Journal of Ethnopharmacology 215 (2018) 191–198

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

In vivo nonspecific immunomodulatory and antispasmodic effects of T

common purslane (Portulaca oleracea Linn.) leaf extracts in ICR mice
⁎
Elena S. Catapa,b, , Markyn Jared L. Khoa, Maria Rexie R. Jimeneza,b
a
Institute of Biology, National Science Complex, University of the Philippines, Diliman, Quezon City 1101, Philippines
b
Natural Sciences Research Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines

A R T I C L E I N F O A B S T R A C T

Keywords: Ethnopharmacological relevance: Portulaca oleracea (common purslane) is used in traditional medicine to cure
Immunomodulation various illnesses. However, its immune-protective properties and antispasmodic effects still need more phar-
Antispasmodic activity macological data if the plant will be utilized in herbal and drug formulations. Therefore, the present study
Portulaca oleracea determined the capacity of this plant species to modulate nonspecific immune responses and to confirm its
Intestinal motility
antispasmodic activity in vivo in ICR mice.
Nonspecific immunity
Materials and methods: Phagocytic activity of peritoneal macrophage, splenic lymphocyte proliferation and
plasma lysozyme levels were measured in mice that were immunosuppressed using cyclophosphamide and
treated with the ethyl acetate extract of Portulaca oleracea. In addition, the charcoal meal transit test was used to
measure intestinal motility using ethanolic (EtOH), hexane (HEX), and ethyl acetate (EA) solvent extracts.
Phytochemical analysis was undertaken and DPPH scavenging properties of the three solvent extracts were also
determined.
Results: The EA extract of P. oleracea exhibited immunoactivity through significant increase in phagocytosis and
higher proliferative response in splenic lymphocytes. Plasma lysozyme level was also higher in EA-treated mice
at high dose but this was not statistically significant. Decreased intestinal motility was also exhibited in mice
treated with the three leaf solvent extracts compared to the negative control and the acetylcholine-treated group.
The antispasmodic activity of the solvent extracts was comparable to that of the atropine-treated group.
Phytochemical analysis showed the presence of tannins in EA extract in addition to alkaloids and steroids. The
EtOH and HEX extracts contain alkaloids, steroids and terpenoids. DPPH scavenging activity was highest in the
EA extract.
Conclusions: The present study showed that the EA extract of P. oleracea leaves ameliorated the im-
munosuppressive action of cyclophosphamide in mice. The results also indicated that the three solvent extracts
of the plant decreased smooth muscle spasms in mice ileum. However, further experiments are warranted to
further isolate the plant's immunoactive component. Also, the mechanisms involved in the immunoactivity and
antispasmodic properties of P. oleracea deserve full elucidation.

1. Introduction Antispasmodic and antispastic agents are used to inhibit or decrease

The use of plant extracts with medicinal and therapeutic values has
gained a lot of attention in recent years due to the serious side-effects
often caused by the use of synthetic drug formulations (Patil et al.,
2012). Presently, interests on immunomodulatory activities of plant-
based extracts have likewise intensified. The complex of compounds in
natural immunomodulators could provide smoother action and produce
less allergic reactions compared to synthetic drugs. Furthermore, these
compounds do not accumulate to toxic levels and thus, they may be
administered for an extended period of time (Vijaya et al., 2011).
the intensity of muscle contractions and muscle tone, and spasms (Van
Tulder et al., 2003). Plant-derived natural products from chamomile,
fennel, peppermint, melissa and caraway have been traditionally used
as antispasmodic agents (Khalighi et al., 1988). Since their usage
started traditionally, their biological activities must be further verified
and confirmed through animal model studies.
Common purselane or Portulaca oleracea (Family Portulaceae) is a
medicinal plant that possess anti-inflammatory, analgesic, antioxidant,
antispasmodic, hypoglycemic, antihypoxic, wound healing, and anti-
tumor activities (Behravan et al., 2011; Chan et al., 2000; Dawei et al.,

⁎
Corresponding author at: Institute of Biology, National Science Complex, University of the Philippines, Diliman, Quezon City 1101, Philippines.
E-mail address: elenacatap@yahoo.com (E.S. Catap).

