Journal of Ethnopharmacology 146 (2013) 243–249

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Journal of Ethnopharmacology

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guyonia

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journal homepage: www.elsevier.com/locate/jep

Immunomodulatory and cellular anti-oxidant activities of an aqueous extractof Limoniastrum guyonianum gall

Mounira Krifa a, Ines Bouhlel a, Leila Ghedira-Chekir b,n, Kamel Ghedira a

a Unite de Pharmacognosie/Biologie Moleculaire, Faculte de Pharmacie de Monastir, Tunisiab Laboratoire de Biologie Moleculaire et Cellulaire, Faculte de Medecine Dentaire de Monastir, Universite de Monastir, Monastir, Tunisia

a r t i c l e i n f o

Article history:

Received 19 October 2012

Received in revised form

9 December 2012

Accepted 25 December 2012Available online 2 January 2013

Keywords:

Limoniastrum guyonianum

Gall aqueous extract

Immunomodulation

Cellular anti-oxidant activity

41/$ - see front matter & 2013 Elsevier Irelan

x.doi.org/10.1016/j.jep.2012.12.038

viations: G extract, Aqueous gall extract; L

num; NO, Nitric oxide; LPS, Lipopolysaccharid

S, Reactive oxygen/nitrogen species

espondence to: Faculte de Medecine Denta

re et Moleculaire, Rue Avicenne, 5000 Monas

1673461832; fax: þ21673461150.

ail address: leila.chekir@laposte.net (L. Ghedi

a b s t r a c t

Ethnopharmacological relevance: Many studies have been performed to assess the potential utility of

natural products as immunomodulatory agents to enhance host responses to disease/infection/etc. or to

ameliorate immune based pathologies (i.e., inflammation, autoimmune associated diseases, etc.). In this

particular study, the immunomodulatory potential of gall aqueous extract from Limoniastrum

guyonianum Boiss. (Zita) was assessed in vitro.

Materials and methods: The effect of G extract on splenocytes proliferation and NK activity were

assessed by MTT test. The induction of NO production and the phagocytic activity of macrophages were

evaluated in vitro. Activation of the cellular anti-oxidant activity in splenocytes was determined by

measuring the fluorescence of the DCF product.

Results: The studies first demonstrated that the extract could enhance lysosomal enzyme activity and

nitrite oxide production in murine peritoneal macrophages, suggesting a potential role in activation of

these cells. In studies to assess potential effects on humoral immunity, the results indicated that the

extract could significantly promote LPS-stimulated splenocyte proliferation implying a potential

activation of B-cells and enhanced humoral immune responses in hosts given this natural product. In

studies to assess any effects of extract on cellular immunity, the results showed that the extract

significantly enhanced the killing activity of isolated NK cells but had negligible effects on mitogen-

induced proliferation of splenic T-cells. Considerable effects were also observed on the cellular anti-

oxidant activity.

Conclusion: We conclude from these studies that aqueous extract from L. guyonianum gall exhibited an

immunomodulator effect which could be ascribed, in part, to its cytoprotective effect via its anti-

oxidant capacity. Furthermore, these results suggest that L. guyonianum gall extract contains potent

components such as flavonoids which should be potentially used to modulate immune cell functions in

physiological and pathological conditions.

& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

A large number of plants used in traditional medicines havebeen shown to possess immunomodulating activities (BenSghaier et al., 2011; Limem et al., 2010). Many of these are beingextensively explored for their potential use in the prevention-treatment of chronic diseases. Modulation of the immune systemdenotes to any change in the immune response that can involveinduction, expression, amplification, or inhibition of any part or

d Ltd. All rights reserved.

. guyonianum, Limoniastrum

e; NK, Natural killer;

ire, Laboratoire de Biologie

tir, Tunis, Tunisia.

ra-Chekir).

phase of an immune response. While several types of immuno-modulatory agents are available, undesirable side effects oftenlimit their use. Recently, complementary or alternative medicineshave become popular for treating different immune disorders.Increasingly among these are extracts from medicinal plants.Evaluation of the immunomodulatory activity of plant extractsis an interesting and growing area of research. However, little isknown about its immunomodulatory activities, in particular, forless frequently studied or previously unknown (medically) plants.

