In Vitro Investigation of the Potential Immunomodulatory and Anti-Cancer Activitiesof Black Pepper (Piper nigrum) and Cardamom (Elettaria cardamomum)

Amin F. Majdalawieh1 and Ronald I. Carr2,*

1Department of Biology and Chemistry, Faculty of Arts and Sciences, American University of Sharjah, Sharjah,United Arab Emirates; and 2Department of Microbiology and Immunology, Faculty of Medicine,

Dalhousie University, Halifax, Nova Scotia, Canada

ABSTRACT Although the immunomodulatory effects of many herbs have been extensively studied, research related to

possible immunomodulatory effects of various spices is relatively scarce. Here, the potential immunomodulatory effects of

black pepper and cardamom are investigated. Our data show that black pepper and cardamom aqueous extracts significantly

enhance splenocyte proliferation in a dose-dependent, synergistic fashion. Enzyme-linked immunosorbent assay experiments

reveal that black pepper and cardamom significantly enhance and suppress, respectively, T helper (Th)1 cytokine release by

splenocytes. Conversely, Th2 cytokine release by splenocytes is significantly suppressed and enhanced by black pepper and

cardamom, respectively. Experimental evidence suggests that black pepper and cardamom extracts exert pro-inflammatory

and anti-inflammatory roles, respectively. Consistently, nitric oxide production by macrophages is significantly augmented

and reduced by black pepper and cardamom, respectively. Remarkably, it is evident that black pepper and cardamom extracts

significantly enhance the cytotoxic activity of natural killer cells, indicating their potential anti-cancer effects. Our findings

strongly suggest that black pepper and cardamom exert immunomodulatory roles and antitumor activities, and hence they

manifest themselves as natural agents that can promote the maintenance of a healthy immune system. We anticipate that black

pepper and cardamom constituents can be used as potential therapeutic tools to regulate inflammatory responses and

prevent=attenuate carcinogenesis.

KEY WORDS: � anti-cancer activity � black pepper � cardamom � immunomodulation � inflammation

INTRODUCTION

Strong religious and mystical beliefs have been asso-ciated with the healing properties of many natural

products.1 The health-promoting properties of many herbsand spices are numerous and well recognized.2,3 There is nodoubt that a healthy, functional immune system is highlycorrelated with a well-balanced nutritional intake.4,5 Im-munonutrition has emerged as a new concept describingdifferent diets containing certain nutrients, including argi-nine, glutamine, fish oil, and nucleotides, that have profoundeffects on the immune system.4,5 Immunomodulation is aninduced modification of immune responses by means of in-troducing natural or synthetic chemical substances thatpossess the ability to regulate the immune system.6–8 Im-munomodulation is considered to be an invaluable tool forpreventing and treating various infectious and noninfectiousdiseases.6–8

A wide range of natural products isolated from herbs andspices have been shown to possess immunomodulatory ef-fects that can be very beneficial in fighting many diseases.9–12

The great potential that some spices possess in terms ofpreventing and treating various diseases, including cancer,cannot be underestimated.13–15 However, relatively little isknown about the potential immunomodulatory and anti-cancer effects of a wide range of commonly used spices, andthe molecular mechanisms underlying such effects are eitherpoorly understood or largely unidentified. In this study, wefocus on investigating the potential immunomodulatory andanti-cancer effects of black pepper (Piper nigrum) andcardamom (Elettaria cardamomum). We also attempt toshed light on the possible molecular mechanisms by whichblack pepper and cardamom extracts exert their immuno-modulatory and anti-cancer effects.

Many studies have previously demonstrated that blackpepper, its extracts, and its major constituents have diversephysiological effects in the gastrointestinal tract, kidney,and liver.16 Black pepper extracts have also been shown toexert antimicrobial activities.17–19 Moreover, some studieshave demonstrated that black pepper extracts and its majorconstituents possess anti-cancer properties in vitro andin vivo.20–24 Indeed, the ability of black pepper to alter the

Manuscript received 7 May 2009. Revision accepted 26 November 2009.

Address correspondence to: Dr. Amin F. Majdalawieh, Department of Biology andChemistry, Faculty of Arts and Sciences, American University of Sharjah, P.O. Box26666, Sharjah, United Arab Emirates, E-mail: amajdalawieh@aus.edu

*Deceased.

