
The effects of DAPP on inflammatory cell behavior was
tested using a transwell migration assay. Each control and
treatment was performed in quadruplicate following the
experimental model. Serial dilutions of DAPP aqueous were
incubated with cells. The plate was allowed to sit for 30 min
with 50 lg/mL fibronection coating the top compartments of
Millipore transwell (3.0 lm pore size) migration plates.
Culture medium containing leukotriene B4 (12 nM) was
placed in the bottom chamber at a volume of 150 lL in the
1206 JENSEN ET AL.
transwell migration plate. Aspiration of the fibronectin from
the top wells was performed before plating of cells. The top
chambers were plated with 50 lL of cells (1 ·10
6
/mL), then
lowered into the bottom plate, and allowed to incubate for
4 h at 37C. The top chambers were removed after the in-
cubation. CyQuant
was used to stain the relative number of
cells that had migrated to the bottom chambers. The Tecan
Spectrafluor plate reader (Durham, NC, USA) was used to
measure the fluorescence intensity. Experiments were re-
peated thrice using PMN cells from three different healthy
donors.
Clinical pilot study
A 12-week clinical pilot study was performed, following
an open-label design. Twelve study participants were
screened and enrolled after written informed consent, as
approved by Sky Lakes Medical Center Institutional Review
Board (FWA 2603). Screening involved an interview to
ensure that subjects met the study inclusion/exclusion cri-
teria, and a prestudy ROM assessment was performed for
two purposes: (1) to ensure that a given study participant had
reduced ROM in specific joints, (2) to accommodate a
learning curve for the detailed ROM assessment, so that data
collection would not be affected at later visits. Inclusion
criteria were as follows: Subjects of either gender, 45–75
years of age, eating a balanced Western diet, with more than
6 months of chronic pain in well-defined area(s). Exclusion
criteria were recent trauma that would affect ROM and pain
scoring, and recent changes in diet, supplements, or medi-
cation that could potentially affect joint health and pain
scores. Daily consumption of over-the-counter pain medi-
cations as well as supplements that may be beneficial to joint
health was not an exclusion criterion; however, subjects
were instructed to maintain this constant during the study.
The study subjects consumed 4.25 g (nine capsules) of
DAPP daily for 12 weeks, spread over three daily doses of
three capsules. Study participants were instructed to keep
diet and lifestyle constant during the study, and were al-
lowed to consume over-the-counter pain medication if
needed. Compliance was evaluated during each visit, in-
cluding product consumption, diet and lifestyle, and medi-
cations. The study participants were monitored at baseline
and after 2, 4, 8, and 12 weeks, where questionnaires, ROM
assessment, and blood draws were performed (Fig. 1). Ser-
um samples were banked at -80C during the clinical phase
of the study, and used for the testing of serum antioxidant
protection capacity in the CAP-e bioassay after the clinical
phase was completed.
ROM assessment using dual digital inclinometry
The evaluation of ROM was conducted using a detailed
protocol where the ROM of the entire vertical weight-
bearing axis of the body was studied from the neck to the
knees, as well as the shoulders. The rationale behind this
detailed assessment is that often a person’s primary com-
plaint (e.g., left knee) will lead to a compensated posture and
ROM of other anatomical areas such as the lower back, as
the person strives to alleviate pressure on the affected joint.
This complete assessment (15 distinct ROM measurements)
optimizes our ability to show significant changes as a result
of consumption of a nutritional product. The ROM assess-
ment was active, that is, the study participants performed
movements and the extent of movement was recorded. This
method is very reliable, as the software provides voice
prompts for whether three readings gave a good average,
and will prompt approximately six readings, if needed, for
each ROM measurement. A screening ROM exam was
performed before study entry to ensure the subject was used
to the examination procedure so subsequent readings were
more accurate and not affected by learning the instructions
and expected motions. All ROM assessments were per-
formed by a single examiner (D.M.A.), using wireless dual
digital inclinometry ( JTECH Tracker Freedom; JTECH
Medical, Salt Lake City, UT, USA).
Pain assessment
Pain is inherently a subjective parameter, as pain per-
ception is affected by many physiological, emotional, and
stress-related factors. A person’s self-reported pain under
different conditions (at rest versus at use when performing
physical activities) is considered the most reliable tool.
17
Scoring in a research setting is optimally performed using
neutral scoring tools, such as an unmarked 100 mm Visual
Analogue Scale (VAS).
