In Vitro culture studies of FlorEssence® on human tumor cell lines

STUDIES OF FLORESSENCE® ON HUMAN TUMOR CELL LINES 107

Copyright © 2005 John Wiley & Sons, Ltd. Phytother. Res. 19, 107–112 (2005)

Copyright © 2005 John Wiley & Sons, Ltd.

Received 29 April 2002Accepted 3 November 2003

PHYTOTHERAPY RESEARCHPhytother. Res. 19, 107–112 (2005)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1532

In Vitro Culture Studies of FlorEssence® onHuman Tumor Cell Lines

Joseph Tai* and Susan CheungCenter for Complementary Medicine Research, BC’s Research Institute for Children’s and Women’s Health, Departments ofPathology and Pediatrics, University of British Columbia, British Columbia, Canada

FlorEssence® (FE) is an herbal tea widely used by patients to treat chronic conditions in North America,particularly cancer patients during chemo- and radiation therapy. Although individual components of FEhave antioxidant, antiestrogenic, immunostimulant and antitumor properties, in vitro evidence of anticanceractivity for the herbal tea itself is still lacking. We studied the antiproliferative effect of FE on MCF7 andMDA-MB-468 human breast cancer, and Jurkat and K562 leukemia cell lines. We found that FE significantlyinhibited the proliferation of both breast and leukemia cells in vitro only at high concentrations, with 50%inhibition of MDA-MB-468 cells at about 1/20 dilution, Jurkat cells at about 1/10 dilution and MCF7 andK562 cells at less than 1/10 dilution. Flow cytometry analysis showed that treatment with a high concentrationof FE induced G2/M arrest in MCF7 and Jurkat cells, with also an increased SubG0/G1 fraction in MCF7cells. MDA-MB-468 cells showed a significantly increased Sub G0/G1 fraction after treatment with 1/10dilution of FE while the cell cycle of K562 was unaffected. When MCF7 and MDA-MB-468 breast cancer cellswere treated with a combination of FE with either paclitaxel or cisplatin, results showed that only the combi-nation of 1/20 dilution of FE with 0.5 µµµµµM cisplatin resulted in a small but significantly higher MCF7 cellsurvival than 0.5 µµµµµM cisplatin treatment alone. FE at 1/20 and 1/50 dilutions did not affect the antiproliferativeproperties of these two commonly used chemotherapeutic agents. The results suggest that FE at high concen-trations show differential inhibitory effect on different human cancer cell lines. Further studies are needed toassess the biological activities of FE. Copyright © 2005 John Wiley & Sons, Ltd.

Keywords: herbal tea; breast and leukemia cancer cell lines; cytotoxicity; cell cycle analysis.

* Correspondence to: Dr J. Tai, BC’s Research Institute for Children’sand Women’s Health Room L306 4480 Oak Street Vancouver, BritishColumbia, Canada V5Z 4H4.E-mail: [email protected]/grant sponsor: Lotte and John Hecht Memorial Foundation.Contract/grant sponsor: Tzu Chi Foundation.

such as phytosterols, anthraquinones, and saponins.Some of them have been reported to show antiprolifera-tive properties against some cancer cell lines. There areno reported studies in the literature of either in vitro oranimal studies on the antiproliferative effects of FE oncancer cells. The purpose of this study was to assess theantiproliferative properties of FE against four humancancer cell lines in vitro.

MATERIALS AND METHODS

High performance liquid chromatography (HPLC) ana-lysis. Three samples of liquid FE preparation (Lot 1051;Lot 1131A, Lot 1144; Flora Ltd., Burnaby B.C.) werepurchased from a natural health products store inVancouver. British Columbia’s Institute of TechnologyForensic Science Centre, Herbal Evaluation and Ana-lysis Laboratory performed the HPLC fingerprint-ing. Each 3-ml sample of FE was extracted with 7 ml of70% aqueous methanol. The solution was mixed byvortex and sonicated for 30 min to ensure completemixing of the solution. An aliquot was removed andsyringe filtered for analysis with a Hewlett Packard 1100series HPLC system, monitored with an Agilent 1100series diode array detector at 300 and 260 nm.