https://doi.org/10.1016/j.jep.2018.01.009
Received 14 October 2016; Received in revised form 29 November 2017; Accepted 6 January 2018
Available online 09 January 2018
0378-8741/ © 2018 Elsevier B.V. All rights reserved.
E.S. Catap et al.
2010; Dkhil et al., 2011; Erkan, 2012; Kumar et al., 2008; Li et al.,
2009; Okwuasaba et al., 1986; Parry et al., 1987; Rashed et al., 2003;
Wang et al., 2012; Yan et al., 2012). Due to its detailed medicinal and
natural therapeutic records, many cultures around the world have
branded it as a “global panacea” (Dweck, 2001). In fact, the World
Health Organization has listed P. oleracea as one of the most commonly
used medicinal plants (Behravan et al., 2011). In the Philippines, the
leaves of the plant have been traditionally used to treat various medical
problems (Pardo de Tavera, 1901).
Most of the medicinal uses of common purslane could be attributed
to the compounds in its various plant parts. For instance, a fraction
from the methanolic crude leaf extract reportedly had high phenolic
acid and flavonoid contents (Erkan, 2012). Its seeds were reported to
possess polyunsaturated fatty acids, flavonoids and polysaccharides (El-
Sayed, 2011). Betacyanins were also obtained from aqueous extracts of
P. oleracea seedlings (Wang and Yang, 2010), while aqueous extract of
its leaves had phenolic compounds, including flavonoids, tannins and
other phenolic compounds, carbohydrates and terpenoids (Zidan et al.,
2014).
Ethnopharmacological significance of P. oleracea as an im-
munomodulator is mostly associated with its anti-inflammatory activity
and wound healing properties as previously reported (Simopoulos,
2004; Abas et al., 2006). Results from the study of Barakat and
Mahmoud (2011) showed the immunomodulatory effect of a mixture of
purslane, pumpkin and flax seeds through an increase in serum IgG and
IgM in cholesterolemic rats. These limited reports warrant the need for
further research on the immune-protective properties of P. oleracea.
Although the antispasmodic effects of this particular plant have
been studied previously in skeletal muscles (Okwuasaba et al., 1986;
Parry et al., 1987, 1993; Habtemariam et al., 1993), there are very
limited studies on its effects on intestinal and vascular smooth muscle
contractions (Parry et al., 1988) and bronchial smooth muscles (Malek
et al., 2004). In the present study, in vivo experiments were undertaken
in order to determine the effect of P. oleracea ethyl acetate extract on
some nonspecific immune responses of cyclophosphamide-treated mice.
In addition, the charcoal meal transit test using mice as experimental
animal was undertaken to validate the antispasmodic activity of three
solvent extracts prepared from the leaves of P. oleracea.
2. Materials and methods
2.1. Plant materials and preparation of solvent extracts
Portulaca oleracea leaves were collected in Dasmariñas, Cavite,
Philippines. The plant specimens were authenticated at the Jose Vera
Santos Memorial Herbarium, Institute of Biology, University of the
Philippines, Diliman, Quezon City, Philippines, where a voucher spe-
cimen has been deposited (Accession No. 3821). The leaves were al-
lowed to air-dry until crisp, and then pulverized using a homogenizer.
For extraction, 115 g of pulverized leaves was soaked in 95% ethanol
(1:30 w/v ratio) for 48 h. The resulting supernatant was collected and
the ethanolic extract (EtOH) was obtained using a rotary evaporator.
The percentage yield for the ethanolic extract was 9.85% (11.33 g of
crude extract/115 g dry material). The hexane (HEX) and ethyl acetate
(EA) solvent extracts were obtained via liquid-liquid partitioning. For
partitioning, 10 g of the crude EtOH extract was dissolved in distilled
water and placed in a separatory funnel. An equal amount of hexane
was then added into the separatory funnel and vigorously mixed
through shaking, occasionally releasing the pressure by opening the
stopcock. Eventually, both layers (HEX and EtOH) were collected in
separate glass containers. The same procedure was done in preparing
the EA fraction from the EtOH extract. The collected extracts were
poured in Petri plates, air dried and stored at 4 °C prior to use in the
tests and assays. Percentage yield for the HEX and EA extracts is 3.7%
and 9.6%, respectively (0.37 g/10 g for HEX and 0.96 g/10 g of crude
extract for EA). Solvents used (ethanol, hexane and ethyl acetate) were
192
Journal of Ethnopharmacology 215 (2018) 191–198
purchased from J.T. Baker (Mallinckrodt Baker, Inc., Phillipsburg, NJ,
USA).
2.2. Phytochemical analysis
The phytochemical components of the three solvent extracts were
analyzed for the following compounds using standard methods:
2.2.1. Test for tannins
2 mg of each sample was dissolved in 10–15 drops of methanol then
mixed with 5 mL of distilled water. Ferric chloride solution was then
added dropwise to the solution (Shah and Seth, 2010).
2.2.2. Test for saponins
5 mg of each sample was dissolved in 10–15 drops of methanol then
mixed with 5 mL of distilled water. The solution was boiled, cooled, and
then shaken vigorously afterwards. Frothing of the solutions indicated
