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12
Borgis
INFLUENCE OF BETALAIN-RICH EXTRACT
ON REDUCTION OF DISCOMFORT
ASSOCIATED WITH OSTEOARTHRITIS**
Zbigniew Pietrzkowski1, Boris Nemzer2, Aneta Spórna3, Paweł Stalica3,
Wayne Tresher2, Robert Keller4, Roxanna Jimenez4, Tadeusz Michałowski3,
*Sławomir Wybraniec3
1Bio-Clinical Research, FutureCeuticals, Inc.
2Chemistry Research, FutureCeuticals, Inc.
3Department of Analytical Chemistry, Institute C-1, Faculty of Chemical Engineering and Technology,
Cracow University of Technology
4NutraClinical, Inc.
Summary
Introduction. Osteoarthritis (OA) subjects typically experience progressive discomfort related to pain, joint stiffness, and general
tiredness. The most common treatment of these conditions includes use of non-steroidal anti-inflammatory drugs (NSAIDS).
However, efficacy of NSAID treatment is generally not completely satisfactory. Therefore, further improvements in management
of OA-associated discomfort are needed.
Aim. The aim was to verify whether a betalain-rich red beet extract at dose range of 35-100 mg twice per day could reduce
discomfort associated with osteoarthritis (OA) conditions.
Materials and methods. Study participants experiencing OA symptoms were treated with red beet extract (RBE) twice per day
for exactly ten days. McGill and Energy Score data were evaluated at days 1, 5 and 10. The serum levels of advanced oxidation
protein products (AOPP) were measured using a commercial kit.
Sera from volunteers treated with RBE were subjected to a cytokines and chemokines array.
Results. Collected data showed that ingestion of RBE for 10 days reduced McGill scores in a time- and dose-dependent manner with
maximum 33% reduction as compared to the first day of the treatment. Interestingly, due to the treatment, serum levels of TNF-alpha
were reduced in subjects whose serum TNF-alpha was greater than 1 pg/mL prior to initiation of the treatment. It was also found that
serum levels of AOPP (proteins oxidized by hypochlorous acid/hypochlorites) were reduced by up to 48% after 10 days of the treatment.
Conclusions. This study showed that ingestion of RBE, at dosages greater than 35 mg, had a beneficial effect on pain associated with
OA conditions. RBE may act by inhibiting protein oxidation typically induced by hypochlorous acid released from active neutrophils.
Key words: betacyanins; betalains; osteoarthritis; antioxidants; hypochlorous acid; chlorinated proteins
INTRODUCTION
Osteoarthritis (OA) subjects typically experience
progressive discomfort related to pain, joint stiffness, and
general tiredness (1, 2). The aetiology of this condition is
complex and a number of factors are reported to contribute
to development and progression of this condition (3).
The most common treatments of these conditions include
use of non-steroidal anti-inflammatory drugs (NSAIDS) (4).
However, efficacy of NSAID treatment is generally not
completely satisfactory. Therefore, further improvements
in management of OA-associated discomfort are needed.
Recently, it was reported by Steinbeck (5) that chlorinated
peptides and elevated levels of myeloperoxidase (MPO)
are associated with early OA conditions. Interestingly,
Deberg published in 2008 a clinical observation showing
that serum MPO was significantly reduced in OA patients
after knee replacement (6). In 1991, it was shown by
Katrantzis that the oxidant hypochlorite, a product of
myeloperoxidase, degrades articular cartilage (7). In
2000, Bellometti followed changes in blood levels of nitric
oxide, myeloperoxidase and glutathione peroxidase in
OA patients subjected to mud baths. Interestingly, mud
bath treatment was associated with reduced blood levels
of MPO and nitric oxide (NO) (8). Altogether, these
publications suggest a possible connection between OA
conditions associated with damage of cartilage, and
increased levels of chlorinated proteins and MPO.
Studies on OA treatment with natural formulations
indicating a beneficial effect of ginger extract on the
progress of osteoarthritis were recently discussed (9).
