The Bioactive Potential of Red Raspberry ( Rubus idaeus L.) Leaves in Exhibiting Cytotoxic and Cytoprotective Activity on Human Laryngeal Carcinoma and Colon Adenocarcinoma

Abstract

In this article, the bioactive potential of red raspberry leaves, a by-product of this widely spread plant, mostly valued for its antioxidant-rich fruits, was determined. The polyphenolic profile and antioxidative properties of red raspberry leaf extract were determined and examined for potential biological activity. Cytotoxic effect, antioxidative/prooxidative effect, and effect on total glutathione concentration were determined in human laryngeal carcinoma (HEp2) and colon adenocarcinoma (SW 480) cell lines. SW 480 cells are more susceptible to raspberry leaf extract in comparison with HEp2 cells. The antioxidative nature of raspberry leaf extract was detected in HEp2 cells treated with hydrogen peroxide, as opposed to SW 480 cells, where raspberry leaf extract induced reactive oxygen species formation. Raspberry leaf extract increased total glutathione level in HEp2 cells. This effect was reinforced after 24 hours of recovery, indicating that induction was caused by products formed during cellular metabolism of compounds present in the extract. Comparison of the results obtained on these two cell lines indicates that cellular response to raspberry extract will depend on the type of the cells that are exposed to it. The results obtained confirmed the biological activity of red raspberry leaf polyphenols and showed that this traditional plant can supplement the daily intake of valuable natural antioxidants, which exhibit beneficial health effects.

Introduction

B ecause of substantial scientific evidence that has emerged regarding the health benefits of medicinal plants, which arise from their rich bioactive profile, the consumption of traditionally used medicinal plants as remedies has recently become a renewed trend. At present, many commercial formulas are available on the market containing red raspberry (Rubus idaeus L.) extracts or its active constituents. Red raspberry has recently received attention from both scientists and consumers because it contains high levels of ellagic acid, a known anticancer agent. Some studies performed provided more comprehensive evidence on the properties assigned to raspberry leaves by folk medicine practices.¹

Although significant attention has been focused on the antioxidant capacity of polyphenols present in red raspberry fruits,^2
–4 there is much less information available on phenolic antioxidants from the leaf tissues of this widely consumed fruit and their biological activity. According to previous studies,^5,6 raspberry leaves are a rich source of flavonoid derivatives, represented by quercetin derivatives, as well as phenolic acids, triterpenes, mineral salts, and vitamin C.^7
–9 The mostly appreciated and well-established bioactive compounds of raspberry leaves are ellagic acid and ellagitannins, whose contents are highly affected by the cultivar and geographical location of the plant's origin.^5,6 Ellagic acid is present in raspberries in three different forms: as ellagitannins, in which hexahydroxydiphenic acid forms esters with a sugar (the main one in terms of content); as free ellagic acid; and as ellagic acid glycosides.^10,11

Although the beneficial activities of raspberry fruit have been established to some extent, there is little information about the biological activity of raspberry leaf. Herbal preparations containing red raspberry leaves, which are used for phytotherapeutic purposes, need more detailed examination in order to confirm their possible functional properties and pharmacological effects. The purpose of this study was to characterize the polyphenolic profile of red raspberry leaf water extract, by using a range of rapid spectrophotometric analyses for the determination of total content of polyphenolic compounds, as well as high-performance liquid chromatography in order to determine the individual polyphenolic compounds, especially ellagic acid. For further characterization, the antioxidant potential of raspberry leaf extract was evaluated using three radical scavenging assays. In order to provide an insight in the antioxidative and prooxidative nature and possible cell specificity response to raspberry extract, two human cell lines established from laryngeal and colon cancers were used: human laryngeal carcinoma (HEp 2) and human adenocarcinoma of the colon (SW 480). Concentrations that were examined are in the range that could be expected to be brought into contact with the gastrointestinal tract. Time of incubation was chosen according to recent findings suggesting that bioactive compounds present in raspberry extract (including phenolic compounds, ellagitannins, and quercetin) are not stable for more than 2 hours in the presence of growth medium and cells.^12
–14 Prooxidative and antioxidative parameters were determined in the cells immediately after treatment, but also in the cells recovered at 24 hours after treatment in order to determine if metabolic products (formed during cellular metabolism of bioactive compounds that were absorbed) affect the events in these two cell lines. The results obtained will contribute to an overall understanding of the antioxidative/prooxidative mechanisms of the compounds present in raspberry leaf extract in relation to the time of exposure of a biological system. Also, the results obtained will show if bioactive compounds and their metabolites present in the raspberry leaf extract can cause different effects on two cell lines established from different tumors originating from the gastrointestinal tract, whose number increases every year and represent a growing problem worldwide. These findings will form the basis for future experimental design in which normal cells and primary cell lines will be used as the biological test system.

