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Journal of Ethnopharmacology 133 (2011) 613–620
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Immunostimulatory effects of Uncaria perrottetii (A. Rich.) Merr. (Rubiaceae)
vinebark aqueous extract in Balb/C mice
Leonora P. Nudoa,b, Elena S. Catapa,b,∗
aInstitute of Biology, College of Science, University of the Philippines, Diliman, Quezon City 1101, Philippines
bNatural Sciences Research Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines
article info
Article history:
Received 30 June 2010
Received in revised form 21 October 2010
Accepted 21 October 2010
Available online 28 October 2010
Keywords:
Immunostimulation
Uncaria perrottetii
Rubiaceae
Vinebark aqueous extract
Balb/C mice
abstract
Aims of the study: Crude extract of Uncaria perrottetii (A. Rich.) Merr. vinebark was evaluated for its
immunomodulating activity in Balb/C mice. Initially, the immunomodulatory potential of the plant
extract was evaluated using in vitro immune response assays at different concentrations of the plant
extract (10 g/mL, 20 g/mL, 50 g/mL and 100 g/mL). Using the optimum concentration determined
in the in vitro assays, the protective effect of the plant extract was assessed against drug-induced immuno-
suppression in vivo.
Materials and Methods: For the in vivo experiment, thirty-six (36) mice were divided into 3 groups of
12 mice each: (1) cyclophosphamide drug-induced (30 mg/kg BW) immunosuppressed mice (Cy group)
served as the positive control; (2) Uncaria perrottetii extract and Cy-treated mice (U+ Cy); and (3) PBS-
injected mice as the negative control group [(−) CTRL].
Results: The optimum concentration was determined to be 50 g/mL in the in vitro assays. At this con-
centration, Uncaria perrottetii extract stimulated peritoneal phagocyte activation, produced a significant
increase in the activity of phagocytic cells from the spleen and promoted splenic cellular proliferation
with or without lipopolysaccharide (LPS) when compared with the PBS-treated cells (negative control).
Moreover, cells treated with 50 g/mL of Uncaria perrottetii increased macrophage respiratory burst
activity that was comparable to that of the phorbol myristate acetate-stimulated splenic macrophages.
In all immune assays undertaken in the in vivo experiment, the Cy-treated mice showed significantly
lower response when compared with the PBS-treated mice. Significant improvement in peritoneal cell
activation, phagocytic activity and cellular proliferation was exhibited by the U+ Cy-treated mice when
compared with Cy-injected mice. The extract from Uncaria perrottetii also significantly enhanced respi-
ratory burst and plasma lysozyme activity compared with the Cy-injected mice.
Conclusions: Based on the results of both in vitro and in vivo trials, Uncaria perrottetii extract has
immunopotentiating activities on the innate immunity of Balb/C mice and the extract could potentially
reverse the immunosuppressive effects of Cy. However, the potential of the plant as source of bioactive
products and metabolites for drug development still has to be fully investigated.
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Modulation of immune response to alleviate disease has been
of interest for a long time (Mehrotra et al., 2002; Bani et al., 2005).
Immunomodulation, either through stimulation or suppression,
could aid in maintaining disease-free state (Ghule et al., 2006).
Extracts of a diverse range of plants have been shown to possess
immunomodulatory properties (Nicholl et al., 2001). The develop-
ment of natural health products that could potentially modulate the
∗Corresponding author at: Institute of Biology, College of Science, University of
the Philippines, Diliman, Quezon City 1101, Philippines. Tel.: +63 2 9205471;
fax: +63 2 9205471.
E-mail addresses: elenacatap@yahoo.com,escatap@gmail.com (E.S. Catap).
immune system provides an alternative source of bioactive agents
with medical significance.
Uncaria perrottetii is an indigenous plant species found in the
vast forest area of Kanawan, Morong, in the province of Bataan,
Philippines. Uncaria perrottetii is a species of liana with a monopo-
dial main shoot and plagiotropic, lateral shoots. The plant is
locally known as sungay kalabaw (carabao’s horn) referring to
thorns or hooks present along the length of its vine. Traditionally,
Uncaria species is used as treatments for wounds and ulcers, fevers,
headaches, gastrointestinal illnesses, and for bacterial/fungal infec-
tions (Heitzman et al., 2005). Extract of Uncaria perrottetii is
traditionally used to treat hematuria as well as a remedy during
the six-week period of post-natal care to prevent puerperal fever
as well as puerperal sepsis (Duke and Beckstrom-Sternberg, 2002).
Recently, a study has demonstrated the antibacterial, antifungal
0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2010.10.044
614 L.P. Nudo, E.S. Catap / Journal of Ethnopharmacology 133 (2011) 613–620
and antiprotozoal effects of this specific plant species (Vital and
Rivera, 2009). However, the traditional use of this plant species by
the Aeta tribe in the Philippines has been mainly anecdotal and
there is still no available report on its immune regulatory proper-
ties.
This study investigated the immunomodulatory effects of
Uncaria perrottetii extract on the enhancement of the innate
immune response by employing a number of immune response
assays. Specifically, it (1) determined the optimum dosage
of plant crude extract that would elicit immunomodula-
tory response in vitro, (2) characterized and evaluated the
in vitro effect of plant extract on macrophage activity in
Balb/C mice, specifically macrophage cell spreading, phagocyto-
sis, white blood cell proliferation, and PMA-induced superoxide
anion production, (3) confirmed whether the observed in vitro
immunomodulatory activity of the plant crude extract would
result to similar effect in vivo using an immunosuppressed
animal model, and (4) assessed the protective effect of the
plant extract against drug-induced immunosuppression in mice
in vivo.
