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Journal of Ethnopharmacology 114 (2007) 406–411

Effect of Rhaphidophora korthalsii methanol extract on humanperipheral blood mononuclear cell (PBMC) proliferation

and cytolytic activity toward HepG2

S.K. Yeap a, N.B. Alitheen a,∗, A.M. Ali a, A.R. Omar b, A.R. Raha c,A.A. Suraini c, A.H. Muhajir d

a Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences,Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

b Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

c Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences,Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

d Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences,Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Received 4 May 2007; received in revised form 10 August 2007; accepted 14 August 2007Available online 19 August 2007

bstract

The study of bioactivity of natural product is one of the major researches for drug discovery. The aim of this finding was to study the prolif-ration effect of Rhaphidophora korthalsii methanol extract on human PBMC and subsequently the cytotoxic effect of activated PBMC towardepG2 human hepatocellular carcinoma. In this present study, MTT assay, cell cycle study and Annexin 5 binding assay were used to study the

mmunomodulatory and cytotoxic effects. In vitro cytotoxic screening of Rhaphidophora korthalsii methanol extract showed that the extract wason-toxic against hepatocellular carcinoma (HepG2). In contrast, the extract was able to stimulate the proliferation of human PBMC at 48 h and 72 hn MTT assay and cell cycle progress study. The application of immunomodulator in tumor research was studied by using MTT microcytotoxicity

ssay and flow cytometric Annexin V. Results indicated that pre-treated PBMC with Rhaphidophora korthalsii methanol extract induced the highestytotoxicity (44.87 ± 6.06% for MTT microcytotoxicity assay and 51.51 ± 3.85% for Annexin V) toward HepG2. This finding demonstrates thathaphidophora korthalsii methanol extract are potent to stimulate the cytotoxic effect of immune cells toward HepG2.2007 Elsevier Ireland Ltd. All rights reserved.

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eywords: Immunomodulator; Cytotoxic; HepG2; PBMC; Rhaphidophora kor

. Introduction

In recent years, focus on use of traditional herb as alterna-ive treatment has been revived all over the world. Public hastarted to use some of the commonly well-known herbs crudextract as daily food supplement. However, a lot of traditional

erbs do not have any extensive scientific review of the effectn the body system. To fill up the gap, researchers have startedo study the biological properties of the traditional herbs. For

∗ Corresponding author. Tel.: +60 3 8946 7471; fax: +60 3 8946 7510.E-mail address: noorjahan@biotech.upm.edu.my (N.B. Alitheen).

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378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2007.08.020

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xample, Zhou et al. (2006) demonstrated that the volatile oil ofinger downregulated the T lymphocytes proliferatioin in vitrond reduced the effect of delayed type of hypersensitivity in vivo.haphidophora (Araceae) is a large genus of a climbing shrubistributed throughout India, Sri Lanka, Cambodia, Venezuela,alaysia, Australia and Indonesia (Kiritikar and Basu, 2001).

haphidophora pertusa in this genus has been studied and wasound that the methanol extract of it showed antioxidant andntimicrobial activity in the dose-dependent manner (Sasikumar

nd Doss, 2006). Also it has been reported that the isolated com-ounds of Rhaphidophora decursiva possess antimalarial andytotoxic activity (Zhang et al., 2001). Rhaphidophora korthal-ii is widely known with the common name “Dragon tail” due to

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he morphology of the plant. Traditionally, this plant is used asnticancer drug and to treat skin disease. Wong and Tan (1996)ho studied this plant found that the ether fraction of Rhaphi-ophora korthalsii showed cytotoxicity in P388, Molt 4, KB andW 620 cancer cell line at the concentration above 50 �g/mL.esides, the same study also showed that the water fraction of thelant can stimulate weak but significant enhancement of spleno-yte proliferation at full range of concentration. The cytotoxicityf the plant may be due to the presence of 5,6-dihydroxyindoleDHI), which showed the cytotoxic effect toward p388. (Toyotand Ihara, 1998). DHI also proved to have the cytotoxic effectn nonmelanocytic cancer cell but not melanocytic cell (Urabet al., 1994).

