Anti-Proliferative Properties of Terminalia sericea Burch . Ex Dc Leaf Extracts Against Caco 2 and HeLa Cancer Cell Lines

Introduction: Terminalia spp. are characterised by their high levels of antioxidant phytochemicals and several species have anticancer activity. This study examines the anti-proliferative activity of T. sericea leaf extracts against Caco2 and HeLa carcinoma cell proliferation. Methods: Solvent extracts were prepared from T. sericea leaves and their antioxidant capacities were determined by the DPPH free radical scavenging assay. Anti-proliferative activities against Caco2 and HeLa cancer cells were determined by an MTS based cell proliferation assay. Toxicity was determined using the Artemia franciscana nauplii bioassay. Results: The methanolic and aqueous T. sericea leaf extracts displayed high antioxidant capacities (equivalent to 150 and 340 mg of ascorbic acid per gram of plant material extracted respectively). In contrast, the ethyl acetate, chloroform and hexane extracts had relatively low antioxidant contents (≤5 mg of ascorbic acid equivalents per gram of plant material extracted). The antioxidant contents of the T. sericea leaf extracts correlated with the ability of the extracts to inhibit proliferation of Caco2 and HeLa cancer cell lines. The high antioxidant methanolic and aqueous extracts were potent inhibitors of cell proliferation, with IC50 values 120-1400 μg/mL. The aqueous T. sericea leaf extract was particularly effective, with IC50 values of 528 and 120 μg/mL against Caco2 and HeLa cells respectively. The methanolic extract also displayed good, albeit substantially less potent, antiproliferative activity against HeLa cells, with an IC50 of 1358 μg/mL. In contrast, the lower antioxidant content extracts generally did not inhibit cancer cell proliferation. Cell imaging studies detected morphological features consistent with apoptosis in Caco2 cells exposed to sub-lethal concentrations of the methanolic and aqueous T. sericea leaf extracts, indicating that these extracts are functioning by cytotoxic mechanisms. The aqueous T. sericea leaf extract displayed low to moderate toxicity in the Artemia franciscana bioassay, with an LC50 value of 737 μg/mL. All other extracts were nontoxic. Conclusion: The antiproliferative activity and low toxicity of the T. sericea methanolic and aqueous leaf extracts extracts against HeLa and Caco2 cancer cell lines indicates their potential in the treatment and prevention of some cancers.