2. Materials and methods
2.1. Plant material
Vines of Uncaria perrottetii were collected in forest area of
Kanawan, Morong, in the province of Bataan, Philippines. The spec-
imens were sent for authentication and identification at the Botany
Division of the National Museum, Manila, Philippines (Control no.:
616, February 22, 2008).
2.2. Preparation of plant extracts
Aqueous extract of Uncaria perrottetii vinebark was obtained
using a decoction method proposed by Bobrowski (2008). Air-dried
vinebark was boiled in water (20 g/L) for 30 min at a tempera-
ture approximately 90–100 ◦C to yield a brown aqueous extract.
To this extract, ethyl acetate was added at 1:1 volume ratio. Fol-
lowing agitation and settling, the ethyl acetate layer was separated
from the aqueous layer. The aqueous layer was subjected to evap-
oration under vacuum to remove the remaining ethyl acetate and
the final extract was subsequently dried. The total aqueous extract
concentrate yield per gram of dried plant material was determined
using the formula: [weight (g) of dried extract]/[dry-weight(g) of
plant material] ×100. A total of 10.6 g of dried aqueous extract of
Uncaria perrottetii was obtained from 57.9 g of vinebark used giving
a percentage yield of 18.31.
A stock solution of the plant extract was prepared by dissolving
5 mg of the plant extract in phosphate-buffered saline solution and
filter sterilized using a 0.2 m syringe filter. Extract concentrations
of 10 g/mL, 20 g/mL, 50 g/mL and 100 g/mL were prepared
for the evaluation of immune parameters in vitro.
2.3. Experimental animals
Laboratory-bred Balb/C mice approximately 6–8 weeks old and
of either sex were used in the in vitro experimental set-up. For
the in vivo experimental set-up, Balb/C mice obtained from the
Marine Science Institute (MSI), University of the Philippines at Dil-
iman and the National Institutes of Health (NIH), University of the
Philippines Manila, were initially acclimated to laboratory condi-
tions for one week prior to experiment proper. The animals were
housed in appropriate cages at the animal house facility of the Natu-
ral Sciences Research Institute (NSRI) under a 12-h light:dark cycle
and were allowed free access to feed pellets (Purina Feeds) and
sterile drinking water ad libitum. The experimental set-up and pro-
cedures were approved by the College of Science Animal Care and
Use Committee (CSACUC), UP Diliman.
2.4. In vitro immune response assays
2.4.1. Peritoneal phagocyte spreading
The protocol of Donaldson et al. (1984) was used in eval-
uating the effect of the plant extract on peritoneal phagocyte
spreading activity with some modifications. Mice (n= 3) were
killed by cervical dislocation and peritoneal exudates were col-
lected by peritoneal lavage with 5 mL RPMI 1640 medium (Gibco
cat. no. 31800-022) supplemented with 10% heat-inactivated FCS
(Gibco cat. no. 16000-044), 50 M 2-mercaptoethanol (Riedel-de
Haën 62736), 100 U/100 g penicillin–streptomycin (Gibco cat. no.
15140-122) and 0.25 g/mL amphotericin B (Gibco cat. no. 041-
95780). The exudates were centrifuged at 1500 rpm, 25 ◦C for
20 min. Peritoneal phagocytic cells were resuspended in complete
RPMI 1640 medium. The cell number and viability was determined
by trypan-blue dye exclusion technique. The cell number was sub-
sequently adjusted to 1 ×106cells/mL of medium.
For the control set-up, a volume of cellular suspension was
layered on clear glass slide and incubated for 1 h at room tempera-
ture (25–28 ◦C) inside a humidified chamber, instead of incubating
the slides at 37 ◦C in dry condition. Such humidified conditions
are usually employed in cell culture studies. The extract-treated
set-up, 50 L of plant extracts was added before incubation. After-
wards, slides were gently rinsed in PBS. The glass adherent cells
were fixed with 2.5% glutaraldehyde (Fluka 49629) and examined
under a light microscope. A total of 200 peritoneal phagocytes
were counted and diameter measured using a calibrated eye-
piece micrometer. Cell diameter was classified according to the
following measurements: 0–5 m, >5–10 m, >10–15 m and
>15–20 m.
2.4.2. Splenocyte phagocytic activity
In assessing the effect of the plant extracts on the phagocytic
activity of splenic phagocytes, the protocol of Zelikoff (1997) was
used with some modifications. Mice were sacrificed and each of
their spleen was dissected out aseptically. Cell suspension was
prepared by placing the spleen tissue on sterile wire mesh and
mechanically dispersed using a syringe plunger in RPMI 1640
medium (RPMI 1640 Sigma cat. no. R8755) supplemented with
10% heat-inactivated fetal bovine serum (FBS) and antibiotics
(penicillin/streptomycin). The viability of splenic phagocytes was
determined by trypan-blue dye exclusion technique. The cell num-
ber was adjusted to of 5 ×106cells/mL.
