Journal of Ethnopharmacology 101 (2005) 9094

In vitro immunomodulatory effect of Pouteria cambodiana

(Pierre ex Dubard) Baehni extract
A. Manosroi a,b, , A. Saraphanchotiwitthaya c , J. Manosroi a,b
b

a Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand

Natural Products for Thai Traditional Medicines Research Unit, Pharmaceutical Cosmetic Raw Materials and Natural Products
Research and Development Center (PCRNC), Institute for Science and Technology Research and Development,
Chiang Mai University, Thailand
c Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences,
Naresuan University, Thailand

Received 6 January 2004; received in revised form 28 March 2005; accepted 28 March 2005
Available online 6 June 2005

Abstract
The methanolic stem bark extract from Pouteria cambodiana (Pierre ex Dubard) Baehni was evaluated for immunomodulating activity
on BALB/c mice. The antioxidant effect was also assessed. The extract presented a good dose-response effect in the peritoneal macrophage
phagocytosis assay with higher activity at 1 mg/ml and an EC50 of 0.02 mg/ml and also activated lysosomal enzyme activity with an EC50 of
0.16 mg/ml. In the splenocyte proliferation assay, the extract without mitogen was active (EC50 , 0.01 mg/ml) while the EC50 of the extract
with lipopolysaccharide (LPS) and pokeweed mitogen (PWM) were 0.02 and 0.41 mg/ml, respectively.
The extract showed low free radical scavenging activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay with an IC50 of
0.24 mg/ml, being less active than ascorbic acid, butylated hydroxytoluene (BHT) and -tocopherol which showed IC50 of 0.08, 0.10 and
0.11 mg/ml, respectively. The extract at doses up to 0.073 mg/ml had no effect on lipid peroxidation. The potent immunological but no antioxidant activity of the extract presented in this study can explain, at least in part, the Thai folklore application of this plant in the treatment of
fever and skin eruption.
2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Pouteria cambodiana (Pierre ex Dubard) Baehni; Immunomodulating activity; Phagocytosis; Proliferation; Sapotaceae; Thai plant

1. Introduction
Pouteria cambodiana (Pierre ex Dubard) Baehni, a parenial plant of the Sapotaceae family is widely distributed in
Asia (Bailey, 1949). It is known as Nom-nang or Tan-nom
in Thailand. The decoction of its bark has been orally taken
daily by breast feeding mothers for lactation promotion in
Thailand. Other parts of this plant have been used in folklore
medicines for the treatment of nausea, vomiting, fever and
back pain (Wuttithamwej, 1996). Bark decoction of other

Corresponding author. Tel.: +66 53 894806; fax: +66 53 894169.

E-mail address: pmpti005@chiangmai.ac.th (A. Manosroi).

0378-8741/$ see front matter 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2005.03.031

plants of the same family including Pouteria campechiana

are used as antipyretic in Mexico as well as to treat skin
eruptions in Cuba. Their seed extract has been employed for
treating ulcers (Morton, 1987). Arginin C, a saponin from
Tieghemella heckelii fruits, another plant of Sapotaceae family strongly inhibited HIV entry into HeLa-CD4 (+) cells in a
cell fusion assay (Gosse et al., 2002). Some of the ethanopharmacological claims of this plant relate to immunomodulating
and antioxidant activities (Labadie, 1993), since some of their
biological mechanism pathways are through these activities.
The present study was undertaken to assess the immunomodulating and antioxidant activities of a methanolic extract
from Pouteria cambodiana in relation with its folklore
use.