JOURNAL OF MEDICINAL FOODJ Med Food 13 (2) 2010, 371–381# Mary Ann Liebert, Inc. and Korean Society of Food Science and NutritionDOI: 10.1089=jmf.2009.1131

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metabolic activity of various enzymes has been proposed asthe mechanism of action by which black pepper inhibitscarcinogenesis.22,23,25,26

Cardamom was shown to play a wide range of health-promoting roles against various conditions such as con-stipation, colic, diarrhea, dyspepsia, vomiting, headache,epilepsy, and cardiovascular diseases.27,28 Recently, carda-mom was reported to exhibit spasmogenic, spasmolytic,blood pressure-lowering, vasodilator, diuretic, and sedativeactivities.29 Antimicrobial properties of cardamom extractshave been documented.30–33 Experimental evidence suggeststhat cardamom extracts display anti-cancer activities.26,34–37

Enzymatic modulation potential26,34–37 and anti-inflammatory,antiproliferative, and pro-apoptotic activities36 have beenproposed as mechanisms underlying the anti-cancer prop-erties of cardamom.

Our findings suggest that black pepper and cardamomare potential immunomodulators of splenocyte proliferationand Th1=Th2 cytokine profiles. Furthermore, our findingsalso suggest that the aqueous extracts of black pepper andcardamom exert pro-inflammatory and anti-inflammatoryroles, respectively. Finally, our study also suggests that thepreviously identified anti-cancer activities of black pepperand cardamom extracts may be mediated via the profoundpotential of such extracts to provoke the cytotoxic activity ofnatural killer (NK) cells. Hence, our study provides com-pelling evidence suggesting that black pepper and cardamomextracts may serve as potential immunoregulators of inflam-matory responses, Th1=Th2 immune responses, and carci-nogenesis. We anticipate that black pepper and cardamomconstituents may serve as natural therapeutic agents to pre-vent=treat diverse inflammatory conditions and various typesof cancer.

MATERIALS AND METHODS

Mice

Age-matched BALB=c and C57=BL6 mice (6–8 weeksold) were purchased from Jackson Laboratories (Bar Har-bor, ME, USA) and were kept on a 12-hour light=dark cyclein the Carleton Animal Care Facility at Dalhousie Uni-versity, Halifax, NS, Canada. Mice were fed chow diet andwere sacrificed by cervical dislocation for splenocyte ormacrophage isolation.

Preparation of aqueous extracts of the spices studied

Whole-seed black pepper and cardamom were washed,dried, and ground in liquid nitrogen. After complete evap-oration of liquid nitrogen, 10 mL of double distilled H2Owas added to the 20 g of ground spice and stirred overnightwith magnet stirrer to allow extraction. The crude spiceextracts were centrifuged at 10,000 g for 15 minutes at roomtemperature. Subsequently, the supernatants were harvestedand subjected to rotatory evaporation. After completeevaporation, a stock concentration of 20 mg=mL of eachextract was prepared, and the extracts were sterilized by

filtration using Nalgene filters (Thermo Fisher Scientific,Rochester, NY, USA) (pore size, 0.22mm).

Reagents and materials

Thioglycollate broth medium was purchased from DIFCO(Detroit, MI, USA). Lipopolysaccharide (LPS) isolated fromEscherichia coli O55:B5 and concanavalin A (ConA) werepurchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium, fetal bovine serum, penicillin-streptomycin,and l-glutamine were purchased from Invitrogen (Burlington,ON, Canada). Interferon-g (IFNg) was purchased from PeproTech (Rocky Hill, NJ, USA). BD OptEIATM enzyme-linkedimmunosorbent assay (ELISA) kits were purchased fromBD Pharmingen (Mississauga, ON, Canada). [3H]Thymidinewas purchased from Amersham Biosciences (Little Chalfont,UK). YAC-1 tumor cells (mouse lymphoma cells) werepurchased from the American Type Culture Collection(Rockville, MD, USA).

Isolation of splenocytes

Splenocyte isolation was performed as previously de-scribed.38 In brief, spleens were isolated from BALB=c mice,cut into several pieces, and gently crushed. Clumps werefurther dispersed by passing the suspension through a 19-gauge needle. Subsequently, cell suspension was filteredthrough a 200-mm mesh nylon screen, and cells were collectedby centrifugation. Erythrocytes were lysed using ACK lysisbuffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM disodiumEDTA), and splenocytes were finally washed and resus-pended in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin,10 mM HEPES, and 50mM b-mercaptoethanol. By trypanblue exclusion, cell counting revealed >98% viability.

Peritoneal macrophage isolation and culture

Thioglycollate-elicited peritoneal macrophages wereisolated from BALB=c mice as previously described.39 Inbrief, mice were injected intraperitoneally with 3 mL ofsterile 3% Brewer’s thioglycollate broth solution (Sigma-Aldrich). Five days later, mice were sacrificed by cervicaldislocation, and peritoneal exudate cells were isolated byperitoneal lavage. Peritoneal exudate cells were obtainedby centrifugation and resuspended in ACK lysis buffer forerythrocyte lysis. Subsequently, cells were centrifuged, re-suspended, and cultured in RPMI-1640 medium supple-mented with 10% heat-inactivated fetal bovine serum,1% penicillin-streptomycin, 10 mM HEPES, and 50mM b-mercaptoethanol. By trypan blue exclusion, cell countingrevealed >98% viability.