INTRODUCTION
Epidemiological studies have shown that a diet rich in antioxidants is associated with a decreased incidence of chronic diseases. 1High antioxidant levels have also been shown to act as a preventative against the devel opment of degenerative diseases such as cancer, 2 cardio vascular diseases, 3 neural degeneration, 4 diabetes and obesity. 5Phenolic phytochemicals are generally strong antioxidants. 6Their primary action involves the protection of cell constituents against oxidative damage through the scavenging of free radicals, thereby averting their deleterious effects on nucleic acids, proteins, and lipids in cells. 6Phenolics interact directly with receptors or enzymes involved in signal transduction, 7 indicating that they play specific roles in human physiology via modulation of the cellular redox state.Common plant phenolic compounds include flavonoids, tannins, anthocyanins, and gallic acid.
The relationship between cellular redox state and cancer progression has been particularly well studied.The induction of cellular oxidative stress has been linked with several types of cancer. 8,9Thus it is possible that high antioxidant contents of many plants may inhibit cancer formation and/or progression.Studies into the antioxidant/prooxidant effects of extracts from various plant species have dem onstrated that the ability of a plant extract to exert antioxidant activity depends on multiple factors.Aloe vera antioxidant components for example may function as either antioxidants or a prooxidants, with their action being dependent upon their con centration. 10The Aloe vera anthraquinone aloe emodin exerts antioxidant behaviour at lower concentrations, yet acts as a prooxidant at high concentrations.In contrast, a different Aloe vera anthraquinone (aloin) has an antioxidant effect at higher concentrations, yet a prooxidant effect at low concentrations.Thus, Aloe vera extracts and components may act as either antioxidants or as pro-oxidants, dependent on differing levels of the various constituents, and on their ratios.Thus, although many plant species have very high antioxidant contents, it is possible that the individual components may act as either antioxidants or as pro-oxidants and thus may also be effective in the treatment of cancer, as well as in its prevention at different concentrations.Similar pro-oxidant effects have been reported for other antioxidant phytochemicals including flavonoids 11 and tannins. 12Furthermore, the presence of transition metal ions such as copper or iron in an extract can further enhance the conversion of the antioxidant to the prooxidant state. 13,14The prooxidant/antioxidant effect of plant extracts is due to a balance between the free radical scavenging activities and reducing power of their phytochemical components.This can be explained using the antioxidant vitamin ascorbic acid as an example.Although ascorbic acid has well characterised antioxidant bioactivities, it acts as a prooxidant at high concentrations. 15This is due to the greater reducing power of ascorbic acid compared to its free radical scavenging activity.In the presence of transition metal ions, ascorbic acid will function as a reducing agent, reducing the metal ions.In this process, it is converted to a prooxidant.Therefore, high dietary intake of ascorbic acid (or other antioxidants) in individuals with high iron levels (e.g.premature infants) may result in unexpected health effects due to the induction of oxidative damage to susceptible biomolecules. 16,17,18lants of the genus Terminalia (family Combretaceae) are characterised by their high antioxidant contents. 19It has previously been postulated that these high antioxidant contents may provide them with therapeutic effects.Furthermore, several individual Terminalia spp., (and traditional medicine combinations containing Terminalia spp.) have been reported to have potent anticancer properties.The Ayurvedic medicine Triphala (which contains Terminalia belliricia (Gawrtn.)Roxb.and Terminalia chebula (Retz.)Lyons) has cytotoxic effects against thymic lymphoma cells 20,21 human breast cancer cell lines. 21,22human prostate cancer cell lines and human pancreatic cancer cell lines. 23Conversely, the cytotoxic effects of Triphala are negligible in normal cell lines. 20,21T. belliricia extracts have also demonstrated growth inhibitory effects towards human A549 lung cancer cell lines and HepG2 hepatocarcinoma cell lines without the presence of the other plant components present in Triphala. 24T. belliricia extracts also have synergistic effects with conven tional anticancer chemotherapeutics, enhancing the cytotoxic activity of cisplatin and doxorubicin towards the cancer cell lines, indicating their potential as synergistic cancer chemotherapeutic agents.Similarly, the Australian species Terminalia ferdinandiana Exell.has broad spectrum antiproliferative activity against a panel of cancer cell lines. 25he proliferation of normal cell lines was also inhibited in that study.The extract activated caspase7 and caspase9 as well as poly (ADPribose) polymerase (PARP), indicating that apoptosis induction is via an intrinsic pathway.Extracts prepared from the leaves of two other Australian Terminalia spp.(T.carpentariae and T. grandiflora) were also recently reported to be good inhibitors of Caco2 and HeLa cell proliferation. 26hat study identified 3 lanostane and 2 pentacyclic triterpenoids in the extracts and postulated that they may contribute to this activity.Despite these previous studies, limited information is available on the anticancer activity of other Terminalia spp.This study screened the Southern African species Terminalia sericea C.T. White for the ability to inhibit proliferation of Caco2 colorectal and HeLa cervical cancer cell lines.