Investigation of the cell proliferation effect of the naturalroduct treated immune cell is the preliminary study for the dis-overy of new immunomodulator from herbs. Human PBMCnd mice splenocyte is commonly used as target cell for thistudy (Swamy and Tan, 2000; Kumar et al., 2004). Other thanhat, plant extract immunomodulatory effect also has been stud-ed extensively in vivo by using C57BL/6 mice that were infectedith Lewis lung carcinoma (Wang et al., 2007). Besides the

imple immune cell proliferation test and complicated in vivotudy, there are some studies that used the co-culture systemo estimate the cytotoxic effect and anti-tumor effect of eitherreated or untreated immune cell. Kim et al. (2004) used theo-culture system of HepG2/Hep3B and NK cell to study theilling mechanism of immune cell toward hepatocellular car-inoma. Microcytotoxicity assay which utilize the concept ofsing attached effector cell and co-incubating with suspensionmmune cell as target was also applied in the co-culture studyWahlberg et al., 2001).

To our knowledge, the proliferation effect of this plant extractn human cell and the application of the plant extract activatedBMC in anticancer study have not yet been reported. In thistudy, the effect of Rhaphidophora korthalsii methanol extractn PBMC proliferation was investigated. In addition, this studynalyzed the cytotoxicity activity of Rhaphidophora korthalsiiethanol extract activated PBMC on HepG2 through Annexinmethod and MTT microcytotoxicity assay.

. Materials and methods

.1. Preparation of plant extract

Leaves of Rhaphidophora korthalsii (Araceae) was collectedn Georgetown, Penang in June 2006, and were identifiedy science officer Lim Chung Lu (Kepong, Selangor) fromorestry Division, Forest Research Institute Malaysia (FRIM).he voucher number of Rhaphidophora korthalsii is FRIM3687. The leaves of the plant was air-dried in shade and finelyowdered. The leaf extract was prepared by keeping the leafowders soaked in 250 mL of methanol (J.T. Baker, USA) for2 h. The extracts were filtered with Whatman filter paper no. 1

nd evaporated to dry under reduced pressure by using Aspirator-3S (EYELA, Japan) at <40 ◦C. The process was repeated three

imes (yield 27.3%, w/w). The dried residue was suspended inMSO (Fisher Scientific, UK) as a plant extract stock. Briefly,

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rmacology 114 (2007) 406–411 407

he concentrated plant extracts were dissolved in dimethyl-ulphoxide (DMSO) (Sigma, USA) to get a stock solution of0 mg/mL. The substock solution of 0.2 mg/mL was preparedy diluting 20 �L of the stock solution into 980 �L serum-ree culture medium, RPMI 1640 (the percentage of DMSO inhe experiment should not exceed 0.5). The stock and substockolutions were both stored at 4 ◦C.

.2. Reagents and chemicals

Concanavalin A (Con A) (Sigma, USA) and lipopolysaccha-ide (LPS) (Sigma, USA) (1 mg/mL) was used as a positiveontrol. This commercial immunomodulator was prepared byissolving it with RPMI medium (Flowlab, Australia). The stocknd substock solutions were prepared as above.

.3. Cell lines

Suspension target cell line, HepG2 (hepatocellular carci-oma) were obtained and thawed from Animal Tissue Cultureaboratory, Department of Cell and Molecular Biology, UPMnd were maintained in RPMI 1640 and DMEM media, respec-ively with 10% FBS (PAA, Austria) at 37 ◦C, 5% CO2 and 90%umidity throughout the study. The cell viability was assessedy the trypan blue exclusion method.

.4. Mononuclear cell isolation

Blood (20–25 mL) was taken from same donor throughouthis research by using the 25 mL syringe. The blood sample wasiluted with the same volume of PBS. After that, the dilutedlood sample was carefully layered on Ficoll-Paque Plus (Amer-ham Biosciences, USA). The mixture was centrifuged undert 400 × g for 40 min at 18–20 ◦C. The undisturbed lympho-yte layer was carefully transferred out. The lymphocyte wasashed and pelleted down with three volumes of PBS for twice

nd resuspended RPMI-1640 media (Flowlab, Australia) with00 IU/mL of penicillin, 100 �g/mL of streptomycin (Flowlab,ustralia), 10%, v/v fetal bovine serum (FBS) (PAA, Austria).ell counting was performed to determine the PBMC cell num-er with equal volume of trypan blue.

