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Okra pods were widely consumed by Indonesians to maintain health. The aim of this study was at investigating the potential of crude polysaccharides from okra pods on immune response in mice infected with Staphylococcus aureus. Thirty male Balb/C mice were divided into six groups: normal control, negative control, and treatment groups (administration of crude polysaccharides at doses of 25, 50, 75, and 100 mg/kg). Crude polysaccharides were administrated for fourteen days. Furthermore, mice were exposed to S. aureus at the fifteenth day. Two weeks after the end of treatment, the parameters were measured. This study showed that crude polysaccharides at a dose of 75 and 100 mg/kg improved phagocytic activity, spleen index, and splenocytes proliferation. Rising of TNF- α levels was shown in groups treated with crude polysaccharides at doses of 25, 50, and 100 mg/kg. All treatment groups showed a decreasing level of IL-17. Crude okra polysaccharides also showed a slight increase in NK cells activity and IFN- γ level. Thus, crude okra polysaccharides could act as an effective material to enhance immune response including phagocytic activity, spleen index, splenocytes proliferation, and control immune responses through cytokine production.
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Research Article
Crude Polysaccharides from Okra Pods (Abelmoschus esculentus)
Grown in Indonesia Enhance the Immune Response due to
Bacterial Infection
Sri Puji Astuti Wahyuningsih , Manikya Pramudya, Intan Permata Putri, Dwi Winarni,
Nadyatul Ilma Indah Savira, and Win Darmanto
Department of Biology, Faculty of Science and Technology, Airlangga University, Surabaya 60115, Indonesia
Correspondence should be addressed to Sri Puji Astuti Wahyuningsih; sri-p-a-w@fst.unair.ac.id
Received 23 March 2018; Revised 24 August 2018; Accepted 19 September 2018; Published 9 October 2018
Academic Editor: Paola Patrignani
Copyright ©2018 Sri Puji Astuti Wahyuningsih et al. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Okra pods were widely consumed by Indonesians to maintain health. e aim of this study was at investigating the potential of
crude polysaccharides from okra pods on immune response in mice infected with Staphylococcus aureus. irty male Balb/C
mice were divided into six groups: normal control, negative control, and treatment groups (administration of crude poly-
saccharides at doses of 25, 50, 75, and 100 mg/kg). Crude polysaccharides were administrated for fourteen days. Furthermore,
mice were exposed to S. aureus at the fifteenth day. Two weeks after the end of treatment, the parameters were measured. is
study showed that crude polysaccharides at a dose of 75 and 100 mg/kg improved phagocytic activity, spleen index, and
splenocytes proliferation. Rising of TNF-αlevels was shown in groups treated with crude polysaccharides at doses of 25, 50, and
100 mg/kg. All treatment groups showed a decreasing level of IL-17. Crude okra polysaccharides also showed a slight increase
in NK cells activity and IFN-clevel. us, crude okra polysaccharides could act as an effective material to enhance immune
response including phagocytic activity, spleen index, splenocytes proliferation, and control immune responses through
cytokine production.
1. Introduction
e human body is surrounded by environment-contained
microbes, including extracellular bacteria, S. aureus. ese
bacteria are able to cause nosocomial infection which can
result in serious infections [1]. Normally, immune-related
cells will inhibit S. aureus transmission but the bacteria also
release components against the immune system. erefore,
the body needs a particular compound to enhance the
immune response.
Dietary phytochemicals from plants may play important
roles in the prevention of many diseases [2]. Plant poly-
saccharides have been known as an important immunos-
timulatory agent with broad spectrum, low toxicity, and few
side effects [3]. If polysaccharides are component of our
daily food, it will give many health benefits for human body.
Okra (A. esculentus) is vegetable crop used as food and
traditional medicine for many diseases such as dysentery and
diarrhea [4, 5]. Okra contains flavonoid and vitamin C as
antioxidant and polysaccharides as an immunomodulator
[6, 7]. A study with cyclophosphamide as an antigen has
revealed that okra polysaccharides increased spleen index,
splenocyte proliferation, and cytokines secretion [7]. e
extract of okra increased IL-12 secretion and decreased IL-10
secretion in dendritic cells [8]. Related with bacterial in-
fection, okra fruit has high tannins that could abolish
bacteria [9].
However, studies have not been reported on the po-
tential of crude polysaccharides from okra pods consumed
in Indonesia to overcome high cases of S. aureus infection.
To further investigate the potential of crude okra poly-
saccharides, the present study explored the effect by
Hindawi
Advances in Pharmacological Sciences
Volume 2018, Article ID 8505383, 7 pages
https://doi.org/10.1155/2018/8505383
evaluating phagocytic activity, cytokine production, spleen
index, splenocytes proliferation, and NK cell activity.
2. Materials and Methods
2.1. Materials and Chemicals. Okra pods were collected from
the traditional market in Surabaya, Indonesia, in May 2017.
e okra pods were packaged 500 g per polyethylene bag and
then stored at 20°C until use. S. aureus (ATCC 25923) was
purchased from Balai Besar Laboratorium Kesehatan, Sur-
abaya, Indonesia. RPMI-1640 was purchased from Gibco
(Invitrogen Co, Massachusetts, USA). Lipopolysaccharide
(LPS) from Escherichia coli 055 : B5, L2880, and lyophilized
powder were purchased from Listlab (List Biological
Labs, Inc., California, USA). 3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) and human hepa-
toma cell line (huh7it) were acquired from Institute of
Tropical Disease, Airlangga University (Surabaya,
Indonesia). Interleukin-17 (IL-17), interleukin-23 (IL-23),
interferon-c(IFN-c), and tumor necrosis factor (TNF)-α,
enzyme-linked immunosorbent assay (ELISA) kit were
purchased from BioLegend (BioLegend, Inc., San Diego,
USA). All other chemicals and solvent used were of ana-
lytical reagent grade.
2.2. Preparation of Crude Polysaccharides from Okra Pods.
According to Ramesh et al. [29] and Chen et al. [7], frozen
okra pods (500 g) were cleaned with distilled water, cut into
small slices, homogenized, and macerated with 500 ml
double-distilled water (ddH2O) overnight. e extract of
okra was filtered and macerated twice again with 300 ml
ddH2O. e supernatants were collected by centrifugation at
4300 rpm for 5 min. e supernatants were precipitated by
the addition of anhydrous ethanol 1X sample volume and
incubated for 24 h at 4°C and then centrifuged again as
above. e precipitated material was then dissolved in
ddH
2
O and dialyzed through cellulose membrane (Sigma-
Aldrich, retaining >Mw 14,000) for 24 h. e aqueous
solution was then collected from the dialysis bag and freeze-
dried to obtain the crude okra polysaccharides.