Chemicals and reagents. FE was diluted and mixedthoroughly with culture medium to the desired concen-trations. Anti-neoplastic agents camptothecin (CAM),paclitaxel (PTX) and cisplatin (CPT) were purchased

INTRODUCTION

Essiac is a mildly bitter herbal tea, said to possess‘cleansing properties’, that has been widely used inCanada for more than 70 years. In the early 1920s, aCanadian nurse, Rene Caisse, obtained the formulationfrom a breast cancer patient who was given the recipeby a First Nations healer. The four herbs in the com-mercial Essiac preparation are: burdock root (Arctiumlappa L.), Turkish rhubarb root (Rheum palmatum L.),sheep sorrel (Rumex acetosella L.) and inner bark ofslippery elm (Ulmus rubra Muhl.) (Stelling, 2000).Caisse and Brush developed an ‘improved’ eight-herbformulation, Flor-Essence (FE), by adding four herbs:watercress (Nasturtium officinale R.Br.), blessed thistle(Cnicus benedictus L.), red clover (Trifolium pratenseL.) and kelp (Laminaria digitata Lmx.) that they be-lieved enhanced the action and improved the taste tothe formula (Tamayo et al., 2000). This tea is one ofseveral herbal tea products taken by cancer patients asa supplement during their radiation or chemo-therapy.Proponents and the manufacturer of FE promote it asa health-enhancing herbal tea (Boik, 1996; Le Moine,1997; Essiac/FlorEssence, 2000). Some of the herbs usedto prepare FE contain biologically active compounds

108 J. TAI AND S. CHEUNG


from Sigma-Aldrich Canada (Oakville, ON). CAM andPTX were dissolved in dimethyl sulfoxide (DMSO) as1 mM stock, CPT was dissolved in phosphate bufferedsaline as 500 µM stock and they were further diluted tothe desired concentrations with culture medium.

Cell lines and culture conditions. Human leukemia K562and Jurkat cell lines were gifts from Dr A. J. Tingle ofBC’s Research Institute for Children’s and Women’sHealth. Human mammary adenocarcinoma MCF7 andMDA-MB-468 cell lines were purchased from theAmerican Type Culture Collection (Rockville, Md).K562 and Jurkat cells were cultured in RPMI1640medium supplemented with 10% fetal bovine serum,2 mM L-glutamine and 50 µg/ml gentamycin. MCF7 cellswere cultured as monolayers in Dulbecco’s MinimalEssential medium supplemented with 10% fetal bovineserum, 2 mM L-glutamine, 1 mM non-essential aminoacid and 50 µg/ml gentamycin. Jurkat, K562 and MCF7cells were cultured in a 5% CO2 humidified incubatorat 37 °C. MDA-MB-468 cells were cultured in L15medium (Sigma-Aldrich, Oakville, ON) supplementedwith 10% fetal bovine serum, 2 mM L-glutamine and50 µg/ml gentamycin in a humidified incubator at 37 °C.Jurkat, K562 and MCF7 cells were subcultured every4 days while MDA-MB-468 cells were subcultured every4–6 days to maintain logarithmic growth.

For testing, tumor cells were cultured in 96-wellplates. Starting cell numbers were 5 × 104 cells per wellfor Jurkat, 2.5 × 104 cells per well for K562 and 104 cellsper well for MCF7 and MDA-MB-468. Cell numberswere determined by hemocytometer counting and vi-ability was monitored by trypan blue exclusion test.The log growth phase of each cell line was establishedand cell counting for subsequent experiments was setat day 2 for Jurkat and K562 cells and day 4 for MCF7and MDA-MB-468 cells. Quadruplicate or triplicatesamples of cells were treated with culture mediumcontaining different concentrations of FE. Mixtures ofFE and PTX (0.5–100 nM) or CPT (0.05–10 µM) weretested on the breast cancer cell lines to investigatewhether the drug combinations could produce additiveor synergistic effects. Cell counts in samples treatedwith the test compounds were normalized to percent ofcontrol and the means and SEMs were calculated fromat least three independent experiments.