the presence of saponins (Trease and Evans, 1989).
2.2.3. Test for terpenoids
2 mg from each sample were dissolved in 2 mL of CHCl3, followed
by addition of concentrated H2SO4. The occurrence of a reddish brown
interface between the two layers indicated the presence of terpenoids
(Shah and Seth, 2010).
2.2.4. Test for flavonoids
2 mg of each sample was dissolved using 1 M NaOH. Afterwards, the
solution was observed for changes from its initial color to a yellow or
orange hue which indicated the presence of flavonoids. The resulting
colorless solution upon the addition of 1 M HCl further confirms the
presence of flavonoids (Harborne, 1998).
2.2.5. Test for cardiac glycosides
2 mg of each sample was dissolved in 10–15 drops of methanol,
followed by addition of 2 mL of distilled water. One percent (1%) FeCl3
was then added dropwise, followed by addition of 1 mL concentrated
H2SO4. The formation of a brown ring confirmed the presence of car-
diac glycosides (Harborne, 1998).
2.2.6. Test for steroids
2 mg of each extract was dissolved in 2 mL acetic anhydride, fol-
lowed by addition of 2 mL of 1 M H2SO4. The change in color of the
solution to green or blue indicates the presence of steroids (Shah and
Seth, 2010).
2.2.7. Test for alkaloids
5 mg of the extract was dissolved in 10–15 drops of methanol, then
mixed with 2 mL distilled water. This was followed by addition of three
drops of Wagner's reagent (2 g of I2 and 6 g of KI dissolved in 100 mL of
distilled water). The formation of blue-black precipitate confirmed the
presence of alkaloids (Shah and Seth, 2010).
2.3. DPPH (α, α-diphenyl-β-picrylhydrazyl) assay for scavenging activity
The DPPH assay method used in this study was described by Mensor
et al. (2001). Increasing concentration (5 µg/mL, 10 µg/mL, 25 µg/mL,
50 µg/mL, 125 µg/mL and 250 µg/mL) of plant extract was prepared by
addition of ethanol to a previously prepared stock solution of the ex-
tract (w/v = 1 mg/mL). One mL of 0.3 mM DPPH solution in ethanol
was added for every 2.5 mL of the solutions. One mL ethanol mixed
with 2.5 mL extract solution served as the blank solution. The negative
control was prepared by mixing 1 mL of 0.3 mM DPPH solution with
2.5 mL of ethanol. The solutions were allowed to react at room tem-
perature (26–28 °C) for 30 min and then were read with a spectro-
photometer at 517 nm. The absorbance readings were used to calculate
the percentage antioxidant activity (%AA) as free radical scavenging
E.S. Catap et al.
activity (FRSA) of the extract using the formula:
[(Abs (sample ) − Abs (blank )) x100]
%AA = 100 −
Abs (control)
2.4. Experimental animals
Two batches of ICR mice used for the in vivo experiments were
obtained from the Food and Drug Administration, Alabang, Muntinlupa
City, Metro Manila, Philippines. The mice were maintained in the an-
imal facility of the Institute of Biology, University of the Philippines.
The animals were given free access to food and water for one week
during acclimatization. The protocol for handling and maintenance of
the animals was approved by the University of the Philippines Diliman-
Institutional Animal Care and Use Committee (UPD-IACUC PAF-IB-
2013-06).
2.5. Experimental designs
2.5.1. In vivo immunomodulation experiment
Prior to the in vivo experiments, an in vitro MTT assay (Mossman,
1983) to test the proliferation of splenic lymphocytes was performed to
determine which extract will be administered to the mice. All the ex-
tracts stimulated lymphocyte proliferation, except HEX at 100–200 µg/
mL which failed to stimulate cellular proliferation, possibly due to some
cytotoxic effects. However, the highest proliferative effect was observed
at concentrations of 50–100 µg/mL for the EA extract, with lower
proliferative effects at concentrations of 200 µg/mL. The proliferative
effects of EA were comparable to that of ConA but higher than that
induced by LPS. Therefore, the EA extract was selected for the in vivo
immunomodulation experiment.
A total of forty-eight (48) mice were divided randomly into four
groups with three replicate cages per group. Each replicate cage con-
tained four mice. The four treatment groups are as follows: negative
control, positive control, and experimental groups, which were ad-
ministered with the P. oleracea EA extract (high and low doses) on a
daily basis via oral gavage. After one (1) h, the mice were administered
with a dose of cyclophosphamide (30 mg/kgBW) via oral gavage on
days 1, 4, 7, 10, and 13 of the experiment. The EA extract treatments
were administered using high (50 mg/kgBW) and low (5 mg/kgBW)
dosages. The negative control group was administered with sterile
phosphate-buffered saline (PBS), while the positive control group was
administered with cyclophosphamide (30 mg/kgBW) on the days pre-
viously indicated. Mice were sacrificed on days 8 and 15 to collect
blood and spleen tissues to be used in the immune assays. Peritoneal
macrophages were collected through peritoneal lavage and used for the
phagocytosis assay. Plasma was collected and used for the lysozyme
