ARTICLE
INTRODUCTION
There is a growing interest in medicinal botanicals as complementary
medicine all over the world.
Sambucus species were reported to have been used for years by
native Americans, mostly against rheumatism and fever [1, 2]. Sambucol
is a natural remedy with antiviral properties, especially against the
human influenza virus. The main ingredient of the formulation is an extract
of the black elderberry (Sambucus nigra L.) [1]. The preparation
also contains smaller amounts of Echinacea and Propolis. The black elderberry,
which is widely used in Europe for juices and preserves, and designated
as a "natural antibiotic product" contains high levels of three bioflavonoids
and anthocyanins [3, 4]. Bioflavonoids possess a wide spectrum of biological
activities, including antioxidant properties. The antioxidant and prooxidant
activities of an elderberry extract on low density lipoproteins have been
demonstrated [5]. Antioxidants disarm free radicals, thus decreasing tissue
damage. They also prevent damage caused by irradiation [6, 7]. Extracts
of plants containing bioflavonoids and purified flavonoids were active
against Herpes virus Type 1, respiratory syncytial virus, parainfluenza
and influenza viruses [7]. Sambucol was shown to inhibit the hemagglutination
and the replication of different human influenza viruses, both type A
and B. It was also shown in vitro to be effective against 10 strains
of the influenza virus [8]. In a double-blind, placebo-controlled, randomized
study, Sambucol reduced the duration of flu symptoms to 3-4 days. Convalescent
phase serum antibodies showed higher antibody levels to influenza virus
in the group treated with Sambucol, as compared to the control group [8].
The prevention of flu like symptoms by Sambucol was demonstrated as well
in a colony of chimpanzees in the Jerusalem Zoo [9].
Echinacea, the purple coneflower, is used as a herbal medicine against
the common cold and bronchitis [1, 10]. Studies have shown that preparations
of this plant affect the phagocytic immune system but not the acquired
immune system or the production of cytokines [11-13].
Propolis is produced by the honey bee from plant resins. It has been
used as an anti-bacterial material for wound cleaning and as an anti-inflammatory
remedy for over 2,000 years. Propolis is considered to be an anti-oxidant,
having a protective effect on the liver [14]. It has also been shown to
reduce damage caused by irradiation [15].
The aim of the present study was to determine the immune enhancing potency
of Sambucol preparations by studying their effect on the production of
inflammatory cytokines by human peripheral blood monocytes of healthy
individuals.
MATERIALS AND METHODS
Healthy donors blood monocyte cultures
Twelve donors (young, healthy students with no inflammatory conditions
and currently receiving no medication) participated in this study. Heparinized
peripheral blood (50 ml) was obtained from each subject and diluted 1:1
with phosphate-buffered saline (PBS). The mononuclear cells were separated
by Ficoll-Hypaque sedimentation (400 g, 30 min) and washed three times
in PBS (175 g, 10 min) to remove platelets. An aliquot of cells was counted
in a hemocytometer, and viability was checked using the trypan blue exclusion
method. The adherent cells were > 90% monocytes by beta-naphthyl acetate
non-specific esterase staining [16]. The cells were suspended in RPMI
1640 media, supplemented to a final concentration of 1 mM sodium pyruvate,
50 U/ml penicillin, 50 mg/ml streptomycin, 2 mM L-glutamine, 1/100 MEM-vitamins
and 2% inactivated human AB serum. The cells were plated in 24-well culture
dishes at a concentration of 4 x 106 cells per well and incubated
for 90 min at 37° C in a humidified atmosphere containing 5% CO2.
Non-adherent cells were removed by aspiration and the wells washed three
times with PBS. The adherent cells were cultured for 24 hours in RPMI
media without the addition of serum. Different Sambucol formulations (1/10
dilution, 10 mul/well) or LPS (100 ng/ml) as a control monocyte stimulator,
were added to the monocytes. At the end of the culture period (24 hours),
the supernatants were harvested, centrifuged (300 g, 10 min) and stored
at - 70° C until assayed for cytokines, as previously reported [17,
18].