In vitro splenocyte proliferation assay

Splenocyte proliferation was assayed as previously de-scribed.40 In brief, 2�105 splenocytes were cultured for48 and 72 hours in medium supplemented with vehicle,10 ng=mL LPS, 1 mg=mL ConA, and aqueous extracts ofblack pepper and cardamom. Subsequently, cultured sple-

372 MAJDALAWIEH AND CARR

nocytes were pulsed with [3H]thymidine (1mCi per well) for16 hours before cell harvest. Cells were harvested using asemiautomated multiwell harvester, and cell lysates weretransferred onto fiberglass filter paper (Skatron Instruments,Lier, Norway). The dried filter paper was subsequentlytransferred to a vial containing 1.5 mL of scintillation fluid(Beckman, Fullerton, CA, USA). Incorporation of [3H]thy-midine was determined using the 1211 Rackbeta scintilla-tion counter (LKB Wallac, Turku, Finland).

Measurement of nitric oxide (NO) productionby macrophages (Griess assay)

NO production by macrophages was assessed by thecolorimetric Griess reaction as previously described.41 Inbrief, 2�105 macrophages were cultured in medium sup-plemented with vehicle, 10 ng=mL LPS, 2 U=mL IFNg, acombination of LPS and IFNg, or aqueous extracts of blackpepper and cardamom in the presence or absence of IFNgand=or LPS for 48 hours. Subsequently, 100mL of super-natant and serial dilutions of NaNO2 standard solution wereplaced in 96-well microtiter plates and then mixed withGriess reagent containing 1% sulfanilamide, 0.1% naph-thylethylenediamide dihydrochloride, and 2.5% H3PO4. Theoptical density was measured at 550 nm using an Emax�

precision microplate reader (Molecular Devices, Sunnyvale,CA, USA), and the amount of accumulated nitrite in thesamples was quantified according to the standard curve.

NK activity assessment by JAM assay

The cytotoxic activity of NK cells was assessed by JAMassay as previously described.42 In brief, YAC-1 tumor cellswere cultured for 4 hours in medium containing 5 mCi=mL[3H]thymidine for labeling. Subsequently, labeled YAC-1tumor cells were cultured in 96-well V-bottom culture platesin the presence or absence of splenocytes (containing NKcells) isolated from C57=BL6 mice at effector:target ratios(E:T ratios) of 200:1, 100:1, and 50:1. YAC-1 tumor cellscultured in the presence or absence of splenocytes weretreated with vehicle or aqueous extracts of black pepper andcardamom. At 4 hours post-incubation, YAC-1 tumor cellswere harvested using a semiautomated multiwell harvester,and radioactivity was measured using the 1211 Rackbetascintillation counter (LKB Wallac). Percentage cytotoxicitywas determined as follows: % cytotoxicity¼ ([vehicle-treatedYAC-1 tumor cells� targeted-YAC-1 tumor cells]=vehicle-treated YAC-1 tumor cells)�100.

Assessment of cytokine secretion by ELISA

For assessment of interleukin (IL)-4, IL-10, and IFNgrelease, 2�105 splenocytes were treated with vehicle,10 ng=mL LPS, 1 mg=mL ConA, and aqueous extracts ofblack pepper and cardamom in the presence or absence of1 mg=mL ConA for 48 hours. For assessment of IL-6 andtumor necrosis factor-a (TNFa) release, 2�105 macro-phages were treated with vehicle, 10 ng=mL LPS, 2 U=mLIFNg, a combination of LPS and IFNg, and aqueous extracts

of black pepper and in the presence and absence of LPS plusIFNg for 48 hours and 12 hours (IL-6 and TNFa, respec-tively). Subsequently, supernatants were harvested, andcytokine concentration was determined using BD OptEIAELISA kits and the Emax precision microplate reader.

Statistical analysis

Data are mean� SEM values of the indicated number ofexperiments. Statistical significance was determined usingStudent’s t test for unpaired observations; *P< .05, **P<.01, and ***P< .001 were considered statistically significant.