assay while splenic lymphocytes were isolated and used for the lym-
phocyte proliferation assay.
2.5.2. Immune response assays
2.5.2.1. Peritoneal macrophage phagocytic activity. A quantitative
measurement of the phagocytic activity of the peritoneal
macrophages was done via microscopic observation and cell counting.
The protocol used is a modified version of that of Zelikoff (1997). The
macrophages were obtained via peritoneal lavage after irrigation of the
peritoneal cavity with 5 mL of RPMI-1640 medium (Gibco cat. no.
31800-022). The exudates were collected and subjected to
centrifugation at 1500 rpm, 25 °C for 20 min prior to resuspension
with 1 mL of RPMI-1640 medium. The viability and cell concentration
was determined through the Trypan-blue dye exclusion technique in
order to obtain a final cell concentration of 1 × 106 cells/mL.
Congo red-coated yeast cells, Saccharomyces cerevisiae, were used as
the test material to be engulfed by the macrophages. The yeast cells
were first coated with Congo red stain to aid in adhesion and inter-
nalization. A mixture containing 250 μL of peritoneal macrophages and
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Journal of Ethnopharmacology 215 (2018) 191–198
50 μL of yeast cells were incubated for 1 h at 37 °C, and from which an
aliquot of 100 μL was placed onto a glass slide and smeared, air-dried,
and fixed with 95% ethanol. The cells were subsequently stained with
Giemsa and counterstained with Eosin Y. A total of 100 macrophages
were counted per slide. Quantification of the phagocytic activity was
calculated by dividing the number of cells containing the engulfed yeast
cell particles by the total number of cell counted.
% Phagocytic activity
number of cells with engulfed yeast particles × 100
=
total number of cells counted
2.5.2.2. Lymphocyte proliferation assay. To measure lymphocyte
proliferation, the protocol used is the MTT assay developed by
Mossman (1983). Mice were sacrificed and the spleen tissues
harvested using aseptic techniques. The spleen tissues were then
mechanically ground using fine mesh wirescreen in Petri dishes
containing PBS, then transferred into 15 mL conical tubes with RPMI-
1640 phenol red-free medium (RPMI-1640 PR- Sigma cat. no. R8755)
supplemented with 10% heat-inactivated fetal bovine serum and
penicillin and streptomycin. The cell suspension was incubated for
10 min in an ice bath and then centrifuged for 10 min at 30,000 rpm.
The cell suspensions were decanted and resuspended in 5 mL Tris Azo
Coupling or TAC buffer (20 mM Tris-HCl, pH 7.2 and 0.82% NH4Cl).
The spleen lymphocyte population was then isolated using density
gradient centrifugation. Percoll (density=1.077, Sigma cat. no. P1644)
was prepared in 15 mL conical tubes and the crude splenocyte
suspension was layered on top and then subjected to centrifugation
for 20 min at 30,000 rpm. After centrifugation, the layer of cells was
obtained using a Pasteur pipette, and washed three times with
unsupplemented RPMI-1640 medium. The cells were resuspended in
1.0 mL of RPMI-1640 medium and the viability was determined using
the trypan blue exclusion technique. The working cell concentration
was adjusted to 1 × 106 cells/mL.
Four centrifuge tubes were prepared for the reaction mixture prior
to distribution in a 96-well plate. Tube 1 served as the negative control
without any mitogen, plant extract and an additional blank tube with
culture medium only was prepared. Lymphocyte proliferation was de-
termined by adding 200 μL of the cell suspension to computed volumes
of plant ethanolic extract and the mitogens (LPS and ConA) as reference
compounds. Ten (10) μL of lipopolysaccharide (LPS, Sigma cat. no.
L3129; stock concentration = 2 mg/mL; final concentration = 10 μg/
mL) and 5 μL of ConcavalinA (ConA, Sigma cat. no. C7275; stock
concentration = 2 mg/mL; final concentration = 10 μg/mL) were then
dispensed into centrifuge tubes 2 and 3, respectively, while the plant
solvent extract were added in tube 4. The final volume of the reaction
mixture in each centrifuge tubes was set at 1.0 mL using the RPMI-1640
PR- medium as diluent. From each tube, 200 μL of the reaction mixture
was transferred into a 96-well microplate in triplicates, before in-
cubation of the plate at 37 °C for 48 h in a humid, 95% CO2 environ-
ment (Anaeropack CO2, Mitsubishi Gas Chemical Co., Inc.). After in-
cubation, the medium in all wells were replaced with a solution
comprising 40 μL of 1x RPMI-1640 PR- (supplemented with 100U/
100 μg penicillin/streptomycin and 0.25 μg/mL amphotericin B) and
20 μL of MTT salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide; Sigma cat. no. M2128; stock concentration = 5 mg/
mL in PBS). After further incubation in similar conditions for 3 h,
100 μL of dimethyl sulfoxide (DMSO) was added into the wells to stop
the reaction. Then, absorbance readings at 595 nm were taken.
Lymphocyte proliferation was computed using the equation:
ODsample – Odcontro l × 100
% Proliferation =
ODcontrol
2.5.2.3. Plasma lysozyme assay. For this assay, blood samples were
E.S. Catap et al. Journal of Ethnopharmacology 215 (2018) 191–198

