Cytotoxic and Cell Cycle Effects Induced by Two Herbal Extractson Human Cervix Carcinoma and Human Breast Cancer Cell Lines

Tatjana P. Stanojkovic,1 Aleksandra Konic-Ristic,2 Zorica D. Juranic,1 Katarina Savikin,3

Gordana Zdunic,3 Nebojsa Menkovic,3 and Milka Jadranin4

1Department of Experimental Oncology, Institute for Oncology and Radiology of Serbia; 2Faculty of Pharmacy,University of Belgrade; 3Institute for Medicinal Plant Research ‘‘Dr Josif Pancic’’; and 4Institute for Chemistry,

Technology and Metallurgy, Belgrade, Serbia

ABSTRACT In recent times interest has increased in the complementary medicine of cancer patients. Two herbal mixtures

were prepared from 17 and 12 plants, respectively. The goal of this study was to examine the in vitro cytotoxic and cell cycle

effects of the aqueous-ethanol extracts (Extract 1 and Extract 2) obtained by maceration of the mixtures. The two extracts

investigated exhibited significant antiproliferative activity toward two human breast cancer cell lines (MDA-MB-361 and

MDA-MB-453) and a human cervix carcinoma cell line (HeLa) with 50% inhibitory concentration (IC50) values ranging from

9.92 to 17.38mL=mL. The extracts did not exert any significant cytotoxicity toward healthy human peripheral blood

mononuclear cells. In vitro antitumor activites were accompanied by an important apoptotic fraction of all cell lines after

treatment with the extracts. The amount of total phenols was similar in both extracts, whereas the concentration of total

tannins was significantly higher in Extract 1. Extract 1 was also found to be a stronger free radical scavenger, with an IC50

value of 13.4 mg=mL. Both extracts contained rosmarinic acid, while ursolic acid was identified in Extract 2.

KEY WORDS: � apoptosis � 1,1-diphenyl-2-picrylhydrazyl � HeLa � MDA-MB-361 � MDA-MB-453 � peripheral blood

mononuclear cells � phenolics

INTRODUCTION

According to the World Health Organization, canceris a leading cause of death worldwide. The most fre-

quent types of cancer among women are breast, lung,stomach, colorectal, and cervical cancer. The use of herbs ascomplementary medicine among women with cancer, es-pecially advanced cancer, has recently increased.1 One newapproach to cancer therapy focuses on anticancer and anti-metastatic agents, with little or no cytotoxic activity. Thisapproach can be used for long-term treatment combined withconventional short-term treatment of cytotoxic anticancerdrugs.2 It was shown that many herbal formulations possessactivity only if the ingredients are combined in a strictlydefined ratio.3,4 As different components of plants may havesynergistic activities, mixtures of herbs might have moretherapeutic or preventive activity than when used alone.5

Phenolic compounds commonly found in most plantspossess a wide spectrum of biological activities, includingeffects on cell proliferation, differentiation, and apoptosis.6,7

It was shown that ursolic acid possesses significant cyto-toxicity against some tumor cell lines8 and induces apo-ptosis in a wide variety of cancer cells, including breastcarcinoma, melanoma, hepatoma, prostate carcinoma, andacute myelogenous leukemia.9 Rosmarinic acid, frequentlyfound as a secondary metabolite in medicinal plants, ex-hibited antimicrobial, antiviral, antioxidative, and anti-inflammatory activities.10 It was also shown that rosmarinicacid inhibits the growth of human gastric adenocarcinoma(MK-1), human uterine carcinoma (HeLa), and murinemelanoma (B16F10) cells in vitro and induces apoptosis inJurkat cells.11

The aim of this study was to evaluate biological proper-ties of two aqueous-ethanol herbal extracts, named Extract 1and Extract 2. Cytotoxic effects on human cervix carcinoma(HeLa) and human breast cancer (MDA-MB-453 andMDA-MB-361) cell lines as well as normal immune com-petent cells in vitro were tested. The cell cycle distributionwas estimated by cytofluorometric analysis. Moreover,radical scavenging activity was tested with 1,1-diphenyl-2-picrylhydrazyl (DPPH). Quantification of total phenolics,tannins, and rosmarinic acid and confirmation of the pres-ence of ursolic acid by liquid chromatography (LC)-massspectrometry (MS) in Extract 1 and Extract 2 were done.