2.3. Determination of Polysaccharides Content in Okra Pods.
Polysaccharides content in okra pods was determined using
phenol sulphuric acid assay. Sample solution of crude okra
polysaccharides was made from stock of crude okra poly-
saccharides (10 µL) and aquadest (90 µL). en, 50 µL of
phenol 5% was added. After being homogenized for 1 min,
2 mL of sulphuric acid was added to the solution and in-
cubated for 10 min in room temperature. e blank solution
was made from 50 µL of phenol 5% and 100 µL of aquadest.
e absorbance was measured at 490 nm.
2.4. Animals. Male BALB/c mice (8–10 weeks old, 30–40 g)
were provided by the Laboratory Animal from Faculty of
Pharmacy, Airlangga University, Surabaya, Indonesia. e
animals were maintained in cages made of a plastic with a lid
made of woven wire cage at 20°C, with 12 h light/12 h dark
cycle, fed and watered by ad libitum. All procedures
involving animal care were approved by Animal Care and
Use Committee (ACUC) of Veterinary Faculty, Airlangga
University, Surabaya, Indonesia, no. 714-KE.
2.5. Experimental Design. After 10 days of acclimatization,
BALB/c mice were randomly divided into six groups (KN:
normal control without any treatment; K: negative control
exposed by S. aureus without okra crude polysaccharides
administration; P1, P2, P3: okra crude polysaccharides doses
25, 50, 75, and 100 mg/kg BW, respectively). Okra crude
polysaccharides were administrated by gavage in fourteen
days. Furthermore, mice were exposed to S. aureus (0.5 Mc.
Farland) once through intraperitoneal at the fifteenth days.
Two weeks after the last administration, the animals were
weighed, blood samples were collected to obtain serum, and
then the animals were killed. e intraperitoneal fluid was
collected. e spleen was excised from the animal and
weighed immediately and placed in cold PBS-penicillin-
streptomycin. e relative was calculated according to the
following formula: spleen index (mg/g) (weight of
spleen/body weight).
2.6. Phagocytic Activity Assay. e mice were injected in-
traperitoneally with 0.2 mL of S. aureus suspension. One
hour later, the mice were killed by ketamine anesthesia, and
3 mL of 3% EDTA was used as an anticoagulant. en, the
intraperitoneal fluid was collected. Intraperitoneal suspen-
sion was smeared on glass slides and air-dried. e smear
was fixed using methanol for 15 minutes and stained with
Giemsa solution for 20 mins. Phagocytic activity was de-
termined by counting the number of phagocytes in a pop-
ulation of 100 phagocytes.
2.7. Serum Cytokine Assay. Whole blood was collected and
centrifuged at 3000 rpm and 4°C for 10 min, while the upper
layer contained the serum. e levels of IFN-c, TNF-α, IL-
17, and IL-23 in the serum were analyzed by commercial
enzyme-linked immunosorbent assay (ELISA) kits (BioL-
egend, Massachusetts, USA) according to the manufacturer’s
protocol. e absorbance was measured using ELISA reader
at 450 nm.
2.8. Splenocytes Isolation. e spleens were gently homog-
enized and passed through a sterilized copper sieve (200-
mesh) to obtain single cell suspensions. Splenocytes sus-
pension was centrifuged at 1000 rpm for 5 minutes. Pellet
containing red blood cells was resuspended in tris-buffered
NH4Cl pH 7.2 and centrifuged at 1000 rpm for 5 minutes
until white pellet was obtained. Splenocytes were washed
with 5 ml of PBS-100 unit/ml penicillin-100 µg/ml strepto-
mycin and resuspended in RPMI 1640-FBS 10% medium.
en, splenocytes were used in splenocytes proliferation
assay and natural killer cell activity assay.
2.9. Splenocytes Proliferation Assay. Cell numbers of sple-
nocytes were counted by haemocytometer. 195 µl of
2Advances in Pharmacological Sciences
splenocytes (3 ×105 cells/well) and 5 µl of LPS (200 µg/ml)
were seeded in 96-well plates. After incubation at 37°C in an
incubator with 5% CO
2
for 48 hours, MTT assay was used.
e absorbance was measured at 560 nm. Splenocytes
proliferation (%) ((OD value of okra-treated cells)/(OD
value of control)) ×100.
2.10. Natural Killer Cell Activity Assay. e splenocytes as
the effector (3 ×105 cells/well) was added to human he-
patocyte cell line as the target cells (6 ×103 cells/well) to give
E/T ratio 50 : 1. ey were cultured in 96-well plate and
incubated at 37°C in an incubator with 5% CO
2
for 48 hours.
e activity of NK cell was evaluated by the MTT assay. e
absorbance was measured at 560 nm. NK cell activity (%)
((OD value of okra-treated cells)/(OD value of control)) ×
100.
2.11. Statistical Analysis. Statistical analysis was performed
by one-way analysis of variance (ANOVA) followed by
Duncan’s post hoc test. All analysis was performed using
IBM SPSS Statistics 24 software. e results were reported as
the mean ±standard deviation (SD) of five repeats. Pvalue
of <0.05 was considered statistically significant.
3. Results
3.1. Determination of Polysaccharide Content in Okra Pods.
Using the polysaccharide standard regression equation, the
polysaccharides content in the stock solution with dose of
100 mg/kg BW was 22.87 mg/mL.
3.2. Phagocytic Activity. Phagocytic activity was significantly
increased in P3 group and P4 group compared to normal
control group and negative control group (P<0.05). e
highest increase on phagocytic activity was shown by P3
group. Meanwhile, P1 group and P2 group increased
phagocytic activity but did not show significant difference
compared with normal and negative control groups
(Figure 1).
3.3. Cytokines Production. Serum levels of TNF-αwere
significantly increased in P1 group (423.20 ±128.66 pg/ml),
P2 group (460.40 ±79.28 pg/ml), and P4 group (282.40 ±
80.38 pg/ml) compared to normal control and negative
control groups (P<0.05). P3 group (175.50 ±79.76) also
showed slight increase of TNF-αlevel compared to normal
control and negative control groups (P>0.05) (Table 1).