RESULTS

Assessment of splenocyte proliferation in the presenceof aqueous extracts of black pepper and cardamom

We hypothesized that aqueous extracts of black pepper andcardamom can potentially enhance splenocyte proliferation. Totest this hypothesis, BALB=c splenocytes were cultured inmedium supplemented with vehicle, ConA (T lymphocytemitogen), LPS (B lymphocyte mitogen), or aqueous extracts ofblack pepper or cardamom at four doses (1, 10, 50, and100mg=mL) for 48 and 72 hours. Subsequently, culturedsplenocytes were subjected to an in vitro proliferation assayusing [3H]thymidine incorporation to assess the potentialmodulatory effects of the aqueous extracts on splenocyte pro-liferation. As shown in Figure 1A and B, the proliferation ofsplenocytes was enhanced in a dose-dependent manner in thepresence of aqueous extracts of black pepper and cardamom at72 hours. Notably, all doses (except 1mg=mL) of black pepperaqueous extract led to significant enhancement of splenocyteproliferation, but significant enhancement by of splenocyteproliferation by cardamom was only observed at 100mg=mL.Noteworthy is that significant enhancement of splenocyteproliferation at 48 hours was only observed with black pepperaqueous extract at 50 and 100mg=mL (data not shown).

Synergistic stimulatory effect of aqueousextracts of black pepper and cardamomon splenocyte proliferation

To determine whether the aqueous extracts of blackpepper and cardamom have any synergistic effect on sple-nocyte proliferation, BALB=c splenocytes were cultured inmedium supplemented with 100mg=mL aqueous extract ofblack pepper, cardamom, or a combination of both. Vehicle,LPS, and ConA were used as experimental controls. At 72hours post-incubation, cultured splenocytes were subjectedto the in vitro splenocyte proliferation assay. As shown inFigure 1C, splenocyte proliferation was significantly greaterin the presence of aqueous extracts of both black pepper andcardamom in combination compared to the effect of eachextract separately. Indeed, the enhancement of splenocyteproliferation in the presence of the aqueous extracts of bothblack pepper and cardamom in combination is comparableto that caused by LPS (P¼ .23). A significant synergisticeffect of the aqueous extracts of black pepper and cardamomon splenocyte proliferation was also observed at lower doses

IMMUNOMODULATION BY BLACK PEPPER AND CARDAMOM 373

(1, 10, and 50mg=mL) of the extracts (data not shown), inwhich the stimulatory action of one extract is enhanced andmagnified in the presence of the other extract. Notably, theaqueous extract of cardamom displayed a significantlygreater potential to enhance splenocyte proliferation com-pared to the aqueous extract of black pepper (P¼ .004) (Fig.1C). Together, these data suggest that not only do aqueousextracts of black pepper and cardamom significantly en-hance the proliferation of splenocytes, but they also interactcooperatively to further augment splenocyte proliferation.

Polymyxin B (PB), which is known to potentially inacti-vate LPS, was used to determine whether the aqueous extractsof black pepper and cardamom were LPS-contaminated. Tothis end, BALB=c splenocytes were cultured in mediumsupplemented with aqueous extracts of black pepper andcardamom at 100mg=mL in the presence or absence of1mg=mL PB and subsequently subjected to the in vitrosplenocyte proliferation assay. Clearly, PB significantly re-duced the ability of LPS to promote splenocyte proliferation,whereas the ability of aqueous extracts of black pepper orcardamom to enhance splenocyte proliferation was not alteredin presence of PB (Fig. 1D). Collectively, these results clearlyrule out the possibility that the aqueous extracts of blackpepper and cardamom enhance splenocyte proliferationbecause of possible LPS contamination.

Aqueous extracts of black pepper and cardamommodulate cytokine release by splenocytesand macrophages

To further evaluate the immunomodulatory effects of theaqueous extracts of black pepper and cardamom, the pro-

duction of IL-4, IL-10, and IFNg by lymphocytes and theproduction of IL-6 and TNFa by macrophages were assessed.To assess IL-4, IL-10, and IFNg secretion, BALB=c spleno-cytes were cultured in medium supplemented with vehicle,LPS, ConA, and aqueous extracts of black pepper and car-damom (1, 10, 50, and 100mg=mL) in the presence or absenceof ConA. As shown in Figure 2A and C, the aqueous extract ofblack pepper had no significant effect on IL-4 and IL-10 re-lease by splenocytes at any of the doses compared to vehicle-treated splenocytes. However, dose-dependent inhibition ofIL-4 and IL-10 release was observed when splenocytes weretreated with the aqueous extract of black pepper in the pres-ence of ConA (Fig. 2A and C). With regard to cardamom,IL-4 and IL-10 release was significantly enhanced whensplenocytes were treated with 50 and 100mg=mL aqueousextract of cardamom compared to vehicle-treated splenocytes(Fig. 2B and D). Likewise, a dose-dependent increase in IL-4and IL-10 release was observed when splenocytes weretreated with aqueous extract of cardamom in the presence ofConA compared to ConA-treated splenocytes (Fig. 2B and D).