Manuscript received 6 April 2009. Revision accepted 21 May 2009.

Address correspondence to: Katarina Savikin, Institute for Medicinal Plant Research‘‘Dr Josif Pancic,’’ Tadeusa Koscuska 1, 11000 Belgrade, Serbia, E-mail: ksavikin@mocbilja.rs

JOURNAL OF MEDICINAL FOODJ Med Food 13 (2) 2010, 291–297# Mary Ann Liebert, Inc. and Korean Society of Food Science and NutritionDOI: 10.1089/jmf.2009.0086

291

MATERIALS AND METHODS

Plant material

Two herbal mixtures were prepared from plant materialobtained from the Institute for Medicinal Plants Research,Belgrade, Serbia. Mixture 1 was composed of 17 herbs inequal amounts: Althaea officinalis, Vaccinium myrtillus,Plantago spp., Rubus fruticosus, Taraxacum officinale,Juglans regia, Asperula odorata, Teucrium chamaedrys,Geranium macrorrhizum, Glechoma hederacea, Origanumvulgare, Thymus serpyllum, Viola tricolor, Viscum album,Phaseolum vulgaris, Petroselinum crispum, and Inula he-lenium. Mixture 2 was prepared from 12 herbs in equalamounts: Helichrysum arenarium, Lavandula officinalis,Olea europaea, Agrimoniae radix, Glycyrrhizae radix,Linum usitatisimum, Taraxacum officinalis, Melissa offici-nalis, Ocimum basilicum, Majorana hortensis, Robiniapseudoacacia, and Arnica montana.

Preparation of herbal extracts

Herbal mixtures 1 and 2 were macerated in hot (378C)distilled water containing 7% ethanol in a closed vesselfor 4 days. Extracts were filtered through a filter paper in aBuchner funnel to obtain 200 mL of Extract 1 and 160 mL ofExtract 2, respectively (drug:extract ratio, 1:1). Fresh liquidExtract 1 and Extract 2 were used for chemical analysis andtesting of biological activities in vitro. Prior to analysis,extracts were filtered (pore size, 0.22mm; Millipore, Bedford,MA, USA).

Determination of the total phenol and tannin contents

The total concentration of phenols was estimated by theFolin-Ciocalteu method with slight modifications.12 Twohundred microliters each of Extract 1 and Extract 2 (diluted1:10) was added to 1 mL of 1:10 diluted Folin-Ciocalteureagent. After 4 minutes, 800mL of sodium carbonate(75 g=L) was added. After 2 hours of incubation at roomtemperature, the absorbance at 765 nm was measured. Gallicacid (0–100 mg=L) was used for calibration of a standardcurve. The results were expressed as gallic acid equivalents=100 mL of the extracts. Triplicate measurements were taken,and mean values were calculated.

Tannin content in extracts was determined quantitativelyby its adsorption on standard hide powder.13 The tannincontent is equivalent to the difference between the totalpolyphenol content and the polyphenol content that re-mained after the tannins were adsorbed by hide powder.

High-performance LC (HPLC) conditions

HPLC analysis was used for the quantification of ros-marinic acid in Extract 1 and Extract 2. Analyses werecarried out on an HP Series 1200 with a diode array detectordetector on a reverse-phase Lichrospher RP-18 analyticalcolumn (250�4 mm i.d.; particle size, 5 mm) (AgilentTechnologies, Schaumburg, IL, USA). Mobile phase A wasH2O containing 1% 0.1 NH3PO4, and phase B was aceto-

nitrile. The gradient was according to the following scheme:90–85% solvent A for 0–10 minutes, 85–70% solvent A for10–20 minutes, 70–30% solvent A for 20–30 minutes, and30–0% solvent A for 30–35 minutes. The flow rate was1 mL=minute, with detection at 260 nm. The amount ofrosmarinic acid was calculated using a calibration curve. Allexperiments were done in triplicate.