In contrast, IL-17 level in P2, P3, and P4 groups was
significantly lower than normal control and negative control
groups (P<0.05). Although the difference was not signifi-
cance, P1 showed decrease level of IL-17. ere was slight
increase in the serum level of IFN-cin P4 group (174.60 ±
64.55 pg/ml) compared to normal control and negative
control groups but did not show a significant difference.
Meanwhile, there was no difference in the result of IL-23
(Table 1).
3.4. Spleen Index. Spleen index was significantly increased in
P2 group (14.84 ±2.76%) and P3 group (14.43 ±3.31%)
compared to normal control group (P<0.05). P4 group at
a dose of 100 mg/kg showed highest spleen index (16.72 ±
3.60%) and increased spleen index significantly compared to
normal control and negative control groups (P<0.05)
(Figure 2).
3.5. Splenocytes Proliferation. Splenocytes proliferation was
significantly increased in the P3 group and P4 group
compared with other groups (P<0.05). e highest increase
of splenocytes proliferation was demonstrated in P3 group
with 157.77 ±11.06% (Figure 3).
3.6. Natural Killer Cell Activity. Kgroup and all the
treatment groups (P1, P2, P3, and P4) did not show sig-
nificant increase of natural killer cell activity compared with
normal control group. P3 and P4 groups showed slight
increase in NK cell activity (104.67 ±15.32% and 106.75 ±
15.32%) compared to normal control, which was similar to
the results of the splenocytes proliferation (Figure 4).
4. Discussion
Immune system gives protection to organism against bac-
terial infection through layered defense, nonspecific im-
munity, and specific immunity. When bacteria pass through
nonspecific defense, the body forms a more complex im-
mune system [10]. ese days, the use of immunomodu-
lators to improve immune responses has been considered
one of the promising alternatives to prevent bacterial in-
fection [11]. One of the potential compounds as immuno-
modulator is polysaccharide. In this study, we used crude
polysaccharides which contain 22.87 mg/mL of poly-
saccharides after phenolic sulphuric acid assay was
performed.
Nonspecific immunity component that firstly recognizes
bacteria is macrophage [12]. Activation of macrophages
0
10
20
30
40
50
60
KN K– P1 P2 P3 P4
Phagocytic activity (%)
Group
∗∗ ∗∗
Figure 1: Effect of crude polysaccharide from okra pods on
phagocytic activity (%). KN: normal control; K: negative control;
P1, P2, P3, and P4 were treated with 25, 50, 75, and 100 mg/kg BW
crude okra polysaccharides, respectively. Each bar represents
means ±SD (n5). ∗∗P<0.05 compared to normal control.
Advances in Pharmacological Sciences 3
plays a key role in nonspecific immunity for developing
a defensive reaction against pathogens via phagocytosis
process [13]. Macrophages release products such as oxygen
radicals and tumor necrosis factor that are harmful to
bacteria [14].
Polysaccharides regulate the host immune system by
activating immune cell-related to lymphocytes, macro-
phages, and NK cells [15]. e present result demonstrated
that crude polysaccharides from okra pods at doses of
75 mg/kg and 100 mg/kg significantly increased phagocytic
activity of intraperitoneal phagocytes. Previous observations
also demonstrated phagocytes activation by polysaccharides
from okra pods both in vitro and in vivo [3, 7]. It has been
reported that crude okra polysaccharides significantly in-
creased NO production on RAW264.7 macrophage [7]. In
this study, the rise of active phagocytes may lead to increase
of proinflammation cytokines production such as TNF-α,
IFN-c, IL-17, and IL-23 and improves host defense against
bacteria. TNF-αand IFN-care cytokines produced by
macrophage and  cell due to all kinds of antigen in-
fections. Macrophage also produced IL-23 to induce acti-
vation of 17. Meanwhile, IL-17 is produced by 17
specifically. Staphylococcus aureus is extracellular bacteria.
Extracellular bacteria specifically induce  na¨
ıve to dif-
ferentiate as 17 [12]. Based on this consideration, we
examine level of TNF-α, IFN-c, IL-17, and IL-23.
Cytokines are small proteins produced by cells such as T
helper, NK cells, and macrophages that regulate the immune
Table 1: Effect of crude polysaccharide from okra pods on cytokines production (pg/ml).
Groups Cytokine (pg/mL)
TNF-αIFN-cIL-17 IL-23
KN 116.70 ±78.23 113.00 ±39.78 208.50 ±85.67 13.348 ±0.11
K157.59 ±41.95 156.80 ±51.00 204.50 ±84.65 13.300 ±0.01
P1 423.20 ±128.66∗∗∗ 98.40 ±50.98 127.50 ±78.77 13.377 ±0.04
P2 460.40 ±79.28∗∗∗ 139.20 ±23.38 73.25 ±22.53∗∗ 13.319 ±0.03
P3 175.50 ±79.76 97.20 ±41.44 78.25 ±50.42∗∗ 13.264 ±0.10
P4 282.40 ±80.38∗∗ 174.60 ±64.55 48.25 ±16.62∗∗ 13.298 ±0.08
KN: normal control; K: negative control; P1, P2, P3, and P4 were treated with 25, 50, 75, and 100 mg/kg BW crude okra polysaccharide, respectively. Values
are represented as mean ±SD (n5). ∗∗P<0.05 compared with KN group. ∗∗∗P<0.05 compared with KN, K, P3, and P4 groups; TNF: tumor necrosis
factor; IFN: interferon; IL: interleukin.
0
5
10
15
20
25
KN K– P1 P2 P3 P4
Spleen index (mg/g)
Group
∗∗ ∗∗
∗∗∗
Figure 2: Effect of crude polysaccharide from okra pods on spleen
index (mg/g). KN: normal control; K: negative control; P1, P2, P3,
and P4 were treated with 25, 50, 75, and 100 mg/kg BW crude okra
polysaccharide, respectively. Each bar represents mean ±SD
(n5). ∗∗P<0.05 compared with normal control. ∗∗∗P<0.05
compared with normal and negative control groups.
0
20
40
60
80
100
120
140
160
180
KK–P1P2P3P4
Splenocyte proliferation (%)
Group
∗∗ ∗∗
Figure 3: Effect of crude polysaccharide from okra pods on
splenocytes proliferation (%). KN: normal control; K: negative
control; P1, P2, P3, and P4 were treated with 25, 50, 75, and
100 mg/kg BW crude okra polysaccharide, respectively. Each bar
represents mean ±SD (n5). ∗∗P<0.05 compared with KN, K,
P1, and P2 groups.