Fifty microliters (50 L) of the plant extract at different con-
centrations (10 g/mL, 20 g/mL, 50 g/mL and 100 g/mL) was
added to 5 ×106splenic phagocytes and 0.05 mL of opsonized yeast
cells (5 ×106cells/mL). Yeast cells were opsonized by coating them
with Congo red stain. The phagocyte–yeast cell mixture was then
incubated for 60 min at room temperature. Then, 20 L aliquot was
placed on glass slide for smearing. Cell smears were air-dried at
room temperature, fixed with 95% ethanol, stained with Giemsa
and counterstained with eosin. A total of 100 cells were counted
per slide at 1000×magnification. Splenic phagocytes without plant
extract served as control. Phagocytic activity was calculated using
the formula: (number of cells with engulfed yeast cell particles/100
cells) ×100%.
2.4.3. Lipopolysaccharide (LPS)-sensitive cell proliferation assay
Another set of mice (n= 3) were sacrificed and each of the
spleen was dissected out aseptically as described in the previous
subsection, however, supplemented RPMI 1640 phenol red-free
medium (RPMI 1640 PR−Sigma cat. no. R8755) was used for
L.P. Nudo, E.S. Catap / Journal of Ethnopharmacology 133 (2011) 613–620 615
this assay. The viability of the splenocytes was determined by
trypan-blue dye exclusion technique. The cell number was adjusted
to1 ×106cells/mL.
The effect of the plant extract was evaluated according to a rapid
colorimetric assay for cellular growth and survival developed by
Mossman (1983) with some modifications. Two hundred micro-
liters of cell suspension was added to a volume of a sample crude
extract and lipopolysaccharide (LPS, Sigma cat. no. L3129; stock
concentration=2mg/mL;final concentration 10 g/mL) in quadru-
plicate wells for each mixture. Lipopolysaccharide was used as a
mitogen to stimulate the proliferation of B lymphocytes in cul-
ture. Volume was adjusted to 1 mL by adding supplemented RPMI
1640 PR−. Wells containing LPS and unsupplemented RPMI 1640
PR−served as positive and negative controls, respectively. Two
hundred microliters of this mixture (0.20 mL) was placed in 96-
well plate, incubated at 37 ◦C for 48h in a humid atmosphere with
5% CO2(Anaeropack CO2, Mitsubishi Gas Chemical Co., Inc.) and
then, the growth culture medium from each well was removed
by aspiration. Forty microliters of 1×RPMI 1640 PR−(supple-
mented with 100 U/100 g penicillin/streptomycin and 0.25 g/mL
amphotericin B) and 20 L of salt MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolim bromide; Sigma cat. no. M2128; stock
concentration=5mg/mLinPBS) were added to all wells, and then
incubated for 3 h at 37◦C in a humid atmosphere. The reaction was
stopped by adding 100 L per well of acid isopropanol (0.04 N HCl in
isopropanol). After a few minutes at room temperature, the optical
density (OD) was measured on a Multiskan EX Thermo Electron Cor-
poration Elisa Reader at 595 nm. Cell proliferation was calculated
using the formula:
% Proliferation =OD sample −OD control
OD control ×100
2.4.4. Splenic macrophage extracellular superoxide anion
production
Assay for the assessment of superoxide anion produced out-
side the mitochondria was performed according to the protocol
used by the group of Zelikoff et al. (1996). Splenic macrophages
were adjusted to a cell concentration of 4 ×106cells/mL. Cell
suspension (125 Lof10
6cells/mL) was added to each of
four previously labeled (1–4) microcentrifuge tubes containing
250 L ferricytochrome C (Sigma cat. no. C7752; stock concen-
tration=4mg/mL in Locke’s mammalian saline solution; final
concentration=2mg/mL).62.5 L of bovine superoxide dismutase
(Sigma cat. no. S2515; stock concentration = 300g/mL; final con-
centration = 37.5 g/mL) was added to the second and fourth tubes.
Ten microliters (10 L) of phorbol 12-myristate 13-acetate (Sigma
cat. no. P8139; stock concentration=1mg/mLDMSO;working con-
centration = 100 g/mL Hank’s balanced salt solution) was added
to the third and fourth tubes at a final concentration of 2 g/mL.
Mammalian physiological saline solution (Locke’s) was added to
each tube to bring the final volume up to 0.5 mL. An additional
tube (labeled as “B”) containing all the reagents, but without cells,
served as the reaction blank. Each tube was vortexed for approx-
imately 30 s and then, 200 L aliquots (2 ×105cells/well) was
placed into the individual wells of a 96-well microtiter plate and the
absorbance measured at 492 nm for up to 1 h. Timepoints suggested
for the measurement include: 0, 15, 30, 45, and 60 min; plates were
incubated at 37 ◦C in a humidified environment between readings.
The rate of superoxide anion radical production can be deter-
mined from measurements taken over time, while OD readings
at a single timepoint (time of peak superoxide anion radical pro-
duction = 60 min) can also be used to make comparisons between
different exposure groups. Change in absorbance was calculated
by subtracting the mean of the “blank” wells and the wells contain-
ing SOD from the absorbance measured in the non-SOD-containing
wells. By multiplying the change in absorbance by 15.87, the nmol
concentration of SOD-inhibitable superoxide anion radical can be
computed. Data were expressed as nmol O2−/2 ×105cells/unit
time.