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2. Materials and methods

2.1. Plant materials
The stem bark of Pouteria cambodiana was collected
in March 1999 from Chiang Mai Province, Thailand. The
specimen was authenticated by the botanist of the Faculty
of Pharmacy, Chiang Mai University, Thailand. A voucher
specimen (No. PC4179509) was deposited at the Pharmaceutical Cosmetic Raw Materials and Natural Products
Research and Development Center (PCRNC), Institute for
Science and Technology Research and Development, Chiang
Mai University, Thailand.
2.2. Preparation of the extracts
Dry powdered bark of Pouteria cambodiana (900 g) was
percolated with 1500 ml of methanol in a percolator until
exhaustion at 26 2 C. The methanolic extract was evaporated under reduced pressure to give a viscous dark brown
mass with a percentage yield of 3.89% (w/w).
2.3. Animals
Female BALB/c mice (56 weeks old) were from the
National Laboratory Animal Center, Mahidol University,
Bangkok, Thailand. The animals were housed under standard
conditions at 25 2 C and fed with standard pellets and tap
water. The experiments were conducted under the surveillance of the Ethic Committee of Institute for Science and
Technology Research and Development, Chiang Mai University, Thailand.
2.4. Preparation of peritoneal mouse macrophages
One milliliter of fetal calf serum (FCS, Biochem KG,
Germany) was injected intraperitoneally into mice as a
stimulant to elicit peritoneal macrophages. Three days
later, the peritoneal exudate was collected by peritoneal
lavage with 8 ml of RPMI 1640 medium (Sigma, Germany)
supplemented with 10% heat-inactivated FCS, 50 M
2-mercaptoethanol (Pharmacia, Sweden), 100 U penicillin,
100 g streptomycin and 0. 25 g/ml amphotericin B
(Sigma, Germany). The exudate was centrifuged at 300 g,
25 C for 20 min. The erythrocytes in the cell pellets were
lysed by hypotonic solution (0.2% NaCl). Isotonicity was
restored with 1.6% NaCl solution. The cell suspension
was centrifuged and the cells were washed twice and
re-suspended in complete RPMI 1640 medium. The cell
number was adjusted to 1 106 cell/ml. The cell number was
determined by counting in a hemocytometer and cell viability
was tested by the trypan-blue dye exclusion technique.
2.5. Preparation of mouse splenocytes
Mice were sacrificed and their spleens were collected
aseptically. Cell suspension was prepared by means of loose

91

potter and flushing. After centrifugation at 300 g, 37 C

for 10 min, erythrocytes were lysed by hypotonic solution
and the cell pellets were washed twice with RPMI 1640. The
cells were resuspended in complete RPMI medium and the
cell number was adjusted to 1 106 cell/ml. The viability of
splenocytes was determined by the trypan-blue dye exclusion
technique.
2.6. Nitroblue tetrazolium (NBT) dye reduction assay
The NBT reduction assay was carried out as previously
described (Rainard, 1986). Briefly, 20 l of the macrophage
suspension and 40 l of RPMI medium were added in a flat
bottom 96-well plate (Nunc , USA). Twenty microliter of
the solution containing the plant extract dissolved in 0.1%
dimethysulfoxide (DMSO) in phosphate buffer saline (PBS)
solution was added in each well to give final extract concentrations of 0.001, 0.01, 0.1 and 1 mg/ml. The 0.1% DMSO in
PBS (without the plant extract) was used as a control. After
incubation for 24 h at 37 C in 5% CO2 humidified atmosphere, 20 l of the heated inactivated yeast (Saccharomyces
cerevisiae) suspension (5 107 particles/ml) and 20 l of
NBT (Sigma, Germany) solution in PBS (1.5 mg/ml) were
added and the mixture was further incubated under the same
conditions.
After incubation for 60 min, the adherent macrophages
were rinsed vigorously with RPMI medium and washed four
times with 200 l methanol. After air-dried, 120 l of 2 M
KOH and 140 l of DMSO were added. The absorbance was
measured at 570 nm by a well reader (Seikagaku SK601,
Japan) and the percentage of NBT reduction was calculated
by the following equation:
NBT reduction (%)
=