0
20
40
60
80
100
120
140
160
KKP1P2P3P4
NK cell activity (%)
Group
Figure 4: Effect of crude polysaccharide from okra pods on NK cell
activity (%). KN: normal control; K: negative control; P1, P2, P3,
and P4 were treated with 25, 50, 75, and 100 mg/kg BW crude okra
polysaccharide, respectively. Each bar represents mean ±SD
(n5).
4Advances in Pharmacological Sciences
response and inflammation [16]. S. aureus presented by
active macrophage induces activation of -17. T helper-17
produced cytokine such as IL-17, IL-22, and TNF-α[12].
Among the cytokines, TNF-αis one of the most important
proinflammatory cytokines against microbe. e present
result showed that serum level of TNF-αsignificantly in-
creased in groups with administration of crude okra poly-
saccharide at doses of 25, 50, and 100 mg/kg BW. e
previous study also reported that crude okra polysaccharides
significantly increased TNF-αlevel in RAW264.7 macro-
phage [7].
From the previous study, okra polysaccharide-induced
cytokine production from macrophages through activation
of the transcription factor NFlB [17]. e transcription
factor NFlB exhibits a potent activity in modulating gene
transcription involving TNF-α. is present study of TNF-α
demonstrated that administration of crude okra poly-
saccharides at a dose of 75 mg/kg did not significantly in-
crease the level of TNF-α.
Contrast with this result, the highest increase of
phagocytic activity was found in the group with at a dose of
75 mg/kg BW. ere is no relation between active macro-
phages that undergo phagocytosis with production of TNF-α
level in this study. is result indicated that TNF-αwas not
dominantly produced by macrophages. Crude poly-
saccharides from okra pods may induce other cells to
produce TNF-α. Zhou et al. [18] reported that crude extract
of T. wilfordii strongly inhibits TNF-αand IL-1 production.
e overproduction of TNF-αis related with development of
various diseases [19]. us, we found this result beneficial
because TNF-αis suppressed in higher doses. Insignificant
level of TNF-αin P3 with dose of 75 mg/kg BW could be due
to its optimal doses. Based on this result, optimal doses of
polysaccharides to increase TNF-αwere lower doses (25 and
50 mg/kg BW). Although P4 showed significant result, the
level of TNF-αdid not raise as high as P1 and P2 groups.
is study also showed increase of serum level of IFN-c
but the difference was insignificant. Meanwhile, serum level
of IL-23 did not show any difference. Contrast with serum
level of TNF-α, all of the treatment groups had decreased
serum level of IL-17 compared with normal control group
and negative control group. IL-17 is one of the proin-
flammatory cytokines [10]. ese results suggested that
crude polysaccharides from okra pods could modulate
immune function through promoting and inhibiting cy-
tokines level which help in killing of microbes and control
proinflammation cytokine level. In this study, crude
polysaccharides from okra pods increased the serum level
of IFN-cand controlled serum level of IL-17 and IL-23
possibly to prevent healthy cells from becoming damaged.
Both IFN-cand IL-17 are proinflammatory cytokines, and
overexpression of proinflammatory cytokines will induce
excessive inflammation.
Lymphocytes circulated in the blood and the lymph are
also found in large numbers in lymphoid tissues or lymphoid
organs. e spleen is a secondary lymphoid organ, a place for
maintaining mature naive lymphocytes [20]. Spleen weight
and spleen index are changed in response to the nonspecific
immunity. It has been reported that immunomodulator can
enhance spleen index [21]. is result showed that crude
polysaccharides from okra pods at doses of 50, 75, and
100 mg/kg significantly increased the spleen index. is result
demonstrated that polysaccharides from okra pods stimulate
the immune system by inducing proliferation of immune
cells. erefore, we investigated the effect of polysaccharides
from okra pods on the splenocytes proliferation.
Splenocytes are immune cells in the spleen. Splenocytes
consist of T cell, B cell, macrophages, and dendritic cells [3].
is result showed that splenocytes proliferation was sig-
nificantly increased in groups treated with polysaccharides
from okra pods at doses of 75 and 100 mg/kg. e previous
study also demonstrated an increase of splenocytes pro-
liferation by administration of polysaccharides from Den-
drobium huoshanense [22]. Recent studies indicated that
crude polysaccharides with higher doses increase splenocyte
proliferation. Splenocyte proliferation response is related to
improved T- or B-lymphocyte immunity which could be an
indicator of immune activation [23]. As we report in this
study that crude polysaccharides from okra pods induced
spleen index, increase of spleen index may occur due to
rising of splenocytes proliferation.
As a member of lymphocyte class, NK cells are best
known for their nonspecific killing of tumor cells, and there
is evidence for their role in controlling infection in the
earliest phases of the body’s immune response [24]. ey can
react against and destroy target cells without the help of
either major histocompatibility complex- (MHC-)
dependent-recognition or prior sensitization, but by exo-
cytosis of perforin-containing granules [25].
erefore, an NK cell activity assay is a routine method
for analysis of a patient’s cellular immune response in vitro
and can also be used to test the antitumor activities of
possible drugs [26]. In this study, we further investigated NK
cells activity using human hepatocytes cell line. Our result
showed that NK cells activity was able to be restored like
normal control group but the difference was not statistically
significant. Possibly, polysaccharides target macrophages
rather than NK cells.
Immunomodulatory activities of polysaccharides may be
due to direct or indirect interaction with immune system
components. Complement proteins and monocytes, mac-
rophages, dendritic cells, neutrophils, and lymphocytes have
been reported as target responding to polysaccharides
[27–29]. Binding of polysaccharides to specific recognition
receptors on immune cells trigger diverse signaling pathway
and responses [30].
e results of the present study all together showed that
crude polysaccharides of the okra pods had the potential to
enhance the immune response of some immune compo-
nents. Treatment groups with higher doses of crude okra
polysaccharides increased phagocytic activity, spleen index,
and splenocytes proliferation. Meanwhile, treatment groups
with lower doses of crude polysaccharides from okra pods
showed the highest significant rising of TNF-αlevel. Con-
trast with other results, treatment groups with higher doses
of crude okra polysaccharides showed decrease level of IL-17
as response to prevent overexpression of proinflammatory
cytokines.
Advances in Pharmacological Sciences 5
Based on this study, crude okra polysaccharides could act
as an immunomodulator. Crude okra polysaccharides had
both immunostimulator activity and immunosuppression
activity. Most of the immune components examined in this
study showed significant increase and decrease at the doses
of 75–100 mg/kg. Crude okra polysaccharide could enhance
immune response, showed with rising of phagocytic activity,
spleen index, splenocytes proliferation, and TNF-αlevel.