LC-MS

LC-MS with electrospray (negative ionization method)was used, along with a diode array detector, to confirm thepresence of ursolic acid in Extract 1 and Extract 2.14 Theseparation was carried out in an HPLC instrument (1200Series, Agilent Technologies, Santa Clara, CA, USA) with abinary pump, an autosampler, a column compartmentequipped with a Zorbax SB-C18 column (particle size,3.5mm; 2.1�30 mm; Agilent Technologies, Santa Clara),and a diode array detector coupled with a model 6210 time-of-flight LC-MS system (Agilent Technologies, SantaClara). The mobile phase consisted of water containing 0.2%formic acid (solvent A) and acetonitrile (solvent B). A gra-dient program was used as follows: initial 0–2 minutes, 5%solvent B; 2–42 minutes, linear change from 5% solvent B to95% solvent B; 42–57 minutes, maintaining 95% solvent B;and 57–58 minutes (stop time), linear change from 95%solvent B to 5% solvent B. Post-time was 5 minutes. Themobile phase flow rate was 0.4 mL=min, and the columntemperature was set at 258C. Spectral data from all peakswere accumulated in the range of 190–450 nm. A personalcomputer system running MassHunter Workstation software(Agilent Technologies, Santa Clara) was used for data ac-quisition and processing. In the atmospheric pressure elec-trospray ionization method, the eluted compounds weremixed with nitrogen in the heated nebulizer interface, andthe polarity was tuned to negative. Adequate calibration ofelectrospray ionization parameters (capillary voltage, gastemperature, nebulizer pressure, and fragmentor voltage)was required to optimize the response and to obtain a highsensitivity of the molecular ion. The selected MS valueswere as follows: capillary voltage, 4,000 V; gas temperature,3508C; drying gas, 12 L=minute; nebulizer pressure, 45 psig;fragmentor voltage, 140 V; and mass range, 100–1,500 m=z.

DPPH radical scavenging capacity

The free radical scavenging capacity of the extracts,based on the stable DPPH radical, was carried out accordingto the procedure described previously by Blois15 with slightmodifications. The antiradical capacity of Extract 1 andExtract 2 was evaluated using a dilutions series, in order toobtain a large spectrum of sample concentrations. Extract 1and Extract 2 (100 mL) in different concentrations weremixed with 1,400mL of a 80mM methanol solution ofDPPH. Absorbance at 517 nm was measured after 20 miutes.The percentage of inhibition was calculated using Eq. 1:

Inhibition ¼ ([A0�Ai] / A0) · 100 (1)

292 STANOJKOVIC ET AL.

where A0 is absorbance of the control and Ai is absorbance ofthe samples. The 50% inhibitory concentration (IC50) valueswere estimated using a nonlinear regression algorithm. Alltest analyses were run in triplicate. Trolox was used as apositive control.

Cell lines and culture conditions

The human breast cancer cell lines MDA-MB-361 andMDA-MB-453 and human cervix adenocarcinoma HeLacells were obtained from the American Type Culture Col-lection (Manassas, VA, USA). All cancer cell lines weremaintained in the recommended RPMI-1640 medium sup-plemented with 10% heat-inactivated (568C) fetal bovineserum, l-glutamine (3 mM), streptomycin (100 mg=mL),penicillin (100 IU=mL), and 25 mM HEPES and adjusted topH 7.2 by bicarbonate solution. MDA-MB-361 and MDA-MB-453 cell lines were grown in medium containing1.11 g=L glucose. Cells were grown in a humidified atmo-sphere of 95% air and 5% CO2 at 378C.

Preparation of peripheral blood mononuclearcells (PBMCs)

PBMCs were separated from whole heparinized blood ofsix healthy volunteers by Lymphoprep� (AXIS-SHIELDPoC AS, Oslo, Norway) gradient centrifugation. Interfacecells, washed three times with Haemaccel� (Aventis Phar-ma, Frankfurt, Germany), were counted and resuspended innutrient medium.