OD sample OD negative control

100
OD negative control

The EC50 value represents the effective concentration

required for 50% enhancement of oxidative burst reduction
activity.
2.7. Cellular lysosomal enzyme activity assay
The cellular lysosomal enzyme activity was used to
determine acid phosphatase in macrophages as previously
described (Suzuki et al., 1988). Briefly, 20 l of macrophage
suspension (1 106 cells/ml), 40 l of RPMI medium and
20 l of the plant extracts dissolved in 0.1% DMSO in PBS
were added in each well of a flat bottom 96-well plate to
obtain final concentrations of 0.001, 0.01, 0.1 and 1 mg/ml.
Some 0.1% DMSO in PBS was used as a control. The culture
was incubated at 37 C in 5% CO2 humidified atmosphere for
24 h. The medium was removed by aspiration and 20 l of
0.1% Triton X-100 (Pharmacia, Sweden), 100 l of 10 mM
p-nitrophenyl phosphate (p-NPP) (Sigma, Germany) solution and 50 l of 0.1 M citrate buffer (pH 5.0) were added

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A. Manosroi et al. / Journal of Ethnopharmacology 101 (2005) 9094

in each well. The plate was further incubated for 30 min,

150 l of 0.2 M borate buffer (pH 9.8) was then added and
the absorbance was measured at 405 nm. The percentage of
lysosomal enzyme activity was calculated by the following
equation:
lysosomal enzyme activity (%)
=

OD sample OD negative control

100
OD negative control

Results are presented as EC50 .

2.8. Mitogen-induced splenocyte proliferation assay
The lymphocyte proliferation assay was carried out
according to the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] method (Mosmann, 1983).
Briefly, 20 l of various concentrations (0.001, 0.01, 0.1 and
1 mg/ml) of the plant extracts dissolved in 0.1% DMSO in
PBS were added to the mixture of 20 l of the splenocyte
suspensions (1 106 cells/ml) and 40 l of RPMI medium
in a 96-well plate. The optimum dose of lipopolysaccharide
(LPS) and pokeweed mitogen (PWM) at 5 g/ml tested preliminary were used as mitogens and 0.1% DMSO in PBS
was used as a control. After incubation at 37 C in humidified 5% CO2 atmosphere for 48 h, 20 l of MTT (5 mg/ml)
in PBS and 40 l of RPMI media were added. The culture
medium was removed by aspiration and 100 l of 0.04 M HCl
in isopropyl alcohol were added to lyse cells. Then, 100 l
of distilled water were added to dilute the solution and the
absorbance was measured at 570 nm. The percentage of proliferation was calculated by the following equation:
OD sample OD control
proliferation (%) =
100
OD control
Results are presented as EC50 .

The free radical scavenging activity of the plant extracts

was expressed as IC50 , which was defined as the extract concentration (mg/ml) required to scavenge the DPPH radicals
by 50%.
2.10. Lipid peroxidation assay
The content of lipid peroxidation products was measured using the thiobarbituric acidtrichloroacetic acid
(TBATCA) reagent according to Heath and Packer (1968).
Briefly, 2.2 mg of the extract and standard radical scavenging
compounds (ascorbic acid, BHT and -tocopherol) were dissolved in 2 ml of ethanol. Some 400 l of the mixture were
then placed into 411 l of pure linoleic acid (Sigma Co.),
800 l of PBS and distilled water to make up 2.4 ml in a 20 ml
tube. After incubation at 40 C for 7 days, 25, 50 or 100 l
of the samples were placed into a 96-well plate and PBS was
added up to 100 l. TCA (100 l) and TBA (50 l) solutions were then added to obtain final extract concentrations
of 0.018, 0.037 and 0.073 mg/ml. The mixture was mixed and
heated at 100 C in a steaming bath for 10 min and cooled to
room temperature (25 C). The percentage of the antioxidative activity was calculated as IC50 :
antioxidative activity (%)
=

OD negative control OD sample

100
OD negative control

2.11. Statistical analysis

All experiments were performed in triplicate and the
results were expressed as mean S.E. Statistical significance
was analyzed using Students t-test. p values less than 0.05
were considered significant.