Crude okra polysaccharides could suppress immune re-
sponse (immunosuppression), showed with decrease of IL-
17 level.
5. Conclusions
We concluded that crude polysaccharides from okra pods
could enhance phagocytic activity, spleen index, splenocyte
proliferation, and TNF-αlevel, but decrease IL-17 level as
a response to prevent overexpression of proinflammatory
cytokines. is study suggests that crude polysaccharides
from okra pods grown in Indonesia could act as an effective
compound to improve immune response.
Data Availability
e data used to support the findings of this study are in-
cluded within the article.
Conflicts of Interest
e authors declare that there are no conflicts of interest
regarding the publication of this paper.
Acknowledgments
is study was financially supported by applied research of
pre-eminent college or Penelitian Terapan Unggulan Per-
guruan Tinggi (PTUPT), fiscal year 2017, No.
004/SP2H/LT/DRPM/IV/2017, April 8th 2017.
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... e approaches have some limitations, including serious side effects when used for a long time, and have limited ability to produce the desired impacts [4,14]. Alternatively, the use of natural products such as compounds from plants and their derivatives have shown promising results because they are less toxic and have little or no side effects [4,14,15]. ...
... Fresh okra heals constipation, leucorrhea, spermatorrhea, diabetes, and jaundice; the mucilage can cure diarrhea, dysentery, gastric ulcer [14,17], and syphilis [3], and when mucilage of okra is mixed with a ripe banana can be used to treat colitis, cystitis, hepatitis, and jaundice. e polysaccharides from okra modulate and improve organisms' immune response due to S. aureus infection [15]. Okra pods contain polyphenols and flavonoids such as quercetin that have higher antioxidant activity [18][19][20] can scavenge free radicals and decrease oxidative stress in the cells. ...
... e blood was left to stand in the microtube for two hours at room temperature, and after forming two phases, the liquid phase was collected. e sample obtained was stored at 4°C for further use [15]. e serum was used to determine the AST, ALT, and ALP activities, and TSP concentration. ...
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Vegetables, drinking water, and preserved meats may contain sodium nitrite (NaNO2), which causes liver disease by inducing oxidative stress. Phytochemicals are highly recommended as an alternative to synthetic drugs and affordable medicines to treat liver disease because they have fewer or no side effects. Therefore, this study aims to determine the antioxidant and hepatoprotective potential of red okra fruit ethanol extract against NaNO2-induced liver damage. Thirty-six male mice were separated into six groups. The normal control group (WA) was given distilled water only, and the NaNO2 (SN) group was given only 50 mg/kg BW NaNO2. The other four groups (P1, P2, P3, and P4) were given NaNO2 and red okra ethanol extract at doses of 25, 50, 75, and 100 mg/kg BW, respectively. Gavage was administered orally for 21 consecutive days. Commercial kits define all biochemical parameters according to the manufacturer’s instructions. Liver tissue staining followed standard protocols using hematoxylin and eosin. The study revealed that NaNO2 induction causes oxidative stress and damages the liver. The activity of antioxidant enzymes (superoxide dismutase and catalase) significantly increased in the groups treated (P2–P4) with ethanol extract of red okra (p
... IL-17 levels decreased in all treatment groups. Crude okra polysaccharides also slightly increased NK cell activity and IFN-γ levels [37]. Further research by the same authors focused on the effect of okra raw polysaccharide extract (ORPE) on immune cells and cytokines in mice with diethyl nitrosamine induced-hepatocarcinogenic conditions. ...
... Okra (Abelmoschus esculentus L) [37] In vivo Polysaccharides Targeted √ -√ - ...
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Many edible plants exhibit immunomodulator activities that have beneficial effects on human health. These activities include the ability to activate, multiply, or suppress elements of the immune response. Some of these plants promote health by strengthening host defences against different diseases. In this article, we provide a comprehensive review of the constituents of several edible plants, their immunomodulatory activity, and mechanism of actions for Carica papaya, Coffea sp, Asparagus cochinchinensis, Dioscorea alata, beans, mushrooms, herbs, spices, and several vegetables. The studies reported here are pre-clinical (in vitro and in vivo) and clinical studies (limited in number). The bioactive compounds responsible for the immunomodulator activity of these plants were yet to be identified. This is because the plant is naturally a complex mixture, whilst the immune system is also an intricate system involving many cells and cytokines/chemokines. Metabolomics is a key tool for conducting global profiling of metabolites in a complex system. Therefore, it offers the ability to identify the presence of compounds in plant extracts associated with their immunomodulation effects. Likewise, metabolomics can also be used to detect any changes to metabolites in the cell as a response to treatment. Therefore, affected metabolic pathways that lead to the activation of certain immune responses can be determined from one single experiment. However, we found in this review that the use of a metabolomics approach is not yet fully developed for an immunomodulator study of food plants. This is important for the direction of future research in this field because unlike medicinal plants, food plants are consumed on a regular basis in small amounts with more obvious effects on the immune system. Information about possible bioactive compounds, their interactions (synergism, antagonism), and how the human body responds to them should be studied in a more holistic way.
... Gao et al. used fractions of okra polysaccharides, as anti-fatigue and observed it may be the main antifatigue remedy among A. esculentus substances [29]. Wahyuningsih et al. found that crude okra polysaccharides could play a role in enhancing the immune response, including phagocytic activity, spleen index, splenocytes proliferation, and control immune responses through cytokine production [30]. Liu et al. found that polysaccharides isolated from okra named (rhamnogalacturonan) possess hypoglycemic activity and are responsible for the hypoglycemia function in OP [31]. ...
... Enhancing immune response including phagocytic activity, spleen index, splenocytes proliferation, and control immune responses through cytokine production [30] 7 Synthesized ZnO nanofilms contains okra mucilage, showed high antibacterial activity against S. aureus than E. coli [34] 8 Exhibited significant in vitro antioxidant activity [16] 9 ...