Sambucol preparations
A standardized extract of the black elderberry, (E.Ex.) is the main
ingredient of different Sambucol preparations. Sambucol Black Elderberry
Syrup (B.E.) contains 38% E.Ex., glucose, raspberry extract, citric
acid and honey. Sambucol Active Defense (A.D.), in the USA known
under the name Immune System Formula, contains also 38% E.Ex.,
glucose, raspberry extract, citric acid, honey, Echinacea angustifolia,
Echinacea purpurea, propolis, ascorbic acid and zinc gluconate. Sambucol
for Kids (Kids) contains half the amount of E.Ex. (19%) used in the
other formulations and in addition - glucose, raspberry extract, citric
acid, E. angustifolia, E. purpurea and propolis.
The anthocyanin content of the Sambucol formulations was assessed by
measuring the absorbance at 526 nm (not less than 0.6 for B.E, A.D. and
0.3 for Kids [1, 7].
Cytokine assays
Levels (pg/ml) of inflammatory cytokines (IL-1beta, IL-6, IL-8 and TNF-alpha)
in supernatants of monocyte cultures were measured by a solid phase ELISA
(R&D, Minneapolis, USA). This assay employs the quantitative "sandwich"
enzyme immunoassay technique. A monoclonal antibody specific for the cytokine
molecule was pre-coated onto the polystyrene microtiter plate. Standards
and samples were introduced into the wells where the immobilized specific
antibodies bind the cytokines. After washing away any unbound proteins,
the second enzyme-linked polyclonal or monoclonal antibody specific for
the cytokine was added to the wells to "sandwich" the cytokine immobilized
during the first incubation. Following a wash to remove any unbound antibody-enzyme
reagent, a substrate solution was added to the wells, causing the development
of a color that was proportional to the amount of cytokine bound in the
initial step. The color development was stopped and the intensity of the
color was measured. A standard curve plotting the optical density versus
the concentration of a given cytokine was prepared, and used to determine
the concentration of the cytokine in unknown samples, as previously reported
[17-20].
Evaluation of Sambucol's effect
The effect of the different formulations of Sambucol on the induction
of inflammatory cytokines production (in pg/ml) was also expressed as:
Statistical analysis
Comparison between groups was performed by Mann-Whitney's test. A value
of p < 0.05 was considered significant.
RESULTS
The Sambucol E.Ex. and its three formulations (B.E., A.D., Kids) significantly
stimulated the production of the inflammatory cytokines IL-1beta, IL-6,
IL-8 and TNF-alpha by normal human monocytes.
The effects of the Sambucol formulations and of LPS (the well known
positive control for monocyte activation) expressed as range (minimum
and maximum induction levels) and as mean ± SE of stimulation indesces,
for each of the cytokines tested by 12 healthy donors' monocytes, are
presented in Table 1: IL-1beta
(Table 1a), TNF-alpha (Table
1b), IL-6 (Table 1c) and IL-8
(Table 1d).
In addition, the effect of Sambucol preparations on the actual amounts
of cytokines produced (expressed as pg/ml) in comparison to the untreated
control monocytes, which represent physiological levels, are presented
in Table 2: IL-1beta (Table
2a), TNF-alpha (Table 2b), IL-6
(Table 2c) and IL-8 (Table
2d).
The highest stimulation index was observed with the E.Ex., followed
by B.E. and A.D. which contain the same amount of elderberry extract (E.Ex.)
and had similar stimulatory effect on these cytokines. Kids, which contains
the lowest amount of extract, showed indeed the lowest stimulation index.
For the E.Ex., the highest mean stimulation index (44.9) was observed
for the induction of TNF-alpha, followed by IL-6 (19.5), IL-1beta (6.4)
and IL-8 (3.3).
The effect of Sambucol preparations and of LPS (tested in 7 out of 12
donors) expressed as mean stimulation indexes, on the production of inflammatory
cytokines for all individual donors, are shown in Figures
1-4:
IL-1beta (Figure 1), TNF-alpha
(Figure 2), IL-6 (Figure
3) and IL-8 (Figure 4).