In this work we try to contribute to find new interest species instudying an endemic plant growing in North Africa: Limoniastrum

guyonianum Boiss. This plant is named locally ‘‘Zita’’ and iswidespread in South Tunisia. Decoction of L. guyonianum gallhas been used in traditional medicines to treat gastric infections.It has also been employed as an anti-bacterial in the treatment ofbronchitis (Le Floch, 1983). Limoniastrum feei has been similarlyused in the treatment of bronchitis and stomach infections

M. Krifa et al. / Journal of Ethnopharmacology 146 (2013) 243–249244

(Belboukhari and Cheriti, 2009). Previous investigations revealedthat methanol extract from L. feei leaves contained potential anti-fungal constituents that could be employed against Candida

albicans and anti-bacterial constituents useful against Escherichia

coli (Belboukhari and Cheriti, 2005).Furthermore, another study reported the antimicrobial activity

of the essential oil extracted from L. guyonianum (Hammami et al.,2011). Recently, it was mentioned that L. guyonianum exhibiteda very high antioxidant activity and the ethyl acetate extract from thisplant contains gallocatechin, epigallocatechin and epigallocatechin-3-O-gallate which showed anti-oxidant activities (Trabelsi et al., 2012).Owing to its frequent use in traditional medicine, we investigated theimmunomodulatory activity and cellular anti-oxidant potential ofL. guyonianum gall aqueous extract. To our knowledge, this is the firstreport of immunomodulatory and cellular anti-oxidant activities ofthis plant collected from the southern region of Tunisia.

2. Material and methods

2.1. Plant material

L. guyonianum samples were collected from El Hama at Gabbes(a region situated in southern Tunisia) in October 2009. Dr. FethiaSkhiri (Department of Botany, Higher Institute of Biotechnologie,University of Monastir) performed sample identification andverification according to the Tunisian Guide on Flora (Pottier-Alapetite, 1979). A voucher specimen (#L.g-10.09) was preservedfor future reference.

2.2. Preparation of plant extract

The collected gall samples were shade dried, powdered, andthen stored in a tightly closed container for further use. Whenneeded, powdered gall (100 g) was extracted in boiling water(1 L) for 15–20 min. After filtration, the aqueous extract wasfrozen and then lyophilized and kept at 4 1C. The total aqueousextract concentrate yield (per gram dried plant material) wasdetermined using the formula: 100�weight (g) of dried extract/dry weight (g) of plant material. The actual percentage yield inthis study was 17.8%. From this material, extract solutionscontaining concentrations of 5, 25, 50, 75 and 100 mg/ml werethen prepared for use in the evaluation of their effects on selectimmune parameters (see below).

2.3. Quantitative analysis of extract

The polyphenol content of L. guyonianum gall aqueous extractwas quantified by the Folin–Ciocalteau method (Chattopadhyayand Kumar, 2006; Yuan et al., 2005). Aliquots of test sample(100 ml) were mixed with 2.0 ml of 2% Na2CO3 and incubated atroom temperature for 2 min. After the addition of 100 ml of 50%Folin–Ciocalteau phenol reagent, the reaction tube was incubatedfor 30 min at room temperature, and finally absorbance was readat 720 nm. Gallic acid (0.2 mg/ml) was used as standard. Poly-phenol content was expressed according to the following formula:

Polyphenols %ð Þ ¼OD extract� 0:2

OD Gallic acid� extract concentration

� �� 100

A known volume of each extract was placed in a 10 mlvolumetric flask to estimate flavonoid content according to themodified method of Zhishen et al. (1999). After addition of 75 mlof NaNO2 (5%), 150 ml of freshly-prepared AlCl3 (10%), and 500 mlof NaOH (1 N), the volume was adjusted with distilled water until2.5 ml. After 5 min incubation, the total absorbance was mea-sured at 510 nm. Quercetin (0.05 mg/ml) was used as a standard.

Flavonoid content was expressed according to the followingformula:

Flavonoids %ð Þ ¼OD extract� 0:05

OD Quercetin� extract concentration

� �� 100

The method described by Pearson (1976) was used for thedetermination of tannin content of samples. Extraction of tanninswas achieved by dissolving 5 g of sample in 50 ml of distilledwater in a conical flask, allowing the mixture to stand for 30 minwith shaking the flask at 10 min intervals, and then centrifugingat 5000g to obtain a supernatant (tannin extract). The extract wasdiluted to 100 ml in a standard flask using distilled water. Fivemilliliters of the diluted extract and 5 ml of standard tannic acid(0.1 g/l) were measured into different 50 ml volumetric flasks.One milliliter of Folin–Denis reagent was added to each flaskfollowed by 2.5 ml of saturated sodium carbonate solution. Thesolutions were made up to the 50 ml mark with distilled waterand incubated at room temperature (20–30 1C) for 90 min. Theabsorption of these solutions was measured against the reagentblank (containing 5 ml distilled water in the place of the extractor the standard tannic acid solution) in a Genesys (Wisconsin,USA) spectrophotometer at 760 nm wavelength. Tannin contentwas calculated in triplicate (Nwabueze, 2007) according to thefollowing formula:

Tannins %ð Þ ¼OD extract

e� L� extract concentration

� �� 100

where e¼molar extinction coefficient (L g�1 cm�1) of tannic acid(¼3.27 L g�1 cm�1) and L¼1 cm.

2.4. Cell preparations from mice

Specific pathogen free BALB/c mice (6–8-wk-old, male, 18–22 g)were obtained from the Pasteur Institute (Tunis, Tunisia). The micewere housed under standard conditions of temperature (22–28 1C),humidity (30–70%), and light (12 h light/dark) in an accreditedpathogen free facility. All animals were provided ad libitum accessto standard rodent chow and filtered water. All experiments wereperformed in accordance with the guidelines for the care and use oflaboratory animals as published by the US National Institute ofHealth. All experiments received the explicit approval of the EthicsAnimal Committee in Tunisia.

Spleen BALB/c lymphocytes were obtained as previouslyreported (Limem et al., 2010). Briefly, mice were euthanized bycervical dislocation and each spleen was isolated aseptically andthen minced with a sterile forceps. Splenocytes were then isolatedby centrifugation (1500 rpm, for 10 min), and any red blood cellspresent were lysed by resuspending the pellet in lysing buffer(144 mM NH4Cl, 1.7 mM Tris Base) and placing on ice for 10 min.Cells were then washed twice with phosphate buffered saline(PBS, pH 7.4) and then resuspended in complete RPMI medium(GIBCO, BRL) containing 10% fetal bovine serum (FBS; GIBCO) and100 mg/ml gentamycin (Gibco-BRL, Paisley, UK).

Other mice were used to provide peritoneal macrophages aspreviously reported (Limem et al., 2010). For this, peritoneal cellswere obtained after intraperitoneal injection of 4 ml sterile PBS,massaging of the peritoneum, and drawing back of the fluid(E4 ml) into the syringe. The obtained cells were washed twiceand resuspended in complete RPMI 1640 medium. Cell viabilitywas assessed using the trypan blue exclusion technique.

2.5. Cell proliferation assay

Assays of lymphocyte proliferation were performed accordingto the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide] method outlined by Mosmann (1983). Splenocyte

M. Krifa et al. / Journal of Ethnopharmacology 146 (2013) 243–249 245

suspension in RPMI 1640 medium (5�106 cells/ml; 100 ml ali-quot/well) was pre-incubated in 96-well plate for 24 h, before theaddition of mitogens (lectin or LPS, each at 5 mg/ml) alone or incombination with increasing concentrations of G extract (0, 25, 50and 100 mg/ml) solubilized in RPMI. Cells were then incubated at37 1C in humidified 5% CO2 atmosphere for an additional 48 h.Thereafter, the plates were centrifuged at 1500 rpm for 10 minand then the cell pellet in each well was resuspended in 50 ml of aMTT (5 mg/ml) in RPMI solution of and incubated for 4 h at 37 1C.After this period, the plate was centrifuged again, the MTT in eachwell was removed, and 100 ml of dimethyl sulfoxide (98% DMSO)was added. After incubation at 37 1C for 15 min, absorbance offormed formazan in each well was measured at 570 nm in amicroplate reader (Thermo Scientific, Vantaa, Finland).

The percentage of proliferation was ultimately calculatedusing the equation: Proliferation (%)¼100� (OD sample�ODcontrol)/OD control (Manosroi et al., 2003).