Materials and Methods

Preparation of red raspberry leaf extract

Dried red raspberry leaves (R. idaeus L.) were purchased at a local herbal store. Extraction was carried out by pouring 200 mL of boiled distilled water over the plant sample (10 g) at room temperature. After extraction (30 minutes), the extract was filtered through a tea strainer and used for the determination of polyphenolic compounds.

In order to determine the biological effects of red raspberry leaves, the previously prepared extract was evaporated to dryness and redissolved (and concentrated) in 2 mL of 10% dimethyl sulfoxide (DMSO). Different concentrations of raspberry leaf extract (ranging from 0.5× to 2.5×) were prepared in growth medium (Dulbecco's modified Eagle's medium). In household preparation of herbal infusions for consumption as teas, the ratio of 2 g of plant sample and 200 mL of hot water is used; the concentration of bioactive compounds present in such an infusion is defined as 1×. The lowest concentration contained 50% of the bioactive compounds that are present in the ordinary prepared water infusion, and the highest concentration contained a 2.5 times higher concentration of all bioactive compounds in comparison with those present in ordinary water extract. All concentrations of diluted raspberry leaf extract used in the experiment were checked for the formation of microscopically apparent crystals to be sure that all components are in soluble form after dilution.¹⁵

Determination of polyphenolic compounds of raspberry leaf extract

Total phenol content was determined spectrophotometrically according to a method modified from that of Lachman et al.,¹⁶ and the results were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight of herb.¹⁷ The content of flavan-3-ols was determined using the vanillin assay and the reaction with p-dimethylaminocinnamaldehyde as described by Di Stefano et al.¹⁸ The content of flavan-3-ols was expressed as milligrams of (+)-catechin per gram of dry weight. Proanthocyanidins (i.e., condensed tannins) were analyzed by the procedure described by Porter et al.,¹⁹ with some modifications. The quantity of condensed tannins was determined from a standard curve of cyanidin chloride treated with an n-butanol–HCl–Fe(III) mixture and expressed as milligrams of cyanidin chloride equivalents per gram of dry weight. For the determination of flavons, flavonols, and isoflavones the method described by Chang et al.,²⁰ was used. The results were expressed as milligrams of quercetin equivalents/g of dry weight. The content of tannins was determined according to a procedure described by Schneider²¹ and expressed as percentage of the mass of dry plant material. The total hydroxycinnamic acid content was determined using a spectrophotometric method previously described in the European Pharmacopoeia ²² for Fraxini folium, with some modifications. The results were expressed as milligrams of caffeic acid equivalents per gram of dry weight.

High-performance liquid chromatography analysis of phenolic compounds

The extract was filtered through nylon membranes (pore size, 0.45 μm) (Supelco, Bellefonte, PA, USA) before high-performance liquid chromatography analysis. Twenty microliters of sample was injected for high-performance liquid chromatography analysis using a Varian (Walnut Creek, CA, USA) Pro Star Solvent Delivery System 230 and a Varian Pro Star 330 photodiode array detector by using a reversed-phase column (Gemini NX C-18 column, Phenomenex, Torrance, CA, USA) (250×4.6 mm, 5 μm i.d.). The solvents consisted of 3% formic acid in acetonitrile (solvent A) and 3% formic acid in water (solvent B) at a flow rate of 0.9 mL/minute. The elution was performed with a gradient starting at 10% A in B, rising to 40% A after 25 minutes and then to 70% A after 30 minutes, and becoming isocratic for 5 minutes. Chromatograms were recorded at 278 nm. Detection was performed with the photodiode array detector by scanning between 200 and 400 nm, with a resolution of 1.2 nm. Phenolic compounds were identified by comparing the retention times and spectral data with those of standards. The data acquisition and treatment were conducted using Varian Star Chromatography Workstation version 5 software. All analyses were repeated three times.

Determination of antioxidant capacity

2,2-Diphenyl-1-picrylhydrazyl radical scavenging assay

Antioxidant capacity of raspberry leaves extract was determined using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^⋅) scavenging assay described by Brand-Williams et al.²³ The free radical scavenging capacity using the DPPH^⋅ reaction was evaluated by measuring the absorbance at 515 nm after 30 minutes of reaction at room temperature. The results were expressed as millimolar Trolox equivalents, using the calibration curve of Trolox (0–1 mM). All measurements were performed in triplicate.

2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay

The Trolox equivalent antioxidant capacity was estimated by the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization assay.²⁴ The results, obtained from triplicate analyses, were expressed as Trolox equivalents and derived from a calibration curve determined for Trolox (100–1,000 μM).

Ferric reducing/antioxidant power

The ferric reducing/antioxidant power assay was carried out according to a standard procedure by Benzie and Strain.²⁵ All measurements were performed in triplicate. Aqueous solutions of FeSO₄·7H₂O (100–1,000 μM) were used for the calibration curve, and the results are expressed as millimolar Fe(II).

