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Bull Vet Inst Pulawy 50, 461-465, 2006

IMMUNOSTIMULATORY EFFECT
OF IMMUNOSTIM PLUS A STANDARDIZED
FIXED COMBINATION OF SCHIZANDRA CHINENSIS
WITH ELEUTHEROCOCCUS SENTICOSUS EXTRACTS
ON LYMPHOCYTE-DEPENDENT CELLULAR IMMUNITY
IN MICE
EWA SKOPISKA-REWSKA, ANDRZEJ K. SIWICKI1, ROMAN WJCIK1,
JOANNA MAACZEWSKA1, SYLWIA TRAPKOWSKA1, JADWIGA NARTOWSKA2,
BARBARA J. BAAN AND ALEKSANDER WASIUTYSKI
Department of Pathology, Medical University of Warsaw, 02-004 Warsaw, Poland
Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine,
University of Warmia and Mazury, 10-917 Olsztyn, Poland
2
Department of Pharmacognosy, Faculty of Pharmacy,
Medical University of Warsaw, 02-097 Warsaw, Poland
ewaskop@hotmail.com

Received for publication January 30, 2006.

Abstract

Material and Methods

The in vivo effects of fructus Schizandrae and radix


Eleutherococci combined extracts on various parameters of
lymphocyte activity in Balb/c mice were studied. Feeding mice
300, 600 or 1200 g of the extracts per day for 7 d, produced
the activation of chemokinetic activity of spleen lymphocytes,
enhancement of proliferative response of splenocytes and
blood lymphocytes to mitogens, stimulation of the graftversus-host activity of splenic lymphocytes, and stimulation of
SRBC antibody production.

Animals and experimental design. The study


was performed on 8-10 week old inbred Balb/c mice,
weighing about 20 g, of both sexes, delivered from a
breeding colony from the Polish Academy of Sciences,
and Warsaw and Mazurian (Olsztyn) universities. The
studied material was Immunostim Plus (Herbapol
Lublin, Poland), 300 mg capsules, composed of dried
extract of Eleutherococcus senticosus radix (155 mg),
dried extract of Schizandrae chinensis fructus (100 mg),
and adjunctive substances (50 mg). The mice received
Immunostim for 7 d in daily dose of 600 g. The dose
corresponded to 300 mg given to a 70 kg person
(applying the counter 7 for differences between the
mouse and human in relation of body surface to body
mass), and was suggested by the producer as a daily
therapeutic dose. This dose was used in all our
experiments. The effect of higher (1 200 g) or lower
(300 g) dose of Immunostim was also evaluated in
some experiments. Mice received orally (feeding with
the use of Eppendorf pipette) Immunostim dissolved in
45 L of water or in 10% ethanol, and the same dose of
10% ethanol or water as corresponding controls.
On day 8, some mice were immunised with
10% SRBC (0.2 mL intraperitoneally), and bled 7 d after
the immunisation. The remaining group of mice was
anaesthetized with chloral hydrate, bled from the
retroorbital plexus, and euthanized. Their spleens and
blood samples were used for cellular immunity tests, and
the Splenocytes were isolated from mice under sterile

Key
words:
mice,
Schizandra,
Eleutherococcus, lymphocytes, cellular immunity,
humoral immunity.
Eleutherococcus senticosus (siberian ginseng)
is a commonly used herbal preparation with
adaptogenic, anti-stress, and immunomodulatory
properties (3, 5, 8, 10, 18, 19). Schizandra chinensis
(Magnoliaceae), Chinese herb, has a long history of its
medical use as adaptogen and anti-oxidant, and because
of its hepato-protective properties. It was effective
against viral and chemical induced hepatitis (12).
However, data concerning the solitary immunotropic
effects of Shizandra, or immunotropic effects from the
combination of Siberian ginseng with Shizandra are
lacking. The aim of this study was to evaluate, for the
first time, the effect of these two medicinal plant extract
combinations, for the important parameters of in vivo
lymphocyte function in mice.