2.5. In vivo experiments
2.5.1. Experimental set-up
Thirty-six (36) mice were divided into 3 groups of 12 mice
each. The average weight of mice used was 21.5 g for the exper-
iments on peritoneal phagocytic cell spreading, phagocytic activity
of splenocytes and plasma lysozyme levels. For the production
of ROS and the MTT assays, the average weight of the mice was
22.0 g. The Uncaria perrottetii extract was injected intraperitoneally
(2.5 mg/kg BW) in one group for a period of ten days. This dose
elicited immunoactivity based on the results of preliminary in vitro
assays on the crude extract. These in vitro assays were undertaken
since the dosage of traditional actual use of Uncaria extracts in the
Philippines is not available. Another group of mice was injected
intraperitoneally with sterile phosphate-buffered saline at a vol-
ume dose of 50 mL/kg body weight (negative control) which was
based on the study of Bin-Hafeez et al. (2001). The last group
was injected with intraperitoneally with 0.2 mL of 30 mg/kg body
weight of cyclophosphamide (Endoxan®, i.p.), at days 1, 4, 7 and
10 of the treatment period. Cyclophosphamide was also adminis-
tered to groups of mice treated with Uncaria perrottetii, 1 h after
each administration of the plant extract. The dosage of cyclophos-
phamide used in the study and the timing of plant extract and Cy
injections were based on the paper of Bafna and Mishra (2006).
The volume dose used during the experiments was consistent for
all three groups of mice. All experimental animals were sacrificed
on the 11th day.
2.5.2. Immune response assays
Immune response assays as described in the previous section
(in vitro experimental set-up) were performed. In addition, plasma
lysozyme activity was determined in this experiment.
2.5.3. Plasma lysozyme assay
Blood samples were extracted from the external jugular vein of
the mice from the three experimental groups and centrifuged at
1000 rpm for 10 min. Plasma was collected and stored at −20 ◦C
until the assay was performed.
Lysozyme activity was determined by a microtitre plate method
which measures the lysis of a suspension of Micrococcus lysodeikti-
cus (75 mg 100 mL−1of 0.1 M phosphate/citrate buffer with 0.09%
NaCl, pH 5.6). One hundred and seventy-five microliters (175 L)
of the bacterial suspension was added at 25 L of each plasma
sample, and to hen egg white lysozyme standard (Sigma, cat.
No. L-6876; 0–50 gmL
−1of 0.1 M phosphate/citrate buffer with
0.09% NaCl, pH 5.8) in flat bottomed, 96-well plates, in duplicate
wells per sample. The plates were incubated for 30 min. The rate
of lysis was determined against Micrococcus lysodeikticus (Sigma,
cat. no. M-3770) blank at 450 nm on a Statfax 2100 multiscan
plate reader. Lysozyme level was calculated from the standard
curve, and data were expressed as g/mg protein (Secombes,
1998). Plasma protein concentration was determined using the
Bio-Rad protein assay kit by following the manufacturer’s instruc-
tions.
2.6. Statistical analysis
Results were expressed as mean ±S.E. Data obtained were
tested for normality (Kolmogrov–Smirnov’s test) and homogene-
ity of variance (Levene’s test) prior to any appropriate statistical
analysis. Nested ANOVA (Mixed Model) was used specifically for
616 L.P. Nudo, E.S. Catap / Journal of Ethnopharmacology 133 (2011) 613–620
Table 1
Effects of various concentrations of Uncaria perrottetii vinebark (A. Rich.) Merr.
extract on in vitro activation and spreading activity of murine peritoneal exudates
cells. Data are the mean percentages of cell activation.
Treatment Uncaria perrottetii PBS (−)
10a20a50a100a
Cell diameter range (m)
0–5 21 22 8*7*38
>5–10 60 65 79 73 60
>10–15 19*12 12 12 2
>15–20 1 1 1.2 0 0
aConcentration (g/mL)
*P< 0.05 vs PBS.
the in vitro and in vivo peritoneal macrophage spreading assay.
ANOVA (parametric) and Kruskal–Wallis test (non-parametric)
were appropriately used to analyze results obtained in other
immune response assays. Pvalue of less than 0.05 was considered
statistically significant. Least Significant Difference (LSD) was used
as a posteriori test to compare the means. Statistical analyses were
performed using SPSS ver. 15.
3. Results
3.1. In vitro immune response assays
3.1.1. Peritoneal phagocyte spreading and activation
Highest percentage of cells were counted within the >0–5 m
and >5–10 m diameter ranges in PBS-exposed peritoneal phago-
cytes. Significantly lower percentage of cells within the >0–5 m
diameter range was detected when incubated with either 50 g/mL
and 100 g/mL of the plant extract as compared with PBS-
incubated cells. Higher percentage of activated cells within the
>5–10 m diameter range were counted at all concentrations of
plant extracts used, however, this was not significantly different
from the PBS-treated cells. Furthermore, at all concentrations of
Uncaria perrottetii, higher percentages of activated cells, within the
diameter range of >10–15 m, were obtained when compared with
PBS-treated cells (Table 1). Compared with PBS-treated cells, signif-
icantly higher percentage of activated cells (>10–15 m diameter)
was obtained when 10 g/mL of plant extract was used. Overall,
Fig. 1. Effects of various concentrations of Uncaria perrottetii (A. Rich.) Merr.
vinebark extract on in vitro phagocytic activity of murine splenic phagocytes. The
plant extract-treated phagocytes significantly showed higher activity compared
with PBS-treated cells (control). Each bar represents the mean ±S.E. of each treat-
ment (*P< 0.05 vs control).