Independently, red-violet betalains and yellow
betaxanthins, the water-soluble nitrogenous pigments
present in members of most families of the plant order
Caryophyllales, were reported to reduce activity of
**This research was financed in part the Polish Ministry of Science and Higher Education for years 2007-2010 (Project
No. N312 3268 33).
13
Influence of betalain-rich extract on reduction of discomfort associated with osteoarthritis
neutrophil-derived myeloperoxidase. As described by
Allegra et al. (10), betanin (one type of betalains) inhibits
myeloperoxidase-mediated oxidation of low density
lipoproteins and may also scavenge hypochlorous acid (11).
Finally, in 2009, it was shown that beetroot juice may inhibit
oxidative metabolism of neutrophils collected from obese
subjects (12). Results presented recently (10-12) show that
betalains may diminish activity of myeloperoxidase,
resulting in reduced generation of hypochloric acid. Other
independent research shows that betalains are well
distributed in vivo after ingestion (13, 14). This characteristic
favours the potential use of betalain-rich natural products
for various health conditions associated with over-activation
of neutrophils, and with involvement of myeloperoxidase
and hypochlorous acid (as described above).
AIM
Following the above rationale, we hypothesized that
ingestion of betalain-rich RBE containing 25% of total
betalains may reduce general discomfort in subjects
experiencing minor forms of OA. Consequently, this
clinical exploratory study was designed to include
measurements using the McGill scoring system and an
Energy Score questionnaire in order to verify whether
RBE may diminish OA discomfort. Study participants
were divided into three experimental groups taking 35,
70 or 100 mg of RBE for 10 days only. Collected results
are presented and discussed in this report.
MATERIALS AND METHODS
Betalain-rich red beet extract (RBE). A novel and
proprietary food-based extract ProLain prepared from red
beet roots was obtained from FutureCeuticals, Inc. USA,
where it was produced using a patent-pending
technology that does not require use of organic solvents
and that significantly reduces amounts of sugar in the
final material.
Betalain analysis. Quantification of betalains was
performed by a spectrophotometric multiple-component
method of Nilsson (15). Betalain profile analysis was
performed according to Wybraniec (16).
Clinical study description and design. This study
was designed to be an open type clinical discovery rather
than a clinical efficacy study. The primary goal was to
verify whether RBE may improve pain and fatigue
associated with osteoarthritis conditions. The secondary
goal of this study was to identify the minimum effective
dose (MED). Therefore, we employed a multiple fixed-
dose type study with three time points: day 1, 5, 10. There
were 8 subjects per experimental group. Each group was
treated with 100 mg (Group 1), 70 mg (Group 2) or 35 mg
(Group 3) twice per day. All participants were asked to
take one capsule of RBE 30 min prior to eating a meal.
Subjects for this study were selected randomly from
a group of people who had been previously diagnosed
with osteoarthritis and who had reported symptoms
characteristic of OA such as joint pain, limited joint
flexibility, and feeling energy-depleted due to chronic pain
and joint problems. We used McGill pain score system
and Energy Score system questionnaires at day 1, 5 and
10 as a means of quantifying symptoms.
Recruitment of subjects, treatments, blood sampling
at day 1, 5 and 10, McGill and Energy Score tests and
blood chemistry were performed by NutraClinical, Inc.
(San Diego, California, USA). Measurements of human
cytokines and chemokines in collected sera were
provided by Quensys, Inc. on a research service basis.
AOPP testing was performed on sera collected from
subjects at day 1 and 10 days after the treatment in
FutureCeuticals’ lab using a commercially available kit
(Cell Biolabs, Inc, USA).
RESULTS
Betalain composition. Red beet (Beta vulgaris L.) is
commonly consumed as a food product and
FutureCeuticals’ RBE is a specially processed extract
obtained from this material. Due to FutureCeuticals’ RBE
production process, this material is depleted of sugars
and enriched in total betalains up to 25%. The relative
betalain composition in RBE was approximately
expressed by chromatographic signal values of main
analysed pigments measured at their λmax: betanin
(12.6), isobetanin (15.6), 17-decarboxy-betanin (0.60),
17-decarboxy-isobetanin (0.44) and neobetanin (3.9).