Flow cytometry. Monolayer cells: MCF7 and MDA-MB-468 cells were cultured at 5 × 105 cells per 25 cm2

flask in 10 ml of culture medium. Test compounds wereadded to the cell cultures the next day. After the cellshave stabilized in cultures, the cultures were then incu-bated for a further 48 h. Single cell suspension was pre-pared by trypsin-EDTA (0.08% trypsin, 0.3 mM EDTA)treatment followed by washing with cold calcium andmagnesium free Hanks’ balanced salt solution (HBSS)and fixation in ice-cold 70% methanol overnight at−20 °C. Fixed cells were pelleted and resuspended in2 µg/ml RNase and 50 µg/ml propidium iodide for20 min at 37 °C to stain the nuclear DNA. Analysiswas performed with a Facscalibur flow cytometer (BDBioscience, Mississauga, ON) and DNA sub-fractionprofiles were analyzed using ModFit LT V2.0 software.Suspension cultures: Jurkat and K562 cells were cul-tured at 2.5 × 105 cells/ml in 25 cm2 flasks with 10 ml ofculture medium. Test compounds were added the next

day after the cells have stabilized in the culture medium.The cells were incubated for a further 24 h then pre-pared for cell cycle DNA determination, as describedfor the monolayer cell lines.

Apoptosis assay. Fluorescein isothiocyanate conjugatedannexin V propidium iodide assay kit (Roche, Quebec)was used to determine apoptosis in the cell cultureafter treatment with different dilutions of FE (Spectoret al., 1998a).

DNA fragmentation. DNA strand fragmentation wasexamined by extracting cellular DNA from the cellsamples and subjecting them to 1% agarose gel electro-phoresis in 40 mM Tris-acetate, 1 mM EDTA bufferpH 7.2 at 10 V/cm (Spector et al., 1998b).

Statistical analysis. The results are expressed as themeans ± SEMs. The Student’s unpaired t-test was usedto compare the means of two groups. Differences wereconsidered significant when p < 0.05.

RESULTS

As demonstrated in Fig. 1, the sample of three dif-ferent lots of FE have similar HPLC profiles at 300 nm.Comparison of the relative peak areas of the HPLCprofiles showed some differences, expected for anyformulation of botanicals. The small variation that is

Figure 1. HPLC-DAD chromatographic profile of three differentlots of Flor-Essence with absorbance at 300 nm. A. Lot number1051, B. Lot 1131A and C. Lot 1144. Batch-to-batch consistencycomparison based on relative peak area abundance show con-sistency of HPLC profiles among the batches.



Figure 2. Effect of Flor-Essence on the proliferation of Jurkat,K562, MCF7 and MDA-MB-468 tumor cells assessed by trypanblue assay. Cells grown in 96-well plates were treated withvarious concentration of the herbal extract for 48 h (Jurkatand K562) or 96 h (MCF7 and MDA-MB-468). Cell growth wasassessed by counting viable cells by trypan blue exclusion witha hemocytometer. The results of at least three independentexperiments were normalized to percent of control and arepresented as mean ± SEM. Key: NS – non significant; * p < 0.05;+ p < 0.01; ^ p < 0.001 and # p < 0.0001 versus control.