collected from the external jugular vein using syringes with heparin. Table 1
The procedure described by Ellis (1990) was used with modifications. Phytochemical analysis of the ethanolic (EtOH), ethyl acetate (EA) and hexane (HEX)
solvent extracts of Portulaca oleracea, common purslane. (+) – present; (−) – absent.
The blood samples were centrifuged for 10 min at 1000 rpm to extract
the blood plasma samples, which were then stored at 20 °C. The assay Phytochemical test EtOH EA HEX
was done using a 96-well microplate, with each well (in triplicate)
containing 175 μL of a suspension of the bacterium Micrococcus Tannins − + −
Saponins − − −
lysodeikticus (75 mg/100 mL of 0.1 M phosphate/citrate buffer with
Terpenoids + − +
0.09% NaCl, pH 5.6) plus 25 μL of either the plasma samples or hen egg Flavonoids − − −
white lysozyme (Sigma cat. no. L6876) standard (0–50 μg/mL of 0.1 M Cardiac glycosides − − −
phosphate/citrate buffer with 0.09% NaCl, pH 5.6). After incubation of Steroids + + +
the plate for 30 min, the lysozyme activity on the bacterium was taken Alkaloids + + +

by absorbance at 450 nm, after which a standard curve was generated

based on the hen egg white lysozyme concentrations. Plasma lysozyme
3. Results
activity was expressed as μg lysozyme/mg protein.
3.1. Phytochemical analysis (Table 1)

2.5.2.4. Total protein determination. Total protein content of blood

Table 1 shows the results of the phytochemical tests on the three
plasma was determined with the use of the BIO-RAD Protein Assay
solvent extracts obtained from the leaves of P. oleracea. All three solvent
Kit (BIO-RAD Laboratories; California, USA) according to the
fractions have steroid and alkaloid components while the EA fraction
manufacturer's instructions. The protein assay kit was based on the
also has tannins. Both the EtOH and HEX fractions also contain terpe-
protocol of Bradford (1976). Bovine serum albumin (BSA), at different
noids.
dilutions, was used as the standard. Ten (10) μL of each standard and
sample solution were added to 96-well microplates. Two hundred
(200) μL of the prepared dye was then added to all wells. The plates 3.2. DPPH assay
were incubated for 5 min at room temperature and absorbance was
read at 570 nm with the use of an ELISA plate reader. Data were Fig. 1 shows the DPPH scavenging activity of the three P. oleracea
expressed as mg/mL. fractions. All three leaf solvent extracts showed scavenging activity but
the EA extract showed clearly a dose-dependent effect with 95–100%
activity at 125–250 µg/mL.
2.5.3. In vivo antispasmodic experiment
Male ICR mice (4–6 weeks old, 54 mice) were used as experimental 3.3. Nonspecific immunomodulatory activity
animals for this experiment. Acetylcholine and atropine (Sigma-Aldrich
Co., St. Louis, MO, USA) as reference drugs and the solvent extracts 3.3.1. Peritoneal macrophage phagocytic activity
(EtOH, HEX and EA) were administered orally via gavage. The charcoal At day 8 (Fig. 2), the cyclophosphamide-treated group exhibited
meal transit test protocol used in the study was based on the works of significantly lower phagocytic activity than the negative control mice.
Rao et al. (1997), Gilani et al. (2006) and Adeyemi et al. (2009) with The EA treatment groups showed significantly increased phagocytic
some modifications. ICR mice were fasted overnight prior to the ex- activity relative to the positive control. At day 15, the cyclopho-
periments, but allowed free access to water. The negative controls were sphamide-treated mice exhibited lower phagocytic activity than the
administered orally with PBS. The reference drug groups were ad- negative control, but this was not statistically significant. The EA
ministered with (acetylcholine as the reference drug for stimulating treatment resulted in increased phagocytic activity relative to the po-
contractions, 5 mg/kg), the third group of mice were treated with sitive control, but only the EA high dose group showed significantly
atropine (as reference relaxant drug, 5 mg/kg). The groups to be treated enhanced phagocytic activity.
with the three plant solvent extracts received two concentrations se-
parately via gavage (5 mg/kg and 50 mg/kg concentrations). Fifteen
3.3.2. Splenic lymphocyte proliferation
minutes after the treatment, charcoal meal (10% charcoal suspension in
In LPS-treated lymphocytes (Fig. 3) at day 8, the positive control
5% gum acacia solution) were orally administered. All animals were
mice exhibited a slightly higher lymphocyte proliferation rate than the
sacrificed fifteen minutes later and the small intestine was immediately
negative control mice. Both EA treatment groups showed increased
dissected out and placed on a clean surface. The intestine was inspected
cellular proliferation relative to the positive control, with the EA high
and the distance traveled by the charcoal meal plug from the pylorus up
group having a higher proliferation rate than the EA low group. For
to the cecum was measured. The distance traveled by the charcoal plug
ConA-treated lymphocytes, the positive control mice exhibited a
was expressed as a percentage of the total length of the small intestine.
slightly lower lymphocyte proliferation rate than the negative control
All treatment groups, including the negative control, reference groups
mice. The EA treatment groups likewise showed higher proliferation of
(acetylcholine and atropine) were set up in two replicates of three mice
each replicate.