Noticeably, treatment of splenocytes with the aqueous extractof black pepper was accompanied by a dose-dependent increasein IFNg release in the presence and absence of ConA comparedto ConA-treated and vehicle-treated splenocytes, respectively(Fig. 2E). As shown in Figure 2F, the aqueous extract of car-damom had no significant effect on IFNg release by splenocytesat any of the doses compared to vehicle-treated splenocytes.However, dose-dependent inhibition of IFNg release was ob-served when splenocytes were treated with the aqueous extractof cardamom in the presence of ConA (Fig. 2F).

To assess IL-6 and TNFa secretion, BALB=c macro-phages were cultured in medium supplemented with vehicle,

FIG. 1. Dose-responsive effect of aqueousextracts of (A) black pepper and (B) carda-mom on splenocyte proliferation 72 hourspost-treatment. The aqueous extracts of blackpepper and cardamom were used at 1, 10, 50,and 100mg=mL. Statistical significance wasdetermined in comparison to vehicle-treatedsplenocytes. (C) Assessment of the synergis-tic effect of aqueous extracts of black pepperand cardamom on splenocyte proliferation72 hours post-treatment. The aqueous extractsof black pepper and cardamom were used at100 mg=mL. Statistical significance was de-termined in comparison to vehicle-treatedsplenocytes. (D) Evaluation of the modulatoryeffects of aqueous extracts of black pepperand cardamom on splenocyte proliferation inthe presence of PB 72 hours post-treatment.For each sample where PB was used, statis-tical significance was determined in compar-ison to the relative sample where splenocyteswere left PB-untreated. Count per minutes(CPM) data are expressed as mean� SEMvalues. **P< .01, ***P< .001.

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LPS, IFNg, combination of LPS and IFNg, and aqueousextracts of black pepper and cardamom (1, 10, 50, and100mg=mL) in the presence or absence of LPS plus IFNg. At48 and 12 hours post-incubation, supernatants were har-vested and subjected to ELISA to measure the concentrationof IL-6 and TNFa, respectively. As shown in Figure 3A andC, IL-6 and TNFa release by macrophages was significantlyenhanced by the aqueous extract of black pepper in thepresence and absence of LPS and IFNg compared to mac-rophages treated with LPS and IFNg and vehicle-treatedmacrophages, respectively. Clearly, the aqueous extract ofcardamom alone had no significant effect on IL-6 and TNFarelease by macrophages at any of the doses (Fig. 3B and D).However, dose-dependent inhibition of IL-6 and TNFa re-

lease was observed when macrophages were treated with theaqueous extract of cardamom in the presence of LPS andIFNg (Fig. 3B and D). Collectively, these findings suggestthat black pepper and cardamom possess immunomodula-tory functions with regard to cytokine release profile insplenocytes and macrophages.

Assessment of NO production by macrophagesin the presence of aqueous extracts of blackpepper and cardamom

To assess the potential ability of aqueous extracts of blackpepper and cardamom to modulate NO production bymacrophages, BALB=c macrophages were cultured in

FIG. 2. Effect of aqueous extracts of (A, C, E)black pepper and (B, D, F) cardamom on the secretionof (A, B) IL-4, (C, D) IL-10, and (E, F) IFNg bysplenocytes. The aqueous extracts of black pepper andcardamom were used at 1, 10, 50, and 100 mg=mL. Forsplenocytes that were treated with the aqueous extractsin the absence of ConA, statistical significance wasdetermined in comparison to vehicle-treated spleno-cytes. For splenocytes that were treated with theaqueous extracts in the presence of ConA, statisticalsignificance was determined in comparison to ConA-treated splenocytes. Data are mean� SEM values.*P< .05, **P< .01, ***P< .001.