Figure 3. Effect of combined treatment of Flor-Essence andcisplatin or paclitaxel on proliferation of MCF7 (A and B) andMDA-MB-468 (C and D) cells. Respective tumor cells grown in96-well plates were cultured with 1/10, 1/20 and 1/50 dilutionsof Flor-Essence combined with various concentrations ofcisplatin or paclitaxel for 96 h. Viable cell numbers were deter-mined by trypan blue exclusion method. The results are mean± SEM from at least three independent experiments. Only thecombination of 1/20 dilution of FE and 0.5 µM cisplatin wassignificantly less effective than 0.5 µM cisplatin alone (p = 0.02)on MCF7 cells.

present between the FE batches indicates lot-to-lotconsistency of the three lots of the formulation tested.

Assessment of cell growth by cell counts

FE has differential antiproliferative properties on thetwo breast and two leukemia cell lines tested (SeeFig. 2). Among these cell lines, MDA-MB-468 wasthe most sensitive to FE treatment. Cell counts aftertreatment with 1/10, 1/20 and 1/50 dilutions of FE were18.9%, 39.4% and 73.2% of control, respectively. Inhibi-tion of cell numbers to ~50% of control was about 1/10dilution of FE for Jurkat and MCF7 cells, and less than1/10 dilution for K562 cells.

Drug combination study

FE did not affect the cytotoxic effect of chemothe-rapeutic agent PTX on MCF7 cells at the concen-trations tested (Fig. 3A). 1/20 dilution of FE slightly,but significantly, reduced the cytotoxic effect of 0.5 µMof CPT on MCF7 cells ( p = 0.02) (Fig. 3B). Since 1/10and 1/20 dilutions of FE alone significantly inhibitedMDA-MB-468 cell growth, combined FE and drug treat-ment on this cell line was performed with 1/50 dilutionof FE only. FE did not affect the cytotoxic effect ofCPT and PTX on MDA-MB-468 cells at this concen-tration (Fig. 3C and 3D).

DNA profile in the cycling cells

Flow cytometry analysis of MCF7 breast cells treatedwith FE at 1/10, 1/20 and 1/50 dilutions showed arrestin G2/M phase and increased SubG0/G1 fraction whencompared with the control cells. A reduction in S phasewas also seen in cells treated with 1/10 dilution of FE.Treatment of MCF7 cells with 0.1 µM PTX, the positivecontrol, resulted in a reduction of cells in G0/G1 phaseand a significant increase in Sub G0/G1 (apoptotic/necrotic/debris) and G2/M populations (Table 1).

Treatment of MDA-MB-468 breast cancer cells with1/10 dilution of FE increased the sub-G0/G1 fraction.

The other fractions were not significantly differentfrom the control. CPT treatment at 1 µM significantlyincreased S phase population and reduced cell popula-tion in G0/G1 and G2/M phases.

Treatment of Jurkat cells with 1/10 and 1/20 dilutionsof FE arrested cells in the G2/M phase. The Sub G0/G1 and G0/G1 fractions were not significantly different



Table 1. Effect of Flor-Essence on cell cycle of human breast and leukemia cell lines*

Cell Line Test Agent Sub G0/G1% G0/G1% S% G2/M%

MCF7 Control 1.6 ± 0.5 53.4 ± 2.6 25.0 ± 2.9 22.2 ± 2.2FE1/10 12.1 ± 6.3* 48.6 ± 3.0 17.8 ± 1.6* 33.5 ± 3.4#FE1/20 11.2 ± 10.7# 48.3 ± 3.6 19.1 ± 3.4 32.7 ± 5.3*FE1/50 8.7 ± 5.4# 45.7 ± 4.2 22.9 ± 2.0 31.1 ± 5.5*PTX 0.1 µM 15.2 ± 10.3# 36.5 ± 8.8# 28.0 ± 10.7 35.5 ± 10.9#

MDA-MB-468 Control 6.1 ± 1.1 50.3 ± 3.6 24.4 ± 1.0 24.4 ± 3.8FE1/10 17.2 ± 2.1# 51.4 ± 5.1 30.1 ± 2.5 18.6 ± 2.6FE1/20 9.1 ± 2.7 54.6 ± 2.3 26.6 ± 2.6 18.8 ± 4.6FE1/50 8.1 ± 1.6 57.0 ± 2.2 24.3 ± 3.7 18.7 ± 4.0CPT 1 µM 7.9 ± 2.9 30.7 ± 1.6* 49.3 ± 13.7 20.0 ± 11.7