Determination of biological activity in vitro

Human cell lines

Both human laryngeal carcinoma (HEp2) and colon cancer (SW 480) cells were provided as a gift by the Rudjer Boskovic Institute, Zagreb, Croatia. Cells were grown as monolayer cultures in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (GIBCO), 4,500 mg/L glucose, and 1% penicillin/streptomycin.

Cytotoxicity assay

Cytotoxicity of raspberry leaf extract was determined by neutral red assay. Cells were treated with different raspberry leaf extracts in concentration ranges (0.5×−2.5× of the concentration present in the originally prepared raspberry leaf extract) for 1 and 2 hours, with and without a subsequent recovery period that lasted for 24 hours. Subsequently, neutral red assay was carried out, as described by Babich and Borenfreund.²⁶ The intensity of absorbance was measured at 540 nm in a microtiter reader (Cecil Instruments Ltd., Cambridge, UK). Each raspberry extract concentration was tested in quadruplicate, and each experiment was repeated three times. For cytotoxicity testing, data sets are presented in log scale to make a valid linear regression. The 50% effective concentraiton (EC₅₀) was determined from the direction of the equation.¹⁵ Cell viability was calculated using the following equation: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} \% \ cell \ viability = {\rm Absorbance}_{\rm sample} / {\rm Absorbance}_{\rm control} \times 100 \% \end{align*} \end{document}

where Absorbance_sample is the absorbance of cells treated with raspberry leaf extract and Absorbance_control is the absorbance of the corresponding vehicle control (growth medium with 0.1% DMSO). The solvent (DMSO) in the highest applied concentration did not cause any biological effects in control experiments.

Reactive oxygen species determination

Reactive oxygen species (ROS) formation in the cells after the treatment with raspberry leaf extract was determined by the dichlorohydrofluorescein (DCF) assay using a microplate reader.^27,28 Cells were cultured and treated with raspberry leaf extract (0.5×−2.5× of the concentration present in the originally prepared raspberry leaf extract) for 1 and 2 hours, with and without a subsequent recovery period of 24 hours. In order to determine raspberry leaf extract's antioxidative/prooxidative nature in cells in which oxidative stress is already induced, cells were treated with 250 μM H₂O₂ for 30 minutes, in order to induce oxidative stress. After the treatment with H₂O₂, cells were treated with the raspberry leaf extract as previously described.

Treated cells were washed twice, and 2′,7′-dichlorodihydrofluorescein diacetate was added at the final concentration of 50 μM. After 30 minutes, 2′,7′-dichlorodihydrofluorescein diacetate was removed, and cells were washed and loaded with 100 μL of phosphate-buffered saline containing 1% bovine serum albumin.²⁹ Finally, the fluorescence of the cells was measured. The excitation wavelength was 485 nm, and the emission wavelength was 520 nm. Data, reported as fluorescence intensity in relation to cell survival, were obtained according to the following equation: DCF arbitrary units=DCF fluorescence intensity/(Absorbance_sample/Absorbance_control), where Absorbance_sample is the absorbance of cells treated with raspberry leaf extract and Absorbance_control is the absorbance of the corresponding vehicle control (growth medium with 0.1% DMSO). The solvent (DMSO) in the highest concentration applied did not cause any biological effects in control experiments. Each raspberry extract concentration was tested in quadruplicate, and each experiment was repeated three times.

Determination of glutathione level

Intracellular glutathione (GSH) content was examined spectrophotometrically, according to the procedure developed by Tietze.³⁰ During a 24-hour period, the cells were grown to confluence and incubated with raspberry leaf extract. Oxidative stress was induced by treatment of the cells for 30 minutes with 250 μM H₂O₂ prior to treatment with raspberry leaf extract. Cells were treated for 1 or 2 hours without and with a subsequent recovery period of 24 hours. Thereafter, cells were collected, counted, lysed, and centrifuged at 3225.6 g for 15 minutes. GSH was determined in supernatants following the reaction with 5,5′-dithiobis(2-nitrobenzoic acid). The measurements of GSH concentrations were performed in triplicates for each treated Petri dish. Total amount of proteins present in the supernatants was determined by the assay of Bradford.³¹

Statistical analysis

Statistical analyses were performed using SPSS version 8.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance was used to determine whether the means obtained with various groups differ significantly from each other. The significance was established using the Dunnett and Scheffé post hoc test. The probability level of P<0.05 was considered significant. All data are expressed as mean±SD values of the results obtained by three independent measurements.

Results and Discussion

Chemical composition of the raspberry leaf extract

The detailed characterization of polyphenolic profile and the confirmation of significant antioxidant potential of red raspberry leaves determined in this study (Table 1) provide information that extends the knowledge of possible mechanisms that underlie the traditional use of this plant.

Table 1.