Fig. 2. Effects of various concentrations (g/mL) of Uncaria perrottetii (A. Rich.) Merr.
vinebark extracts on in vitro proliferation of murine spleen cells with lipopolysaccha-
rides (LPS, 10 g/mL, black bars), and without LPS (white bars). Cells in RPMI 1640
served as the control; cells +LPS only at 10 g/ml (light gray bar). Data expressed as
mean ±S.E. (*P< 0.05 vs control).
50 g/mL and 100 g/mL of Uncaria perrottetii extracts induced
remarkable in vitro peritoneal phagocyte activation.
3.1.2. Splenocyte phagocytic activity
Uncaria perrottetii extract induced significant phagocytic stimu-
lation in a relatively dose-dependent response compared with the
PBS-treated cells (Fig. 1). The highest test concentration exhibited
the highest phagocytic activity.
3.1.3. Proliferation of lipopolysaccharide (LPS)-sensitive
splenocytes
Enhanced cell proliferation was obtained at 20 g/mL, 50 g/mL
and 100 g/mL extract concentration without LPS when compared
with the control set-up (RPMI only). For the set-ups with LPS, lower
cell proliferation occurred in 50 g/mL and 100 g/mL of Uncaria
perrottetii extract-treated cells (Fig. 2).
3.1.4. Splenic macrophage production of extracellular superoxide
anion
Extracellular superoxide anion production of murine
macrophages peaked after 30 min. In splenic macrophages
treated with 50 g/mL of Uncaria perrottetii extract, the observed
Fig. 3. Effects of various concentrations Uncaria perrottetii (A. Rich.) Merr. vinebark
extract on in vitro superoxide anion production of murine macrophages at 30 min
incubation. The effect of the extract is comparable to that of phorbolmyristate
acetate-stimulated cells. Each value is expressed as nmol O2−/2 ×105cells/30 min.
L.P. Nudo, E.S. Catap / Journal of Ethnopharmacology 133 (2011) 613–620 617
Table 2
Effects of 50 g/mL of Uncaria perrottetii (A. Rich.) Merr. vinebark extract on murine
peritoneal macrophage cell spreading activity. Mice intraperitoneally injected with
30 mg/kg BW of cyclophosphamide (Cy only) as an immunosuppressant served as
positive control while mice injected with PBS served as negative control. Data are
mean percentages of activated peritoneal exudates cells.
Cy only PBS (−)U+Cy
Cell diameter range (m)
0–5 98 90*89*
>5–10 2 10*11*
>10–15 0 0 0
>15–20 0 0 0
*P< 0.05 vs Cy only.
increase of 1.2 nmol O2−/2.5 ×105cells/30 min was comparable
to that of 1.6 nmol O2−/2.5 ×105cells/30 min in PMA-stimulated
splenic macrophages (Fig. 3). However, it should be noted that
significantly higher extracellular superoxide production was
detected when splenic macrophages were exposed to the plant
extracts when compared with unstimulated cells (P< 0.05).
3.2. In vivo experimental set-up
3.2.1. Peritoneal phagocyte spreading and activation
As shown in Table 2, cells with the smallest diameter range
(>0–5 m) comprised 98% of the total number of cells counted in
the immunosuppressed mice (positive control), which was signifi-
cantly higher when compared with the PBS-treated mice (negative
control) and the Uncaria perrottetii extract-treated mice. Moreover,
significantly higher number of cells with >5–10 m diameter were
counted in PBS-treated and Uncaria perrottetii-treated mice com-
pared with the positive control. There were no cells observed with
a bigger diameter range (>10–15 m and >15–20 m).
3.2.2. Splenocyte phagocytic activity
A significant decrease in the ability of splenic phagocytes
to engulf opsonized yeast cells was observed in immunosup-
pressed (Cy) mice compared with the negative control (PBS) mice
(Fig. 4). The plant extract significantly enhanced the phagocytosis
in immunosuppressed mice (U+ Cy) at P< 0.05. Uncaria perrottetii
significantly increased the mean percentage phagocytic activity to
27% from that of Cy-treated group (4.33%).
Fig. 4. Effects of 50 g/mL of Uncaria perrottetii (A. Rich.) Merr. vinebark extracts on
phagocytic activity of splenic phagocytes. Each bar presents the mean ±S.E. of treat-
ment. The plant extracts significantly enhanced the phagocytic activity compared
with cyclophosphamide (Cy)-treated mice (*P< 0.05 vs Cy; **P<0.05 vs Cy).
Fig. 5. Effects of 50 g/mL of Uncaria perrottetii (A. Rich.) Merr. vinebark extract
on cell proliferation activity of LPS-sensitive cells from spleen. The plant extracts,
without LPS (white bars), and with LPS (black bars), significantly reversed the effect
of cyclophosphamide (Cy) on cell proliferation. Each bar represents the mean ±S.E.
of treatment (*P< 0.05 vs Cy; **P<0.05 vs Cy).