2.9. DPPH assay

3. Results

The free radical scavenging activity of the plant extract

was assessed by the decoloration of the stable 1,1-diphenyl2-picrylhydrazyl (DPPH) free radical (Cotelle et al., 1996).
Briefly, 2.2 mg of the extract and standard compounds (ascorbic acid, butylated hydroxytoluene (BHT) and -tocopherol)
were dissolved in 2 ml of ethanol. Some 40, 60 or 80 l
of samples were pipetted into a 96-well plate and 80 l of
DPPH solution (1.25 104 M) was then added. The total
volume of 240 l was adjusted by acetate buffer (0.1 M, pH
5.5) to obtain final extract concentrations of 0.183, 0.275
and 0.367 mg/ml. The absorbance was determined at 570 nm
using an Elisa reader at initial (t0 ) and after 5 min (t5 ). The
percentages of the residual absorbance rate (RRA, %) were
calculated as follows:

3.1. Phagocytic activity

RRA (%) =

OD sample (t5 )
100
OD sample (t0 )

As shown in Fig. 1, the methanolic extract of Pouteria cambodiana enhanced the NBT reduction at 0.01, 0.1
and 1 mg/ml by 47% (p < 0.01), 67% (p < 0.01) and 80%
(p < 0.01), respectively, with an EC50 value of 0.02 mg/ml.
The extract also activated the lysosomal enzyme activity by
57% at 0.1 mg/ml and by 84% (p < 0.05) at 1 mg/ml with an
EC50 of 0.16 mg/ml. The dose response curve was markedly
presented.
3.2. Mitogen-induced splenocyte proliferation
For proliferation assay in the absence of mitogen, the
extract at 1 mg/ml enhanced the proliferation by 175%
(p < 0.01) compared to the control. In the presence of LPS,
the extract elicited an increase of splenocyte proliferation

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93

4. Discussion and conclusions

Fig. 1. Effects of a methanolic Pouteria cambodiana (Pierre ex Dubard)

Baehni stem bark extract on in vitro phagocytic assay of normal Balb/c
macrophages: (A) nitroblue tetrazolium test and (B) lysosomal enzyme activity test. Each value represents the mean S.E. of the triplicates comparing
to the control; ** p < 0.05, * p < 0.01.

by 163% (p < 0.05), 151% (p < 0.01) and 169% (p < 0.01) at
0.01, 0.1 and 1 mg/ml, respectively. When treated with PWM,
the extract at 0.1 and 1 mg/ml enhanced the proliferation by
56% (p < 0.01) and 191% (p < 0.05) of the control, respectively (Fig. 2). The EC50 values of splenocyte proliferation
stimulation without mitogen, with LPS and PWM were 0.01,
0.02 and 0.41 mg/ml, respectively.
The extract in concentration up to 1 mg/ml was not toxic
to mouse macrophages and splenocytes (survival rates higher
than 90 and 80%, respectively).
3.3. Antioxidant activity assay
The free radical scavenging effect of the methanolic
extract of Pouteria cambodiana was determined using the
DPPH assay as well as the inhibition of linoleic acid oxidation. The IC50 value of the extract on DPPH activity was
0.24 mg/ml while ascorbic acid, BHT and -tocopherol presented IC50 values of 0.08, 0.10 and 0.11 mg/ml, respectively.
In the lipid peroxidation assay, the extract was inactive while
BHT at 0.017 mg/ml inhibited oxidation by approximately
52% (p < 0.01).

In this study, the phagocytic activity of the methanolic

extract of Pouteria cambodiana was tested on oxidative burst
reduction and acid phosphatase activity of macrophages. The
higher reduction in NBT assay represented higher activity of
oxidase enzyme reflecting the stimulation of phagocytes in
proportion to the foreign particles ingested (Rainard, 1986).
The enhanced transformation of p-nitrophenyl phosphate
(p-NPP) to a color compound by the membrane associated
acid phosphatase activity of the treated macrophages in the
lysosomal enzyme activity assay is related to the stimulation
effect (Suzuki et al., 1990). The mitogenic responses of
mouse splenocytes by the extract together with the optimum
dose of LPS (a mitogen for T-cell independent B-cell
proliferation) and PWM (a mitogen for T-cell dependent
proliferation) were also evaluated. The presence of mitogens
in the system can postulate the possible proliferation
activation pathway of the extracts.
The extract exhibited high activity on the oxidative burst
reduction, presenting intracellular killing, and the enhancement of lysosomal enzyme activity, showing the activity on
degranulation of macrophages. The bark extract of Manilkara
achras (Mill.) Forsberg, a plant belonging to the same family
as Pouteria cambodiana demonstrated antiprotozoal activity (Muelas-Serrano et al., 2000). This activity appears to be
related to the macrophagelymphocyte defense system, as
our extract in this study.
In the present study, the maximum phagocytic activity
of the extract on the NBT dye reduction was the same as
the lysosomal enzyme activity. However, the EC50 value
from the former was less than the latter indicating a stronger
potency on superoxide production than lysosomal enzyme
activity. It should be stated that the extract might contain
some constituents responsible for intracellular killing more
than degranulation. These constituents may be phenolic compounds since they were detected after spraying a FeCl3