18
For this study, each person’s an-
atomical areas of primary and secondary joint stiffness and
reduced function were identified before the study start. At
each visit, pain levels for both the primary and secondary
areas were scored for ‘‘pain at rest’’ and ‘‘pain at use,’’
using VAS. General pain at the time of the visit was also
tracked. The VAS were 100 mm without increment marks,
FIG. 1. Diagram showing the study design for the 12-
week open-label study. Range of motion (ROM) along
the vertical weight-bearing column (neck to knees) and
shoulders was performed using dual digital in-
clinometry. Additional data involved questionnaire-
based data collection pertaining to pain levels in areas
of primary and secondary pain areas identified at study
start. Blood draws for serum testing was performed at
baseline, 2, 4, 8, and 12 weeks.
DRIED APPLE PEEL IMPROVES JOINT RANGE OF MOTION 1207
where one end was labeled ‘‘no pain,’’ and the other end was
labeled ‘‘intense pain.’’ The score was measured on the
scale in millimeters and scored in percentage.
Statistical analysis
For the in vitro testing, average and standard deviation for
each data set was calculated using Microsoft Excel. Statis-
tical analysis of in vitro data was performed using the two-
tailed, dependent t-test. For the evaluation of ROM and pain
scores, a comparison of arithmetic means was performed
using Student’s t-test, using Microsoft Excel (Microsoft,
Redmond, WA, USA). Statistical significance of changes
from baseline to later assessments was evaluated by
‘‘within-subject’’ analysis performed using the two-tailed
paired t-test. Statistical significance was indicated if P<.05,
and a high level of significance was indicated if P<.01.
RESULTS
Clinical pilot study
The 12-week open-label pilot study involved 12 people of
both genders with well-defined reduced joint function in sev-
eral anatomical locations. These locations were identified at
study startas the primary and secondary complaints (Table 1).
Improved ROM was seen rapidly for some joints, and
more slow improvements were seen for other areas (Fig. 2).
The lumbar area and shoulders showed rapid improvements,
where some ROM measures were significantly increased
already at 2 weeks, when compared with baseline ROM for
each motion (P<.05). Particularly, the right shoulder ad-
duction/abduction reached a high level of significance when
compared with baseline at 8 and 12 weeks (P<.01). Both
the cervical lateral ROM and thoracic ROM showed im-
provements at 4 weeks, where the improvement in cervical
lateral motion was statistically significant compared with
baseline ROM (P<.05). Cervical lateral ROM, thoracic and
lumbar rotation, hip ROM, as well as all ROM associated
with both shoulders were significantly improved after 12
weeks of DAPP consumption when compared with baseline
ROM (P<.05). The improvements in cervical rotation,
thoracic and lumbar rotation also reached a high level of
significance (P<.01), with the lumbar rotation already
showing significance after 2 weeks of consumption.
For eight of the study participants, the anatomical area of
primary pain was in a joint where ROM was being measured
during the study. For seven of these eight people, the ana-
tomical area of primary pain showed improved ROM, and
for six of these people the improvement was robust.
Improved antioxidant status was measured after 2 weeks
of DAPP consumption, and the serum antioxidant protection
status continued to improve throughout the initial 8 weeks of
the study. After the 8 weeks, a plateau was seen, and the
serum antioxidant protection status at week 12 was similar
to week 8, and significantly above baseline (P<.05) (Fig. 3).
Reduction of chronic pain associated with each person’s
primary and secondary complaint areas was reported al-
ready at the first return visit at 2 weeks, and reached sta-
tistical significance at 4 weeks for both the primary and
secondary complaint areas (P<.05). The pain reduction
continued throughout the 12 week study and reached a high
level of statistical significance at study exit (P<.01) (Fig. 4).
Antioxidant capacity of DAPP
The antioxidant capacity of DAPP was evaluated by the
measurement of the ORAC assay (Table 2). The antioxidant
capacity of DAPP was primarily seen in the hydrophilic
compounds, with a small contribution from lipophilic anti-
oxidant compounds.
Cellular antioxidant protection by DAPP
Given the high level of antioxidant capacity in the water-
soluble fraction of DAPP, an aqueous extract of DAPP was
tested for its ability to provide cellular antioxidant protec-
tion in the CAP-e bioassay. A clear dose response was seen,
where measureable cellular protection from oxidative stress
was provided by DAPP starting at a dose of 1 g/L (Fig. 5).
Reduced production of ROS by PMN cells
in the presence of DAPP
The PMN cell type comprises *70% of all circulating
white blood cells in humans. The cell type is inflammatory
in nature, and has the capacity to, on an inflammatory
stimulus, very rapidly produce robust levels of free radicals,
including ROS. Many polyphenol-rich natural products
significantly reduce the production of ROS.