2.6. Statistical analysis

All data were analyzed using Kolmogorov-Smirnov's test and

homogeneity of variance prior to one-way ANOVA. Tukey's test was
used as post hoc test for homogenous data and Games-Howell test for
non-homogenous data. Non-normal data were analyzed using Kruskal-
Wallis and Mann-Whitney test. The software package SPSS ver. 20 was
used for data analysis. Fig. 1. Antioxidant activity of Portulaca oleracea leaf EtOH, EA and HEX extracts mea-
sured through scavenging of DPPH (α, α-diphenyl-β-picrylhydrazyl) compound. Data
obtained from three trials.

194
E.S. Catap et al. Journal of Ethnopharmacology 215 (2018) 191–198

80% 0.9
70% b 0.8
b b
b ab ab
0.7

(μg lysozyme/mg protein)

60%

Lysozyme Concentraon
a
Phagocyc Acvity

50% 0.6
a
Negave Control 0.5
40% Negave Control
Posive Control
30% 0.4 Posive Control
EA Low
20% 0.3 EA Low
EA High
0.2 EA High
10%
0.1
0%
Day 8 Day 15 0
Day 8 Day 15
Fig. 2. Effects of Portulaca oleracea EA leaf extract on the phagocytic activity of murine
peritoneal macrophages at day 8 and day 15 of experiment 2. Each bar represents the Fig. 5. Effects of Portulaca oleracea EA leaf extract on the blood plasma lysozyme activity
mean ± SEM percent (%) phagocytic activity per treatment. Different letters indicate at day 8 and day 15 of experiment 2. Each bar represents the mean ± SEM lysozyme
significant difference at P < 0.05; n = 3. concentration (μg lysozyme/mg protein). Significance was set at P < 0.05; n = 3.

positive control. At day 15 sampling, the positive control exhibited

lower lysozyme activity than the negative control. Both dosages of the
EA extract showed increased lysozyme activity relative to the positive
control.

3.4. Antispasmodic activity

Compared to the acetylcholine-treated group of mice (89.71%),

intestinal motility was lower in mice treated with all the doses of all P.
oleracea solvent fractions. The higher dose of the fractions (EtOH:
60.35%, EA: 53.94%, HEX, 57.84%) was also found to induce sig-
nificantly slower motility than the lower dose (EtOH: 66.83%, EA:
69.62%, HEX: 72.26%), except EtOH fraction that was comparable to
the effect of atropine (Fig. 6).
Fig. 3. Effects of Portulaca oleracea EA leaf extract on the cell proliferation activity of
splenic lymphocytes at day 8 of the experiment. Each bar represents the mean ± SEM
percentage (%) lymphocyte proliferation. Significance was set at P < 0.05; n = 3. LPS, 4. Discussion
lipopolysaccharide; ConA, Concanavalin A.
Portulaca oleracea has been reported to possess a number of bioac-
700% tive compounds such as alkaloids, flavonoids, glycosides, and triterpene
600%
acids, phenols, β-carotene, α-linolenic acid, palmitic acid, oleic acid,
ascorbic acid and many other compounds such as polysaccharides
500% (Chowdhary et al., 2013; Naeem and Khan, 2013; Kamal Uddin et al.,
Lymphocyte Proliferaon

