Journal of Ethnopharmacology 101 (2005) 90–94

In vitro immunomodulatory effect ofPouteria cambodiana(Pierre ex Dubard) Baehni extract

A. Manosroia,b,∗, A. Saraphanchotiwitthayac, J. Manosroia,b

a Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailandb 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 2005Available online 6 June 2005

Abstract

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The methanolic stem bark extract fromPouteria cambodiana(Pierre ex Dubard) Baehni was evaluated for immunomodulating acn BALB/c mice. The antioxidant effect was also assessed. The extract presented a good dose-response effect in the peritonealhagocytosis 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.16 mg/ml. In the splenocyte proliferation assay, the extract without mitogen was active (EC50, 0.01 mg/ml) while the EC50 of the extracith 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 an50 of

.24 mg/ml, being less active than ascorbic acid, butylated hydroxytoluene (BHT) and�-tocopherol which showed IC50 of 0.08, 0.10 an

.11 mg/ml, respectively. The extract at doses up to 0.073 mg/ml had no effect on lipid peroxidation. The potent immunological butdant activity of the extract presented in this study can explain, at least in part, the Thai folklore application of this plant in the treever and skin eruption.

2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Pouteria cambodiana(Pierre ex Dubard) Baehni; Immunomodulating activity; Phagocytosis; Proliferation; Sapotaceae; Thai plant

. Introduction

Pouteria cambodiana(Pierre ex Dubard) Baehni, a pare-ial plant of the Sapotaceae family is widely distributed insia (Bailey, 1949). It is known as ‘Nom-nang’ or ‘Tan-nom’

n Thailand. The decoction of its bark has been orally takenaily by breast feeding mothers for lactation promotion inhailand. Other parts of this plant have been used in folkloreedicines for the treatment of nausea, vomiting, fever andack 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).

plants of the same family includingPouteria campechianare used as antipyretic in Mexico as well as to treateruptions in Cuba. Their seed extract has been employetreating ulcers (Morton, 1987). Arginin C, a saponin fromTieghemella heckeliifruits, another plant of Sapotaceae faily strongly inhibited HIV entry into HeLa-CD4 (+) cells incell fusion assay (Gosse et al., 2002). Some of the ethanophamacological claims of this plant relate to immunomodulaand antioxidant activities (Labadie, 1993), since some of thebiological mechanism pathways are through these activThe present study was undertaken to assess the immunulating and antioxidant activities of a methanolic extfrom Pouteria cambodianain relation with its folkloreuse.

378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2005.03.031

A. Manosroi et al. / Journal of Ethnopharmacology 101 (2005) 90–94 91

2. Materials and methods

2.1. Plant materials

The stem bark ofPouteria cambodianawas collectedin March 1999 from Chiang Mai Province, Thailand. Thespecimen was authenticated by the botanist of the Facultyof Pharmacy, Chiang Mai University, Thailand. A voucherspecimen (No. PC4179509) was deposited at the Phar-maceutical Cosmetic Raw Materials and Natural ProductsResearch and Development Center (PCRNC), Institute forScience and Technology Research and Development, ChiangMai University, Thailand.

2.2. Preparation of the extracts

Dry powdered bark ofPouteria cambodiana(900 g) waspercolated with 1500 ml of methanol in a percolator untilexhaustion at 26± 2◦C. The methanolic extract was evapo-rated under reduced pressure to give a viscous dark brownmass with a percentage yield of 3.89% (w/w).

2.3. Animals

Female BALB/c mice (5–6 weeks old) were from theNational Laboratory Animal Center, Mahidol University,B dardc tapw rveil-l andT Uni-v

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potter and flushing. After centrifugation at 300×g, 37◦Cfor 10 min, erythrocytes were lysed by hypotonic solutionand the cell pellets were washed twice with RPMI 1640. Thecells were resuspended in complete RPMI medium and thecell number was adjusted to 1× 106 cell/ml. The viability ofsplenocytes was determined by the trypan-blue dye exclusiontechnique.

2.6. Nitroblue tetrazolium (NBT) dye reduction assay

The NBT reduction assay was carried out as previouslydescribed (Rainard, 1986). Briefly, 20�l of the macrophagesuspension and 40�l of RPMI medium were added in a flatbottom 96-well plate (Nunc®, USA). Twenty microliter ofthe 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 concen-trations of 0.001, 0.01, 0.1 and 1 mg/ml. The 0.1% DMSO inPBS (without the plant extract) was used as a control. Afterincubation for 24 h at 37◦C in 5% CO2 humidified atmo-sphere, 20�l of the heated inactivated yeast (Saccharomycescerevisiae) suspension (5× 107 particles/ml) and 20�l ofNBT (Sigma, Germany) solution in PBS (1.5 mg/ml) wereadded and the mixture was further incubated under the sameconditions.