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Background Vegetables with edible parts like flowers, fruits, stems, leaves, fibers, roots, and seeds are rich sources of essential vitamins, minerals, and trace elements with various medical functions. Many diseases such as osteoporosis, diabetes, high cholesterol, obesity, heart diseases, and stroke are caused by poor, healthy lifestyle or nutrition. Therefore, generation of new biological functions from vegetables will increase the interests of scientific research and applications. Main body Okra is an edible vegetable which contains vitamins, fiber, carbohydrates, protein, and minerals. The bioactive compounds of okra possess various biological activities such as anti-inflammation, antibacterial, anticancer, and antifungal. Polysaccharides from vegetables or medicinal plants are important large molecules with various biological applications. In this review, we will focus on the biological properties and nanoparticle uses of polysaccharides isolated from okra and the extraction methods of polysaccharides. Conclusion This review will enhance the scientific research findings of okra polysaccharides and recommend future prospective of polysaccharides for biological uses.
... The in vitro study of a human breast cancer cell line conducted by Monte et al. [11] revealed that the lectin protein isolated from okra could induce up to 63% cell growth inhibition. Accordingly, Wahyuningsih et al. [12] showed that the administration of an okra polysaccharide extract increased the immune response through the increase in TNF-α, IFN-γ, and Natural Killer (NK) cell activity in mice with inflammation caused by bacterial infection. Despite such evidence, a study of the activity of an ethanol extract of red okra pods as an anti-breast cancer agent has not been performed. ...
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Background and aim: Breast cancer is the most frequent malignancy in women. The consumption of phytochemical components from plants may play an essential role in preventing and treating this cancer. This study aimed to investigate the anti-cancer activity of an ethanolic extract of red okra pods (EEROP) in rats (Rattus norvegicus) induced by N-methyl- N-nitrosourea (MNU). Materials and methods: The experimental animals were divided into six groups (n=5/group), namely, KN (normal control, without any treatment), K- (negative control, exposed to MNU without EEROP), K+ (positive control, exposed to MNU and Methotrexate), and the treatment Groups P1, P2, and P3 (exposed to MNU and EEROP at doses of 50, 100, and 200 mg/kg body weight [BW], respectively). Intraperitoneal delivery of MNU and EEROP oral administration was carried out for 8 weeks. After the end of treatment, the parameters of cytokines, CD4+ and CD8+ T cells, and mammary gland histology were measured. Results: The results showed that EEROP at doses of 100 and 200 mg/kg BW significantly downregulated interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, IL-17, IL-10, and tumor growth factor-β (p<0.05). In addition, doses of 200 mg/kg BW significantly increased the activity of CD4+ and CD8+ T cells, prevented the proliferation of mammary gland epithelial cells, and yielded a significantly thinner epithelium of the mammary gland (p<0.05). Conclusion: It can be concluded that EEROP was an effective anti-cancer agent by modulating the immune response. Further studies using a nanoparticle system are warranted to achieve optimal working conditions for these bioactive compounds.
... They showed an enhanced immune response with increased NK and T-helper cells. The enhance immune study on okra pods (Abelmoschus esculentus) by (Wahyuningsih et al., 2018) against bacterial infection (Staphylococcus aureus) revealed a slight increase in NK cell activity and IFN-γ levels and decrease level of IL-17 in all treatment groups. Extracts of crude polysaccharide extract in doses of 25, 50, 75 and 100 mg/kg enhance the immune response which regulates phagocytic activity, spleen index, splenocytes proliferation, and control immune responses through cytokine production. ...
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... Okra mucilage especially has demonstrated functional health properties through in vitro and in vivo studies, such as antitumor, antioxidant, antimicrobial, hypoglycemic, and antiulcerogenic capacities, as well as the ability to bind cholesterol and bile acids, removing toxins from the liver [40][41][42][43][44][45][46]. Most studies on the functional properties of okra mucilage also focused on its potential biological activity in controlling the biochemical factors of type 2 diabetes [47][48][49][50][51]. Matazu et al. [52] developed a nutraceutical formulation using okra seeds and peels, rich in mucilage, and evaluated its antidiabetic and antioxidant properties. ...
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Abelmoschus esculentus has fruit popularly known as okra and belongs to the Malvaceae family. It is commonly used in cooking but also in traditional medicine in the treatment of worms, dysentery, inflammation, and also irritation of the stomach, intestines, and kidneys, as it is a potential functional food. Its mucilage is a highly viscous polysaccharide that is mostly composed of monosaccharides D-galactose, L-rhamnose, and galacturonic acid, as well as proteins and minerals. The functional properties of okra mucilage have been widely studied, mainly for its potential antidiabetic activity; thus, its use as adjuvant or nutraceutical therapy for diabetes is very promising. Due to its rheological properties, it is a potential resource for pharmaceutical and food applications. Okra mucilage can be extracted by several methods, which can directly influence its physicochemical characteristics and biological activity. Features such as low cost, non-toxicity, biocompatibility, and high availability in nature arouse the interest of researchers for the study of okra mucilage. The survey of research on the applications of okra mucilage highlights the importance of using this promising source of bioactive compounds with interesting technological properties. The potential of okra as a functional food, the properties of okra mucilage, and its technological applications are discussed in this review.
... Okra pectin from may also exhibit antibacterial properties via their antiadhesive effects against Helicobacter pylori (Thöle, Brandt, Ahmed, & Hensel, 2015). Also, they may be potentially tailored to serve as novel immunomodulators, as it has been shown to exert a range of immunomodulating activities including, for example, increase in proliferation of various types of cells (Chen et al., 2016;Trakoolpolpruek, Moonmangmee, & Chanput, 2019;Wahyuningsih et al., 2017;Wahyuningsih et al., 2018). Okra polysaccharides, have been also found to reduce inflammatory responses in the intestinal mucosal barrier (Xiong et al., 2021) whereas anti-inflammatory activity of baobab polysaccharides has been suggested by its moderate inhibition of cyclo-oxygenase enzymes (Ibrahim, Mahmoud, & Asker, 2014). ...
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The structure, technological and biological functionality of polysaccharides extracted from members of Malvaceae family plants have been evaluated. In addition to glycosidic linkages that are regularly observed in pectic polysaccharides, several members of this family, including lacebark, grewia, durian, and some fractions of kola have high levels of 3,4-linked galacturonic acid residues. This specific linkage is commonly found in RG-II structures casting doubts over the true macromolecular characteristics of rhamnogalacturonan segments in these plants, commonly presumed as RG-I. Several members of the family exhibit unique linkages and functionalisation not encountered in other species, such as 4-linked α-galactose that is unique to lacebark leave polysaccharides or terminal α-galactose in the kola tree bark and okra pectin. Polysaccharides from lacebark and grewia are only acetyl esterified as opposed to those from okra that are both acetyl and methyl esterified whereas those from baobab, durian and cacao have very low amounts of acetyl groups. Polysaccharides from Malvaceae family present opportunities for further technological development in the area of biomaterial science as they have a broad range of functionality allowing them to be used as emulsifiers, film-forming agents, controlled delivery vehicles, or rheology modifiers. Research on the biological activity of polysaccharides from Malvaceae is still nascent but early findings are reassuring, as a broad range of functionalities (e.g., anti-inflammatory, antioxidant or modulation of metabolism) have been reported.