IMMUNOMODULATION BY BLACK PEPPER AND CARDAMOM 375

medium supplemented with vehicle, IFNg, LPS, or aqueousextracts of black pepper or cardamom at four different doses(1, 10, 50, and 100mg=mL) in the presence or absence ofIFNg. Subsequently, cultured macrophages were subjectedto Griess assay to measure NaNO2 production. As shown inFigure 4, LPS, IFNg, and a combination of both causedmacrophages to produce about 9, 40, and 99mM NaNO2,respectively, compared to 6 mM NaNO2 in vehicle-treatedperitoneal macrophages. Whereas the aqueous extract ofcardamom had no significant effect on NO production bymacrophages at any of the doses under unstimulatory con-ditions (Fig. 4B), the aqueous extract of black pepper at100mg=mL significantly enhanced NO production by mac-rophages compared to vehicle-treated macrophages underunstimulatory conditions (Fig. 4A). In the presence of IFNg,the aqueous extract of black pepper significantly enhancedNO production by macrophages at 50 and 100mg=mL com-pared to IFNg-treated macrophages (Fig. 4A). Likewise,treatment of macrophages with 50 or 100mg=mL blackpepper extract in the presence of LPS and IFNg led to asignificant increase in NO production compared to macro-phages treated with LPS and IFNg (Fig. 4A). These findingssuggest that the aqueous extract of black pepper mimics theLPS potential to prime macrophages. Interestingly, however,the aqueous extract of cardamom significantly suppressed

NO production at 50 and 100mg=mL doses in the presence ofIFN compared to IFNg-treated macrophages (approximatelytwofold suppression) (Fig. 4B). In the presence of both LPSand IFNg, however, 100mg=mL cardamom extract was theonly dose capable of causing significant suppression of NOproduction by macrophages (Fig. 4B).

The aqueous extract of cardamom is capable of potentlysuppressing NO production by IFNg-treated macrophages aswell as IFNg- and LPS-treated macrophages even in presenceof the aqueous extract of black pepper (Fig. 4C). Indeed, thecombined effect of the aqueous extracts of black pepper andcardamom on NO production is very comparable to the effectof the aqueous extract of cardamom alone in unstimulated,IFNg-treated, and IFNg- and LPS-treated macrophages(P¼ .22, P¼ .35, and P¼ .96, respectively) (Fig. 4C). Thisobservation indicates that cardamom is a much more potentmodulator of NO production than black pepper.

Evaluation of the cytotoxic activity of NK cellsin the presence of aqueous extracts of blackpepper and cardamom

To further assess the immunostimulatory effects of blackpepper and cardamom, the cytotoxic activity of NK cellsagainst YAC-1 tumor cells was evaluated in the presence of

FIG. 3. Effect of aqueous extracts of (A, C)black pepper and (B, D) cardamom on thesecretion of (A, B) IL-6 and (C, D) TNFaby peritoneal macrophages. The aqueous ex-tracts of black pepper and cardamom wereused at 1, 10, 50, and 100 mg=mL. For mac-rophages that were treated with the aqueousextracts in the absence of LPS and IFNg,statistical significance was determined incomparison to vehicle-treated macrophages.For macrophages that were treated with theaqueous extracts in the presence of LPS andIFNg, statistical significance was determinedin comparison to LPS- and IFNg-treatedmacrophages. Data are mean� SEM values.*P< .05, **P< .01, ***P< .001.

376 MAJDALAWIEH AND CARR

aqueous extracts of black pepper and cardamom. YAC-1tumor cells were cultured in medium supplemented withvehicle, 100mg=mL aqueous extract of black pepper orcardamom, and effector cells (NK cells) at E:T ratios of200:1, 100:1, and 50:1. In addition, YAC-1 tumor cells weretreated with 1, 10, 50, and 100mg=mL aqueous extracts ofblack pepper and cardamom in the presence of effector cellsat the 200:1 E:T ratio. As shown in Figure 5A, the aqueousextract of black pepper significantly enhances the cytotoxicactivity of NK cells at 50 and 100mg=mL doses. With regardto cardamom, significant dose-dependent stimulation of thecytotoxic activity of NK cells is observed at all doses (1, 10,50, and 100mg=mL) (Fig. 5B). Importantly, the aqueousextracts of black pepper and cardamom possess no directcytotoxic activity against YAC-1 tumor cells (Fig. 5A andB). As expected, the enhanced cytotoxic activity of NK cellsagainst YAC-1 tumor cells by aqueous extracts of blackpepper and cardamom is proportional to the E:T ratio (datanot shown). Interestingly, the aqueous extracts of blackpepper and cardamom display synergistic stimulatory effecton the cytotoxic activity of NK cells against YAC-1 tumorcells (Fig. 5C), in which NK cytotoxic activity is muchgreater in the presence of both extracts compared to theseparate action of each extract. Notably, the aqueous extractof cardamom displays a more potent stimulatory effect onthe cytotoxic activity of NK cells compared to that of blackpepper (Fig. 5C), but this differential ability to enhancethe cytotoxic activity of NK cells did not reach statistical

significance (P¼ .13). Taken together, these data stronglysuggest that black pepper and cardamom have the potentialto markedly enhance the anti-cancer activity of NK cells.