15,17,18
This is
not only a matter of antioxidant protection but also likely
involves signaling events, programming the PMN cell to a
less inflammatory behavior.
19
The PMN cell type is subject
to individual variation between blood donors, depending on
the donor’s nutritional and inflammatory status; therefore,
we present data on the cellular ROS production after DAPP
treatment from three healthy donors (Fig. 6). The treatment
of PMN cells with DAPP before triggering an inflammatory
response resulted in much reduced production of ROS in cell
Table 1. Demographics and Areas of Reduced
Range of Motion at Study Start
Anatomical area with limited ROM at study start
Vol no. Gender Age Primary Secondary
V01 F 72 Hands Right knee
V02 F 48 Knees Fingertips
V03 M 73 Shoulder Neck
V04 F 62 Feet Hips
V05 M 61 Hands Achilles tendon
V06 M 56 Neck/shoulders Knees
V07 F 61 Right shoulder Hips
V08 M 56 Left knee Right hand
V09 M 68 Right ankle Back
V10 F 63 Lower back/hip Knees
V11 M 61 Lower back Upper back spine area
V12 F 56 Neck Back
ROM, range of motion.
1208 JENSEN ET AL.
cultures from all three donors, across a wide dose range of
DAPP. The reduction was statistically significant compared
with untreated cells at all doses of DAPP tested (P<.05).
Inhibition of inflammatory cell migration
in response to leukotriene B4
The recruitment of inflammatory cells into tissue involves
the process of chemotaxis where the inflammatory cells sense
a chemotactic compound and initiate directional migration
toward it. A bioassay using transwell migration plates was
used to test the effect of DAPP on this cellular behavior. The
inflammatory chemotactic compound leukotriene B4 was
added to cell culture medium in the bottom chambers, and
cells pre-treated with various doses of DAPP were added to
the top chambers of the transwell plates. The separating bar-
rier between the two chambers had 3 lm pores to enable di-
rectional cellular transmigration. Cells pretreated with DAPP
FIG. 2. Changes in joint ROM during 12 weeks of consumption of dried apple peel powder (DAPP). The ROM was evaluated along the vertical
weight-bearing column (neck to knees) as well as shoulders, using dual digital inclinometry. Rapid improvements were seen in lumbar and
shoulder ROM, where some types of ROM improvements were statistically significant already at 2 weeks, when compared with baseline ROM
(P<.05). The cervical lateral ROM and thoracic ROM showed improvements at 4 weeks, where the cervical lateral ROM was significantly
improved compared with baseline ROM (P<.05). Cervical lateral ROM, thoracic and lumbar rotation, hip ROM, as well as all ROM associated
with the left and right shoulders were significantly improved after 12 weeks of DAPP consumption when compared with baseline ROM (P<.05).
The level of significance, as calculated by the paired t-test (‘‘within-subject’’ analysis), is indicated by * if P<.05 and by ** if P<.01.
DRIED APPLE PEEL IMPROVES JOINT RANGE OF MOTION 1209
showed a mild reduction in migratory behavior. Importantly,
the LMW fraction of DAPP showed a stronger inhibition of
inflammatory migration across a wider doserange (down to the
low dose of 25 lg/L). The inhibition by DAPP was predomi-
nantly associated with the <3 kDa LMW fraction, where the
inhibition was highly significant when compared with cells
exposed to leukotriene B4 in the absence of DAPP LMW,
across a dose range of 0.025–3.2mg/L (P<.01) (Fig. 7).
Inhibition of inflammatory enzymes COX-2
and lipoxygenase by DAPP
DAPP was tested for its ability to inhibit two enzymes
involved in inflammatory processes: COX-2 and lipox-
ygenase, comparing a crude water extract of DAPP with an
LMW fraction (DAPP LMW). Both COX-2 and lipox-
ygenase enzymes were inhibited by DAPP at a dose of
10 g/L (Fig. 8). Data from the comparison of the crude and
LMW fractions suggest that several synergistic compounds
act on both enzymes, where the LMW fraction provided
better inhibition of COX-2 than lipoxygenase when com-
pared with the crude water extract, suggesting that com-
pounds below 3 kDa are involved in the inhibition of
COX-2. The LMW fraction showed less inhibition than
crude DAPP, but still provided robust inhibition of lipox-
ygenase enzyme activity, suggesting that lipoxygenase in-
hibitors are present in both the high- and LMW fractions.