.5. Cell viability assay

The effect of the extract on cell viability of PBMC, andepG2 was first determined by using a colorimetric technique,hich is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumromide (MTT) assay. Briefly, 100 �L of DMEM media with0% of FBS was added into the all the well except row A inhe 96-well plate (TPP, Switzerland). Then, 100 �L of dilutedxtract at 200 �g/mL was added into row A and row B. A seriesf twofold dilution of extract was carried out down from row Bntil row G. The row H was left untouched and the excess solu-

ion (100 �L) was discarded and 100 �L of lymphocyte (fromice spleen, thymus or human peripheral blood) with cell con-

entration at 5 × 105 cells/mL was added into all wells in the6-well plate and incubated in 37 ◦C, 5% CO2 and 90% humid-

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ty incubator for selected period (24, 48 and 72 h). After theorresponding period (either 24, 48, or 72 h), 20 �L of MTTSigma, USA) at 5 mg/mL was added into each well in the 96-ell plate and incubated for 4 h in 37 ◦C, 5% CO2 and 90%umidity incubator. One hundred and seventy microlitres ofedium with MTT was removed from every well and 100 �LMSO (Fisher Scientific, UK) was added to each well to extract

nd solubilize the formazan crystal by incubating for 20 min in7 ◦C, 5% CO2 incubator. Finally, the plate was read at 570 nmavelength by using � Quant ELISA Reader (Bio-Tek Instru-ents, USA). The results of the plant extract were comparedith the result of ConA (1 �g/mL) and LPS (1 �g/mL) and with-ut mitogens or extract by using same method. Each extract andontrol was assayed in triplicate for three times. The percentagef proliferation was calculated by the following formula:

Proliferation = OD sample − OD control

OD control× 100

.6. Cell cycle study

The perturbations in the distribution of cells in the differenthases of the cell cycle were determined by flow cytometry.ell concentration of PBMC was adjusted to 5 × 105 cells/mLnd the extracted human PBMC (1 mL) was treated with sameolume of plant extract at 50 �g/mL (1 mL) in a 6-well cul-ure plate (TPP, Switzerland). Control cultures without extractnd positive control with ConA (1 �g/mL) and LPS (1 �g/mL)ere prepared simultaneously. The culture was then incubated

or respective time (24, 48 and 72 h). After the correspondingeriod, the samples were washed and transferred to a centrifugeube (BD Bioscience, USA). The cells were pelleted and fixedith 80% ethanol and incubate at 4 ◦C for 2 h. Then, the cellsere pelleted again and washed with PBS buffer twice. The

ell pellet was finally dissolved and stained in PBS buffer con-ist of 0.1% triton X-100, 10 mM EDTA, 50 �g/mL RNase and�g/mL propidium iodide (PI) in dark. The cell was then incu-ated for half an hour in 4 ◦C and should be read with COULTERPICS ALTRA flow cytometer (Beckman Coulter, USA) under10BP within 24 h. Each extract and control was assayed inriplicate.

.7. Co-culture MTT microcytotoxicity assay

HepG2 (Effector-E) was seeded into the 96-well plate andulture at 37 ◦C in an atmosphere of 5% CO2 and absoluteumidity for 24 h to allow their adherence. PBMC (Target-T)as exposed to the plant extract at 25 �g/mL for 72 h in 96-ell plate. Then, the activated PBMC was cultured with the

dherent HepG2 at ratio of T/E at 2/1 and incubated in 37 ◦C,% CO2 and 90% humidity for 24 h. Control cultures with onlyBMC and HepG2 were prepared simultaneously. After the co-ulture incubation period, 170 �L the media and the suspended

ell were discarded and washed with 200 �L PBS. Equal volume200 �L) of new media was added and incubated for 2 h. Afterhat, 20 �L of MTT (Sigma, USA) at 5 mg/mL was added intoach well in the 96-well plate and incubated for 4 h in 37 ◦C,

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rmacology 114 (2007) 406–411

% CO2 and 90% humidity incubator. One hundred and seventyicrolitres of medium with MTT was removed from every well

nd 100 �L DMSO (Fisher Scientific, UK) was added to eachell to extract and solubilize the formazan crystal by incubated

or 20 min in 37 ◦C, 5% CO2 incubator. Finally, the plate wasead at 570 nm wavelength by using � Quant ELISA ReaderBio-Tek Instruments, USA).