DISCUSSION

A search for new drugs designed for promoting optimalimmune function is the main focus of many researchersworldwide, and natural products used in traditional medi-cines have been the source of many medically beneficialdrugs.43,44 In search of such natural products, we investi-gated the potential immunomodulatory functions of blackpepper and cardamom. In this study, we provide experimen-tal evidence demonstrating that aqueous extracts of blackpepper and cardamom are potentially capable of modu-lating the function of various immune cells. Indeed, theproliferation of splenocytes is significantly enhanced in thepresence of aqueous extracts of black pepper and carda-mom in a dose-dependent (Fig. 1A and B) and cooperative(Fig. 1C) fashion. This suggests that the aqueous extractsof black pepper and cardamom contain constituents thatare capable of promoting the proliferative signaling path-ways in splenocytes. Consistently, piperine, an active al-kaloid component of black pepper, was shown to enhancemurine splenocyte proliferation.45 Similarly, eugenol, anactive component of cardamom, has been reported tosignificantly enhance cell-mediated lymphocyte prolifera-tion in vitro.46

FIG. 4. Dose-responsive effect of aqueousextracts of (A) black pepper and (B) cardamomon NO production by macrophages. The aque-ous extracts of black pepper and cardamomwere used at 1, 10, 50, and 100 mg=mL. (C)Assessment of the synergistic effect of aqueousextracts of black pepper and cardamom on NOproduction by macrophages. The aqueous ex-tracts of black pepper and cardamom were usedat 100 mg=mL. For macrophages that weretreated with the aqueous extracts in the absenceof LPS and IFNg, statistical significance wasdetermined in comparison to vehicle-treatedmacrophages. For macrophages that were trea-ted with the aqueous extracts in the presence ofIFNg, statistical significance was determined incomparison to IFNg-treated macrophages. Formacrophages that were treated with the aqueousextracts in the presence of LPS and IFNg, sta-tistical significance was determined in compar-ison to LPS- and IFNg-treated macrophages.Data are mean� SEM values. *P< .05, **P<.01, ***P< .001.

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The immunomodulatory effects of spices on the release ofmajor cytokines by splenocytes and macrophages have notbeen widely investigated. In our in vitro studies using pri-mary splenocytes, the release of the Th2 cytokines IL-4 andIL-10 has been shown to be suppressed by black pepperextract and enhanced by cardamom extract (Fig. 2). Con-versely, the release of the Th1 cytokine IFNg has beenshown to be enhanced and suppressed by aqueous extracts ofblack pepper and cardamom, respectively (Fig. 2). Con-sistently, piperine was shown to induce the secretion of Th1cytokines IL-2 and IFNg from splenocytes45 while inhibit-ing Th2 cytokine secretion.47 Moreover, and in agreementwith our findings suggesting that the cardamom extract in-hibits Th1 cytokine secretion and enhances Th2 cytokinesecretion in splenocytes, eugenol was shown to be a potentinhibitor of the major Th1 cytokine IL-2.48 Because Th2cells are responsible for type-I hypersensitivity responses(i.e., allergic reactions), it is conceivable that the aqueousextract of cardamom, but not black pepper, may have thepotential to induce type-I hypersensitivity reactions, devel-opment of Th2 cells, and establishment of Th2 immuneresponses. Interestingly, two studies have suggested thatcardamom powder may be associated with contact andsystemic contact-type dermatitis.49–51

It is evident that the release of the pro-inflammatory cy-tokines IL-6 and TNFa by macrophages is enhanced by theaqueous extract of black pepper (Fig. 3), indicating that blackpepper promotes macrophage pro-inflammatory responsive-ness. It is noteworthy that our data clearly indicate that theaqueous extract of black pepper significantly enhances NOproduction by macrophages only in the presence of IFNg(Fig. 4A). This suggests that black pepper extract con-

tains constituents that mimic the ability of LPS to primemacrophages for enhanced NO production. Conversely, theaqueous extract of cardamom impedes macrophage pro-inflammatory responsiveness, as reflected by the suppressiveeffects of the cardamom extract on IL-6, TNFa, and NOrelease by macrophages (Fig. 3). In agreement with theseresults, it was proposed that eugenol leads to inhibited se-cretion of the proinflammatory mediators IL-1b and IL-6,52

inhibitory NO synthase and NO,53,54 and cyclooxygenase-2.55 Interestingly, oral administration of the aqueous extractof cardamom is accompanied by a significant reduction incyclooxygenase-2 and inhibitory NO synthase expression inmurine models of colon cancer.36 Moreover, cardamom wasshown to have anti-inflammatory activity against acutecarrageenan-induced plantar edema in albino rats.56 Takentogether, the aqueous extract of black pepper exhibits pro-inflammatory activities, whereas the aqueous extract of car-damom exhibits anti-inflammatory activities in macrophages.