DISCUSSION
The importance of maintaining joint mobility with aging
is a major factor for remaining physically active and inde-
pendent of caregivers. The resulting cost savings pertaining
to overall physical and mental health, social functioning,
and reduced costs for assisted living are undeniable. The
interest in nutritional products that help maintain healthy
joint function is on a rise, and was an incentive for the study
we are reporting here.
FIG. 3. Changes in serum antioxidant protection during 12 weeks
of consumption of DAPP. Serum samples collected during the study
were tested in a modified cellular antioxidant protection (CAP-e)
bioassay, where the antioxidant protection data reflect whether serum
contained antioxidants of a chemical composition that are available to
enter into and protect cells from oxidative stress. An improvement in
serum antioxidant protection status was statistically significant al-
ready after 2 weeks of consumption, and continued to increase until 8
weeks, after which a plateau was seen. The improvement from
baseline remained statistically significant at all time points (*P<.05).
FIG. 4. Pain scores during 12 weeks of consumption of DAPP.
Joint pain was evaluated at baseline and at all the next visits using
Visual Analogue Scales. Pain reduction was observed already after 2
weeks of DAPP consumption, and it was statistically significant after
4 weeks of consumption (*P<.05). The improvement continued
throughout the 12 week study, at which time the improvement from
baseline was highly significant (**P<.01).
Table 2. Antioxidant Capacity of Dried Apple Peel Powder
ORAC
hydro
lm TE/g 327
ORAC
lipo
lm TE/g 3
ORAC
total
lm TE/g 330
H-ORAC lm CAE/g 57
N-ORAC lmTE
2
/g 23
SOD kU SOD eq/g 1.8
ORAC, oxygen radical absorbance capacity; TE, trolox equivalent; CAE,
caffeic acid equivalent; SOD, superoxide dismutase.
FIG. 5. Cellular antioxidant protection provided by DAPP was
measured using the CAP-e bioassay. Human erythrocytes were
treated with serial doses of DAPP before being exposed to oxidative
stress. The fluorescence intensity of a reporter dye reflected the level
of intracellular oxidative damage. The inhibition of intracellular
damage is shown as the average +standard deviation of duplicate data
points, in reference to negative and positive controls, each of which
are performed in hexaplicate. The protective effects provided by
antioxidants in DAPP, able to enter into and protect the living cells,
were dose dependent, with an IC50 around 20 mg/mL.
1210 JENSEN ET AL.
The daily consumption of a DAPP in an older population
with some joint mobility limitations resulted in rapid and
sustained improvements in joint function. This improvement
was not limited to each person’s identified problem areas;
rather, a general improvement in the function of many joints
was seen. Over the course of the study, the general upper and
lower back ROM improved, with significant improvements
in thoracic, lumbar, and hip rotation (P<.05). Furthermore,
even though only three study participants complained about
reduced function of shoulders and neck, cervical lateral
motion (the bending of the head forward and backward), as
well as shoulder adduction/abduction (arching of the
shoulder/arm from above the head to down across the body)
showed significant improvements when compared with
baseline, with some improvements reaching statistical sig-
nificance already after 2 weeks. The highly significant im-
provement of ROM for the right shoulder at 8 and 12 weeks
(P<.01), given a predominantly right-handed population, is
noteworthy in light of a general tendency to shoulder and
neck tension due to poor posture during work-related tasks.
The improvement in joint ROM (Fig. 2) was parallel to
an improvement in serum antioxidant protection status
(Fig. 3), as measured by significant improvements in serum
CAP-e results already at 2 weeks. The importance of the
test results using the CAP-e bioassay is that the improve-
ment is not solely a reflection of an improved level of
serum antioxidants, but it is also associated with an im-
proved ability of serum to provide antioxidants of a com-
position that are able to enter into and protect live cells
from free radical damage.
The changes in ROM and antioxidant status were also
paralleled by reduced pain scores. The reduced pain levels
FIG. 6. Changes in intracellular production of reactive oxygen
species (ROS) were evaluated using an in vitro bioassay with in-
flammatory polymorphonuclear (PMN) cells from three healthy do-
nors. Pretreatment of PMN cels with DAPP before an inflammatory
insult was introduced resulted in reduction of the levels of ROS in
cells from all three donors, across the dose range of 0.0001–0.1 mg/mL
DAPP (P<.05). The level of significance, as calculated by the in-
dependent two-tailed t-test, is indicated by * if P<.05 and by ** if
P<.01.