Cytotoxic assays

Cell survival was determined in vitro, using assays formeasuring the metabolism of a tetrazolium substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT), according to the method of Mosmann16 and theKenacid BlueR (KBR) dye binding method.17 The indi-vidual cell lines were exposed for 72 hours to Extract 1and Extract 2 at final concentrations ranging from 2 to20mL=mL of culture medium. All experiments were per-formed in triplicate.

Acridine orange=ethidium bromide staining of cells

For fluorescence microscopy, adherent HeLa, MDA-MB-361, and MDA-MB-453 cells were cultured on coverslipsfor 24 hours and then treated with two different concentra-tions (IC50 and 2�IC50) of Extract 1 and Extract 2 for 24hours. After this time, the cells were examined for mor-

phological features of apoptosis and necrosis by fluores-cence microscopy using acridine orange and ethidiumbromide stains.

Flow cytometry

The cell cycle distribution was estimated from the DNAfrequency histograms of different cell lines after 24, 48, and72 hours of cell growth in the presence of two differentconcentrations (IC50 and 2�IC50) of Extract 1 and Extract 2.As a control, cell cycle distribution was estimated in thesame conditions, but without addition of Extract 1 and Ex-tract 2. After incubation with propidium iodide, DNA con-tent and cell cycle distribution were analyzed using a flowcytometer. Cytofluorometric analysis was performed using aCellQuest� (Becton Dickinson, San Jose, CA, USA) on aminimum of 30,000 cells per sample.

Statistical analysis

Results were expressed as mean� SD values. Statisticaldifferences were assessed by Student’s unpaired t test, withP< .05 as statistically significant.

RESULTS AND DISCUSSION

Content of total phenolics and tannins

Both Extract 1 and Extract 2 contained phenol and tannincompounds (Table 1). The amount of total phenols wassimilar in both extracts, but the concentration of total tan-nins was significantly higher in Extract 1 (Table 1). Suchresults were expected regarding the composition of herbalmixtures 1 and 2 that were used for the preparation of Ex-tract 1 and Extract 2, where Extract 1 contained more plantspecies known to possess high tannin content.

HPLC and LC-MS analysis

According to the fingerprint HPLC profiles, rosmarinicacid was found to be one of the dominant compounds in bothExtract 1 and Extract 2 (Fig. 1). A slightly higher amountwas detected in Extract 2 (1.28� 0.06%) obtained frommixture 2, which contained mostly Lamiaceae speciesknown to possess high amount of rosmarinic acid (Table 1).

The presence of ursolic acid in Extract 1 and Extract 2was investigated using LC-MS methods by comparison ofretention time and mass spectrum with those of the stan-dard substance. Ion (M-H)� equal to 455.35293 (error�0.30 ppm) corresponding to ursolic acid (molecular formula

Table 1. Total Contents of Phenolics, Tannins, and Rosmarinic Acid

and DPPH Radical Scavenging Activity of Extract 1 and Extract 2

SampleTotal

tannins (%)Total phenolics (mg of

GAE=100 mL of extracts)Rosmarinic

acid (%)Radical scavenging

activity (IC50 [mL=mL])

Extract 1 3.41� 0.64 32.9� 2.7 1.07� 0.03 13.4� 0.5Extract 2 0.09� 0.02 36.9� 3.1 1.28� 0.06 24.8� 0.9

GAE, gallic acid equivalents.

HERBAL EXTRACTS: CYTOTOXIC=CELL CYCLE EFFECTS 293

C30H48O3; exact molecular mass, 456.36035) was observedonly in Extract 2.