After incubation for 60 min, the adherent macrophagesw fourtK wasm 601,J latedb

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angkok, Thailand. The animals were housed under stanonditions at 25± 2◦C and fed with standard pellets andater. The experiments were conducted under the su

ance of the Ethic Committee of Institute for Scienceechnology Research and Development, Chiang Maiersity, Thailand.

.4. Preparation of peritoneal mouse macrophages

One milliliter of fetal calf serum (FCS, Biochem Kermany) was injected intraperitoneally into mice atimulant to elicit peritoneal macrophages. Three dater, the peritoneal exudate was collected by peritoavage with 8 ml of RPMI 1640 medium (Sigma, Germaupplemented with 10% heat-inactivated FCS, 50�M-mercaptoethanol (Pharmacia, Sweden), 100 U penic00�g streptomycin and 0. 25�g/ml amphotericin BSigma, Germany). The exudate was centrifuged at 300×g,5◦C for 20 min. The erythrocytes in the cell pellets w

ysed by hypotonic solution (0.2% NaCl). Isotonicity westored with 1.6% NaCl solution. The cell suspenas centrifuged and the cells were washed twice

e-suspended in complete RPMI 1640 medium. Theumber was adjusted to 1× 106 cell/ml. The cell number waetermined by counting in a hemocytometer and cell viabas tested by the trypan-blue dye exclusion technique.

.5. Preparation of mouse splenocytes

Mice were sacrificed and their spleens were colleseptically. Cell suspension was prepared by means of

ere rinsed vigorously with RPMI medium and washedimes with 200�l methanol. After air-dried, 120�l of 2 MOH and 140�l of DMSO were added. The absorbanceeasured at 570 nm by a well reader (Seikagaku SK

apan) and the percentage of NBT reduction was calcuy the following equation:

BT reduction (%)

= OD sample− OD negative control

OD negative control× 100

The EC50 value represents the effective concentraequired for 50% enhancement of oxidative burst reducctivity.

.7. Cellular lysosomal enzyme activity assay

The cellular lysosomal enzyme activity was usedetermine acid phosphatase in macrophages as prevescribed (Suzuki et al., 1988). Briefly, 20�l of macrophaguspension (1× 106 cells/ml), 40�l of RPMI medium and0�l of the plant extracts dissolved in 0.1% DMSO in Pere added in each well of a flat bottom 96-well platebtain final concentrations of 0.001, 0.01, 0.1 and 1 mgome 0.1% DMSO in PBS was used as a control. The cuas incubated at 37◦C in 5% CO2 humidified atmosphere fo4 h. The medium was removed by aspiration and 20�l of.1% Triton X-100 (Pharmacia, Sweden), 100�l of 10 mM-nitrophenyl phosphate (p-NPP) (Sigma, Germany) solion and 50�l of 0.1 M citrate buffer (pH 5.0) were add

92 A. Manosroi et al. / Journal of Ethnopharmacology 101 (2005) 90–94

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 andthe absorbance was measured at 405 nm. The percentage oflysosomal enzyme activity was calculated by the followingequation:

lysosomal enzyme activity (%)

= OD sample− OD negative control

OD negative control× 100

Results are presented as EC50.

2.8. Mitogen-induced splenocyte proliferation assay

The lymphocyte proliferation assay was carried outaccording to the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method (Mosmann, 1983).Briefly, 20�l of various concentrations (0.001, 0.01, 0.1 and1 mg/ml) of the plant extracts dissolved in 0.1% DMSO inPBS were added to the mixture of 20�l of the splenocytesuspensions (1× 106 cells/ml) and 40�l of RPMI mediumin a 96-well plate. The optimum dose of lipopolysaccharide(LPS) and pokeweed mitogen (PWM) at 5�g/ml tested pre-liminary were used as mitogens and 0.1% DMSO in PBSwas used as a control. After incubation at 37◦C in humidi-fied 5% CO atmosphere for 48 h, 20�l of MTT (5 mg/ml)i remi 0o thea of prol

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The free radical scavenging activity of the plant extractswas expressed as IC50, which was defined as the extract con-centration (mg/ml) required to scavenge the DPPH radicalsby 50%.