Fig. 2. Effects of a methanolic Pouteria cambodiana (Pierre ex Dubard) Baehni stem bark extract on in vitro proliferation of normal Balb/c splenocytes: (A)
without mitogen, (B) with lipopolysaccharide (5 g/ml) and (C) with pokeweed mitogen (5 g/ml). Each value presents the mean S.E. of the triplicates
comparing to the control; ** p < 0.05, * p < 0.01.

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A. Manosroi et al. / Journal of Ethnopharmacology 101 (2005) 9094

solution on the TLC plates (data not shown). Phenolic compounds have been previously shown either to stimulate or
suppress the immune system due to the OH groups in
their structure. This will have an effect on the enzyme or
electron-transferring system which gives an immunomodulating property, especially on phagocytic activity (Labadie,
1993). A dose response relationship of our extract on phagocytic activity was observed. The opsonin (complement) in
fresh serum might be added into the test systems in order to
obtain a clearer dose effect (Rainard, 1986).
Due to the similar maximum effect on splenocyte proliferation stimulation of the extract without mitogen and with
LPS or PWM, the extract might contain active components
that involve equally both to T- and B-cell proliferation
stimulation. The EC50 value of the extract when with LPS
exhibited potent activity more than with PWM. Some active
compounds in the extract may involve in B-cell proliferation
stimulation, related to the humoral mediated immunity
(HMI). The extract demonstrated stronger effect on the
T-cell independent (in the case of LPS addition) than in the
T-cell-dependent (in the case of PWM addition) pathway.
The toxicity studies on dominant lethal test in rats treated
with an aqueous extract of Pouteria cambodiana has indicated no toxic effect on male reproductive and progeny outcome (Aritajat et al., 2000). The oral LD50 of the crude drug
in rat is 15 g/kg of body weight (unpublished data).
For free radical scavenging assay, the stable radical DPPH
lost its characteristic purple color when supplied electron or
hydrogen ion to the system. The capacity of the tested substances to donate electrons can be estimated from the degree
of color fading. Ascorbic acid, BHT and -tocopherol were
used as standard radical scavengers. The extract gave only
minor effect on DPPH radical scavenging activity and had no
effect on lipid peroxidation. On the other hand, the ethanolic and aqueous extract of Pouteria cambodiana gave the
trolox equivalent antioxidative capacity (TEAC) of about 0.3
(Suttajit et al., 2000). More polar compounds in the extract
may be responsible for the antioxidative activity. The phytochemical screening of our methanolic plant extract disclosed
the phenolic compounds as 3,4-dihydroxy benzoic acid. The
low antioxidative activity may be related to the low concentration of this compound in the extract.
In summary, this study shows that the methanolic extract
from Pouteria cambodiana (Pierre ex Dubard) Baehni
stem bark presents no antioxidant activity, but potent
in vitro immunomodulatory activity of mouse immune
system for both macrophage phagocytosis and splenocyte
proliferation. The present observations appear to give some
support to the traditional use of Pouteria cambodiana
in Thai traditional medicine for fever and skin eruption,
symtoms partly affected through the immune systems. The

immunomodulatory mechanism of the extract is still unclear.

Further study on active constituent isolation and its mode of
immune action will be our next report.

Acknowledgements
This work was partially supported by grants from the
Graduate School, Chiang Mai University and the Institute
of Thai Traditional Medicines, Ministry of Health, Bangkok,
Thailand.

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