Effect of RBE on pain feelings as measured using
McGill test. This study was performed to verify our
hypothesis that betalain-rich food-grade material RBE
may reduce discomforts associated with painful and
swollen joints in people suffering osteoarthritis.
Collected results show that all subjects reported
reduced pain level in a dose-dependent manner as
measured by using the McGill Questionnaire. Detailed data
are provided in table 1. Following these data, it is clearly
noticeable that treatment with RBE resulted in a significant
improvement of the sensory part of the questionnaire.
Less improvement was observed for the affective
aspects, and no effect was observed for the evaluative
part of the questionnaire. Following this trend, treatment
with 70 mg of RBE resulted in 41% reduction of pain as
evaluated by the sensory part, but the total score for the
McGill Questionnaire in this experimental group yielded
a 33% reduction after day 10. It is also interesting to note
that 5 days of treatment with 70 mg of RBE already
resulted in 33% reduction of pain (total McGill score).
This indicates that treatment with RBE may provide a
moderately rapid effect (although not as acute as the
effects of painkiller drugs such as NSAIDS). Exit interviews
of study participants revealed that the first subjective
improvements in pain were noticed after 3 days of the
treatment. This observation strongly suggests that a
3-day period should be included in any future RBE clinical
efficacy study protocol to follow rapid activity and effect
on improvement of OA pain-related conditions.
Blood chemistry analysis. Standard serum
biochemistry analysis was performed on each serum
collected at day 1 and day 10. No unusual changes were
noted in any parameters. All parameters were within the
normal range (data not shown).
14
Zbigniew Pietrzkowski et al.
Subjective energy tests. In parallel to the McGill
Questionnaire, all participants were required to answer
4 questions pertaining to their energy feeling rate (Q1),
awareness rate (Q2), endurance rate (Q3) and mood rate
(Q4). Rates for all these questions were scaled 1-4.
This questionnaire (described in this article as Energy Score)
was performed at day 1, 5 and 10. The highest number
indicated a generally elevated level of the feeling rate.
All detailed data of this questionnaire are presented
in Table 2. These data show that all participants reported
feelings of increased awareness, energy, endurance
and mood in a dose-dependent manner after treatment
with RBE. Treatment with 70 mg resulted in 122%
improvement over day 1 (tab. 2), whereas the treatment
with RBE at a dose of 100 mg resulted in 81%
improvement, indicating that treatment with the lower
dose of 70 mg was optimal. Therefore, as was noted
when analyzing the McGill data, the 70 mg dose seems
to be the most potent for improvement of parameters
listed in Table 2. Also, the lowest dose of 35 mg still
provided significant Energy Score increases of up to 74%
after 10 days of the treatment. In comparison, the McGill
data for the same dose resulted in only 11% improvement.
This may indicate that a primary effect of RBE is to
modulate feelings of energy, mood, endurance and
awareness, since a dose as low as 35 mg caused
improvement of Energy Score up to 74%. These results
were rather unexpected, since the energy score was
puorG esoD
serocSlliGcM
traPyrosneStraPevitceffAtraPevitaulavE
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gm001-1puorG
egarevA78.85*00.14*00.0421.426.3*52.273.6126.5178.51
DtS13.304.412.708.191.284.124.338.454.4
xaM367415664 00.0200.1200.12
niM355303 111 00.0100.800.8
EtS71.155.145.236.077.025.002.117.175.1
muS174823023339281131521721
lliGcM
erocS 21.85:01yaD;42.06:5yaD;63.97:1yaD
gm07-2puorG
egarevA26.95*73.44*5.5352.200.257.105.5173.3157.41
DtS75.586.0127.413.296.107.098.369.318.2
xaM863664663329181
niM355323 111 21901
EtS79.177.366.118.095.052.073.104.199.0
muS774553482816141421701811
lliGcM
erocS 00.25:01yaD47.95:5yaD;73.77:1yaD
gm53-3puorG
egarevA52.3621.6526.4552.221.273.278.1252.0257.12
DtS20.768.995.907.038.015.058.282.308.3
xaM570766 333 526282
niM557353112616171
EtS84.284.393.352.092.081.000.161.143.1
muS605944734817191571261471
lliGcM
erocS 48.87:01yaD;94.87:5yaD;73.78:1yaD
Table 1. Effect of RBE on McGill score after 5 and 10 days of treatment of OA subjects.