3.2.3. Proliferation of lipopolysaccharide (LPS)-sensitive
splenocytes
Fig. 5 shows that Cy-treatment (positive control) significantly
decreased the mean percentage of cell proliferation to almost 10%
from that of PBS-treated group (negative control) with (58.46%)
or without (63.92%) lipopolysaccharide (LPS). The plant extracts
reversed the immunosuppressive effect of Cy-treatment. Results
obtained from Uncaria perrottetii vinebark extract-treated group
showed that without a mitogen (LPS), the activity was enhanced to
29.61% (P> 0.05), and with LPS, the increase in the mean percentage
was 44.57% (P> 0.05).
3.2.4. Splenic macrophage production of extracellular superoxide
anion
As shown in Fig. 6, cyclophosphamide decreased the production
of superoxide anion compared with PBS-treated mice. Uncaria per-
rottetii extract markedly stimulated the production of superoxide
anion compared with the immunosuppressed group (P< 0.05).
Fig. 6. Effects of 50 g/mL of Uncaria perrottetii (A. Rich.) Merr. vinebark extracts
on O2−production of murine macrophages at 30 min incubation. The extract from
Uncaria perrottetii significantly induced respiratory oxidative burst among the
treatments. Each value is expressed as nmol O2−/2 ×105cells/30 min. Black bars,
PMA-stimulated; white bars, unstimulated (*P< 0.05 vs Cy; **P<0.05 vs Cy).
618 L.P. Nudo, E.S. Catap / Journal of Ethnopharmacology 133 (2011) 613–620
Fig. 7. Effects of 50 g/mL of Uncaria perrottetii (A. Rich.) Merr. vinebark extract on
plasma lysozyme activity. Each value is expressed as g lysozyme/mg protein. Each
bar represents the mean ±S.E. (*P<0.05 vs Cy; **P< 0.05 vs Cy).
3.2.5. Plasma lysozyme assay
Results (Fig. 7) showed that significantly higher levels of plasma
lysozyme were detected in PBS-treated mice (negative control)
compared with Cy-treated mice (positive control). Significantly
higher lysozyme levels were induced by Uncaria perrottetii treat-
ment in immunosuppressed mice.
4. Discussion
A large number of plants used in traditional medicines have
been shown to possess nonspecifically immunomodulating activ-
ities (Choi et al., 2004) and are being extensively explored for
their potential in the prevention and treatment of chronic dis-
eases. One of the most well-studied plant species in terms of
medicinal natural products is Uncaria tomentosa, found in Peru,
from which the largest number of compounds (alkaloids, ter-
penes, quinovic acid glycosides, flavonoids, etc.) has been identified
(Heitzman et al., 2005). Our preliminary study on the immunomod-
ulatory effects of Uncaria perrottetii, a species found in southeast
Asia, was patterned after some of the studies done on Uncaria
tomentosa. However, unlike Uncaria tomentosa, there were no pub-
lished reports on the traditional actual dose of Uncaria perrottetii
that is used in the Philippines. The use of Uncaria perrottetii was
largely based on folkloric and anecdotal reports by the Aeta tribe
in Kanawan, Morong, Bataan forest area, regarding its effects on
would healing (pers. comm.). As such, series of in vitro trials were
undertaken to determine the appropriate dose of the extracts
to be used in the in vivo immunosuppression experiment of the
study.
In this study, the optimum dosage of the Uncaria perrottetii
extract was determined to be 50 g/mL based on the results of
in vitro experiments. This concentration strongly enhanced the
immune response activities of murine peritoneal exudate cells
in terms of cell growth as evidenced by cell spreading and acti-
vation. Likewise, the ability to phagocytose by splenocytes and
the pathogen-degradation efficiency via lysozyme release into the
plasma and oxidative burst were enhanced. Moreover, the plant
extract stimulated the proliferation of B lymphocytes in culture.
This dose was therefore used in the subsequent in vivo experiment
and proved to be effective in alleviating immunosuppression in
mice injected with cyclophosphamide, as shown by results of the
immune response assays.
Macrophages are an important part of the innate immune sys-
tem and thus play an important role in the defense mechanism
against host infection and the killing of tumor cells. The modula-
tion of antitumor properties of macrophages by various biological
response modifiers is closely related to immunomodulating activ-
ity (Manosroi et al., 2003). Macrophages are also involved in the
initiation of adaptive immune responses. To fulfill these functions,
macrophages classically produce and release cytokines and super-
oxide anions (Togola et al., 2008). They are activated through signals
from two factors; first is the cytokine IFN-␥and the second signal is
the presence of tumor necrosis factor (TNF) or any TNF inducer such
as lipopolysaccharide. Thus, macrophages are activated by expo-
sure to IFN-␥and via exposure to microbes or microbial compounds
like LPS (Mosser, 2003).
Phagocytosis represents an important innate defense mecha-
nism by which leukocytes ingest particulate ligands whose size
exceeds about 1 m including whole pathogenic microorganisms.