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Okra (Abelmoschus esculentus L.) was commonly found in tropical and sub-tropical country. It contains high level of polyssacharide and secondary metabolites. Hence, no in vivo studies have explored okra’s ability as immunomodulatory agent toward hepatocarcinogenic condition. This study aims to evaluate the effect of okra raw polysaccharide extract (ORPE) on cytokine levels and cell apoptosis and necrosis of mice with hepatocarcinogenic condition induced by diethylnitrosamine (DEN). A total of 36 adult male mice (BALB/C) were acclimatized for 14 days and were randomly divided into six groups: the normal control group without any treatment (CN), negative control induced by DEN with no ORPE administration (C-), positive control induced by DEN with 10mg/kg BB doxorubicin administration (C+), and ORPE treatment of dose 50mg/kg BW (P1), 100mg/kg BW (P2) and 200mg/kg BW (P3) which previously have been injected with DEN. The level of interferon-gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) were measured using the ELISA method. The percentage of apoptosis and necrosis were analyzed using flow cytometry. The administration of ORPE significantly increased the level of TNF-α in DEN-induced liver cancer mice. While it did not cause significant changes in the percentage of apoptosis in mice hepatocytes. These results suggest that ORPE has an immunomodulatory effect on the cytokine of hepatocarcinogenic mice which can be used as a cancer therapy in nutraceutical and pharmaceutical industries.
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Demodex infection gradually develops to Demodex blepharitis, which is characterized as chronic inflammation of the eyelid and meibomian gland (MG) and ultimately leads to MG dysfunction. In the present prospective study, the anti-demodectic effects of an okra eyelid patch in patients Demodex blepharitis were investigated. A total of 52 patients with Demodex blepharitis with ocular discomfort were recruited. Patients were randomized to receive either an okra eyelid patch treatment (treatment group, n=27) or tea tree oil (TTO) eye care patch treatment (control group, n=25) for three months. The Demodex count, the ocular surface disease index (OSDI) score, MG expressibility (MGE) and meibum quality, Schirmer I test (SIT), tear break-up time (TBUT) and corneal fluorescein staining (CFS) were determined prior to treatment and after 1 and 3 months of treatment. Changes in the parameters were compared between the treatment group and control group after 1 and 3 months of treatment. The average survival time in the okra group was 115.25±11.87 min, which was significantly lower compared with the average ST of 378.75±37.94 min in the blank group (P<0.01). After 3 months of okra eyelid patch treatment, the Demodex count was significantly reduced from 10.15±4.53 to 1.30±1.41 (P<0.01) and the OSDI score of the patients was reduced by 16.84±10.17 (P<0.01). There was no significant difference in the Demodex count (P=0.716) and OSDI (P=0.873) between the treatment and control groups. The rate of complete Demodex eradication in the treatment group (11/27, 40.74%) was slightly lower than that in the control group (12/25, 48%), but there was no significant difference between the two groups (χ2=0.277, P=0.598). Regarding the other ocular parameters, no significant difference was observed in the TBUT, meibum quality and MGE between the two groups (P<0.05). TTO group has a significantly improvement compared with Okra group in terms of SIT (P=0.035) and CFS (P=0.023). In conclusion, okra eyelid patch treatment is able to significantly eradicate ocular Demodex as well as markedly alleviate ocular symptoms. Due to causing less irritation than TTO, the okra eyelid patch may be more suitable for sensitive patients with Demodex blepharitis, such as the elderly and children. The study was registered as a clinical trial in the Chinese Clinical Trial Registry (ChiCTR) in November 2018 (registration no. ChiCTR-1,800,019,466).
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Green tea is the second-most consumed beverage in the world (water is the first one) and has been used medicinally for centuries in Indiaand China. The active substances in the green tea are polyphenols (catechins) and flavonols which possess a potent antioxidant activity. Epigallocatechin gallate (EGCG) is one of the four major green tea catechins. Using the Ames test, micronucleus test, comet assay, chemiluminescence test, and blastic transformation test, we examined the antimutagenic effects of chemoprotective substance epigallocatechin gallate (EGCG) in the pure form on the mutagenicity induced by three reference mutagens: aflatoxin B<sub>1</sub> (AFB<sub>1</sub>), 2-amino-3-methylimidazo [4,5-f] qui-noline (IQ), and N-nitroso-N-methylurea (MNU), and the effect of EGCG on the immunosuppression caused by these mutagens. Using the Ames test the dose dependent antimutagenic activity of EGCG was proved against indirect mutagens AFB<sub>1</sub> and IQ, but not against the direct mutagen MNU. In the micronucleus test, EGCG had antimutagenic effect upon all three mutagens. EGCG decreased the level of DNA breaks induced by AFB<sub>1</sub> in bone marrow cells and colon epithelium, and the level of DNA breaks induced by MNU in colon cells to the level found in control. The reparatory effect of EGCG on immunosupression induced by all three carcinogenic compounds was proved using chemiluminescence and blastic trasformation tests.
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Cytokines play an important role in the immune system. Any disorder in the regulation of cytokines can lead to the development of inflammatory diseases. Tumor necrosis factor-α (TNF-α) is one of the most important inflammatory cytokines that controls different types of cell functions. The overproduction of TNF-α is linked with the development of various diseases such as asthma, rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease, septic shock, diabetes and atherosclerosis. Plants are considered as excellent sources of pharmacologically active compounds. Currently, scientists are searching for natural products with anti-TNF-α properties for the treatment of various inflammatory disorders. At present, protein-based drugs are available for the inhibition of TNF-α, however these have some limitations. Plant might provide an alternative and cost-effective source of drugs that can regulate TNF-α levels. This review briefly highlights the physiological and pathological roles of TNF-α along with a description of plant-derived compounds capable of interfering with TNF-α activity and production.