Plant source and extraction
The Terminalia sericea Burch.Ex DC.Leaves used in this study were supplied by Professor Sandy van Vuuren of Witwatersrand University, South Africa and were sourced from the Walter Sisulu Botanical Gardens in Johannesburg, South Africa.The plant materials were identified by Andrew Hankey, chief botanist at the Walter Sisulu Botanical Gardens in Johannesburg, South Africa.Voucher specimens are stored in the School of Natural Sciences, Griffith University, Australia.All plant material was thoroughly air dried and ground into a coarse powder.The dried plant material was stored at 30 o C until use.Individual 1 g quantities of the powdered T. sericea were weighed into separate tubes and 50 mL of methanol, deionised water, ethyl acetate, chloroform or hexane were added.All solvents were obtained from Ajax Chemicals, Australia and were AR grade.The ground T. sericea leaf material was extracted in each solvent for 24h at 4 o C with gentle shaking.The extracts were filtered through filter paper (Whatman No. 54) under vacuum.The solvent extracts were dried in a vacuum oven and the aqueous extract was dried by freeze drying.The resultant dry extract was weighed and redissolved in 10 mL deionised water (containing 1 % DMSO).

Qualitative phytochemical studies
Phytochemical analysis of the extracts for the presence of saponins, phenolic compounds, flavonoids, polysteroids, triterpenoids, cardiac glycosides, anthraquinones, tannins and alkaloids was conducted by previously described assays. 27,28

Antioxidant capacity
The antioxidant capacity of each sample was assessed using the DPPH free radical scavenging method 29 with modifications.Briefly, DPPH solution was prepared fresh each day as a 400 µM solution by dissolving DPPH (Sigma) in AR grade methanol (Ajax, Australia).The initial absor bance of the DPPH solution was measured at 515 nm using a Molecular Devices, Spectra Max M3 plate reader and did not change significantly throughout the assay period.A 2 mL aliquot of each extract was evapo rated and the residue resuspended in 2 mL of methanol.Each extract was added to a 96well plate in 5, 10, 25, 50, 75 µL volumes in triplicate.Methanol was added to each well to give a volume of 225 µL.A volume of 75 µL of the fresh DPPH solution was added to each well for a total reaction volume of 300 µL.A blank of each extract concentration, methanol solvent, and DPPH was also performed in triplicate.Ascorbic acid was prepared fresh and examined across the range 025 µg per well as a reference and the absorbances were recorded at 515 nm.All tests were performed in triplicate and triplicate controls were included on each plate.The antioxidant capacity based on DPPH free radical scavenging ability was determined for each extract and expressed as µg ascorbic acid equivalents per gram of original plant material extracted.

Cancer cell lines
The Caco2 and HeLa carcinoma cell lines used in this study were obtained from American Type Culture Collection (Rockville, USA).The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies), supplemented with 20 mM HEPES, 10 mM sodium bicarbonate, 50 µg/mL streptomycin, 50 IU/mL penicillin, 2 mM glutamine and 10 % foetal calf serum (Life Technologies).The cells were maintained as monolayers in 75 mL flasks at 37 o C, 5 % CO 2 in a humidified atmosphere until approximately 80 % confluent.

Evaluation of cancer cell antiproliferative activity
Antiproliferative activity of the extracts was assessed as previously described. 30Briefly, 1 mL of trypsin (Sigma) was added to the culture flasks and incubated at 37 o C, 5 % CO 2 for 15 min to dislodge the cancer cells.The cell suspensions were then transferred to a 10 mL centrifuge tube and sedimented by centrifugation.The supernatant was discarded and the cells were resuspended in 9 mL of fresh media.Aliquots of the resuspended cells (70 µL, containing approximately 5000 cells) were diluted in artificial seawater for use in the Artemia franciscana nauplii bioassay.

Artemia franciscana nauplii toxicity screening
Toxicity was tested using a modified Artemia franciscana nauplii lethality assay. 31 34Briefly, 400 µL of seawater containing approximately 53 (mean 53.1, n = 125, SD 12.3) A. franciscana nauplii were added to wells of a 48 well plate and immediately used for bioassay.A volume of 400 µL of diluted plant extracts or the reference toxin were transferred to the wells and incubated at 25 ± 1 o C under artificial light (1000 Lux).A negative control (400 µL seawater) was run in triplicate for each plate.All treat ments were performed in at least triplicate.The wells were checked at regular intervals and the number of dead counted.The nauplii were considered dead if no movement of the appendages was observed within 10 seconds.After 24 h, all nauplii were sacrificed and counted to deter mine the total % mortality per well.The LC 50 with 95% confidence limits for each treatment was calculated using probit analysis.