.8. Co-culture Annexin V binding assay

PBMC (Target-T) was exposed to the plant extract at5 �g/mL for 72 h in a 6-well plate. Then, the activated PBMCas cultured with equal volume of HepG2 (Effector-E) at ratiof T/E at 2/1 and incubated in 37 ◦C, 5% CO2 and 90% humidityor 24 h. Control culture with only PBMC and HepG2 was pre-ared simultaneously. At the end of the co-culture incubation,ells were harvested, washed and stained with Annexin V FITCBD Biosciences, USA) with propidium iodide (PI). Briefly, theulture was washed with PBS and resuspended in 500 �L totalolume that contain 100 �L of cells (5 × 105 cells/mL), 10 �LI (Sigma, USA), 5 �L Annexin V-FITC (BD Bioscience, USA)nd 400 �L binding buffer (BD Bioscience, USA). After 15 minncubation in dark, the cells were analyzed with COULTERPICS ALTRA flow cytometer (Beckman Coulter, USA) withinalf an hour. Each extract and control was assayed in triplicate.

. Results

.1. Proliferation effect of Rhaphidophora korthalsiictivated PBMC

To study the proliferation stimulatory effect of Rhaphi-ophora korthalsii activated PBMC in various dosages andime point, MTT assay was used to check for the cell viabil-ty. The increase of cell viability shows that the extract is notoxic to the immune cell and is potentially modulates the cellu-ar immune response (Swamy and Tan, 2000). Only the viablend metabolically active cell can cleave the MTT to produceormazan (Mosmann, 1983). The methanol extract of Rhaphi-ophora korthalsii did not display strong cytotoxic effect onepG2 with viability of 94.27 ± 0.11% at 25 �g/mL. No IC50

an be obtained at the range of concentration (the result is nothowed). On the other hand, the extract showed less sensitiveroliferation effect on PBMC at 24 h with the highest prolifera-ion effect was observed at 100 �g/mL with 11.76 ± 4.88% moreells compared to the untreated PBMC culture. The prolifera-ion effect increased significantly for both treatment at 50 �g/mL40.98 ± 8.15%) and 25 �g/mL (44.36 ± 8.95%) at 48 h. For2 h, 25 �g/mL showed the highest proliferation effect with1.93 ± 0.03% more cells compared to the control. Interestingly,he activation of the plant extract at all the high concentrationroduced significant proliferation responses.

To clarify the role of the extract on the increase of cell number

n MTT assay, flow cytometric cell cycle analysis was per-ormed. In cell cycle study, PI which is a highly water-solubleuorescent compound will penetrate and intercalate between theases in double stranded nucleic acids of exposed nuclei after

S.K. Yeap et al. / Journal of Ethnopharmacology 114 (2007) 406–411 409

Table 1Effects of Rhaphidophora korthalsii methanol extract (25 �g/mL) on PBMC cell cycle progression

Time (h) Treatment Cell cycle (24 h)