462
conditions by straining through stainless sieve and
cotton gauze, and then centrifuged on Lymphoprep in
order to remove erythrocytes.
Spleen cell chemokinesis (spontaneous
migration) assay in vitro was performed according to
the Sandberg method (9) with our own modifications
(2). Briefly, isolated splenocytes were resuspended in a
Parker culture medium with 5% inactivated foetal calf
serum (FCS), at the final concentration of 30x106
cells/mL. Afterwards, siliconized capillary tubes were
filled with cell suspension, sealed with plasticine,
centrifuged (5 min, 450 g), and then fixed on the glass
plates. After 24 h incubation (370C, 5% CO2), the
distances of migration were measured in millimetres at a
magnification of 6.5 x, and presented as migration units
(1 M.U.= 0.18 mm).
Examination of antibody level. The antibody
level was evaluated by using the haemagglutination
assay, in inactivated (560C, 30 min) sera. After
performing serial serum dilutions, 0.5% SRBC was
added, and the mixture was incubated for 60 min at
room temperature, then centrifuged (10 min, 150 g) and
finally shaken. The Haemagglutination titer was
evaluated under a light microscope; and the last dilution
where at least 3 cell conglomerates were present in at
least 3 consecutive fields at objective magnification 20x,
were considered positive.
Influence on the graft-versus-host reaction.
Local graft-versus-host reaction (lymphocyte-induced
angiogenesis test, LIA) was performed according to
Sidky and Auerbach (11). Briefly, mice were fed
Immunostim Plus, as described above, next the spleens
were dissected, and spleen cell suspensions were grafted
intradermally (5 x 105 cells in 0.05 mL of Parker
medium per graft) into F1 mice (Balb/c x DBA2).
Before performing the injections, the mice were
anaesthetised with 3.6% chloralhydrate (0.1 mL per 10 g
of b. w.). Both flanks of each mouse were finely shaved
with a razor, and 2-3 injections were localised on each
flank. Cell suspensions were supplemented with 0.05
mL/mL of 0.01% trypan blue, in order to facilitate
recognition of injection sites later on.
The grafted spleen cells recognised DBA2
antigens, and produced many immunological mediators
including proangiogenic growth factors (immunological
angiogenesis). A number of newly formed blood vessels
were the indirect measure of this aspect of lymphocyte
reactivity.
After 72 h, the mice were treated with a lethal
dose of Morbital (Biowet, Puawy, Poland). All newly
formed blood vessels were identified and counted under
microscopic dissection on the inner skin surface, at 6x
magnification, using criteria suggested by the authors of
the method. Statistical analysis was done by Student's ttest.
Isolation of cells from blood. Three groups of
mice were bled (control and mice fed 600 g or 1 200
g of Immunostim Plus), and blood from 5 mice was
pooled in each group. Leukocytes were isolated from
blood by centrifugation at 200 g for 30 min at 40C on the
Gradisol L gradient (Aqua-Medica, Poland), washed 3
times in PBS, and then resuspended in RPMI 1640

medium (Sigma), supplemented with 10% FCS (GibcoBRL) at a stock concentration of 2 x 106 cells/mL of
medium. The viability of the cells was checked by
supravital staining with 0.1 % w/v of trypan blue.
Proliferative response of splenic and blood
lymphocytes. Proliferative responses of lymphocytes
stimulated by mitogen concanavaline A (ConA),
phytohaemagglutinin (PHA), or lipopolysaccharide
(LPS) were determined by MTT assay (7, 13, 20). MTT
[3-(4,5-dimethyl thiazol-2-yl) 2,5-diphenyl-tetrazolium
bromide] (Sigma) was dissolved in PBS at concentration
of 5 mg/mL and filtered. On 96-well culture plates
(Costar, USA) 100 L of blood lymphocytes in RPMI
1640 containing 10% FCS, 2 mmol L-glutamine, 0.02
mmol 2-mercaptoethanol, 1% Hepes buffer, and
penicillin/streptomycin (100 U/100 g/mL), were mixed
with 100 L of RPMI 1640 containing mitogens ConA
(5 g/mL), PHA (10 g/mL) or LPS (20 g/mL). Three
cultures from each pool of leukocytes were established.
After 72 h incubation at 370C in 5% carbon dioxide
atmosphere (Asab Incubator, Sweden), 50 L of MTT
solution was added into each well, and the plates were
incubated for 4 h at 370C. After incubation, the plates
were centrifuged (140 g, 150C, 5 min), supernatants
were removed, 100 L of DMSO (Sigma) was added
into each well, and then incubated for 15 min at room
temperature. After incubation, the solubilized reduced
MTT was measured colorimetrically at 620 nm in a plate
microreader (MRX 3 Dynatech).
The results from 3 cultures were pooled. The
mean values and standard errors from pooled
experiments were used for comparison between the
groups by Students t-test.

Results
The results from the effect of Immunostim Plus
feeding on chemokinetic activity of mouse spleen
lymphocytes in vitro, and on the graft-versus-host
activity in vivo, after grafting these cells intradermally to
F1 hybrids, are presented on the Table 1. As can be seen
from the Table, highly significant stimulation of
splenocyte migration in in vitro cultures, and significant
in vivo stimulation in local graft-versus-host response
(LIA tests), for both tested Immunostim doses were
found. Highly significant stimulation of splenic and
blood lymphocyte proliferative response to mitogens
and stimulation of SRBC antibody production, were also
observed (Table 2 and Fig.1). The presented results
indicate that Immunostim Plus may be used to improve
antibody production and lymphocyte-mediated cellular
immunity. All the tested doses displayed stimulatory
properties.