Jurkat Control 2.3 ± 0.5 41.0 ± 4.3 34.9 ± 3.6 24.1 ± 4.5FE1/10 3.6 ± 0.8 34.6 ± 0.7 22.0 ± 1.5 43.4 ± 1.2*FE1/20 2.3 ± 0.6 35.8 ± 2.1 21.9 ± 0.3 42.0 ± 0.6*FE1/50 1.9 ± 0.4 37.7 ± 2.1 25.3 ± 5.3 37.0 ± 1.8CAM 1.5 µM 59.6 ± 10# 25.1 ± 10.3 50.5 ± 7.0* 24.2 ± 4.1

K562 Control 5.5 ± 2.5 38.3 ± 1.7 40.4 ± 3.1 21.3 ± 2.1FE1/10 6.0 ± 4.4 42.9 ± 0.4 29.6 ± 0.4 27.3 ± 0.1FE1/20 7.6 ± 4.6 44.3 ± 4.2 29.2 ± 2.0 25.5 ± 7.4FE1/50 7.7 ± 6.1 42.9 ± 4.7 29.3 ± 1.4 27.9 ± 6.1CPT 10 µM 6.1 ± 2.6 30.0 ± 12.1 61.6 ± 9.8* 8.6 ± 6.2*

* The cells were treated with culture medium alone or medium containing different concentrations of FE. 24 h (Jurkat and K562) or48 h (MCF7 and MDA-MB-468) later, the cells were processed and stained for DNA with propidium iodide as described in materialsand methods and cell cycle distribution was then determined by FACS analysis. FE treatment induced G2/M arrest in MCF7 andJurkat cells with increase in SubG0/G1 fraction in MCF7 cells. MDA-MB-468 cells showed significantly increased Sub G0/G1 fractionafter treatment with 1/10 dilution of FE while the cell cycle of K562 was unaffected. Positive controls include paclitaxel (PTX), cisplatin(CPT) and camptothecin (CAM). Results are expressed as mean ± SEM, * p < 0.05; # p < 0.003.

Table 2. Apoptosis detection by Annexin V assay in breast carcinoma cells*

Cells Treatment Live % Apoptotic % Apoptotic/necrotic % Dead %

MCF7 Control 99.2 0.43 0.14 0.22FE 1/10 99.0 0.73 0.11 0.15FE 1/20 98.3 1.43 0.16 0.24FE 1/50 98.3 1.41 0.07 0.18

MDA-MB-468 Control 98.0 1.92 0.03 0.03FE 1/10 99.2 0.82 0.01 0FE 1/20 98.4 1.54 0.06 0.02FE 1/50 98.5 1.44 0.03 0

* MCF7 and MDA-MB-468 cells were cultured with different dilutions of Flor-Essence (FE) for 48 h then assayed for annexin V bindingto externalized membrane phospholipid phosphatidylserine with annexin V propidium iodide assay kit. There was no significantdifference between the FE treated cells and the controls.

from the control preparation. CAM at 1.5 µM, the posi-tive control, arrested cells in S phase as well as greatlyincreased cell population in Sub G0/G1 while reducedthose in G0/G1 phases. Treatment of K562 cells withall three dilutions of FE, 1/10, 1/20 and 1/50 did notsignificantly alter the cell cycle profiles. CPT, the posi-tive control, arrested the cells in S phase and signific-antly reduced the G2/M fraction.

Apoptosis assay

Table 2 shows that treatment of MCF7 and MDA-MB-468 cells with 1/10 dilution of FE for 48 hours did notsignificantly increase apoptosis in these cells as shownby annexin V binding assay (see Table 2).