E.Ex. was the strongest activator for all 4 cytokines and was statistically
significantly different from the other Sambucol formulations, except for
IL-8 (p < 0.05). However, there were no statistically significant differences
between effects of B.E., A.D. and Kids. LPS induction of all cytokines
was lower than that induced by E.Ex (p < 0.05). For IL-8, the effect
of LPS was similar to that of E.Ex. and a little higher than the other
3 formulations. For IL-6, the effect of LPS was higher than B.E., A.D.
and Kids. For IL-1beta, the effect of LPS was higher then B.E., A.D. and
Kids. For TNF-alpha the effect of LPS was a little higher than that of
B.E., A.D. and Kids. There was no significant difference between levels
of cytokines activated by B.E. and A.D.
A representative assay performed on monocytes from one single donor
(K.L.) showing cytokine production is presented in Figure
5.
When the Sambucol preparations were mixed together with LPS to activate
the monocytes, a synergistic effect was found, especially for E.Ex. (data
not shown).
DISCUSSION
The results of this study show that all Sambucol elderberry formulations
have a strong stimulatory effect on inflammatory cytokine production by
human normal monocytes. With the exception of IL-8, the stimulatory activity
was dose-dependent, i.e., the E.Ex. with the highest extract concentration
of Sambucol showed the highest cytokine stimulation, while the less concentrated
B.E. and A.D. caused a less pronounced stimulation. The less concentrated
formulation, Kids, had the lowest effect on the production of inflammatory
cytokines. The most impressive effect of E.Ex. was on TNF-alpha
production. The extremely high amounts of TNF-alpha produced by Sambucol,
were even higher than those produced by LPS. TNF-alpha is a multipotential
mediator of cellular immune responses with a wide variety of biological
activities. Under different conditions, TNF-alpha can exhibit favorable
or unfavorable effects on the host immune response. The unfavorable effects
may occur with high concentrations of TNF-alpha or with low concentrations
- for longer periods [21]. The Sambucol preparations, especially the E.Ex.,
when added together with LPS, had a synergistic inducing effect.
Echinacea, propolis, ascorbic acid and zinc gluconate, which
are included in the A.D. formulation, but not in the B.E., did
not enhance the stimulatory effect of the E.Ex. and even appeared to reduce
it (data not shown).
Our results are in accordance with the studies which showed that the
extracts of Echinacea are not able to induce increased cytokine
production [10, 11]. Levels of the cytokines (IL-1alpha, IL-1beta, IL-2,
IL-6, TNF-alpha and IFN-gamma) were measured in culture supernatants of
stimulated whole blood cells derived from 23 tumor patients undergoing
a 4-week oral treatment with an Echinacea complex. No significant
alteration in the production of cytokines was detected [11]. In healthy
volunteers, two out of five studies performed with different preparations
of Echinacea showed an enhancement in the phagocytic activity of
polymorphonuclear neutrophil granulocytes [21].
Propolis was shown to enhance murine macrophage spreading and mobility
[22]. Its influence on the production of cytokines has not yet been reported.
In addition to Sambucol's antiviral properties [7, 8], the E.Ex.
and the other Sambucol formulations were shown in this study to activate
the immune system by strongly increasing inflammatory cytokines production.
In preliminary studies, we have shown that Sambucol formulations significantly
induce the production of both Th1( IFN-gamma and sIL-2R) and Th2 (IL-10)
cytokines, as well as the production of the hematopoietic growth factor
GM-CSF by monocytes and lymphocytes from healthy individuals. (unpublished
results).
Sambucol may therefore be beneficial to the stimulation of the immune
system in healthy individuals, as well as in patients with influenza,
cancer and HIV, which have decreased immune functions. As we have shown
previously, breast cancer patients undergoing chemo/hormonotherapy and
provided with an addition of interferons, which activated the immune system,
had a better therapy response concomitant to changes in cytokine production
[24]. We believe therefore, that Sambucol may be beneficial to cancer
patients receiving chemotherapy. The immunoprotective or immunostimulatory
effects of Sambucol, in various patients will be assessed in future clinical
trials.
CONCLUSION
Acknowledgements. This study was not funded in any way by the
company which produces and markets the product of Sambucol in Israel.
As the Israeli Cytokine Standartization Laboratory, we have performed
many studies dealing with various activation pathways of human cytokines.
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