Statistical analysis
Data are expressed as the mean ± SEM of at least three independent experiments.One way ANOVA was used to calculate statistical signifi cance between control and treated groups with a P value < 0.01 considered to be statistically significant.

Liquid extraction yields and qualitative phytochemical screening
Extraction of 1 g of the dried T. sericea powder with solvents of varying polarity yielded dried plant extracts ranging from 38 mg (ethyl acetate T. sericea leaf extract) to 220 mg (methanolic T. sericea leaf extract) (Table 1).The higher polarity methanol and water solvents generally extracted relatively high yields of plant material whilst the mid to lower polarity solvents extracted considerably less material.The dried extracts were resuspended in 10 mL of deionised water (containing 1 % DMSO), resulting in the extract concentrations shown in Table 1.Qualitative phytochemical studies (Table 1) showed that methanol and water extracted the widest range of phytochemicals.Both showed moderate to high levels of phenolics (both water soluble and insoluble added to the wells of a 96 well plate.A volume of 30 µL of the test extracts or cell media (for the negative control) was added to individual wells and the plates were incubated at 37 o C, 5 % CO 2 for 12 h in a humidified atmosphere.A volume of 20 µL of Cell Titre 96 Aqueous One solution (Promega) was subsequently added to each well and the plates were incubated for a further 3 h.Absorbances were recorded at 490 nm using a Molecular Devices, Spectra Max M3 plate reader.All tests were performed in at least triplicate and triplicate controls were included on each plate.The antiproliferative activity of each test was calculated as a percentage of the negative control using the following formula: A ct is the corrected absorbance for the test extract (calculated by sub tracting the absorbance of the test extract in media without cells from the extract cell test combination) and A cc is the corrected untreated control (calculated by subtracting the absorbance of the untreated control in media without cells from the untreated cell media combination).

Cell imaging studies
Cell morphological changes were measured using bright field micro scopic imaging without staining.Briefly, Caco2 cells were seeded into individual wells of a 96 well plate at a density of 5 x 10 3 cells / 100 µL in a 96 well plate.Cells were allowed to adhere overnight.Selected T. sericea extracts were added to the wells at predetermined concentrations (previously determined to be the highest concentration which induced <10 % change in proliferation compared to the untreated control).Cells were incubated for 24 h at 37 °C in a humidified 5 % CO 2 , 95 % air atmo sphere.Each assay was performed in triplicate and the cell morphology was qualitatively appraised after 24 h treatment by using an inverted phase microscope with camera (Olympus IX70) with an optical zoom of 20X.The images were captured via computer and analysed to scale.

Reference toxin for toxicity screening
Potassium dichromate (K 2 Cr 2 O 7 ) (AR grade, ChemSupply, Australia) was prepared as a 1.6 mg/mL solution in distilled water and was serially   phenolics), flavonoids, tannins and triterpenoids as well as low levels of saponins.Ethyl acetate extracted similar classes of compounds, albeit at lower levels.The chloroform and hexane extracts were much less complex, with only low to moderate relative abundances of flavonoids detected in both extracts.Phytosterols, alkaloids, cardiac glycosides and anthraquinones were not detected in any extract.

Antioxidant content
Antioxidant capacity (expressed as ascorbic acid equivalents) for the T. sericea extracts are shown in Table 1.The antioxidant capacity ranged from a low of 0.4 mg ascorbic acid equivalents per gram of dried plant material extracted (T.sericea hexane extract) to a high of 340 mg ascorbic acid equivalents per gram of dried plant material extracted (aqueous T. sericea extract).The T. sericea methanol extract also had a high anti oxidant capacity of 150 mg ascorbic acid equivalents per gram of dried plant material extracted.The ethyl acetate extract had a moderate anti oxidant capacity (22 mg ascorbic acid equivalents).