Polyphenolic Profile and Antioxidant Capacity of Raspberry Leaf Extract

Raspberry leaf
Composition
Total polyphenols (mg of GAE/g of dry weight)	40.64±1.32
Total flavonoids (mg of GAE/g of dry weight)	24.42±0.89
Flavan-3-ols (vanillin index) (mg of CE/g of dry weight)	3.48±0.21
Flavan-3-ols (p-DAC) (mg of CE/g of dry weight)	1.69±0.19
Total tannins (%)	1.43±0.04
Total proanthocyanidins (mg of CyE/g of dry weight)	1.69±0.07
Total flavones, flavonols, and isoflavones (mg of QE/g of dry weight)	3.43±0.34
Hydroxycinnamic acids (mg of CAE/g of dry weight)	10.41±0.76
Caffeic acid (mg/g of dry weight)	0.55±0.08
Chlorogenic acid (mg/g of dry weight)	0.70±0.07
Σ Ellagic acid derivatives (mg/g of dry weight)	3.79±0.19
Σ Quercetin derivatives (mg/g of dry weight)	2.67±0.15
Antioxidant capacity
ABTS (mM Trolox)	4.00±0.89
DPPH (mM Trolox)	6.84±0.22
FRAP [mM Fe(II)]	20.77±1.92

ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); CAE, caffeic acid equivalents; CE, catechin equivalents; CyE, cyanidin chloride equivalents; DAC, dimethylaminocinnamaldehyde; DPPH, 2,2-diphenyl-1-picrylhydrazyl; FRAP, ferric reducing/antioxidant power; GAE, gallic acid equivalents; QE, quercetin equivalents.

The total phenol content of raspberry leaf amounted to 40.64 mg of GAE/g of dry weight, which is significantly higher than total phenols obtained by Dvaranauskaite et al.,⁵ who found a range of 0.3–2.2 mg of GAE/g of total phenols in different dry raspberry leaf cultivars, and Venskutonis et al.,³² who determined up to 11.8 mg of GAE/g of dry weight of total phenols in raspberry leaves derived from different geographical locations. Compared with the polyphenolic content of green tea, which is considered to contain 30–42% polyphenols on a dry weight basis,³³ the polyphenolic content of raspberry leaves obtained in our study exhibits up to 10-fold lower concentration. However, compared with other medicinal herbs, raspberry leaf has been ranked on a high ninth position based on the total phenol content among 70 other medicinal plants.³⁴

The content of total flavonoids was 24.42 mg of GAE/g of dry weight, which indicates that flavonoid polyphenolics account for slightly more than half of total phenolic compounds. This is in accordance with the previously established composition of raspberry leaf polyphenols,^5,6 which revealed the prevalence of quercetin derivatives—ellagic acid and ellagitaninns—as the predominant polyphenolics. The high contents of total flavones, flavonols, and isoflavones (3.43 mg of quercetin equivalents/g of dry weight) indicate that quercetin derivatives account for a high portion of flavonoids, which was also confirmed by the high-performance liquid chromatography analysis of red raspberry leaf extract. Because raspberry leaves contain numerous quercetin and ellagic acid derivatives, the identification of every specific compound is difficult owing to the lack of standard compounds that could elucidate their exact structure. Therefore in this study the sum of ellagic acid and quercetin derivatives was provided, which was obtained by expressing the unknown quercetin derivative as a quercetin compound. The sum of obtained concentrations of quercetin derivatives was 2.67 mg/g of dry weight, which is slightly lower than the spectrophotometrically obtained value for total flavones, flavonols, and isoflavones. Compared with the range of tannin content (2.62–6.87% of dry weight) for different raspberry leaves cultivars obtained by Gudej and Tomczyk,⁶ the content of red raspberry leaf tannins determined in our study is significantly lower (1.34%). The low total content of hydroxycinnamic acids (10.41 mg of caffeic acid equivalents/g of dry weight) and a low content of flavan-3-ol indicate a poorer distribution of these polyphenolic compounds in raspberry leaves. Despite the rich profile of polyphenolic compounds in red raspberry leaf extracts, which include the presence of caffeic acid (0.55 mg/g of dry weight) and chlorogenic acid (0.70 mg/g of dry weight), ellagic acid and its derivatives are the predominant phenolic compounds (3.79 mg/g of dry weight). High concentrations of ellagic acid and quercetin derivatives, as well as caffeic and chlorogenic acid, in red raspberry leaves indicate the strong bioactive potential of raspberry leaf extract, which is in compliance with some previously reported findings.^35,36

Cytotoxicity

Raspberry leaf extract is a complex mixture of many compounds that have been proven to be unstable, so the cells were treated with raspberry leaf extracts for a maximum of 2 hours. The effect of metabolic products formed after absorption and cellular metabolism of the main constituents of raspberry leaf extract was determined in cells that were incubated for 24 hours after complete removal of extract. It is well documented that the stability of numerous bioactive compounds varies with the cell culture conditions (pH 7.0–7.4). Under these conditions, the half-life of polyphenols is less than 2 hours in the presence of cells. Kern et al.³⁷ recorded that anthocyanidins are degraded under cell culture conditions for 30 minutes. As a consequence, phenolic acids are formed and rapidly degraded, with formation of H₂O₂. Long et al.¹² determined that phenolic compounds in the presence of culture medium (including Dulbecco's modified Eagle's medium, which was used in this work) autooxidize and form H₂O₂. Furthermore, Larrosa et al.¹³ determined that ellagitannines are rapidly degraded to ellagic acid and taht the biological effect of ellagitannines is actually a consequence of the biological effect of newly formed ellagic acid. Van der Woude et al.¹⁴ proved that quercetin degrades in phosphate buffer with a half-life of 10 hours. In cell culture growth medium, the half-life of quercetin is 2 hours.