The above results indicated that Extract 1 and Extract 2can serve as a source of bioactive compounds such as ros-marinic or ursolic acid. It was shown that ursolic acidblocked the cell cycle progression in the G1 phase and couldtrigger apoptosis determined by a DNA fragmentationassay.8 Additionally, rosmarinic acid inhibited several im-portant steps of angiogenesis, including proliferation, mi-gration, adhesion, and tube formation of human umbilicalvein endothelial cells in a concentration-dependent manner.18

It was also effective in increasing the expression of apoptosis-related genes and apoptosis inducing in HepG2 cells.19

DPPH radical scavenging activity

Protection against oxidative damage is one of the mostwidely described attributes of plant polyphenols and isconnected with their antiradical activity. The antiradicalproperties of Extract 1 and Extract 2 were evaluated by theDPPH radical scavenging assay. Both extracts were able toscavenge DPPH radical in a concentration-dependent man-ner. Extract 1 was found to be a stronger free radical scav-enger, with an IC50 value of 13.4 mL=mL (Table 1). Resultsshowed that differences in phenol content did not neces-sarily correspond to the same differences in activities towardthe DPPH radical.

Cytotoxic effects of extracts on malignant cellsand normal PBMCs

The investigated target cell lines were exposed to Extract1 and Extract 2 at various concentrations (0, 2, 4, 8, 10, and20 mL=mL) for 72 hours. Cell survival was evaluated by the

MTT assay and the KBR dye binding method. The in vitroassays showed that both Extract 1 and Extract 2 significantlydecreased cell survival in all tested cell lines. The IC

50

values in the MTT assay ranged from 11.28� 2.51 to17.38� 2.77mL=mL, whereas in the KBR dye bindingmethod IC

50values ranged from 9.92� 2.88 to

17.07� 2.20mL=mL (Table 2). Generally, Extract 2 wasmore potent in all cell lines tested. Moreover, the cytotoxicactivity of Extract 1 and Extract 2 was not in correlation withtheir antioxidant activity. This may indicate that their anti-oxidant activity was probably not responsible for the cyto-toxicity.

The effect of Extract 1 and Extract 2 on human PBMCproliferation in vitro was also tested. It was shown thatExtract 1 and Extract 2 did not exert a significant anti-proliferative effect toward healthy human PBMCs, thus in-dicating that Extract 1 and Extract 2 might not be toxic tohumans.

Examination of cell morphology

It was found that the morphology of the cultured cellssignificantly changed upon treatment with Extract 1 andExtract 2 (Fig. 2). Both extracts, in the corresponding con-centrations, effectively induced morphologic changes typi-cal for apoptosis, in all cell lines, after 24 hours ofcontinuous action. In addition, the presence of cells withnecrosis and rounding and detachment of adherent cells wereevident by fluorescence microscopy when cancer cell lineswere exposed to Extract 1 and Extract 2. Apoptosis is animportant homeostatic mechanism that balances cell divi-sion and cell death, and induction of apoptosis in cancer cellswas one of the strategies for anticancer drug development.3

FIG. 1. Fingerprint HPLC chromatograms of Extract 2, which contained the greater amount of rosmarinic acid.

Table 2. Concentration of Extracts Producing 50% Decrease of Cell Survival on the Investigated Cell Lines

IC50 (ml=mL)

MTT assay KBR assay

Cell line Origin Extract 1 Extract 2 Extract 1 Extract 2

HeLa Cervix adenocarcinoma 14.66� 1.21 13.91� 1.94 12.68� 4.55 10.42� 0.62MDA-MB-453 Breast adenocarcinoma 15.74� 1.04 11.42� 1.98 14.90� 2.35 9.92� 2.88MDA-MB-361 Breast adenocarcinoma 17.38� 2.77 11.28� 2.51 17.07� 2.20 13.42� 0.84

IC50 values were expressed as the mean�SD determined from the results of MTT or KBR assay in three or five independent experiments.

294 STANOJKOVIC ET AL.

Cell cycle effects

A representative cell cycle analysis of cancer cell linestreated with Extract 1 and Extract 2 for 24, 48, and 72hours is given in Table 3. We observed that in vitro anti-tumor activity was accompanied by an important apoptoticfraction of all cell lines after treatment with both Extract 1and Extract 2. Changes in the percentage of cells in thesub-G1 phase for all cell lines are listed in Table 3. Adecrease of cells in the G2=M phase led to an increase inapoptosis, but it was obvious that after 24 hours both Ex-tract 1 and Extract 2 induced apoptosis in a dose-dependentmanner for all cell lines. Extract 1 showed the strongestincrease in the percentage of the sub-G1 population in theMDA-MB-453 cell line, whereas Extract 2 was the mostpotent in the HeLa cell line. The lowest effect was obtainedin the MDA-MB-361 cell line exposed to Extract 2. Thus,while exposure to Extract 1 led to an increase in apoptosistoward all cell lines tested, in MDA-MB-361 cells exposure