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Crude okra (Abelmoschus esculentus L.) polysaccharide (RPS) was obtained by water extraction and alcohol precipitation. Three purified fractions of RPS, designated RPS-1, RPS-2, and RPS-3, were fractioned by diethylaminoethyl (DEAE)-cellulose chromatography. Their molecular weights, monosaccharide compositions, infrared (Fourier transform infrared [FT-IR]) spectra, and nuclear magnetic resonance (NMR) spectra were analyzed. Their immunomodulatory activity was evaluated with an in vitro cell model (RAW264.7 cells). In vivo immunomodulatory activity of RPS-2 was evaluated in normal and cyclophosphamide-induced immunosuppressed mice. The results showed that the molecular weights of RPS-1, RPS-2, and RPS-3 were 600, 990, and 1300 kDa, respectively. RPS-1 and RPS-2 were mainly composed of galactose, rhamnose, galacturonic acid, and glucuronic acid, while RPS-3 was mainly composed of galactose, rhamnose, galacturonic acid, glucuronic acid, and glucose. FT-IR and NMR spectrum data indicated a rhamnogalacturonan I characteristic of polysaccharide. Both RPS and its purified fractions RPS-1, RPS-2, and RPS-3 significantly increased RAW264.7 cell proliferation, nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression, and tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-10 secretion (P < .05). The purified fraction RPS-2 also increased the spleen index, splenocyte proliferation, and cytokine secretion in vivo. These results indicate that okra polysaccharides may potentially serve as novel immunomodulators.
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Immunostimulatory polysaccharides are compounds capable of interacting with the immune system and enhance specific mechanisms of the host response. Glucans, mannans, pectic polysaccharides, arabinogalactans, fucoidans, galactans, hyaluronans, fructans, and xylans are polysaccharides with reported immunostimulatory activity. The structural features that have been related with such activity are the monosaccharide and glycosidic-linkage composition, conformation, molecular weight, functional groups, and branching characteristics. However, the establishment of structure-function relationships is possible only if purified and characterized polysaccharides are used and selective structural modifications performed. Aiming at contributing to the definition of the structure-function relationships necessary to design immunostimulatory polysaccharides with potential for preventive or therapeutical purposes or to be recognized as health-improving ingredients in functional foods, this review introduces basic immunological concepts required to understand the mechanisms that rule the potential claimed immunostimulatory activity of polysaccharides and critically presents a literature survey on the structural features of the polysaccharides and reported immunostimulatory activity. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Modulation of immune response to alleviate diseases has long since been of interest. Plant extracts have been widely investigated for their possible immunomodulatory properties. We have evaluated the immunomodulatory activity of aqueous extract of Rhodiola rhizome in human peripheral blood mononuclear cells (PBMCs) and mouse macrophage cell line RAW 264.7. The Rhodiola extract was found to stimulate production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in human PBMCs as well as RAW 264.7 cell line. It also increased production of nitric oxide synergistically in combination with lipopolysaccharide (LPS) in RAW 264.7. Rhodiola at 250 μg/ml increased the p-IκB expression in human PBMCs. Aqueous extract of Rhodiola (250 μg/ml) also activated the nuclear translocation of NF-κB in human PBMCs, which is comparable to the positive stimulant LPS. Thus, our present study suggests that Rhodiola most likely activates proinflammatory mediators via phosphorylated inhibitory kB and transcription factor NF-kB. Our study demonstrates immunostimulatory potential of aqueous extract of Rhodiola rhizome, that can be used for upregulation of immune response in patients with inadequate functioning of the immune system.
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A neutral polysaccharide fraction (SMPA) prepared from the roots of Salvia miltiorrhiza by DEAE-cellulose and Sephadex G-100 chromatography was tested for its immune enhancing function in N-methyl-N′-nitro-nitrosoguanidine (MNNG)-induced gastric cancer rats by intragastric administration. SMPA (200 mg/kg) treatment not only increased the body weight, but also improved the immune organ indices. Furthermore, studies of various immunological activities in gastric cancer rats revealed that SMPA significantly stimulated splenocyte proliferation, promoted anti-inflammatory cytokines (IL-2, IL-4 and IL-10) production, inhibited pro-inflammatory cytokine (IL-6 and TNF-α) secretion, augmented the killing activity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), and increased phagocytotic function of macrophages in gastric cancer rats. In addition, SMPA administration evidently elevated total intracellular granzyme-B and IFN-γ levels produced by splenocytes in gastric cancer rats. Taken together, these results suggested that SMPA could act as an effective immunomodulator and might be explored as a potential supplemental source for gastric cancer therapy.
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Schisandra chinensis (Turcz.) Baill has been used in traditional Chinese medicine for centuries(1). Previous studies have shown that Schisandra polysaccharide (SCPP11) has robust antitumor activity in vivo. In this study, the immunomodulatory activity and mechanisms of action of SCPP11 were investigated further to reveal its mechanism of action against tumors. Results showed that SCPP11 increased the thymus and spleen indices, pinocytic activity of peritoneal macrophages, and hemolysin formation in CTX-induced immunosuppressed mice. Moreover, SCPP11 significantly increased immunoglobulin levels, cytokines levels in vivo and induced RAW264.7 cells to secrete cytokines in vitro. RAW264.7 cells pretreated with SCPP11 significantly inhibited the proliferation of HepG-2 cells. In addition, SCPP11 promoted both the expression of iNOS protein and of iNOS and TNF-α mRNA. TLR-4 is a possible receptor for SCPP11-mediated macrophage activation. Therefore, the data suggest that SCPP11 exerted its antitumor activity by improving immune system functions through TLR-4-mediated up-regulation of NO and TNF-α.
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To evaluate the immunomodulating responses in intestine, spleen and liver, 50 to 200mg/kg of DHP were orally administrated to mice without or with methotrexate. The proliferation of marrow cells, which was performed with the addition of the supernatant of small intestinal lymphocytes isolated from the mice administrated orally with DHP, showed that the intestinal immune response was significantly enhanced in all DHP-treated groups. For the immune response in spleen, all tested doses of DHP remarkably promoted the proliferation of splenic cells and increased the secretion of interferon-γ (IFN-γ). For the immune responses in liver, DHP not only significantly stimulated the proliferation of hepatic cells and the secretion of IFN-γ at all tested doses of DHP, but also significantly elevated the secretion interleukin-4 (IL-4) at the doses of 100 and 200mg/kg. Moreover, DHP could recover methotrexate-injured small intestinal immune function (100 and 200mg/kg) and promoted cell proliferation and IFN-γ production (200mg/kg) in spleen and liver of methotrexate-treated mice. These results suggested that DHP after oral administration possessed immunomodulating effects both in small intestine immune system and in systemic immune system, which were further proved by the mRNA expression of IFN-γ and IL-4.