Survival curves and the highest nontoxic concentrations of raspberry leaf extract were determined after 1 and 2 hours of cell treatment because in this period of time, biologically active compounds are stable enough to accomplish their biological effect,^38,39 without the formation of secondary metabolites or spontaneous degradation products, which could influence the results overall.¹⁵ The concentrations investigated were chosen according to the expected quantities of biologically active compounds that can be introduced into an organism by periodical and/or everyday consumption of raspberry leaf extract. Concentration 1× indicates the concentration of biologically active compounds obtained in a water infusion obtained after extraction of 2 g of raspberry leaves with 200 mL of hot water.

The cytotoxicity of raspberry leaf extract proved to be cell type specific.^35

–39 Raspberry leaf extract proved to be more toxic to SW 480 than HEp2 cells after a prolonged time of exposure. After 1 hour of exposure, no toxic effect was seen (Fig. 1c), but during the subsequent 24-hour recovery period, the cytotoxic effect of the raspberry leaf extract can be seen (Fig. 1a). The EC₅₀ value calculated from the survival curve after linear regression (Table 2) indicates that SW 480 cells are more resistant to raspberry leaf extract (EC₅₀=3.25× estimated value) than HEp2 cells (EC₅₀=2.34×). These findings point out that during 1 hour of incubation, irreversible events take place, and consequences (meaning cytotoxic effect of higher concentrations) are obvious after 24 hours (i.e., after a period of one cell cycle).

FIG. 1.

Survival of human laryngeal carcinoma (HEp2) and human colon cancer (SW 480) cells following (a) 1-hour treatment with raspberry leaf extract after subsequent recovery period (24 hours), (b) 2-hour treatment with raspberry leaf extract after subsequent recovery period (24 hours), (c) 1-hour treatment with raspberry leaf extract, and (d) 2-hour treatment with raspberry leaf extract. Results are shown as log values obtained in three experiments. *Represents how many times the extract is diluted (or concentrated) in comparison with the original (1×). The cell viability obtained after treatment with the control was considered to be 100%. Relative cell viability obtained after treatment with other samples was obtained by comparison with the control. Estimated cytotoxicity 50% effective concentration values of raspberry leaf extract in human laryngeal carcinoma cells are as follows: 2.39× for HEp2 cells and 3.25× for SW 480 cells after 1 hour of incubation and 2.13× for HEp2 cells and 1.48× for SW 480 cells after 2 hours of incubation.

Table 2.

50% Effective Concentration Values Obtained After Treatment of HEp2 and SW 480 Cells with Different Concentrations (0.5–2.5×) of Raspberry Leaf Extract

	EC₅₀ (×)
Cell line, treatment	Without recovery period	With 24-hour recovery period after treatment
HEp2
1 hour	—	2.34×
2 hours	2.33×	2.13×
SW 480
1 hour	—	3.25×
2 hours	3.25×	1.14×

The 50% effective concentration (EC₅₀) values were analyzed by linear regression and determined from the direction of the equation.

A similar relationship can be seen between the cells treated for 2 hours without recovery; during 2 hours of incubation, raspberry leaf extract caused a cytotoxic effect on both cell lines. The HEp2 cell line is still more sensitive to the cytotoxic effect of the compounds present in the extract (EC₅₀=2.33×) in comparison with the SW 480 cell line (EC50=3.25× estimated value) (table 2). This relationship is changed during the recovery period, during which increased cytotoxicity of raspberry leaf extract can be seen in SW 480 cells (Fig. 1b; EC₅₀=1.14×) in comparison with HEp2 cells (EC₅₀=2.13×) (Table 2).