Antiproliferative activity
Aliquots of each extract were tested for the ability to inhibit proliferation of Caco2 (Figure 1) and HeLa (Figure 2) cell lines.Three of the T. sericea leaf extracts displayed significant (p<0.01)antiproliferative effects against the Caco2 cells.The aqueous extract was the most potent inhibitor, with proliferation inhibited by approximately 83% compared to the negative control cell proliferation.Inhibition of proliferation by the aqueous extract was dose dependent, with the level of inhibitory activity decreasing at lower concentrations (Table 2).The methanolic and ethyl acetate extracts also significantly inhibited proliferation, albeit by substantially lower levels (28 and 36% inhibition of proliferation respectively).The dose dependent proliferative activity of these extracts were not evaluated in this study.The chloroform and hexane extracts were completely devoid of antiproliferative activity against Caco2 cells.The extracts were similarly effective at inhibiting HeLa cancer cell proliferation.Three of the T. sericea leaf extracts had significant (p<0.01)antiproliferative effects against the HeLa cell line.As seen for the Caco2 cells, the methanolic, aqueous and ethyl acetate extracts inhibited HeLa carcinoma cell growth significantly (p<0.01).The aqueous extract was the most potent inhibitor, with proliferation inhibited by approximately 96% compared to the negative control cell proliferation.The methanolic extract was also had potent antiproliferative effects, inhibiting HeLa cell proliferation by approximately 62% compared to the negative control cell proliferation.Inhibition of proliferation by the aqueous and metha nolic extracts was dose dependent, with the level of inhibitory activity decreasing at lower concentrations (Table 2).The ethyl acetate extracts also inhibited proliferation, albeit by only approximately 12% compared to the negative control cell proliferation.The chloroform and hexane extracts were completely devoid of antiproliferative activity against HeLa cells.The antiproliferative efficacy was further quantified by determining the IC 50 values for each extract which inhibited cell proliferation against each cell line (Table 2).The aqueous extracts were particularly effective at inhibiting both Caco2 and HeLa cancer cell proliferation at low con centrations, with IC 50 values of approximately 500 µg/mL and 100 µg/mL respectively.The methanolic T. sericea extract was also a moderate to good inhibitor of HeLa, with an IC 50 value <1500 µg/mL.Whilst the methanolic and ethyl acetate extracts also significantly inhibited Caco2 cell proliferation, IC 50 values could not be determined as the % inhibition of proliferation did not exceed 50% at any concentration tested.

Cell imaging studies
The antiproliferative studies presented in this manuscript demonstrate that the methanolic and aqueous T. sericea extracts have profound effects on Caco2 and HeLa cellular proliferation (Figures 1 and 2).These large decreases in cell proliferation compared to the untreated control cells indicated that these extracts may not just inhibit growth by blocking cell cycle progression, but may instead act via cytotoxic/apoptotic mecha nisms.However, further studies are required to test this hypothesis and cell imaging studies were undertaken to confirm this finding.The Caco2 cell line was chosen for cell imaging studies as it is a commonly used cell line in similar studies (allowing for comparisons), has well defined cell morphology, has a reasonable doubling time compared with many other cell lines and was responsive to the T. sericea extracts.
The key organelles for comparison between the treated and untreated cells in these studies are the nucleus, cytoplasm, vacuoles and cell membrane, all of which were clearly visible at 20X optical magnification of the cell in the untreated control cell image (Figure 3a).The outline of the cell is clear, distinct and compact without abnormalities in size and density.Stress granules were not visible at this magnification.These are normally indicators of cellular stress, if it is present.Typical apoptotic phenomena were noted in the morphology of the cells tested against the methanolic (Figure 3b) and aqueous (Figure 3c) extracts when tested at sublethal concentrations.These images are consistent with the findings of the cell proliferative assays which showed that both the methanolic and aqueous T. sericea extracts strongly inhibited Caco2 proliferation.This correlates with the morphological characteristics noted in this study.Apoptosis is generally characterised by the presence of an intact mem brane, marginalisation of the cytoplasm and chromatin condensation. 35hese characteristics were clearly visible in the Caco2 cells treated with the methanolic and aqueous T. sericea extracts (Figure 3b and Figure 3c).Marginalisation of the cytoplasm of cells undergoing apoptosis also produces an enormously large centrally placed vacuole which is clearly visible in both figures.The presence of stress granules in the visible fields (Figures 3a and 3b) indicates that the cells are under stress.'Blebbing' is the appearance of 'spherical bubbles' on the surface of the membrane and is a typical characteristic apparent in apoptotic cells.'Blebbing' is also seen in cells tested against the methanolic and aqueous T. sericea extracts (Figures 3a and 3b).These cells also displayed a loss in membrane integrity with no clear outline of the cell visible, extensive chromatin condensation and the presence of numerous vacuoles.Also visible for both treatments were the 'ghosts' of cell where cellular contents are no longer present within cell.The morphological changes observed with both treatments are indicative of the induction of apoptosis.