to Extract 2 resulted in G1 cell cycle block. These dataindicate that the G1 phase cell cycle arrest, observed inMDA-MB-361 cultures, most likely involved engagementof the G1 checkpoint that is mediated by p53. This is notsurprising because the cell’s response to most antitumordrugs in terms of cell cycle arrest is known to be mediatedthrough p53, and its mutation leads to defective check-points.3

CONCLUSIONS

The strategy of preparation of herbal mixtures tradi-tionally used to treat patients with cancer diseases has oftenrelied on a combination of different ingredients, which mayhave synergistic effects.20 Each of the herbs in the mixture1 or mixture 2 has been reported to be biologically activeand may contribute to the observed effects.21–24 Our re-sults revealed that both Extract 1 and Extract 2 expressedsignificant cytotoxic activity in vitro and did not exert a

FIG. 2. Morphology of cultured cells. The in-vestigated HeLa, MDA-MB-453, and MDA-MB-361 cells were incubated in nutrient mediumalone and with corresponding concentrations ofExtract 1 or Extract 2 (IC50 or 2�IC50). After 24hours of treatment, cells were stained with ethi-dium bromide-acridine orange as described inMaterials and Methods and viewed under thefluorescence microscope. (Inset) Nonadherent,detached cells were harvested by aspirating su-pernatants from cultures of extract-treated cellsfollowed by centrifugation and stained in thesame way as adherent cells. Color images avail-able online at www.liebertonline.com=jmf.

HERBAL EXTRACTS: CYTOTOXIC=CELL CYCLE EFFECTS 295

significant antiproliferative effect toward healthy humanPBMCs. Also, both extracts induced apoptosis in severaltumor lines in all measured time points (24, 48, and 72hours) with a dose-dependent increase in the percentage ofthe sub-G1 population. The perturbation of cell cycle pro-gression, however, was distinctly different in MDA-MB-361 and HeLa or MDA-MB-453 cells. Our results showedthat Extract 1 and Extract 2 could be of interest for furtherinvestigations.

ACKNOWLEDGMENT

The authors acknowledge their gratitude to the Ministryof Science of Serbia for grant support (Project Number145006).

AUTHOR DISCLOSURE STATEMENT

There exist no competing financial interests, nor anycommercial associations that might create a conflict of in-terest in connection with the submitted article.

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Table 3. Change in Percentage of Cells in Sub-G1 Phase as a Result of Exposure to Herbal Extracts

Apoptotic cells (sub-G1) (%)

Extract 1 Extract 2

Cell line Concentration 24 hours 48 hours 72 hours 24 hours 48 hours 72 hours

HeLa Control 4.3 4.3 3.3 4.4 5.5 3.3IC50 8.7 28.9 24.7 11.9 23.6 30.72�IC50 17.9 20.7 38.6 23.2 31.4 48.0

MDA-MB-453 Control 5.9 2.4 4.9 5.3 2.1 4.6IC50 12.1 20.1 43.7 16.8 22.6 34.12�IC50 20.2 29.3 53.8 22.5 44.8 44.3

MDA-MB-361 Control 4.5 5.1 2.8 4.5 5.1 4.6IC50 12.5 11.9 31.3 3.9 6.9 6.82�IC50 35.5 36.5 32.9 3.8 14.5 7.3

Cells were exposed to herbal extracts for 24, 48, and 72 hours. The changes in percentage of cells in the sub-G1 phases of the cell cycle, as shown, represent the

values taken from one of three independent experiments.

296 STANOJKOVIC ET AL.

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HERBAL EXTRACTS: CYTOTOXIC=CELL CYCLE EFFECTS 297