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Table 1
Stimulatory in vivo effect of IMMUNOSTIM-PLUS on the in vitro chemokinetic
and in vivo graft- versus- host activites of Balb/c mice splenic lymphocytes
Chemokinetic activity

G-v-h activity

IMMUNOSTIM dose
(g/mouse)

Number of tests

Mean migration distance


SE

Number of
tests

Mean number
of newly-formed vessels
SE

24

5,67 0,34

26

13,8 0,33

300

24

11,29 0,55***

17

16,8 0,27**

600

24

9,58 0,42***

17

15,1 0,47*

*P<0.05

**P<0.01

*** P<0.001

Table 2
Stimulatory in vivo effect of IMMUNOSTIM-PLUS on the proliferation activity of mice splenic
and blood lymphocytes in the in vitro 72 h cultures with mitogens (mean OD 620 nm SE)
Daily dose (g/mouse)
Spleen

Blood

600

1200

PHA

0.306 0.002

0.362 0.01 ***

0.468 0.01***

ConA

0.209 0.004

0.282 0.003***

0.304 0.004***

LPS

0.315 0.003

0.387 0.006***

0.418 0.006***

PHA

0.212 0.004

0.295 0.004***

0.291 0.002***

ConA

0.192 0.003

0.250 0.005***

0.278 0.006***

LPS

0.207 0.006

0.278 0.007***

0.282 0.005***

***P<0.001

***

Serum dilution

80
70
60
50

***

Controls
600 g
1200 g

40
30
20
10
0
N=

10

10

10

***P<0.001

Fig. 1. Stimulatory effect of IMMUNOSTIM-PLUS on antibody production in mice.

464

Discussion
In our previous studies, a highly significant
increase in granulocyte activity, without influence on
leukocyte number in the blood in mice and pigs fed
Immunostim Plus was observed (14, 15). These
experiments revealed, for the first time, that the
combination
of
Schizandra
chinensis
and
Eleuterococcus senticosus extracts proved to be a strong
immunostimulator of non-specific cellular defence,
dependent on the first line cells, granulocytes. We also
have found that Immunostim Plus, administered after
tumour cell grafting, significantly diminished
neovascular reaction induced in mice by syngeneic
tumour cells (15).
Previously, we have also studied the effect of
Eleutherococcus senticosus alone on lymphocytedependent cellular and humoral immune responses in
mice (8). We have shown that this plant has
immunomodulatory properties. The immunomodulatory
effect of Eleutherococcus on lymphocyte-dependent
immunity was also described by others (3, 10, 18).
In the present study, we obtained further
evidence for strong immunostimulatory in vivo action of
herbal remedy composed of Schizandra chinensis and
Eleutherococcus senticosus extracts. This remedy
augmented the most important classic parameters of
lymphocyte-dependent immunity such as antibody
production, and proliferative response to mitogens,
chemokinetic activity, and the development of growth
factors in the course of graft-versus-host reaction. We
have studied this last effect using in vivo cutaneous test,
which is a good and reliable method for the evaluation
of angiogenic potential of various biological materials,
such as immune or tumour cells, sera, and isolated
growth factors. This test is also a good method to study
the effect of various substances on proangiogenic
activity (15-17).
In our earlier study, we reported the influence
of Eleuterococcus senticosus on cellular and humoral
response in mice. In some tests, the kinds of effect were
dependent on initial activity of lymphatic cells (8). Also
Schmolz et al. (10) suggested Eleutherococcus
senticosus having immunomodulatory potency. They
observed an inhibition at higher and stimulation at lower
Eleutherococcus concentrations of G-CSF, IL-6, and IL13 production in in vitro whole human blood cultures,
enhancement of RANTES synthesis over a wide range
of concentrations, and inhibition of the release of IL-4,
IL-5, and IL-12. In the in vivo placebo-controlled human
study, Bohn et al. (3) observed general enhancement of
the activation state of T lymphocytes. Cao (4) reported
stimulation of lymphokine-activated killer (LAK) cells
by radix Eleuterococcus extract.
However, we have not found papers concerning
the effect of Schizandra alone, or in a combination with
Eleutherococcus on lymphocyte-dependent cellular
immunity. There is one paper describing the use of a
combination of these extracts in clinical studies.
Amaryan et al. (1) performed a double blind, placebocontrolled, and randomised clinical trial of herbal
ImmunoGuard in patients with familial Mediterranean

fever. It was a combination of four herbal extracts,


among them of Schizandra and Eleutherococcus. The
authors obtained significant improvement of all features
(duration, frequency, severity of attacks) in the verum
group as compared with the placebo.

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