aSum of all values in the group
15
Influence of betalain-rich extract on reduction of discomfort associated with osteoarthritis
followed only as an additional subjective parameter to
supplement the McGill pain score.
Analysis of advanced oxidation protein products.
The serum levels of advanced oxidation protein products
(AOPP) were measured using a commercial kit (Cell
Biolabs, Inc., #STA318). This assay measures serum
proteins modified by chloramine or hypochlorous acid.
The detailed collected data are summarized in table 3.
The data show a significant broad range (max and min)
in baseline of AOPP at day 1 in each experimental group.
Interestingly, this range was significantly reduced in each
group after 10 days of the treatment. Resulting sum data
show 36.3, 47.6 and 30.9% reduction in groups 1, 2 and
3, respectively. However, due to the broad range of AOPP
values at day 1, StD is relatively high.
Cytokines and chemokines array. Sera from
volunteers treated with RBE were subjected to a cytokines
and chemokines array as offered by Qynsys Inc. Collected
data showed that prior to treatment only 10 participants
out of 24 were found to have TNF-alpha above the
detection limit of 1 pg/mL per ELISA assay. However,
treatment with RBE caused reduction of TNF-alpha in
Table 2. Effect of treatment with RBE on Energy Score data.
1yaD5yaD01yaD
1Q2Q3Q4Q1Q2Q3Q4Q1Q2Q3Q4Q
1puorG
egarevA41.182.182.124.124.141.217.184.241.282.275.275.2
DtS73.084.084.035.035.073.084.087.096.059.079.079.0
xaM 222223243444
niM 111112121112
EtS41.054.054.002.002.041.081.092.062.053.063.063.0
muS 999 010151217151618181
DtS-/+muSegarevAerocSygrenE
5.0-/+52.91.3-/+5.31 5.1-/+57.61
1yadrevoesaercni%18
2puorG
egarevA21.121.152.121.100.200.252.200.273.256.205.205.2
DtS53.053.064.053.003.160.188.000.004.181.129.029.0
xaM 222244424444
niM 111111121122
EtS21.021.061.021.064.073.013.000.004.181.123.023.0
muS890196161816191120202
DtS-/+muSegarevAerocSygrenE
18.0-/+0.90.1-/+5.618.0-/+0.02
1yaDrevo%221
3puorG
egarevA41.182.182.175.175.124.117.141.200.241.224.241.2
DtS73.084.084.035.035.035.084.096.05.073.079.096.0
xaM 222222233343
niM 111111111211
EtS41.081.081.081.002.002.081.062.012.041.063.062.0
muS899111101215141517151
DtS-/+muSegarevAerocSygrenE
5.1-/+57.861.2-/+0.21 52.1-/+52.51
1yaDrevo%77
16
Zbigniew Pietrzkowski et al.
Table 3. Effect of treatment with RBE on serum level of AOPP [ěM]
tluseR
1puorG2puorG3puorG
1yaD01yaD1yaD01yaD1yaD01yaD
egarevA9.022.314.128.212.9112.51
dtS9.412.74.213.70.89.4
xaM8.057.320.444.529.334.52
niM8.78.67.80.55.82.01
muS6.4412.291717.984515.601
seulavegarevaninoitcudeR%
9.632.058.02
seulavmusninoitcudeR%
3.636.749.03
Table 4. Effect of RBE on blood level of selected cytokines and chemokines in subjects with TNF-alpha blood level >1 pg/mL.