Quantification of toxicity
All extracts were initially screened at 2000 µg/mL in the assay (Figure 4).For comparison, the reference toxin potassium dichromate (1000 µg/mL) was also tested in the bioassay.Figure 4 shows the % mortality induced by each extract and by the control toxin following 24 h exposure.The potassium dichromate reference toxin was rapid in its onset of mortality, inducing mortality within the first 3 h of exposure and 100 % mortality was evident following 45 h (unpublished results).Similarly, the metha nolic and aqueous T. sericea extracts displayed 100 % mortality rates at 24 h and were thus deemed to be toxic.All other extracts induced only low levels of mortality which was not significantly different to the mortality determined for the negative control.These extracts were therefore deemed to be nontoxic.To further quantify the effect of extract concentration on the induction of mortality, the extracts were serially diluted in artificial seawater to test across a range of concentrations in the Artemia nauplii bioassay.Table 2 shows the LC 50 values of the extracts towards A. franciscana.No LC 50 values are reported for the ethyl acetate, chloroform or hexane T. sericea extracts as less than 50 % mortality was seen for all concentrations tested.Whilst the methanolic extract initially appeared to be toxic in the screening study, determination of its LC 50 value (1088 µg/mL) demonstrated that it was nontoxic as extracts with an LC 50 > 1000 µg/mL towards Artemia nauplii are defined as being nontoxic. 34In contrast, an IC 50 value of the 737 µg/mL was determined for the aqueous T. sericea leaf extract, indi cating moderate toxicity.However, it is noteworthy that Terminalia spp.extract often have very high antioxidant content which may be due to high levels of ascorbic acid. 19As high ascorbic acid contents significantly decrease the assay pH and Artemia nauplii are sensitive to pH changes, this may be a fallacious result.

DISCUSSION
Maintenance of the cellular redox state is required for the normal func tioning of living systems.This redox maintenance involves a regulated balance between oxidants and antioxidants.Cells use a variety of different antioxidant mechanisms to help maintain this redox state. 36,37These cellular defences include enzymatic antioxidants (superoxide dismutase, catalase, thioredoxin, thioredoxin reductase, glutathione peroxidase and glutathione peroxidase) and nonenzymatic antioxidants (glutathione, vitamins A, C and E).Deregulation of the redox balance has been implicated in numerous diseases including atherosclerosis, diabetes, cirrhosis, 16  cancer activity. 52Individual extract components responsible for the anti proliferative activity of the T sericea leaf extracts were not identified in the current study.Whilst the use of high levels of antioxidants may be an attractive option in the prevention and treatment some cancers, oxidative stress is not the sole factor in the development and progression of cancer.Genetic factors, dietary habits and environmental factors may all contribute to cancer development.Furthermore, even in oxidative stress induced cancers, dietary antioxidants are of limited value as it is difficult to supply enough of the beneficial antioxidants directly in the food to function effectively as chemoprotectants and/or chemotherapeutics.Instead, high antioxidant extracts are required to obtain a high enough dosage to be effective.Despite this, numerous studies have reported the beneficial effects of using high levels of phytochemical antioxidants in cancer prevention and treatment.Therefore, high antioxidant plant extracts such as the aqueous T sericea leaf extract examined in this report may also prove to be useful anticancer agents.The findings reported here also demonstrate that the majority of the T sericea leaf extracts were nontoxic towards Artemia franciscana nauplii.Of the extracts tested, only the aqueous extract displayed an LC 50 value <1000 µg/mL.This extract was also determined to have a very high antioxidant capacity (equivalent to 340 µg ascorbic acid per g of dried T sericea leaf extracted).As Artemia nauplii are susceptible to pH changes 34 such as those seen with the addition of ascorbic acid (the most prevalent antioxidant in the leaves), it is possible the mortality induced by these extracts is due to their high ascorbic acid contents.All other extracts examined in this study were nontoxic, although further studies using human cell lines are required to verify the safety of these extracts for therapeutic use.