G0/G1 S G2/M

24 Rhaphidophora korthalsii (25 �g/mL) 87.99 ± 0.86 3.62 ± 0.50 3.80 ± 0.34Control 93.0 ± 0.20 1.90 ± 0.10 1.90 ± 0.10

48 Rhaphidophora korthalsii (25 �g/mL) 43.16 ± 3.46 9.22 ± 0.12 25.87 ± 0.99Control 86.00 ± 0.60 3.85 ± 0.70 5.50 ± 0.80

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he cells were fixed with ethanol and permeabilized (Lima etl., 2002). The proliferative potency of the extract in this assays indicated by a change in percentage of cells in Gap2 (G2)nd Mitosis (M) phases of the cell cycle. In cell cycle progresstudy, only the one concentration was tested which is 25 �g/mLecause at this concentration the highest proliferation effect waseen at both 48 and 72 h. Table 1 shows the distribution of PBMCn the different cell cycle phases after 24, 48 and 72 h of Rhaphi-ophora korthalsii methanol extract (25 �g/mL) treatment andntreated culture. Analysis of the proliferative effect of extracty evaluating the cell cycle G2/M phase clearly showed that thextract induced an accumulation of cell in G2/M phase of the cellycle (Table 1). Extract treatment for 48 h resulted in an increasen the percentage of cells in the G2/M phase from 3.80 ± 0.34%o 25.87 ± 0.99. The percentage of cells in G2/M phase fur-her increase to 35.12 ± 2.50% after 72 h of treatment. Thus,ow cytometry DNA analysis of the treated culture revealed

hat Rhaphidophora korthalsii methanol extract stimulated moreBMC enter into G2/M phase in the time-dependent manner.

.2. Co-culture cytotoxic effect of Rhaphidophoraorthalsii activated PBMC

To evaluate the proliferation effect from Rhaphidophoraorthalsii activated PBMC especially the role in anticancertudy, the co-culture of Rhaphidophora korthalsii activatedBMC with HepG2 was performed in the 2:1 of T:E ratio and

he viability of the effectors adherent HepG2 was tested bysing MTT microcytotoxicity assay. The MTT microcytotoxic-

ty assay involves two different cell lines, which are suspendedffector cells and adherent target cells. Wahlberg et al. (2001)roved the effectiveness of this high throughput, simple, eco-omical and non-radioactive assay on measuring the NK cell

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BMC control 6.39BMC treated with Rhaphidophora korthalsii 2.09epG2 control 7.95epG2 treated with Rhaphidophora korthalsii 8.59BMC and HepG2 43.36BMC treated with Rhaphidophora korthalsii and HepG2 51.51

he values were the means ± S.E. of three experiments.

50.99 ± 2.10 9.92 ± 0.92 35.12 ± 2.5083.80 ± 1.40 3.60 ± 0.30 6.60 ± 0.60

ctivity. Due to HepG2 is an anchorage-dependent cell line, it isossible to separate the suspended PBMC and the detach deathells from the viable HepG2. Thus, the metabolically activeepG2 was detected by using MTT assay. Fig. 2 showed that

he co-culture between the activated PBMC and HepG2 gave theest cytotoxic effect with only 55.13 ± 6.06% of viable HepG2n the culture. However, the short term (102.64 ± 13.68% ofell viability) (Fig. 2) and long-term (94.27 ± 0.11% of celliability) (result not showed) cytotoxic effect study for Rhaphi-ophora korthalsii methanol extract toward HepG2 showed thathe extract was not sensitive toward this hepatocellular carci-oma.

To confirm the results of MTT microcytotoxicity assay,nnexin V was performed by using the same co-culture sys-

em between PBMC and HepG2. Annexin V is a method, whichllows the study of cell killing mechanism by binding to thexpose phospholipids phosphatidylserine during the apoptosis.he necrotic cells with the internal and external membranes dis-

uption will be stained by the vital dye PI (Dedoussis et al.,005). Table 2 shows the cytolytic effect of Rhaphidophoraorthalsii activated PBMC in different co-culture system withBMC and/or HepG2 as the control to set the gating. Cytolyticctivity of Rhaphidophora korthalsii activated PBMC on HepG2s better than non-activated PBMC. The cytotoxic effect ofhaphidophora korthalsii extract on HepG2 is 8.59 ± 1.69% ofell apoptosis and 0.91 ± 0.24% of necrosis (Table 2). Withoutny treatment, the PBMC already can cause 43.36 ± 2.9% ofpoptosis and 1.92 ± 0.33% of necrosis to HepG2. The effects more significant where Rhaphidophora korthalsii extract

ctivated PBMC can cause 51.51 ± 3.85% of apoptosis and.92 ± 0.72% of necrosis to HepG2. The differential sensitiv-ties of the treatment toward HepG2 cytotoxicity for both MTT

icrocytotoxicity assay and Annexin V were in the following

opulation of PBMC and HepG2 cells in a ratio 2:1

tosis Necrosis Viable

± 0.15 0.20 ± 0.15 93.40 ± 0.24± 0.42 0.07 ± 0.04 97.84 ± 0.45± 1.8 1.27 ± 0.39 90.78 ± 1.53± 1.69 0.91 ± 0.24 90.50 ± 1.72± 2.9 1.92 ± 0.33 54.72 ± 3.15± 3.85 3.92 ± 0.72 44.64 ± 3.97

4 opharmacology 114 (2007) 406–411

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rder: Rhaphidophora korthalsii activated PBMC > untreatedBMC > Rhaphidophora korthalsii methanol extract.