Our study also provides experimental evidence suggest-ing that the aqueous extracts of black pepper and cardamomenhance the cytotoxic activity of NK cells (Fig. 5A and B).Interestingly, the aqueous extracts of black pepper andcardamom cooperate to robustly augment the cytotoxic ac-tivity of NK cells to reach about 67% cytotoxicity (Fig. 5C).It is important to mention that neither the aqueous extract ofblack pepper nor that of cardamom had any direct cytotoxiceffect against YAC-1 tumor cells (Fig. 5). These datastrongly suggest that black pepper and cardamom possessimmunostimulatory effects towards NK cytotoxic activity.Our findings regarding the anticarcinogenic effects of theblack pepper and cardamom extracts are in agreement withother in vitro and in vivo studies. In one study, oral ad-

FIG. 5. Dose-responsive effect of aqueousextracts of (A) black pepper and (B) cardamomon the cytotoxic activity against YAC-1 lym-phoma cells. The aqueous extracts of blackpepper and cardamom were used at 1, 10, 50, and100 mg=mL, unless indicated otherwise. An E:Tratio of 200:1 was used. (C) Assessment of thesynergistic effect of aqueous extracts of blackpepper and cardamom on the cytotoxic activityagainst YAC-1 lymphoma cells. The aqueousextracts of black pepper and cardamom wereused at 100 mg=mL. For samples where cyto-toxicity was assessed in the presence of theaqueous extracts at an E:T ratio of 200:1, sta-tistical significance was determined in compari-son to vehicle-treated cells at an E:T ratio of200:1. Data are mean� SEM values. *P< .05,**P< .01, ***P< .001.

378 MAJDALAWIEH AND CARR

ministration of black pepper extract significantly improved(by *65%) the life span of mice with Ehrlich ascites tu-mors.20 In other studies, histopathological analyses haverevealed that the rate of inflammatory cell infiltration intothe submucosa, the incidence of papillae, and changes inthe cytoplasm were decreased when rats with experimen-tally induced colon carcinogenesis were fed black pep-per extract.21,23 A recent study has shown that piperinehas antiproliferative effects on human colon cancer cells.24

Additionally, piperine was demonstrated to have antitumoractivity in vivo.57–62 As for the proposed anticarcinogeniceffects of cardamom, several studies have demonstrated thateugenol inhibits tumor formation in vivo.63–66 Consistentwith our findings, eugenol has been shown to significantlyenhance the NK cytotoxic activity, suggesting that carda-mom exerts immunotoxic effects.67

Although a large body of research has revealed that var-ious spices and their chemical constituents could potentiallyplay anticarcinogenic roles, the basis for such anticarcino-genic effects has been attributed to enzymatic modula-tion26,34–37 and anti-inflammatory, antiproliferative, andpro-apoptotic activities.36 Indeed, enzymatic modulationhas been deemed a mechanism of action by which blackpepper manifests its anticarcinogenic roles.22,23,25,26 Like-wise, enzymatic modulation26,34–37 and anti-inflammatory,antiproliferative, and pro-apoptotic activities36 have beenproposed as mechanisms underlying the anti-cancer prop-erties of cardamom. Our findings demonstrate that the an-ticarcinogenic effects of the black pepper and cardamomextracts may be attributed to the immunostimulatory po-tential of the spices’ constituents to promote NK cytotoxicactivity against cancer cells.

In conclusion, our study provides experimental evidencesuggesting that black pepper and cardamom have a greatpotential to serve as immunomodulatory agents. Blackpepper seems to play pro-proliferative, pro-inflammatoryfunctions, while cardamom manifests itself as a potentsuppressor of inflammation. In addition, our study demon-strates that black pepper and cardamom extracts exert anti-carcinogenic effects via promoting the cytotoxic activityof NK cells. Although the exact molecular mechanismsunderlying the immunomodulatory effects exerted by theextracts of black pepper and cardamom on splenocytes,macrophages, and NK cells are still unknown, elucidationof the specific signaling pathways involved in this im-munomodulation is currently underway. Finally, we antici-pate that the active constituents of black pepper andcardamom may serve as potential molecular tools for de-veloping new therapeutic strategies to modulate inflamma-tory responses and prevent=treat various types of cancer.

ACKNOWLEDGMENTS

We are grateful to Dr. Fredrick Palmer (Dalhousie Uni-versity, Halifax, NS, Canada) for allowing us to use theirrotatory evaporators in the preparation of the spice extracts.We thank Hana James, Jillian Tarrant, Wendy Hughes, andBruce Musgrave for their invaluable technical assistance.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

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