CONCLUSION
The results of this study demonstrate the potential of the aqueous and methanolic T. sericea leaf extracts to block proliferation of some cancer cells.The low toxicity of the extracts further indicates their potential in the treatment and prevention of some cancers.Purification and identi fication of the bioactive components is needed to examine the mecha nisms of action of these agents.autoimmune disease and chronic inflammation, 38 neurodegeneration 16 and cancer. 36,37An understanding of redox regulation is important in identifying new targets for the development of new drugs for cancer prevention and cancer therapy.Epidemiological studies have shown that a diet rich in antioxidants is associated with a decreased incidence of chronic diseases. 38Individuals with elevated dietary intakes of nonenzymatic antioxidants such as vitamins A, C and E are less likely to suffer many chronic illnesses and some forms of cancer. 38,39High antioxidant levels have also been shown to prevent the development of neural degeneration. 40Interestingly, the most potent antiproliferative activity against cancer cells in our study correlated with the highest antioxidant capacity extracts, indicating that the extracts may function via modulation of cellular redox status.High antioxidant plant extracts may contain many types of phythochem icals, of which phenolic compounds are prevalent.Phenolic compounds are generally strong antioxidants. 6Their primary action involves the protection of cell constituents against oxidative damage through the scavenging of free radicals, thereby averting their deleterious effects on nucleic acids, proteins, and lipids in cells. 6Phenolics may interact directly with receptors or enzymes involved in signal transduction, 7 clearly indicating that they play a specific role in human physiology.Therefore, consumption of plant material or extracts with high antioxi dant capacities may contribute to reducing oxidative damage via non enzymatic mechanisms and the scavenging of free radicals.However, studies into the medicinal effects of antioxidants have proved confusing, with some studies showing therapeutic effects, 41,42 whilst other studies indicate that these antioxidants may themselves be toxic. 33,43The effects of antioxidants are dose dependent, with low doses behaving as antioxi dants, while high doses themselves induce toxicity through the induction of oxidative stress . 44ulticellular organisms require a reducing environment for cell prolif eration.This is accomplished when there are high reduced glutathione (GSH) levels and low levels of antioxidant proteins such as superoxide dismutase (SOD), glutathione peroxidise (GPx) and catalase. 45,46The redox environment of the cell also plays a significant role in cell differen tiation.In contrast to proliferation, differentiation requires an oxidising environment.Low levels of antioxidants induce cell proliferation whilst high levels inhibit it. 47Therefore, proliferation favours a reducing envi ronment whilst differentiation requires an oxidizing environment. 48,49OS may also play an important physiological role as secondary messen gers. 50and in the maintenance of cellular redox balance.Consumption of phenolic antioxidants may help to reduce the oxidative stress of the cell, and of the whole organism.The antiproliferative efficacy of T. sericea leaf extracts was examined against two cancer cell lines in vitro; CaCo2 (colorectal) and HeLa (cervical).Interestingly, the observed antioxidant activity was related to the antiproliferative activity of the cancer cells.All of the extracts with high antioxidant content demonstrated antiproliferative activity against both cancer cell lines.Conversely, extracts with low antioxidant capacity lacked antiproliferative activity.A previous study on the antioxidant activity of fresh apples reported a similar relationship between antioxi dant activity and antiproliferative activity against Caco2 cells. 51It was suggested that the antiproliferative activity was due to the combination of phenolic acids and flavonoids. 51This correlates with the results observed in the qualitative phytochemical analysis of this report.Phenolic and flavonoid compounds were observed to be present in high levels in all of the extracts that displayed antiproliferative activity, and in much lower levels in the extracts that did not inhibit cellular proliferation.Flavonoids are believed to protect cells from disease by shielding lipids, proteins and DNA from oxidative damage. 52Previous in vitro bioactivity studies of flavonoids have demonstrated antiinflammatory, antioxidant, and anti