2.10. Lipid peroxidation assay

The content of lipid peroxidation products was mea-sured using the thiobarbituric acid–trichloroacetic acid(TBA–TCA) reagent according toHeath and Packer (1968).Briefly, 2.2 mg of the extract and standard radical scavengingcompounds (ascorbic acid, BHT and�-tocopherol) were dis-solved in 2 ml of ethanol. Some 400�l of the mixture werethen 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 mltube. After incubation at 40◦C for 7 days, 25, 50 or 100�lof the samples were placed into a 96-well plate and PBS wasadded up to 100�l. TCA (100�l) and TBA (50�l) solu-tions were then added to obtain final extract concentrationsof 0.018, 0.037 and 0.073 mg/ml. The mixture was mixed andheated at 100◦C in a steaming bath for 10 min and cooled toroom temperature (25◦C). The percentage of the antioxida-tive activity was calculated as IC50:

antioxidative activity (%)

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2n PBS and 40�l of RPMI media were added. The cultu

edium was removed by aspiration and 100�l of 0.04 M HCln isopropyl alcohol were added to lyse cells. Then, 10�lf distilled water were added to dilute the solution andbsorbance was measured at 570 nm. The percentage

iferation was calculated by the following equation:

roliferation (%)= OD sample− OD control

OD control× 100

esults are presented as EC50.

.9. DPPH assay

The free radical scavenging activity of the plant extas assessed by the decoloration of the stable 1,1-diph-picrylhydrazyl (DPPH) free radical (Cotelle et al., 1996).riefly, 2.2 mg of the extract and standard compounds (aic acid, butylated hydroxytoluene (BHT) and�-tocopherolere dissolved in 2 ml of ethanol. Some 40, 60 or 8�lf samples were pipetted into a 96-well plate and 80�l ofPPH solution (1.25× 10−4 M) was then added. The toolume of 240�l was adjusted by acetate buffer (0.1 M,.5) to obtain final extract concentrations of 0.183, 0nd 0.367 mg/ml. The absorbance was determined at 57sing an Elisa reader at initial (t0) and after 5 min (t5). Theercentages of the residual absorbance rate (RRA, %)alculated as follows:

RA (%) = OD sample (t5)

OD sample (t0)× 100

-

= OD negative control− OD sample

OD negative control× 100

.11. Statistical analysis

All experiments were performed in triplicate andesults were expressed as mean± S.E. Statistical significancas analyzed using Student’st-test.p values less than 0.0ere considered significant.

. Results

.1. Phagocytic activity

As shown inFig. 1, the methanolic extract ofPoute-ia cambodianaenhanced the NBT reduction at 0.01,nd 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/mhe extract also activated the lysosomal enzyme activi7% at 0.1 mg/ml and by 84% (p< 0.05) at 1 mg/ml with aC50 of 0.16 mg/ml. The dose response curve was markresented.

.2. Mitogen-induced splenocyte proliferation

For proliferation assay in the absence of mitogen,xtract at 1 mg/ml enhanced the proliferation by 17p< 0.01) compared to the control. In the presence of Lhe extract elicited an increase of splenocyte prolifera

A. Manosroi et al. / Journal of Ethnopharmacology 101 (2005) 90–94 93

Fig. 1. Effects of a methanolicPouteria cambodiana(Pierre ex Dubard)Baehni stem bark extract on in vitro phagocytic assay of normal Balb/cmacrophages: (A) nitroblue tetrazolium test and (B) lysosomal enzyme activ-ity test. Each value represents the mean± S.E. of the triplicates comparingto the control;** p< 0.05,*p< 0.01.

by 163% (p< 0.05), 151% (p< 0.01) and 169% (p< 0.01) at0.01, 0.1 and 1 mg/ml, respectively. When treated with PWM,the extract at 0.1 and 1 mg/ml enhanced the proliferation by56% (p< 0.01) and 191% (p< 0.05) of the control, respec-tively (Fig. 2). The EC50 values of splenocyte proliferationstimulation 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 toxicto mouse macrophages and splenocytes (survival rates higherthan 90 and 80%, respectively).

3.3. Antioxidant activity assay

The free radical scavenging effect of the methanolicextract ofPouteria cambodianawas determined using theDPPH assay as well as the inhibition of linoleic acid oxi-dation. The IC50 value of the extract on DPPH activity was0.24 mg/ml while ascorbic acid, BHT and�-tocopherol pre-sented IC50 values of 0.08, 0.10 and 0.11 mg/ml, respectively.In the lipid peroxidation assay, the extract was inactive whileBHT at 0.017 mg/ml inhibited oxidation by approximately52% (p< 0.01).