. Discussion

Immunomodulator is a compound or extract, which maynterfere with the body immune system. Normally, it involveshe stimulation of non-specific system. Rhaphidophora korthal-ii water extract was proved to have the immunomodulatoryffect toward the mice splencoytes (Wong and Tan, 1996). How-ver, the immunomodulatory effect of Rhaphidophora korthalsiiethanol extract and its application have not been defined. In

his study, Rhaphidophora korthalsii methanol extract showedhe mitogenic effect on the dose- and time-dependent patent.he extract was able to stimulate the human PBMC for prolifer-tion though the proliferation index was much less compared tohe control ConA, LPS and PWM at 24 h. However, the extractas more mitogenic than ConA and LPS at 100–25 �g/mL

ange after 72 h. The proliferation effect of lipopolysaccharideLPS) (B cell stimulator) (Nam et al., 2003) was less com-ared to concanavalin A (ConA) (T cell activator) (Berger,979) and pokeweed mitogen (PWM) (T and B cell mitogens)Yokoyama et al., 2007) because PBMC contain more T cell than

cell and NK cell with the ratio 80%:10%:10% (Abbas et al.,000).

To confirm the MTT assay result, the concentration of thextract that gave the best proliferation effect was subjected toell cycle progress study. Unlike the MTT assay screening, flowytometric cell cycle analysis allows a more sensitive evaluationf proliferative responses in time. The cell cycle progress ofhe activated PBMC showed that there was sixfold potential ofroliferation effect from 24 to 48 h of the plant extracts activatedBMC. This phenomenon increased to 35.12 ± 2.50% of cellndergo G2/M phase at 72 h. On the other hand, the numberf untreated PBMC entering G2/M phase did not change much

rom 48 to 72 h. These results were supported the MTT assayesults (Fig. 1) where 50% more cells can be obtained from thextract activated PBMC compare to the control. The cell cyclef lymphocytes is around 15 h (Lezama et al., 2006). Thus, the

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ig. 2. HepG2 viability in the different culture system. (a) HepG2 with media (1025 �g/mL) (102.64 ± 13.68% of cell viability). (c) HepG2 co-culture with PBMC forhaphidophora korthalsii (25 �g/mL) activated PBMC for 24 h at a ratio 1:2 (55.13 ±

ig. 1. Proliferation effect of Rhaphidophora korthalsii methanol extract oritogens on human PBMC at 24, 48 and 72 h.

timulation of the extract was expected to start after the secondr third cell cycle.

Hepatocellular cancer (HCC) is the fifth most common solidumor worldwide (Zhang et al., 2007). The present therapeu-ic for hepatocellular cancer is less effective especially forhe patients who are in the mid- or final stages (Liau et al.,005). Thus, there is the need to find new and effective curativeethods for hepatocellular cancer treatment. Besides immuno-

roliferative effect study, the cytotoxic effect of Rhaphidophoraorthalsii methanol extract was also screened by using MTTssay. This was to check the cytotoxic effect of the extract towardepG2. Long (result not showed) and short-term in vitro exper-

ments have indicated that Rhaphidophora korthalsii methanolxtract was neither directly toxic to tumor cells nor inhibits cellroliferation (Fig. 2 and Table 2). These results have indicatedhe involvement of the immune system in the reduction of theancer cell viability in the co-culture of activated or untreated

BMC with HepG2. Besides, the extract activated PBMChowed higher cytotoxic effect on HepG2 compared to untreatedBMC (Fig. 2 and Table 2). In this context, it is believed that

he immunomodulatory effect of the plant extract up-regulated

0 ± 4% of cell viability). (b) HepG2 culture with Rhaphidophora korthalsii24 h at a ratio 1:2 (75.67 ± 9.13% of cell viability). (d) HepG2 co-culture with6.06% cell viability).