Caffeic, chlorogenic, and ellagic acids, together with quercetin, are the most abundant compounds present in raspberry leaf extract. According to the results obtained by Lee et al.,⁴⁰ the cytotoxic effect of caffeic acid was observed at the concentration of 25 μM, following the 24-hour incubation, whereas chlorogenic acid was cytotoxic at millimolar concentrations for human oral squamous carcinoma cells. Fjaeraa and Nånberg⁴¹ demonstrated time- and concentration-dependent inhibitory effects of ellagic acid (1–100 μM) on human neuroblastoma cell number during a 96-hour period. Tasaki et al.³⁶ proved that ellagic acid has no toxic effect in an in vivo subchronic toxicity study using F344 rats. Sergediené et al.⁴² reported that quercetin caused a strong cytotoxic effect accompanied by prooxidant events at the concentration of 300 μM. In our study, the concentrations of caffeic acid (152.6 μM), ellagic acid derivatives (560.27 μM), and quercetin derivatives (883.42 μM) determined in raspberry leaf extract were much higher than those for which a cytotoxic effect was proved. Additionally, the raspberry leaf extract investigated in this study is a mixture of polyphenolic and non-polyphenolic compounds, which can interact with each other and cause different biological effects, depending on the time of exposure and concentration, so a synergistic effect of different compounds in raspberry leaf extract must be taken into account. Mertens-Talcot et al.⁴³ determined that ellagic acid significantly potentiated the effects of quercetin (at 5 and 10 μmol/L each) in the reduction of proliferation and viability and the induction of apoptosis in human leukemia cells. A possible but insufficiently investigated mechanism of polyphenol cytotoxicity may be related to their prooxidant properties because the same polyphenol compounds could behave as both prooxidants and antioxidants, depending on the concentration.⁴²

Antioxidant/prooxidant activity of raspberry leaf extract

An overview of the results obtained in this study indicated that red raspberry leaves posses high antioxidant capacity owing to its rich and diverse polyphenolic profile (Table 1). The antioxidant capacity of raspberry leaves determined with the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay amounts to 4.00 mM Trolox, or 6.84 mM Trolox determined with the DPPH assay, whereas the reducing power determined by using the ferric reducing/antioxidant power assay amounts to 20.77 mM Fe(II). These results are comparable to the ones obtained for the antioxidant capacity of green teas,⁴⁴ indicating the high antioxidant potential of red raspberry leaf extract.

Biological activity of polyphenols is considered to be connected with its antioxidant properties, which are mainly due to its ability to scavenge free radicals and ROS and to form complexes with metal ions, thus preventing oxidation of metals with oxygen yielding ROS.⁴² Also, polyphenols can autooxidize in aqueous medium and form highly reactive OH^⋅ radicals in the presence of transition metals once they get to the cell. In that case, polyphenols may act as substrates for peroxidases and other metalloenzymes, yielding quinone- or quinomethide-type prooxidant and/or alkylating products.⁴² Experiments conducted on human cell lines revealed a strong relationship among concentration, cell type, time of exposure, and antioxidant/prooxidant character of raspberry leaf extract. According to our results, raspberry leaf extract did not induce ROS formation in HEp2 cells treated for 1 hour (Fig. 2a) except at the highest concentration, which caused significant increase in ROS formation. After 24 hours of recovery, no ROS formation can be determined (Fig. 3a). After 2 hours of exposure, raspberry leaf extract caused a sigificant increase in ROS formation in comparison with the control (Fig. 2b), whereas no difference was observed in the cells that were allowed to recover for 24 hours after treatment (Fig. 3b). Because ROS are formed continuously in cells as a consequence of both oxidative biochemical reactions and external factors, the cells were treated with H₂O₂ prior to treatment with raspberry extract. The presence of free radicals originating from H₂O₂ did not increase the prooxidant nature of raspberry leaf, but, on the contrary, as the concentration of raspberry extract increased, the presence of ROS decreased. This effect was especially noticed in the cells that were allowed to recover for 24 hours (Fig. 3b). The highest concentration (2.5×) of raspberry leaf extract induced ROS formation (Figs. 2b and 3), so this result is in accordance to the findings of Sergediené et al.,⁴² who reported that the cytotoxic effect of polyphenols is associated with the prooxidant events. The SW 480 cell line exhibited a lower basal level of ROS in the cell in comparison with HEp2 cells. Non-cytotoxic concentrations of raspberry leaf extract slightly increased ROS formation in a dose-dependent manner after 1 hour of incubation (Fig. 2a), and this effect was more obvious in the case of 2 hours of incubation (Fig. 2b). Previous treatment with H₂O₂ caused a significant increase of ROS formation in the cells treated for 1 hour with 0.5× and 0.75× raspberry leaf extract, whereas this effect was diminshed after treatment with higher concentrations of raspberry leaf extract. This effect was noticed for numerous compounds such as dopamine that act as an antioxidant at concentrations less than 500 μM but at higher concentrations act as a prooxidant.²⁷ Because prolonged incubation of SW 480 cells after the recovery period of 24 hours caused a significant increase in the toxicity of raspberry leaf extract, one of the possible explanation for such an effect could be a cytotoxic effect of phenoxyl radicals formed during cell incubation with raspberry leaf extract.

FIG. 2.

Comparison of reactive oxygen species formation in HEp2 and SW 480 cells treated with raspberry leaf extract for (a) 1 hour and (b) 2 hours with and without previous treatment with 250 μM H₂O₂. Pooled data were obtained from three experiments and are mean at the point±SD values. *P≤.05, statistically significant increase compared with the control.

FIG. 3.