4. Discussion and conclusions

In this study, the phagocytic activity of the methanolicextract ofPouteria cambodianawas tested on oxidative burstreduction and acid phosphatase activity of macrophages. Thehigher reduction in NBT assay represented higher activity ofoxidase enzyme reflecting the stimulation of phagocytes inproportion to the foreign particles ingested (Rainard, 1986).The enhanced transformation ofp-nitrophenyl phosphate(p-NPP) to a color compound by the membrane associatedacid phosphatase activity of the treated macrophages in thelysosomal enzyme activity assay is related to the stimulationeffect (Suzuki et al., 1990). The mitogenic responses ofmouse splenocytes by the extract together with the optimumdose of LPS (a mitogen for T-cell independent B-cellproliferation) and PWM (a mitogen for T-cell dependentproliferation) were also evaluated. The presence of mitogensin the system can postulate the possible proliferationactivation pathway of the extracts.

The extract exhibited high activity on the oxidative burstreduction, presenting intracellular killing, and the enhance-ment of lysosomal enzyme activity, showing the activity ondegranulation of macrophages. The bark extract ofManilkaraachras(Mill.) Forsberg, a plant belonging to the same familyasPouteria cambodianademonstrated antiprotozoal activ-ity (Muelas-Serrano et al., 2000). This activity appears to ber m, aso

ivityo e astf ngerp ymea tains oret com-p eCl

F aehni es: (A)w keweed sc

ig. 2. Effects of a methanolicPouteria cambodiana(Pierre ex Dubard) Bithout mitogen, (B) with lipopolysaccharide (5�g/ml) and (C) with poomparing to the control;** p< 0.05,*p< 0.01.

elated to the macrophage–lymphocyte defense systeur extract in this study.

In the present study, the maximum phagocytic actf the extract on the NBT dye reduction was the sam

he lysosomal enzyme activity. However, the EC50 valuerom the former was less than the latter indicating a strootency on superoxide production than lysosomal enzctivity. It should be stated that the extract might conome constituents responsible for intracellular killing mhan degranulation. These constituents may be phenolicounds since they were detected after spraying a F3

stem bark extract on in vitro proliferation of normal Balb/c splenocytmitogen (5�g/ml). Each value presents the mean± S.E. of the triplicate

94 A. Manosroi et al. / Journal of Ethnopharmacology 101 (2005) 90–94

solution on the TLC plates (data not shown). Phenolic com-pounds have been previously shown either to stimulate orsuppress the immune system due to the OHgroups intheir structure. This will have an effect on the enzyme orelectron-transferring system which gives an immunomodu-lating property, especially on phagocytic activity (Labadie,1993). A dose response relationship of our extract on phago-cytic activity was observed. The opsonin (complement) infresh serum might be added into the test systems in order toobtain a clearer dose effect (Rainard, 1986).

Due to the similar maximum effect on splenocyte prolif-eration stimulation of the extract without mitogen and withLPS or PWM, the extract might contain active componentsthat involve equally both to T- and B-cell proliferationstimulation. The EC50 value of the extract when with LPSexhibited potent activity more than with PWM. Some activecompounds in the extract may involve in B-cell proliferationstimulation, related to the humoral mediated immunity(HMI). The extract demonstrated stronger effect on theT-cell independent (in the case of LPS addition) than in theT-cell-dependent (in the case of PWM addition) pathway.

The toxicity studies on dominant lethal test in rats treatedwith an aqueous extract ofPouteria cambodianahas indi-cated no toxic effect on male reproductive and progeny out-come (Aritajat et al., 2000). The oral LD50 of the crude drugin rat is 15 g/kg of body weight (unpublished data).

PPHl n orh sub-s egreo eu e onlym d noe ano-lt 0.3( ctm yto-c osedt Thel en-t

tractf nis tenti nes ocytep omes ai on,s The

immunomodulatory mechanism of the extract is still unclear.Further study on active constituent isolation and its mode ofimmune action will be our next report.

Acknowledgements

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

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L Rus-, and

M tes:

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W kok,

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ic and aqueous extract ofPouteria cambodianagave therolox equivalent antioxidative capacity (TEAC) of aboutSuttajit et al., 2000). More polar compounds in the extraay be responsible for the antioxidative activity. The ph

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n Thai traditional medicine for fever and skin eruptiymtoms partly affected through the immune systems.

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