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he cytotoxic effect of the immune cell to kill the HepG2. Co-ulture MTT microcytotoxicity assay showed that both activatednd non-activated PBMC could induce the immune cell cyto-oxic effect on HepG2. Wahlberg et al. (2001) showed that theK cells were the only effector cells which actively involve

n killing of tumor cell target although there are some otherotentially cytotoxic anticancer immune effector cells such asumor-specific cytotoxic T lymphocytes and cytotoxic NK-Tells. Thus, the reduction of the HepG2 viability in the MTTicrocytotoxicity assay may be due to the NK cell cytolytic

ctivity and the cytolytic activity increase for the treated PBMCay be due to the activation of the plant extract toward the NK

ell population. NK cell is the distinct subset of large granu-ar lymphocytes, which kill the tumor or virus infected targetell by using two mechanisms in the innate immunity (Li etl., 2005). The first mechanism is by secreting granules, whichontain perforin and granzymes that induce necrosis and apop-osis, respectively. The second mechanism is by Fas ligand (Fas)/Fas pathway (Li et al., 2004). Previous study showed thatepG2 was resistant toward the NK cell Fas L/Fas and granular

nduce apoptosis pathway (Kim et al., 2004). However, the sameesearch also indicated that the HepG2 was still sensitive to NKell granule-dependent necrotic death where ∼30% of cytotox-city was observed after incubation of HepG2 with NK cell for4 h (Kim et al., 2004). Table 2 showed the killing mode of theifferent culturing system. Both treated and untreated PBMCnduced more apoptosis compare to necrosis. Thus, the HepG2as susceptible to kill by secreted granzymes of either activatedr non-activated NK cells because granzymes induce apoptosisut perforin induce fast necrosis to the cancer (Li et al., 2005).

Although Rhaphidophora korthalsii methanol extract did nothow in vitro cytotoxic effect on HepG2, the present resultshowed that the immunomodulatory effect of the plant extractight stimulate the cytotoxic effect of immune cells towardepG2. However, the mechanism of the cytotoxic effect on the

xtract activated PBMC remains unclear. Thus, further worko isolate the single population immune cell from the activatedBMC and to clarify its mode of action is under study.

eferences

bbas, A.K., Lichtman, A.H., Pober, J.S., 2000. Cellular and MolecularImmunology, 4th ed. W.B. Saunders Company, Philadelphia, p. 19.

erger, S.L., 1979. Lymphocytes as resting cells. Methods in Enzymology 58,486–494.

edoussis, G.V.Z., Kaliora, A.C., Andrikopoulos, N.K., 2005. Effect of phenolson natural killer (NK) cell-mediated death in the K562 human leukemic cellline. Cell Biology International 29, 884–889.

im, H.R., Park, H.J., Park, J.H., Kim, S.J., Kim, K.h., Kim, J., 2004. Char-

acteristics of the killing mechanism of human natural killer cells againsthepatocellular carcinoma cell lines HepG2 and Hep3B. Cancer ImmunologyImmunotherapy 52, 461–470.

iritikar, K.R., Basu, B.D., 2001. Indian Medical Plant, vol. 11., 2nd ed. OrientalEnterprises, Dehradum, pp. 3578–3622.

Z

rmacology 114 (2007) 406–411 411

umar, R.A., Sridevi, K., Kumar, N.V., Nanduri, S., Rajapopal, S., 2004. Anti-cancer and immunostimulatory compounds from Andrographis paniculata.Journal of Ethnopharmacology 92, 291–295.

ezama, R.V., Aguilar, R.T., Ramos, R.R., Avila, E.V., Gutierrez, M.S.P., 2006.Effect of plantago major on cell proliferation in vitro. Journal of Ethnophar-macology 103, 36–42.

i, Q., Nakadai, A., IIshizaki, M., Morimoto, K., Ueda, A., Krensky, A.M.,Kawada, T., 2005. Dimethyl 2,2-dichlorovinyl phosphate (DDVP) markedlydecreases the expression of perforin, granzyme A and granulysin in humanNK-92CI cell line. Toxicology 213, 107–116.