Figure 1 :
Figure 1: Anti-proliferative activity of the T. sericea extracts and untreated controls against Caco2 cancer cell lines measured as percentages of the untreated control cells.NC = untreated control; M = methanolic T. sericea extract; W = aqueous T. sericea extract; E = ethyl acetate T. sericea extract; C = chloroform T. sericea extract; H = hexane T. sericea extract; PC = cisplatin control (50 µg/mL).Results are expressed as mean percentages ± SEM of at least triplicate determinations.* indicates results that are significantly different to the untreated control (p<0.01).

Figure 2 :
Figure 2: Anti-proliferative activity of the T. sericea extracts and untreated controls against HeLa cancer cell lines measured as percentages of the untreated control cells.NC = untreated control; M = methanolic T. sericea extract; W = aqueous T. sericea extract; E = ethyl acetate T. sericea extract; C = chloroform T. sericea extract; H = hexane T. sericea extract; PC = cisplatin control (50 µg/mL).Results are expressed as mean percentages ± SEM of at least triplicate determinations.* indicates results that are significantly different to the untreated control (p<0.01).

Figure 3 :
Figure 3: Effects of T. sericea extracts on the morphological characteristics of Caco2 cells: (a) viable Caco2 cells without exposure to extracts; (b) Caco2 cells exposed to the methanolic T. sericea extract; Caco2 cells exposed to the aqueous T. sericea extract.

Figure 4 :
Figure 4: The lethality of the T. sericea extracts (2000 µg/mL) and the potassium dichromate control (1000 µg/mL) towards Artemia nauplii following 24 h exposure.M = methanolic T. sericea extract; W = aqueous T. sericea extract; E = ethyl acetate T. sericea extract; C = chloroform T. sericea extract; H = hexane T. sericea extract; NC = negative control; PC = positive control (1000 µg/ml potassium dichromate).All tests were performed in at least triplicate and the results are expressed as mean ± SEM. * indicates results that are significantly different to the negative control (p<0.01).

Table 1 : The mass of dried extracted material, the concentration after resuspension in deionised water, qualitative phytochemical screenings and antioxidant capacities of the T. sericea extracts.
+++ indicates a large response; ++ indicates a moderate response; + indicates a minor response; indicates no response in the assay; M = methanolic T. sericea extract; W = aqueous T. sericea extract; E = ethyl acetate T. sericea extract; C = chloroform T. sericea extract; H = hexane T. sericea extract.Antioxidant capacity was determined by DPPH reduction and is expressed as mg of ascorbic acid equivalents per gram of dried plant material extracted.

Table 2 : The IC 50 values (µg/mL) of Caco2 colorectal cancer and HeLa cer- vical cancer cell lines and the LC 50 values (µg/mL) for Artemia franciscana exposed to the T. sericea extracts.
T. sericea extract; W = aqueous T. sericea extract; E = ethyl acetate T. sericea extract; C = chloroform T. sericea extract; H = hexane T. sericea extract; CND = could not determine as inhibition of proliferation or mortality did not exceed 50% at any concentration tested; indicates no inhibition of proliferation was noted at any concentration tested.