Comparison of reactive oxygen species formation in HEp2 and SW 480 cells treated with raspberry leaf extract for (a) 1 hour and (b) 2 hours after a subsequent recovery period (24 hours) with and without previous treatment with 250 μM H₂O₂. Pooled data were obtained from three experiments and are mean at the point±SD values. *P≤.05, statistically significant increase compared with the control.

Prolonged incubation of the SW 480 cells treated with H₂O₂ resulted in a significant increase in ROS formation, but the level of detected ROS did not differ from that in the cells treated only with raspberry leaf extract (Fig. 2b). During the recovery period, ROS induction was decreased, and all measured values were in the range of ROS measured in nontreated cells (Fig. 3b). This effect can be explained by findings published by Sakihama et al.,⁴⁵ who proposed that polyphenols can act as antioxidants by donating electrons to different enzymatic and nonenzymatic systems in the cell for the detoxification of H₂O₂ produced under stress conditions.

The prooxidative nature of polyphenols present in raspberry leaf extract could be responsible for apoptosis induction, as well as ROS can induce DNA fragmentation.⁴⁶ The ability of polyphenols to bind to DNA and to cause ROS formation is similar to antitumor therapy.⁴⁷ Furthermore, polyphenols can act as receptors or enzymes; they can inhibit or stimulate different signal pathways.⁴⁷ Young et al.⁴⁸ analyzed the level of lipid peroxidation in the blood of volunteers who consumed a polyphenol-rich juice for 7 days. The level of malondialdehyde decreased with time of consumption. At the same time, the level of 2-adipine semialdehyde, which is a biomarker of protein oxidative damage, increased during the testing period, pointing out that polyphenols have antioxidative and prooxidative effects at the same time, but also in the blood plasma there are different levels that can give an opposite response to compounds that were ingested by food. These effects bring into question the global prooxidative/antioxidative nature of the compounds present in blood. Specific cell structures can be damaged or protected by the same compound. Which event will happend depends upon specific interactions among cell constituents and compounds absorbed from food.⁴⁸

Determination of GSH level

According to our findings, during 1 hour of incubation of HEp2 cells, lower concentrations of raspberry leaf extract increased the total level of GSH in comparison with the control. In cells treated with H₂O₂ and raspberry leaf extract the level of GSH significantly decreased in comparison with the control treated with H₂O₂. During 2 hours of incubation, higher concentrations of raspberry leaf extract (0.75×, 1×, and 2.5×) induced the total level of GSH in HEp2 cells treated with raspberry leaf extract and in cells that were previously treated with H₂O₂. The highest concentration of raspberry leaf extract decreased GSH level. Exposure of the HEp2 cells to raspberry leaf extract for 1 hour followed by 24 hours of recovery caused a decrease in GSH level. The highest concentrations of the extract caused an increase in total GSH level after 2 hours of incubation with recovery. In SW 480 cells, biphasic modulation of GSH level was noticed; during 1 and 2 hours of incubation, lower concentrations of raspberry leaf extract (0.5× and 0.75×) increased the level of total GSH, whereas higher concentrations caused a decrease in total GSH level. During the subsequent recovery period, induction of GSH was observed in the cells treated with 0.5× raspberry leaf extract, whereas other concentrations did not influence GSH level. Recent findings suggest that low concentrations of quercetin lead to a small increase of cellular thiols and total antioxidative activity, but higher concentrations lead to a progressive decrease in the total antioxidative activity.⁴⁹ In our previous study,⁵⁰ we had shown that constitutive levels of GSH are higher in cisplatin-resistant CK2 cells of laryngeal carcinoma than in the parental HEp2 cells, but also that quercetin induces a significant increase in the GSH levels of both cell lines, again pointing toward the importance of cellular status wherever the response to flavonoid exposure is an issue. H₂O₂ caused a decrease in total level of GSH in HEp2 cells treated with raspberry leaf extract for 1 hour, but in the cells treated for 2 hours, the level of GSH increased with the increase of raspberry leaf concentration. After ROS induction in SW 480 cells, raspberry leaf extract did not affect the total level of GSH, except the highest concentrations (1× and 2.5×), which caused a decrease of antioxidant status. This effect was noticed in cells treated for both 1 and 2 hours.

In the HEp2 cells that were allowed to recover for 24 hours after the treatment with H₂O₂ and raspberry leaf extract, it can be seen that the highest concentrations of extract (1× and 2.5×) caused induction of total GSH after 2 hours of incubation, whereas in SW 480 cells, 1 hour of incubation with higher concentrations (0.75×, 1×, and 2.5×) induced GSH. Two hours of incubation caused the opposite effect, resulting in a drastic decrease in antioxidant status in SW 480 cells (Table 3).

Table 3.