2.6. Activity of natural killer (NK)

NK cell activity was measured as previously described (Sarangiet al., 2006), with minor modification. Briefly, spleens prepared asdescribed above were used as the source of effector cells; isolatedsplenocytes were seeded into 96-well microtiter plates at5�106 cells/ml. The cells were then stimulated at 37 1C bydifferent concentrations of extract for 24 h. To eliminate directeffects of the extract on target cells, spleens were washed oncewith RPMI 1640 and then target K562 cells (5�104 cells/ml;yielding a 100:1 expected effector-target ratio) were added toeach well in 100 ml aliquots. The plates were then incubated for4 h at 37 1C in 5% CO2 atmosphere. An aliquot (50 ml) of MTTsolution (5 mg/ml) was then added to each well and the plate wasincubated a further 4 h. After that, the plate was centrifugedagain, the MTT in each well was removed, and 100 ml of dimethylsulfoxide (98% DMSO) was added. After incubation at 37 1C for15 min, absorbance of formed formazan in each well wasmeasured at 570 nm in a microplate reader (Thermo Scientific,Vantaa, Finland).

Three kinds of control measurements were performed: targetcell control, blank control and effector cell control. NK cell activitywas calculated as follows: NK activity (%)¼100%� (ODT�(ODS�ODE))/ODT, where ODT¼optical density value of target cellscontrol, ODS¼optical density value of test samples, and ODE¼optical density value of effector cells control.

2.7. Assessment of lysosomal enzyme activity

Lysosomal enzyme activity (reflected by acid phosphatase [AP]activity) in macrophages was measured as previously describedby Manosroi et al. (2005), with some modification. Briefly,macrophage suspensions (100 ml aliquot of 6�106 cells/ml stockpreparation) were seeded into flat-bottom 96-well plates, treatedwith different concentrations of extract and incubated at 37 1C ina 5% CO2 humidified atmosphere for 48 h. The medium was thenremoved and 20 ml of 0.1% Triton X100 (Sigma, St. Louis, MO),100 ml of 100 mM p-nitrophenyl phosphate solution (Sigma) and50 ml of citrate buffer (pH 5.0, 0.1 M) were added to each well. Theplate was then incubated for 30 min at 37 1C before 150 ml ofborate buffer (pH 9.8, 0.2 M) was added to each well and theabsorbance then measured at 405 nm. The percentage of lysoso-mal enzyme activity in treated cultures relative to that in controlcells was calculated as previously reported (Manosroi et al., 2003)using: Lysosomal enzyme activity (%)¼100� (OD sample�ODcontrol)/OD control.

2.8. Nitrite determination

The amount of NO released by macrophages was measured bydetermining the amounts of accumulated nitrite (NO2

�) in cellfree supernatants via the Griess reaction (Green et al., 1982). Inbrief, cells were incubated for 48 h in the presence of increasingconcentrations of extract. Nitrite was then measured by adding100 ml Griess reagent (1% sulfanilamide and 0.1% naphthylene-diamine in 5% phosphoric acid) to 100 ml of harvested culturesupernatant. The optical density at 570 nm (OD570) was thenmeasured in a microplate reader (Thermo Scientific, Vantaa,Finland). NO concentrations were calculated by comparison withthe OD570 of a standard solution of sodium nitrite diluted inculture medium and placed in parallel wells in the assay plates.

2.9. Cellular anti-oxidant activity (CAA) assay

A cellular anti-oxidant activity (CAA) assay, developed byWolfe and Liu (2007), was employed to measure effects of theanti-oxidant potentials of the extract. Briefly, splenocytes wereseeded at a density of 6�104 cells (in 100 ml RPMI) in a 96-wellmicroplate and incubated for 24 h. The medium was thenremoved and the wells were washed with PBS. Triplicate wellswere then treated for 1 h with 95 ml extract along with 5 ml of a25 mM solution of 20,70-dichlorofluorescin (DCFH) in medium.After this incubation, all wells were washed with 100 ml PBSand a 100 ml aliquot of 600 mM ABAP (Sigma Aldrich, Steinheim,Germany) in RPMI was applied to the cells. The ABAP is anexogenous source of peroxyl radicals used to oxidize DCFH-DAto the fluorescent product DCF. Accordingly, cells treated withextracts that have any anti-oxidant activity should have lowerfluorescence compared to untreated cells. Fluorescence in eachwell was read every 5 min for a total of 1 h in a fluorescencemicroplate reader (Biotek, Winooski, USA) using 538 nm emissionand at 485 nm excitation filters. Each plate included triplicatecontrol and blank wells: control wells contained cells treated withDCFH-DA and ABAP; blank wells contained cells with DCFH-DAand RPMI without ABAP. Fluorescence values for the blanksamples and initial fluorescence values were subtracted fromthe sample values. The area under the fluorescence vs. time curvewas integrated at each time point to calculate the CAA units usingthe following equation:

CAAðUÞ ¼ 100�

ZSA=

ZCA

� �� 100

whereR

SA andR

CA are the integrated areas under the fluores-cence vs. time curves for sample and control curves, respectively(Wolfe and Liu, 2007).