2014). Because of these various compounds and secondary metabolites,

400%
300%
200%
100%
0%
LPS ConA
-100%
Fig. 4. Effects of Portulaca oleracea EA leaf extract on the cell proliferation activity of
splenic lymphocytes at day 15 of the experiment. Each bar represents the mean ± SEM
percentage (%) lymphocyte proliferation. Significance was set at P < 0.05; n = 3. LPS,
lipopolysaccharide; ConA, Concanavalin A.
lymphocytes. In LPS-treated lymphocytes (Fig. 4) at day 15, the positive
control mice exhibited a lower lymphocyte proliferation rate than the
negative control mice. Both EA treatment groups showed increased
cellular proliferation relative to the positive control, with the EA high
group having a higher proliferation rate than the EA low group. Similar
results were observed in the ConA-treated lymphocytes.
3.3.3. Plasma lysozyme concentration
At day 8 sampling (Fig. 5), the positive control exhibited higher
lysozyme activity than the negative control. The EA low dose group
showed slightly higher lysozyme activity than the positive control,
while the EA high dose group had lower lysozyme activity than the
Negave Control this plant species was reported to possess antioxidant and medicinal
Posive Control properties (Lim and Quah, 2007; Erkan, 2012; Kamal Uddin et al.,
EA Low 2014). This paper further confirms the antioxidant properties of P.
EA High oleracea based on the DPPH scavenging activity, which was highest in
the EA extract. This activity could be due to the tannins and alkaloids
present in the extracts (Zijuan et al., 2009; Dey et al., 2015). Zijuan
et al. (2009) reported that phenolic alkaloids from P. oleracea has
higher DPPH radical scavenging activity than ascorbic acid and α-to-
copherol. Siriamornpun and Maitree (2010) attributed the antioxidant
activity of P. oleracea to the flavonoid content of the leaf aqueous ex-
tract. However, we failed to detect flavonoids in our extracts. This could
be due to the type of solvent that we used for our extract since some
studies have obtained higher polyphenolic content in water and me-
thanolic extracts (Lim and Quah, 2007). It is also possible that our
extract had very low flavonoid content that was beyond the detection
limit of our test. The difference could also be due to geographical lo-
cations of the plant source since it was reported that the phenol content
of some Malaysian cultivars of ornamental P. oleracea differs to that of
wild P. oleracea (Lim and Quah, 2007).
In the present study, the results showed that treatment with the EA
fraction of P. oleracea leaf extract ameliorated the effects of cyclopho-
sphamide, an immunosuppressant agent, indicated by higher non-
specific immune response than in mice without P. oleracea extract
treatment.
In the study of Zheng et al. (2014), the presence of the poly-
saccharides galactose and rhamnose from the aqueous extract of okra