This phylogenetically conserved process is critical for innate immu-
nity (Greenberg and Grinstein, 2002). Macrophages recognize
pathogens through their cell-surface receptors that could distin-
guish the surface molecules exhibited by the pathogens and those
by the host. These receptors include the mannose receptor, scav-
enger receptors and the CD14 receptor that binds with bacterial LPS
which could dictate the interactions of phagocytes and pathogens
(Janeway et al., 2001). This could explain the results in our in vitro
trial wherein concentrations greater than 10 g/mL did not elicit
further increase in phagocytosis of opsonized yeast cells. It is likely
that the phagocyte–yeast interactions in the in vitro assay sys-
tem were hindered by the number of phagocyte receptors, and
any increase in the extract concentration will not induce further
interactions between phagocytes and yeast cells. However, it also
possible that that the plant extract used has a biphasic effect on
macrophages wherein it is more effective at lower concentrations,
and becomes less stimulatory at higher concentrations. In a study
done on an aqueous extract of Uncaria tomentosa, 50–200 mg/kg
dose induced increasing anti-inflammatory effects but this activity
decreased when 500 mg/kg concentration was used (Aguilar et al.,
2002).
The production of reactive oxygen species observed in vitro
reflected stimulation of cellular spreading and phagocytosis in rela-
tion to extracellular killing similar to reports of Ji et al. (2007).
Stimulation of normal phagocytic cells elicits a non-mitochondrial
burst of respiration in which there is a huge increase in oxygen
consumption and the production of remarkable amounts of super-
oxide anion in polymorphonuclear (PMN) cells (Borregaard, 1985).
The respiratory burst is a distinguishing feature of phagocytes
that serves as an important defense mechanism against invading
pathogens necessary for effective microbiocidal action (Ji et al.,
2007). The cytotoxic systems used by defense cells are multiple
but a very important component is the nicotinamide adenine din-
ucleotide phosphate (NADPH) oxidase system, a transmembrane
electron transport chain that reduces oxygen to superoxide. This
system can be activated by phagocytosis and, with the aid of other
cytotoxic mechanisms, helps the host to clear the infection. In
non-physiological terms, the NADPH oxidase system can also be
activated by some compounds (such as phorbol myristate acetate
and synthetic chemotactic peptides), which are also able to trigger
respiratory burst (Lunardi et al., 2006).
Uncaria perrottetii extract, with or without lipopolysaccharide
(LPS), promoted cell proliferation in vitro compared with the control
splenic cells. The results from our study showed that the addi-
tion of plant extract alone induced higher percentage of cells. This
appears to be contrary to the reported anti-inflammatory effects of
another Uncaria species. In a study done by Åkesson et al. (2003a),
they found dose-dependent increase in spleen cell numbers in mice
supplemented with a hot water extract of Uncaria tomentosa. How-
ever, it was suggested that the increased number of lymphocytes
was not due to proliferation but because of the significantly pro-
L.P. Nudo, E.S. Catap / Journal of Ethnopharmacology 133 (2011) 613–620 619
longed lymphocyte survival (anti-apoptotic effect) in peripheral
lymphoid organs (such as the spleen) when treated with the hot
water plant extract. Prolonged survival of cells led to enhanced
opportunities of DNA repair and consequently to immune stim-
ulation, anti-inflammation and cancer prevention (Åkesson et al.,
2003b; Sheng et al., 2005). It would be worthy to study this aspect
using our crude extract of Uncaria perrottetii to explain the possible
mechanism on the higher percentage of cells we obtained in our
experiment.
Among several B-cell activators, LPS is known as an effec-
tive B-cell mitogen leading to B-cell development. Therefore, LPS
specifically activates clonal proliferation of B lymphocytes (von
Andrian and Sallusto, 2007). As stated earlier, LPS also activates
macrophages; however, it induces the release of inflammatory
factors rather than induce clonal proliferation as what usually
happens among B lymphocytes. Unfortunately, this study did not
use a mitogen to determine the effect of the plant extract on T
lymphocytes. Therefore, future studies on the immunoregulatory
properties of Uncaria perrottetii should include its effects on T lym-
phocytes.
LPS is the major component of the outer membrane of Gram
negative bacteria and consists of a bisphosphorylated lipid (lipid
A) and a hydrophilic polysaccharide (Kim et al., 2006). In our pre-
liminary trials to optimize the MTT assay, we were able to obtain
the highest percentage of cellular proliferation when LPS con-
centration (without the extract) was 10 g/mL as compared to
2g/mL and 5 g/mL (unpub. data). This concentration was there-
fore used in the succeeding in vitro and in vivo experiments. Based
on our results, the addition of LPS and 10 g/mL and 20 g/mL
in the splenocytes resulted to significantly higher percentage of
cellular proliferation compared to the control cells in the in vitro
experiment. However, the percentage of cellular proliferation when
higher doses of the extracts (50 g/mL and 100 g/mL) and the
addition of LPS was not significantly different from that of the con-
trol cells. It appears that the presence of LPS and higher doses
of the Uncaria perrottetii extract only produced cellular prolifer-
ation comparable only to the control splenocytes. In a study by
Sandoval et al. (2000), the production of TNF␣, an inflammatory
response cytokine, was induced by LPS; but the presence of a water
extract of Uncaria tomentosa partially inhibited TNF␣, which con-
firmed its anti-inflammatory effect. It should be noted however that
the extract did not fully suppress LPS-induced TNF␣production
since there was 65–85% inhibition over a wide dose range. LPS is
reportedly an aggressive stimulant of transcription and could acti-
vate other transcription factors aside from NK-B, which is linked
with cell proliferation. Thus, it seems that the extract could not
completely inhibit TNF␣production since other transcriptional fac-
tors could still function with LPS. This could possibly explain the
non-significant increase on cellular proliferation results from our
study when LPS was added with higher doses of Uncaria perrottetii
extract.