2.10. Statistical analysis

All data were expressed as mean (7SD) and compared using aStudent’s t-test. Statistical significance was assigned at p valueso0.05. All data were analyzed using SPSS 11.0 software (SPSSINC; Illinois, USA).

3. Results

3.1. Quantitative analysis of G extract

G extract from L. guyonianum was the subject of a chemicalstudy with the aim of having a global idea in their composition. Itshowed the presence of flavonoids, tannins and polyphenols. Infact, quantitative phytochemical analysis showed that flavonoids,polyphenols and tannins are the major compounds present in the

M. Krifa et al. / Journal of Ethnopharmacology 146 (2013) 243–249246

tested extract, at 48.11%, 15.45% and 0.555% respectively. Othercompounds were undetermined.

3.2. Splenocyte proliferation

To test whether aqueous gall extract (G) from L. guyonianum

promoted or inhibited cell proliferation, splenocytes were iso-lated and cultured with extract alone, extractþ lectin, orextractþLPS. In the absence of mitogen, extract at 25, 50, and75 mg/ml was able to induce splenocyte proliferation (Fig. 1A). Inthe studies with lectin, lower cell proliferation occurred in a

Fig. 1. In vitro effects of aqueous gall extract (G extract) from L. guyonianum on

splenocyte proliferation responses. Cells were incubated for 48 h with: (A)

increasing concentrations of extract without mitogen; (B) lectin (5 mg/ml) in the

absence/presence of extract; or, (C) LPS (5 mg/ml) in the absence/presence of

extract. Control cells were incubated with RPMI 1640 only. Cell proliferation was

assessed using an MTT test. Data shown are mean percentage proliferation (7SD)

from three independent experiments. naValue significantly different compared

with that of negative (RPMI) control (po0.05). nbValue significantly different

compared with that of mitogen (LPS or lectin) treated cells (po0.05).

presence of 75 and 100 mg/ml of G extract (Fig. 1B). In contrast,the extract inversely (dose-dependently) up-regulated LPS-induced proliferation (Fig. 1C).

3.3. NK cell activity

NK cells represent the first line of defense against manypathogens. Measures of the cytotoxic activity of splenic NK cellsagainst NK sensitive tumor cells (i.e., K562 line) revealed that,compared with the control cells, the L. guyonianum extract couldsignificantly enhance NK cell activity at different concentrations,especially at 25 and 50 mg/ml of G extract (Fig. 2). However, thisactivity appeared to decrease with extract concentrations above50 mg/ml.

3.4. Peritoneal macrophage lysosomal activity

The effects of the L. guyonianum extract on lysosomal enzymeactivity in macrophages are demonstrated in Fig. 3. The aqueous

Fig. 2. Effect of gall aqueous extract (G extract) at various concentrations, on mice

natural killer (NK) cells in vitro. Splenocytes were cultured with different doses of

extract for 24 h before K562 target cells were added (at expected 100:1 E:T ratio).

NK activity was then measured using an MTT assay. Values shown are mean

(7SD) % cytotoxicity from six different observations. nnData significantly different

from control cells at po0.01.

Fig. 3. Stimulation of mouse peritoneal macrophages lysosomal enzyme activity

by aqueous gall extract from L. guyonianum. Macrophages (6�105 cells/well) were

incubated in the presence of increasing concentrations of extract for 48 h. Controls

cells were incubated with RPMI 1640 only. Lysosomal enzyme activity was then

assessed as indicated in Materials and methods. Data shown are mean (7SD)

percentages of lysosomal enzyme activity from three independent experiments.nValue significantly different from that of control cells at po0.05.

Fig. 6. Dose-response curve for inhibition of oxidation. Dose-response curve for

oxidation of DCFH to DCF in splenocytes using a cellular anti-oxidant activity

assay in the presence of aqueous gall extract (mean (7SD); n¼3).

M. Krifa et al. / Journal of Ethnopharmacology 146 (2013) 243–249 247

extract modulated phagocytic activity in an inversely dependantmanner. Concentration of 25 mg/ml of G extract exhibited themaximum potency (34.54%).