Concentration of Glutathione in HEp2 and SW 480 Cells, Obtained After 1 and 2 Hours of Incubation with Different Concentrations of Raspberry Leaf Extract With and Without Previous Treatment with 250 μM H₂O₂ and Without and After the Recovery Period (24 Hours)

	Glutathione (nM/mg of protein)
	After recovery period (24 hours)				Without recovery
	1 hour	1 hour+H₂O₂	2 hours	2 hours+H₂O₂	1 hour	1 hour+H₂O₂	2 hours	2 hours+H₂O₂
HEp2 cells
Control	0.392±0.056^{b c d e}	0.14±0.018^{b c d e}	0.74±0.088^{b c d e}	0.215±0.06^{d e}	0.29±0.023^{b c d}	0.91±0.041^{b c d e}	1.25±0.08^{c d e}	1.39±0.125^{b c d e}
0.5×	0.412±0.019^{a c e}	0.16±0.015^{a c d e}	0.45±0.012^{a c d e}	0.2±0.21^{d e}	0.46±0.158^{a d}	0.46±0.016^{a c}	1.39±0.125^{c d e}	2.303±0.136^a,e
0.75×	0.132±0.028^{a b d}	0. 78±0.242^{a b e}	0.31±0.009^{a b d e}	0.2±0.021^{d e}	0.603±0.042^{a d}	0.6±0.04^{a b d e}	1.656±0.19^{a b e}	2.394±0.09^{a d e}
1×	0.13±0.027^{a c e}	0.72±0.005^{a b e}	0.415±0.06^{a b c e}	1.12±0.067^{a b c e}	0.515±0.114^{a d}	0.476±0.09^{a c}	1.81±0.102^{a b e}	1.908±0.09^{a c e}
2.5×	0.212±0.04^{a b e}	0.33±0.024^{a b c d}	0.29±0.013^{a b c d}	1.02±0.064^{a b c d}	0.275±0.021^{b c d}	0.408±0.034^{a c}	3.59±0.413^{a b c d}	2.89±0.319^{a b c d}
SW 480 cells
Control	1.77±0.202^{b c}	2.0±0.067^{c d e}	1.52±0.28^b	1.43±0.33^{b d}	1.05±0.0.128^{b c}	1.31±0.24^{d e}	1.51±0.098^{b c d e}	2.1±0.094^{d e}
0.5×	3.21±0.11^{a c d e}	1.9±0.125^{c d e}	2.32±0.19^{a c d e}	1.68±0.21^{a d e}	2.42±0.11^{a c d e}	1.28±0.124^{d e}	2.25±0.19^{a c e}	2.1±0.099^{d e}
0.75×	1.856±0.12^{a b}	2.48±0.22^{a b e}	1.4±0.14^b	1.53±0.214^{d e}	3.09±0.45^{a b d e}	1.139±0.306^{d e}	3.35±0.25^{a b d e}	2.11±0.15^{d e}
1×	2.1±0.11^{a b}	2.41±0.31^{a b e}	1.62±0.157^b	0.67±0.066^{a b c}	1.09±0.18^{b c}	1.019±0.15^{a b c e}	2.07±0.25^{a c}	1.32±0.13^{a b c e}
2.5×	1.94±0.14^b	3.54±0.62^{a b c d}	1.62±0.34^b	0.71±0.007^{a b c}	1.23±0.14^{b c}	1.582±0.18^{a b c d}	1.85±0.091^{a b c}	1.49±0.18^{a b c e}

Pooled data were obtained from three experiments and are mean at the point±SD values.

Statistically significant difference in comparison with control.

Statistically significant difference in comparison with 0.5×.

Statistically significant difference in comparison with 0.75×.

Statistically significant difference in comparison with 1×.

Statistically significant difference in comparison with 2.5×.

Conclusions

This research is a contribution to the characterization of polyphenolic compounds and antioxidant properties of red raspberry leaf and their biological activity on cell cultures in terms of cytotoxicity, ROS formation, and GSH induction. Quercetin derivatives were recognized as the most abundant phenolic compounds, followed by ellagic acid derivatives and caffeic and chlorogenic acids. Raspberry leaf extract exhibits cytotoxic effects on both HEp2 and SW 480 cell lines, and cell survival decreases with the incubation interval. SW 480 cells are more susceptible to raspberry leaf extract in comparison with HEp2 cells. Raspberry leaf extract did not induce ROS formation in HEp2 cells, and its antioxidative nature was detected in HEp2 cells that were treated with H₂O₂. In contrast, raspberry leaf extract induced ROS formation in SW 480 cells, and its antioxidative nature was not detected on this cell line. Raspberry leaf extract increased total GSH level in HEp2 cells. This effect was reinforced after 24 hours of recovery, indicating that induction was caused by products formed during cellular metabolism of compounds present in the extract.

In SW 480 cells, biphasic modulation of the GSH level was noticed; lower concentrations of extract increased, whereas higher concentrations decreased, total GSH level. These results indicate that expected cell response to raspberry leaf extract is time, concentration, and cell type dependent.

Footnotes

Acknowledgments

This work was supported by projects 058-0000000-3470 and 058-0582261-2246 from the Ministry of Science, Education and Sports, Republic of Croatia.

Author Disclosure Statement

No competing financial interests exist.

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