All other cytokines and chemokines are presented as concentration pg/mL. Upper number in row represents level of
measured peptide at day 1, bottom number at day 10. DOD1= change in peptide level at day 10 over day 1 and expressed
as % of change.
these 10 subjects after 10 days of the treatment (tab. 4).
The same sera were additionally tested for changes in the
levels of other cytokines and chemokines. This screening
123456
tcejbuSpuorGahpla-FNT6-LIahpla-ORGSETNAR
1
1
52.1
00.1
77.1
26.1
8.813
0.532
244152
013812
242.03
34.82
9.641
9.531
5.321
6.46
24573
90172
367.2
61.1
9.3
2.2
5.752
0.722
72163
84233
1DOD%0.82%7.32%0.92%6.61
N3333
4
2
50.3
24.1 DN 5.311
7.62 00832
99001
534.1
73.1
56.6
07.5
9.07
3.15
28503
12832
642.2
11.1
95.2
68.1
3.405
9.603
799083
183112
770.611
99.08
2.674
5.973
2.021
4.91
23972
74652
1DOD%0.53%0.22%2.75%7.33
N4344
8
3
21.143.13.37.4
3.37
9.36
25251
51131
916.107.12.12.2
5.84
4.23
27663
00671
0153.271
88.171
2.392
5.862
2.531
5.611
62851
56201
1DOD%3.8%3.82%0.12%0.43
N3333
yielded data showing that treatment with RBE reduced
serum level of IL6, GRO-alpha, and RANTES levels after
10 days of the treatment (tab. 4).
17
Influence of betalain-rich extract on reduction of discomfort associated with osteoarthritis
DISCUSSION
Data collected in the form of Energy and McGill scores
show that RBE may indeed provide relief for conditions
associated with OA. According to the working hypothesis
mentioned at the beginning of this article, betalains may
improve OA conditions due to their inhibitory effect on
the chlorination of protein by hypochlorous acid released
from activated neutrophils. This hypothesis was based
on two rationales: 1) that betalains can reduce the amount
of hypochlorous acid generated by activated neutrophils;
and 2) that chlorinated proteins may contribute to onset
of osteoarthritis and associated conditions.
In order to begin testing this hypothesis, the serum levels
of AOPP were measured. The results presented herein are
only indicative but justify further clinical investigation on OA
subjects with increased serum AOPP levels.
AOPP is known as a pro-inflammatory factor and
inducer of TNF-alpha release from monocytes. Therefore
it was reasonable to verify whether treatment with RBE
may result in reduction of blood TNF-alpha levels. In order
to further investigate possible actions of betalains, sera
from volunteers treated with RBE were subjected to a
cytokine and chemokine array. In addition, it was found
that treatment with RBE also resulted in reduction of
blood level of IL-6, RANTES, and GRO-alpha. Due to the
rather limited number of participants per group showing
serum level of TNF-alpha higher than 1 pg/mL, data in
Table 4 are presented as indicative rather than definite.
Further work on a higher number of OA subjects is
required in order to confirm these observations and to
understand any mechanisms of these effects.
It should be stressed that subjects with initial serum
levels of TNF-alpha below 1 pg/mL also reported
reduction of McGill score and improvements in Energy
Score. This observation suggests that RBE may improve
McGill and Energy scores in OA subjects in a TNF-alpha-
independent manner. The investigators find this to be an
intriguing observation that requires further research.
CONCLUSIONS
1. Betalains present in RBE may reduce the
detrimental effect of hypochlorous acid released from
active neutrophils in human subjects by inhibiting protein
chlorination typically induced by hypochlorous acid.
2. Chlorinated proteins may contribute to onset of
osteoarthritis and associated conditions.
3. These promising results are presented as new
preliminary clinical observations that justify further clinical
efficacy studies.
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Received: 16.10.2009
Accepted: 12.11.2009 Correspondence to:
*Sławomir Wybraniec
Department of Analytical Chemistry, Institute C-1,
Cracow University of Technology
Warszawska Str 24, 31-155 Cracow, Poland
phone: +48 12 628 30 74, fax: +48 12 628 20 36
e-mail: swybran@chemia.pk.edu.pl