3.5. Peritoneal macrophage NO production

The effects of G extract on NO production by peritonealmacrophage are shown in Fig. 4. Macrophages treated withvarious concentrations of extract (25, 50, 75, and 100 mg/ml) for48 h appeared to display a significant dose related increased inNO production. Release of NO by macrophages increased from10.7% to 35.3% as extract concentration increased from 25 to100 mg/ml.

3.6. Cellular anti-oxidant activity (CAA)

The CAA assay is designed to trigger an acute oxidative stressin cells (by adding the radical initiator ABAP to the cell medium)and to fluorimetrically follow subsequent increases in formation

Fig. 4. Production of the nitrite by mouse peritoneal macrophages in response to

aqueous gall extract (G extract) from L. guyonianum. Macrophages (6�105 cells/

well) were incubated in RPMI 1640 medium in the absence\presence of increasing

concentrations of extract for 48 h. Cells treated with 5 mg/ml LPS alone were used

as positive control. Data shown are mean (7SD) percentage of NO production

from three independent experiments. Cells incubated in RPMI 1640 medium alone

did not produce NO. nValue significantly different compared with that of negative

(RPMI) control (po0.05).

Fig. 5. Kinetics of inhibited oxidation. The kinetics of the inhibition of oxidation of

DCFH in splenocytes using a cellular anti-oxidant activity assay in the presence of

aqueous gall extract (means; n¼5).

of an intracellular (radical-sensitive) fluorescent dye. The CAAassay was used here to quantitatively evaluate the anti-oxidantactivity of the gall aqueous extract in splenocyte. The resultsindicate that the extract could be absorbed into splenocytes andexert anti-oxidant activity. The production of fluorescence byABAP mediated oxidation of DCFH to DCF was dose-relatedlyreduced by the extract over a concentration range of 5–50 mg/ml(Fig. 5). The mean effective concentration (EC50) was calculatedfrom dose-response curve (Fig. 6) to be 10.03 mg of G extractper ml.

4. Discussion

The immune system is the human’s ultimate defense againstinfectious diseases, tumor and cancer growth. A healthy immunesystem contains elements that are in balance with one anotherand if this balance is broken, our immune system will be unableto protect the body against harmful agents or processes. For abovereasons, we investigated several aspects of immunomodulatoryactivity.

A colorimetric MTT assay was used to assess splenocyteproliferation. The results show that lymphocyte subpopulationswere differentially activated by gall aqueous extract (at varyinglevels), and that proliferation decreased with increasing concen-trations. The effects of extract on both LPS and lectin stimulatedcell proliferation were also analyzed. It is generally known thatlectin stimulates T-cells and LPS stimulates B-cells to proliferate(Han et al., 1998; Manosroi et al., 2005). At low concentrations(i.e., 25 and 50 mg/ml), G extract up-regulated LPS-induced cellproliferation. These results suggested potential synergistic effectsof the extract upon the mitogens. The extract may also containsome factors that are acting as mitogens. By enhancing LPS-induced cell proliferation, the gall aqueous extract might act asmodulator of B-cell function and, consequently, of cellularimmune responses in situ. In the presence of lectin and the gallaqueous extract, ‘stimulation’ of cell proliferation was decreased;such results suggested potential antagonistic or competitoreffects from the extract upon the lectin.

Cellular immunity is also mediated by T-cells, includingnatural killer (NK) cells. NK cells play a major role in defendingthe host from both tumor and virally-infected cells. With sponta-neous cell-mediated cytotoxicity, NK cells are functionally similarto cytotoxic T-lymphocytes (CTL). Unlike CTLs however, killing byNK cells is non-specific, and NK cells do not need to recognizeantigen/MHC on the target cell and NK cells can react against/destroy target cells without prior sensitization. NK cell

M. Krifa et al. / Journal of Ethnopharmacology 146 (2013) 243–249248

differentiation and maturation (to killing states) are each acti-vated by interferon and other cytokines. In the acute study, it wasdemonstrated that the gall aqueous extract significantlyenhanced NK cell function. Whether this activation was a resultof some direct stimulation by components within the extract orby a product of extract induced effects on other cells in thesplenic preparations (so as to, in turn, activate the NK cells)remains to be resolved in ongoing studies.