DNA fragmentation

Agarose gel electrophoresis of the DNA samples fromFE treated Jurkat, K562, MCF7 and MDA-MB-468cells showed that FE did not cause DNA strand frag-mentation even at 1/10 dilution, whereas Jurkat cellstreated with 1.5 µM of CAM consistently showedsmeared DNA fragments in agarose gel eletrophoresisruns (Figure not shown).

DISCUSSION

Cancer is a major health problem in the world. Al-though the majority of patients will respond initially



to radiation and chemotherapy, once metastatic tumorsfail to respond to conventional treatment, these patientsoften seek help in complementary or alternative medi-cine treatments. A large variety of herbal products isavailable in natural health products stores as dietarysupplements for cancer patients. Unfortunately, notmuch is known about their efficacy, active principles,and mode of action, side effects and possible adverseinteractions with conventional antitumor drugs. There-fore, testing of herbal agents in vitro on cancer celllines to understand their mechanism of action may behelpful in preventing their undesirable effects as wellas enhancing their effectiveness.

Many breast cancer patients are currently using FE,a modified formulation of Essiac, as a self prescribedherbal supplement. It has not been subjected to formalclinical trials that would provide rigorous testing of itsquality, toxicity and efficacy. An unpublished Canadianstudy in the late 1970s ‘found no evidence of an effecton the cancer process but some subjective improve-ments in quality of life’ (Kaegi, 1998). Studies atMemorial Sloan Kettering laboratories in 1959 andfrom 1973 to 1976 were inconclusive (Canadian BreastCancer Research Initiative, 1996). One of the mainconcerns regarding multi-component herbal extracts isproduct consistency. According to the manufacturer ofFE, the composition of each batch of the extract iscontrolled for reproducibility by HPLC. Our independ-ent HPLC analysis confirmed the consistency of threesample lots of FE used in this study. The presence ofseveral discrete peaks and the difference in heightsof the respective peaks to each other provide a charac-teristic HPLC fingerprint pattern of FE. However, thechemical composition of the individual peaks awaitsidentification.

Since the effect of FE on tumor cell lines in vitro hasnot been reported, the main objective of the presentstudy was to determine its in vitro effect on both breastand leukemia cancer cells. In the preliminary study,MTT was used to determine the effect of FE on cellproliferation as described by Mosmann (1983). How-ever, comparisons of viable cell numbers by actualcounting and by MTT assay revealed that in manyinstances, MTT readings did not change despite theobvious reduction in viable cell numbers. This dis-crepancy in viable cells estimated by these two assaymethods was reported earlier in MCF7 cells treatedwith ursolic acid and genistein (Pagliacci et al., 1993;Es-Saady et al., 1996). Therefore, MTT assay may notbe a reliable marker for cell growth and viability whenassessing herbal compounds that affect MTT reduc-tion to formazan salt (York et al., 1998). In view of thispitfall of MTT assay, actual cell counts were used forthe rest of the study.

We observed that although FE inhibited the prolifera-tion of MCF7, MDA-MB-468, Jurkat and K562 cells,this effect was consistently seen only at the highestconcentration, 1/10 dilution, tested. Cell cycle analysesrevealed that FE treated MCF7 and Jurkat cells werearrested in the G2/M phase. In both MCF7 and MDA-MB-468 cells, high concentration FE treatment alsoincreased the Sub G0/G1 fraction. The mechanism(s)by which FE predominantly affected G2/M, rather thanthe other regulatory steps, require further studies.The ‘active principles’ of FE are still unknown. It hasbeen reported that sheep sorrel root contains emodin,