Immunostimulatory effects of Uncaria perrottetii extract in
vivo significantly increased immunoactivity of macrophages and
lymphocytes in an immunosuppressed animal model. Immuno-
suppression, particularly of humoral immunity, is a common
consequence of long-term cyclophosphamide (Cy) chemotherapy
in cancer patients (Bin-Hafeez et al., 2001). Cyclophosphamide is
known to cause leucopenia as it is cytotoxic not only to cancer cells
but also to leukocytes as well. A single dose of Cy could inhibit
the proliferative response of B lymphocytes and has a particularly
intense effect on short-lived lymphocytes known to include a great
proportion of B-cells (Bafna and Mishra, 2006). Chemotherapy-
induced leukopenia leads to significant morbidity and mortality,
which is a major limiting factor in clinical chemotherapy without
efficacious remedies (Zhu et al., 2007).
Cyclophosphamide treatment impaired macrophage phagocy-
tosis, consistent with the previously reported findings of Zhu et al.
(2007). Cy, likewise, suppressed the release of lysozyme in the
plasma as compared to the PBS-treated mice. The immunosup-
pressive effects of the Cy treatment that we used further validated
the dose used by Bafna and Mishra (2006) in their previous study.
In vivo treatments with low dose of Uncaria perrottetii (2.5 mg/kg
body weight) ameliorated the immunosuppression in Cy-treated
mice through enhancement of phagocytic activity, improvement
in the production of reactive oxygen species as well as significant
improvement of plasma lysozyme level.
Lysozyme is mainly expressed in activated macrophages
and released during phagocytosis. Activation of peritoneal
macrophages as well as enhancement of splenic macrophage
phagocytic activity induced by Uncaria perrottetii provides corre-
lation between the significantly higher levels of plasma lysozyme
and macrophage activation detected in extract-treated mice in vivo.
An activated macrophage expresses more major histocompatibility
complex II proteins on their surface that contains more lysosomes
and lysosomal enzymes (Orsolic et al., 2004). The relation of extra-
cellular release of lysozymes to varying degrees of phagocytic
challenge has been demonstrated experimentally in vitro. Extra-
cellular release of lysozyme increases with increasing phagocytic
challenge (Wright and Malawista, 1972).
Preliminary results of phytochemical screening revealed the
presence of alkaloid through Mayer’s and Dragendorff’s tests and
the presence of hydrolyzable tannins through Ferric Chloride Test
(Vital, 2008). It is likely that these biologically active substances
could have induced the observed immuno-potentiating activities
of Uncaria perrottetii crude extract. Oxindole alkaloids have been
reported to promote phagocytosis (Lemaire et al., 1999) and the
alkaloids identified by Keplinger et al. (1999) to be present in the
vinebark extract of the two Peruvian species of Uncaria are most
probably the same alkaloid components detected in Uncaria per-
rottetii vinebark extract. However, further phytochemical analysis
on the crude extract of Uncaria perrottetii should be undertaken
in order to compare its bioactive components with that of Uncaria
tomentosa, which is one of the most documented medicinal plants.
The presence of polyphenols, such as tannins in the extract
could also have induced immunoactivation. According to some
reports, the presence of hydrolyzable tannins stimulates iodination
of myeloperoxidase-rich granulocytic cells such as polymorphonu-
clear cells (Sakagami et al., 1990; El Abbaouyi et al., 2004).
Moreover, tannins stimulate reactive oxygen intermediates pro-
duction and mediate the release of a neutrophil chemotactic factor.
Also, tannins enhance nitroblue tetrazolium (NBT)-reducing activ-
ity of macrophages (Sakagami et al., 1991), activate morphological
change (spreading) of peritoneal macrophages and induce various
antitumor cytokines such as tumor necrosis factor ␣(TNF␣) and
interleukin 1 (IL-1) (Feldman et al., 1999; El Abbaouyi et al., 2004).
In summary, this study assessed the immunomodulatory poten-
tial of Uncaria perrottetii that will provide baseline information
regarding the potential use of this plant species as an immuno-
nutritional supplement. Results revealed the immunopotentiating
activities of Uncaria perrottetii extract on the innate immunity of
Balb/C mice in vitro. Also, the study also showed that Uncaria per-
rottetii could potentially reverse the immunosuppressive effects
of Cy in vivo. The protective effects of the plant extracts to the
immune system were confirmed in this study. The results of
this study on Uncaria perrottetii are very encouraging; and future
studies on Uncaria perrottetii warrants the complete characteriza-
tion and profile of the bioactive compounds present in the crude
extracts, which could be used as standard when studies on the
different mechanisms of its immunomodulatory properties are
undertaken.
620 L.P. Nudo, E.S. Catap / Journal of Ethnopharmacology 133 (2011) 613–620
Acknowledgments
The authors are grateful to the UP Natural Sciences Research
Institute, the UP System Research Grant, through the OVPAA,
and the National Research Council of the Philippines, Department
of Science and Technology for the research and master’s thesis
grants.
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