The proliferation of splenocytes in response to L. guyonianum

gall extract could be ascribed to several compounds. Preliminaryresults of phytochemical screening revealed a presence of numer-ous polyphenols and tannins, a class of agents already known tobe active immunomodulatory substances (Feldman et al., 1999).The stimulation of lymphocytes could also be attributed toflavonoids present in the extract. Flavonoid compounds havebeen previously shown to modulate the immune system mechan-istically, due to hydroxyl groups in their structures (Lopez-Posadaset al., 2008). Furthermore, it was reported that L. guyonianum ethylacetate extract contains flavonoids such gallocatechin, epigalloca-techin and epigallocatechin-3-O-gallate (Trabelsi et al., 2012),which is a potential immunomodulatory agent (Matsunaga et al.,2002).

One of the most important non-specific immune activitiesin situ is phagocytosis performed by macrophages (as well as byother types of leukocytes). Macrophages play an important role inthe defense mechanism against host infection and the killing oftumor cells. The modulation of the antitumor properties ofmacrophages by various biological response modifiers, is an areaof active interest in cancer chemotherapy and is closely-associated with immunomodulatory activity of test drugs (Kanget al., 2002). Phagocytosis is accompanied by the release oflysosomal enzymes like acid phosphatase (AP) used for the killingand digesting of microbial pathogens. The higher the augmenta-tion in AP activity, the greater the phagocytic stimulation andintracellular killing capacity (Rainard, 1986). With respect tolysosomal enzyme activity, transformation of p-NPP by AP instimulated macrophages correlates with degranulation processes(Suzuki et al., 1990). The results of the analyses here indicatedthat the test extract significantly enhanced cellular lysosomalenzyme activity. It should be stated that L. guyonianum extractcontains some constituents, such as flavonoids, tannins andpolyphenol that could be responsible for this particular immuno-modulatory effect (Lopez-Posadas et al., 2008).

Macrophage phagocytosis is also accompanied by release offree radicals and other reactive oxygen/nitrogen species (ROS/RNS, the latter including NO) involved in pathogen killing(Manosroi et al., 2005). Compared with LPS, a potent inducer ofmacrophage NO production (MacMicking et al., 1997), the studieshere showed that gall aqueous extract could induce NO synthesis,by peritoneal macrophages, even without addition of any otherexogenous stimuli. The increased release of NO reflects phagocy-tic stimulation; as such, the extract could act as an immunosti-mulant of innate immunity. Many plants (and their products)have been shown to influence lysosomal activity and NO synthaseactivity in macrophages (Bao et al., 2001; Limem et al., 2010; Sunet al., 2008). This result could be explained by the presence oftannins and flavonoids in the gall extract. Tannins are known toenhance the nitroblue tetrazolium (NBT) reducing activity ofmacrophages (Sakagami et al., 1991), to activate morphologicalchange (spreading) of peritoneal macrophages, and to inducevarious antitumor cytokines including tumor necrosis factor(TNF) and interleukin (IL)-1 (El Abbouyi et al., 2004).

Lastly, it is well-known that changes in cell oxidant status canimpart a stress that, in turn, impacts upon function; with regardto immune system cells, this could give rise to changes that arereflected as immunomodulation. In cells, ROS can react quickly to

damage various biomolecules, including proteins, DNA, and lipids(Fang et al., 2009). Under physiological conditions, to overcomethe potential toxicity from ROS, cells have evolved a series of anti-oxidative defense systems that contribute to scavenging of freeradicals such that cellular insults caused by ROS are reduced to a‘non toxic’ level (Demple and Harrison, 1994). In the currentstudy, it was shown that the L. guyonianum gall extract could leadto a reduction in the intracellular oxidation of DCFH. This impliedthat either agents in the extract were direct anti-oxidants or thatthey could trigger processes in the cell that augmented anti-oxidant monitoring processes. With regard to the former, poly-phenols (like those known to be in the extract) are considered tobe some of the most active anti-oxidant derivatives found inplants (Bors et al., 2001).

Based on the results presented here, we conclude that theL. guyonianum gall extract has anti-oxidant properties as well asimmunomodulating effects upon splenocytes, NK cells, andmacrophages. While the results are encouraging, a completecharacterization and profile of the bioactive compounds presentin the crude extract is warranted in future studies. The pharma-cologically-active ingredients in L. guyonianum and the signalingpathways involved in the modulation of both macrophage andlymphocyte responses also remain to be further elucidated.

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