195
E.S. Catap et al.
(Abelmoschus esculentus) flowers enhanced macrophage activity through
activation of the NF-κB nuclear pathway. Similarly, Portulaca oleracea
has been found to contain an abundance of polysaccharides (El-Sayed,
2011). It is likely then, that the ameliorative property of Portulaca
oleracea could be attributed to plant-derived polysaccharides, as in-
dicated by significant increase in phagocytic activity in mice treated
with the low and high doses of EA fractions.
Moreover, numerous studies have also indicated that plant-derived
polysaccharides have proliferative effect on lymphocytes. Behravan
et al. (2011) reported that crude ethanolic extracts of Portulaca oleracea
shoots protected against oxidative DNA damage in lymphocytes, ef-
fectively allowing them to proliferate. There have been in vivo assays
using mice administered with these polysaccharides from some plants
species, and the results indicated that they promote lymphocyte pro-
liferation (Li et al., 2014; Shen et al., 2013; Chen et al., 2009). Although
the effect of the EA solvent fraction on splenic lymphocyte proliferation
was not significant in this study, it is still remarkable to note that the
increase in proliferation relative to the cyclophosphamide-treated mice
was dose-dependent. It is very likely then that the EA extract contains
immunoactive compounds that could induce proliferation of splenic
lymphocytes.
Aside from the potential immunoactive role of polysaccharides that
were reported to be present in P. oleracea, the present study indicates
that the presence of tannins and alkaloids in the EA extract could have
produced the immunomodulatory effects in lymphocytes and macro-
phages. In a study by Dey et al. (2015), the addition of tanniferous leaf
meal from Ficus infectoria in the diet of lambs resulted to enhanced cell-
mediated immunity and improved antioxidant status. Similar results
were obtained in another study using sheep that were infected with the
worm, Haemonchus contortus, and fed with a diet supplemented with
condensed tannins (Pathak et al., 2016). Plant-derived tannins have
been reported to enhance innate immunity through proliferation of γδ T
lymphocytes, especially those located in the intestinal mucosa layer,
which provide host protection against pathogens (Holderness et al.,
2007, 2008). In addition, tannins have also been suggested to activate
macrophages, especially during infections, as indicated in an in vitro
study using Leishmania-infected RAW 264.7 cells (Kolodziej and
Kiderlen, 2005). Likewise, alkaloids present in the EA extract could
have induced the proliferation of lymphocytes, albeit, not at a sig-
nificant level. Biphasic effects of alkaloids in mitogen-induced lym-
phocyte proliferation had been reported. Using high concentration of
alkaloid mixture from Tylophora indica, lymphocyte proliferation was
suppressed possibly due to IL-2 inhibition but lower concentration of
the alkaloid mixture induced lymphoproliferation (Ganguly et al.,
2001). It is suggested then, that further studies be undertaken in order
196
Journal of Ethnopharmacology 215 (2018) 191–198
Fig. 6. Intestinal transit (%) of charcoal in ICR mice treated with
acetylcholine, atropine, and different concentrations of the leaf
extracts of P. oleracea. Each bar represents the mean ± SEM of
each treatment. Different letters indicate significant difference at
P < 0.05. PBS, phosphate-buffered saline; PO, Portulaca oleracea;
EtOH, ethanol; EA, ethyl acetate; HEX, hexane.
to optimize the effective dosage that could be employed in im-
munotherapeutic applications of P. oleracea extracts.
Lysozyme activity is an indication of macrophage activation (Yu
et al., 2013). However, results in the present study do not correspond to
the peritoneal macrophage phagocytic activity discussed earlier. This
may be due to inhibition of the enzyme by some phenolic compounds,
which are abundant in Portulaca oleracea (Feng et al., 2012). Com-
pounds such as tannin, terpenoids and alkaloids have the capacity to
bind to proteins such as enzymes (Naczk et al., 1996; Varman and
Singh, 2012; Jash and Kumar, 2014). Moreover, some degree of lyso-
zyme inhibition could also be due to the interference of other types of
lysozymes present but were not detected by the assay used in the study
(Wang et al., 2005). It should be noted, however, that the mice in EA
low dose group at both day 8 and day 15 had relatively higher lysozyme
levels than the positive control or the immunosuppressed group. For the
EA high dose group, it is only at day 15 when the EA extract-treated
group exhibited higher lysozyme activity than the positive control
group.
In the charcoal meal transit experiment, the three solvent extracts
from the leaves of P. oleracea were able to exhibit antispasmodic ac-
tivity. Compared to acetylcholine-treated mice, the higher dose of the
hexane and ethyl acetate extracts decreased smooth muscle contrac-
tions. The present data obtained for P. oleracea therefore provides fur-
ther pharmacological evidence for the use of this plant in traditional
medicine. This plant can also be potentially used to ameliorate the
symptoms of gastrointestinal disorders such as gut inflammation, which
is characterized by increased gut motility. The extracts, however, did
not exhibit a dose-dependent decrease in contraction suggesting the
need to further investigate the most effective and standardized dose.
A number of plants used in traditional or folk medicine have been
validated to decrease smooth muscle contractions that include Ficus
exasperata Aloysia polystachya, Aloysia gratissima, Buddleja scordioides,
Buddleja perfoliata, Vitex negundo and Morinda citrifolia (Anowi et al.,
2012; Consolini et al., 2011; Cortes et al., 2006; Gilani et al., 2010;
Khan et al., 2013). The aqueous extract of Ficus carica showed a dose
dependent inhibition of the K+ induced contractions in the rabbit je-
junum (Gilani et al., 2008), while Zanthoxylum armatum induced anti-
spasmodic effect either via the blocking of the calcium ion channels or
blockage of the release of calcium stores (Barkatullah et al., 2013).
It has been reported that plant-derived tannins could block calcium
ion channel (Shah et al., 2011). In a previous study, the aqueous extract
of P. oleracea leaves has been found to induce relaxation in the rat
skeletal muscle due to calcium ion interference and increase in po-
tassium ion concentration (Parry et al., 1987; Okwuasaba et al., 1986;
Habtemariam et al., 1993). It should be noted that most of the studies
E.S. Catap et al.
on the antispasmodic activity of P. oleracea are focused on the skeletal
muscle. Very few studies investigated the smooth muscle as in the study
of Parry et al., (1988, 1993), which suggested that the aqueous extract
caused relaxation in the guinea pig fundus and rabbit jejunum and this
was attributed to an increase in potassium ion levels. In the study of
Habtemariam et al. (1993), the increase in potassium concentration was
attributed to the polarity of the solvent used. This could also explain the
decreased intestinal motility in mice treated with the EA fraction in this
study. However, the antispasmodic mechanism of the P. oleracea leaf
fraction still in intestinal muscles remains to be elucidated completely.
5. Conclusions
Results showed that the EA fraction of P. oleracea leaf extract
ameliorated the nonspecific immune response of mice which were im-
munosuppressed through the use of cyclophosphamide. Moreover, the
present study showed that the three (EtOH, HEX and EA) solvent
fractions of this plant species have potentially effective antispasmodic
components. These activities could possibly be attributed to some ac-
tive compounds present in the P. oleracea leaf fractions, which also
contributed to its high antioxidant property. However, additional stu-
dies are still needed to explain the complete mechanisms by which the
active compounds present in this plant could induce im-
munomodulatory and antispasmodic effects. Also, additional tests could
also be undertaken to identify other chemical components (e.g. lipids
and fatty acids) that may be present in the leaf extracts of P. oleracea.
Acknowledgments
The authors are grateful to the Natural Sciences Research Institute
(Project Code: BIO-13-2-03) for the support given to E.S. Catap, and the
UPD-Institute of Biology for the use of laboratory equipment. We are
also grateful to Ms. Charmaine Peredas for her technical assistance.
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Elena S. Catap, Ph.D. – Associate Professor, main proponent of the research funded by
the Natural Sciences Research Institute, did data analysis and completed writing of the
manuscript. Research interests include immunomodulatory response in vertebrates,
bioactivity of natural products, histopathology and toxicology.
Mr. Markyn Jared L. Kho – performed the immunomodulation experiments, assays,
statistical analysis. He is presently taking his medical studies.
Ms. Maria Rexie R. Jimenez – Research Assistant of the research project, performed the
immunomodulation assays and the antispasmodic experiment; she also did the statistical
analysis and wrote specific sections of the manuscript.