FIG. 7. Cellular migration in response to the inflammatory chemo-
attractant leukotriene B4 (LTB4) was evaluated using PMN cells in
vitro. Pretreatment of PMN cells with DAPP before cells were placed
within range of the chemotactic compound LTB4 in transwell mi-
gration chambers resulted in a reduction in migratory behavior of the
cells. The <3 kDa low-molecular-weight fraction of DAPP (DAPP
LMW) showed high potency at very low doses where cellular mi-
gration in response to LTB4 was reduced to baseline levels (UT:
untreated cells). Asterisks indicate doses where the results were sta-
tistically signficant when compared with the positive control (LTB4)
where cells were exposed to LTB4 in the absence of DAPP or DAPP
LMW (P<.05). The level of significance was calculated by the in-
dependent two-tailed t-test and is indicated by * if P<.05 and by ** if
P<.01.
FIG. 8. DAPP was tested for its ability to in-
hibit two enzymes involved in inflammatory
processes: Cyclooxygenase-2 (COX-2) and li-
poxygenase. The tests compared a crude water
extract of DAPP to a <3 kDa DAPP LMW. The
crude extract showed inhibition of both enzymes
at a dose of 10 mg/mL. The <3 kDa LMW frac-
tion provided a proportionally better inhibition of
COX-2 than lipoxygenase when compared with
the crude water extract, suggesting that com-
pounds below 3 kDa are involved in the inhibi-
tion of COX-2.
DRIED APPLE PEEL IMPROVES JOINT RANGE OF MOTION 1211
were seen for both primary and secondary pain areas, and
they were preceded by the improved antioxidant status,
suggesting an association between antioxidant uptake and
subsequent pain reduction. Improved antioxidant status
reached statistical significance above baseline already at 2
weeks, whereas the reduced pain scores reached significance
after 4 weeks (P<.05), and reached a high level of signifi-
cance after 8 weeks (P<.01).
In terms of understanding possible underlying mecha-
nisms of action of DAPP that may help put the clinical data
in perspective, several data from in vitro testing point to
antioxidant properties, as well as to some anti-inflammatory
activity. The cellular antioxidant protection by DAPP seen
in serum samples from people consuming DAPP was mir-
rored when DAPP was added to cells in culture using the
CAP-e bioassay, verifying that DAPP contains antioxidants
of a chemical composition that are capable of entering into
living cells and protecting these cells from free radical
damage both in vitro and in vivo. In parallel to that activity,
DAPP triggered a reduction in free radical formation by
PMN cells. DAPP pretreatment also resulted in a reduced
migratory activity of cells in response to the inflammatory
chemokine leukotriene B4. DAPP was shown to contain
compounds that are able to inhibit the enzymatic activity of
both COX-2 and lipoxygenase enzymes. The LMW fraction,
containing water-soluble compounds smaller than 3 kDa,
showed the strongest inhibition of cellular migration, and
also strongly contributed to COX-2 inhibition, pointing to
the presence of small molecules with the ability to quench
inflammatory responses, and due to the COX-2 inhibition,
likely contribute to pain reduction. Taken together, these
data point to multifaceted mechanisms of action that may
help explain the observed improvements in joint function,
antioxidant status, and pain reduction in older people con-
suming DAPP.
In conclusion, the improvements in joint function asso-
ciated with consumption of DAPP were not limited to an
isolated joint problem. The general improvement seen in
this exploratory pilot study suggests that a general im-
provement in antioxidant status may have led to improve-
ments in joint function in this population. In future studies,
the ROM assessments should include an assessment before
study start, to accommodate the learning aspect of per-
forming the motions, such that an improvement at later visits
is not simply associated with an increased familiarity with
the testing method. Further studies are warranted, and
should include a placebo-controlled dose study to evaluate
at which daily dose improvements in joint function and
antioxidant status can be detected, and should incorporate
tracking of diet and exercise, as well as frequency of addi-
tional adjunct therapies. Future studies may also include
assessment of joint health support in younger populations,
including athletes.
ACKNOWLEDGMENTS
The study was conducted at NIS Labs, an independent
contract research laboratory specializing in natural products
research. The Leahy DAPPproduct was provided by
Leahy Orchards, Inc (Franklin Centre, Quebec, Canada).
The study was sponsored by Michael Leahy, CEO Leahy
Orchards, Inc.
AUTHOR DISCLOSURE STATEMENT
All authors are associated with NIS Labs and have no
competing financial interest in the subject matter.
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