i, Q., Nakadai, A., Takeda, K., Kawada, T., 2004. Dimethyl 2,2-dichlorovinylphosphate (DDVP) markedly inhibits activities of natural killer cells,cytotoxic T lymphocytes and lymphokine-activated killer cells via the Fas-ligand/Fas pathway in perforin-knockout (PKO) mice. Toxicology 204,41–50.

iau, K.H., Ruo, L., Shia, J., Padela, A., Gonen, M., Jarnagin, W.R., 2005. Out-come of partial hepatectomy for large (>10 cm) hepatocellular carcinoma.Cancer 104, 1948–1955.

ima, T.M., Kanunfre, C.C., Pompeia, C., Verlengia, R., Curi, R., 2002. Rankingthe toxicity of fatty acids on Jurkat and Raji cells by flow cytometric analysis.Toxicology In Vitro 16, 741–747.

osmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:Application to proliferation and cytotoxic assay. Journal of ImmunologyMethods 65, 55–63.

am, B.H., Hirono, I., Aoki, T., 2003. Bulk isolation of immune response-related genes by expressed sequenced tags of Japanese flounder Paralichthysolivaceus leucocytes stimulated with Con A/PMA. Fish & ShellfishImmunology 14, 467–476.

asikumar, J.M., Doss, P.A., 2006. In vitro antioxidant and antibacterial activityof Rhaphidophora pertusa stem. Fitoterapia 77, 605–607.

wamy, S.M.K., Tan, B.K.H., 2000. Cytotoxic and immunopotentiating effectsof ethanolic extract of Nigella sativa L. seeds. Journal of Ethnopharmacology70, 1–7.

oyota, M., Ihara, M., 1998. Recent progress in the chemistry of nonmonoter-penoid indole alkaloids. Natural Product Report. 307–340.

rabe, K., Aroca, P., Tsukamoto, K., Mascagna, D., Palumbo, A., Prota, G.,Hearing, V.J., 1994. The inherent cytotoxicity of melanin precursors: arevision. Biochimica et Biophysica Acta (BBA)—Molecular Cell Research1221, 272–278.

ahlberg, B.J., Burholt, D.R., Kornblith, P., Richards, T.J., Bruffsky, A., Her-berman, R.B., Vujanovic, N.L., 2001. Measurement of NK activity by themicrocytotoxicity assay (MCA): a new application for an old assay. Journalof Immunological Methods 253, 69–81.

ang, G., Zhao, J., Liu, J., Huang, Y., Zhong, J., Tang, W., 2007. Enhancementof IL-2 and IFN-� expression and NK cells activity involved in the anti-tumoreffect of ganoderic acid Me in vivo. International Immunopharmacology 7,864–870.

ong, K.T., Tan, B.K.H., 1996. In vitro cytotoxicity and immunomodulatingproperty of Rhaphidophora korthalsii. Journal of Ethnopharmacology 52,53–57.

okoyama, K., Terao, T., Osawa, T., 2007. Carbohydrate-binding specificity ofpokeweed mitogens. Biochimica et Biophysica Acta 538, 384–396.

hang, H.J., Tamez, P.A., Hoang, V.D., Tan, G.T., Hung, N.V., Xuan, L.T.,Huong, L.M., Cuong, N.M., Thao, D.T., Soejarto, D.D., Harry Fong, H.S.,Pezzuto, J.M., 2001. Antimalarial Compounds from Rhaphidophora decur-siva. Journal of Natural Products 64, 772–777.

hang, J.F., Liu, J.J., Lu, M.Q., Cai, C.J., Yang, Y., Li, H., Xu, C., Chen, G.H.,

2007. Rapamycin inhibits cell growth by induction of apoptosis on hepato-cellular carcinoma cells in vitro. Transplant Immunology 17, 162–168.

hou, H.L., Deng, Y.M., Xie, Q.M., 2006. The modulatory effects of the volatileoil of ginger on the cellular immune response in vitro and in vivo in mice.Journal of Ethnopharmacology 105, 301–305.