chrysophanic acid, chrysophanein, and 1,8 dihydroxy-3-methyl-9-anthrone; Turkish rhubarb root containsemodin and chrysophanic acid; blessed thistle containsstigmasterol; watercress contains vitamin A, C and Dand red clover blossom contains biochanin, genistein,daidzein, trifolirhizin and pratensein. Emodin, genistein,daidzein, chrysophanic acid and stigmasterol have beenreported to have cytostatic, antitumorigenic or anti-oxidant properties (Yim et al., 1999; Zhang et al., 1999).Emodin and chrysophenols are anthraquinone deriva-tives with cytotoxic activities similar to a class ofantitumor drugs, podophyllotoxins. These are weaknon-binding DNA intercalators with targeted actionon topoisomerase II (Kong et al., 1992). Genistein anddaidzein are isoflavinoids, which are also non-bindingDNA intercalators with actions on topoisomerase II,but exert their effect at a different phase of the cellcycle (Smith and Wiltshire, 2001). The actions of theseDNA intercalators can cause cell cycle arrest in late Sand G2 phase (Shao et al., 2000). This may explain theperturbation in the cell cycle profiles of FE treatedJurkat and MCF7 cells. However, it is premature topostulate any mechanism of action without first identi-fying the potentially active ingredients present in theFE herbal tea mixture.

One of the many mechanisms of action of anti-neoplastic agents on cancer cells is by direct binding toDNA strands causing strand breaks during cell cycleprogression. The effect of FE observed in the four celllines tested here may not be a direct result of DNAstrand breaks, since agarose gel electrophoresis failedto show DNA fragmentation in FE treated cells.Annexin V assay data supported this observation. Onepossibility is that the cell lines used in this study,in particular K562 and MCF7, are more resistant toDNA strand breaks (Specter et al., 1998a; Vadgamaet al., 2000). Another possibility may be due to theculture condition in which the cells were maintained.In a study showing DNA strand breaks in MDA-MB-468 cells treated with PTX, culture medium with re-duced serum was used (Fang et al., 2000). In this study,the cells were maintained in medium supplementedwith 10% serum.

One concern regarding herbal medicine is the possibleadverse interactions with conventional antitumor drugs.An important observation of this study was that gener-ally FE did not affect the antiproliferative property ofantineoplastic agents like PTX and CPT on MCF7 andMDA-MB-468 cells. The only combination that reducedthe antiproliferative effect of CPT on MCF7 cells waswith 0.5 µM of CPT and 1/20 dilution of FE.

It is difficult to extrapolate the results of the in vitrostudies to in vivo conditions because of the lack ofinformation about the identity and concentrations ofthe ‘active principles’ in FE and the pharmacokineticdata of the ‘active principles’ following FE ingestion.Furthermore, the concentrations of FE used in the invitro studies were significantly higher than the expectedlevel present in patients’ circulation and tissues. Forexample, MCF7 cells were suppressed only at a veryhigh concentration, namely 1/10 dilution, of FE in theculture medium. Even with full absorption of the activeprinciples of the orally administered preparation, itis estimated that patients who take this herbal pre-paration at the dosage, 30–60 ml once or twice daily,recommended by the manufacturer, would unlikely



achieve the effective concentrations. Despite this, manyfactors could influence the bioavailability of the ‘activeprinciples’. Information on the extent of absorption andmetabolism of putative bioactive principles present inFE is essential to allow correct interpretation of thein vitro data, to develop appropriate hypothesis and toperform future experiments (Shao et al., 2000).

In the present study, we examined the in vitro effectof FE on the growth and cell cycle of human tumor celllines. Other mechanisms whereby FE can affect cellgrowth in vivo, such as by stimulating the patient’s ownimmune system, have not been evaluated. As FE con-tains a number of potentially ‘active principles’, we

cannot preclude that one or more of these componentscan alter tumor cell growth in vivo by direct inhibitionand/or indirectly through the host’s immune defensemechanism. Further research on this popular herbalagent is needed.

Acknowledgements

The authors gratefully acknowledge funding from the Lotte and JohnHecht Memorial Foundation and Tzu Chi Foundation. Thanks arealso due to Paula Brown of BCIT and Christopher Lowe for technicalassistance.

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In Vitro culture studies of FlorEssence® on human tumor cell lines

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