Journal of Natural Medicines

, Volume 67, Issue 3, pp 446–451

Effect of phenylalanine on Taxol production and antioxidant activity of extracts of suspension-cultured hazel (Corylus avellana L.) cells

Authors

  • Ebrahim Bemani
    • Department of Plant Biology, Faculty of Biological ScienceTarbiat Modares University (TMU)
    • Department of Plant Biology, Faculty of Biological ScienceTarbiat Modares University (TMU)
  • Ayatollah Rezaei
    • Department of Plant Biology, Faculty of Biological ScienceTarbiat Modares University (TMU)
  • Mitra Jamshidi
    • Department of Plant Biology, Faculty of Biological ScienceTarbiat Modares University (TMU)
Original Paper

DOI: 10.1007/s11418-012-0696-1

Cite this article as:
Bemani, E., Ghanati, F., Rezaei, A. et al. J Nat Med (2013) 67: 446. doi:10.1007/s11418-012-0696-1

Abstract

Taxol is produced by a few microorganisms and plants such as yew (Taxus sp.). Recent researches have shown that hazel (Corylus avellana L.) is also able to produce Taxol. In the present study, effects of different concentrations of phenylalanine (Phe) on the production of Taxol, antioxidant activity, and cytotoxic effects of extracts of suspension-cultured hazel cells were investigated. The cells were treated with different concentrations of Phe on day 7 of subculture and were harvested on day 14. The results showed that the amounts of Taxol and antioxidant activity were increased by increasing the phenylalanine supply. Interestingly, the cytotoxic effects of hazel cell extract were even stronger than that of pure Taxol (standard), suggesting hazel cell extract as a novel and suitable probe for treating human cancer. Application of phenylalanine to hazel cells exaggerates their effects.

Keywords

Antioxidant activityCorylus avellanaCytotoxicityPhenylalanineTaxol

Introduction

Taxol (generic name, paclitaxel) is a diterpenoid alkaloid which was first isolated from Taxus brevifolia [1]. Since 1992, Taxol has been approved by the Food and Drug Administration (FDA) against a wide range of tumors [2]. A problem with the extraction of Taxol from the yew tree is the fact that yew is one of the slowest growing trees in the world, and is therefore an environmentally protected species. Damage to three mature trees is a prerequisite for obtaining enough Taxol to treat only one patient. Therefore, finding an alternative source for Taxol seems to be necessary [3].

There is a possibility of increasing the production of secondary metabolites in suspension cultures via selection of a suitable cell line, optimizing the culture conditions [4], application of different elicitors [5, 6], and improving the methods for isolation of secondary metabolites [7]. Elicitors have been found to improve the production and accumulation of secondary metabolites not only in the intact plants but also in cell and tissue cultures [8]. The stimulation of Taxol production in Taxus cultures by the addition of different elicitors to the culture medium has been reported by several authors [911] suggesting a phytoalexin function for Taxol in Taxus plants.

There are many strategies for improving the production of secondary metabolites in plant cell cultures and feeding a precursor is an effective one. According to information on biosynthetic pathways, synthesis of the plant secondary metabolites can be improved by addition of precursors or intermediates to the culture media. Efforts to increase production of certain products by adding ingredients to the medium have been effective in many cases. Ouyang [12] reported that accumulation of phenylethanoid glycosides was promoted by feeding cell cultures of Cistanche deserticola with phenylalanine (Phe) and tyrosine, which are precursors involved in biosynthesis of phenylethanoid glycosides. Feeding cell cultures of Taxus cuspidata with aromatic carboxylic acid and amino acid improved the biosynthesis and excretion of Taxol [13].

Recently, a few studies have also introduced hazelnut as a Taxol (and related taxane) producing species among flowering plants. Hazel (Corylus avellana L.) is an individual plant among angiosperms which has been reported to be able to produce Taxol and other taxanes [14, 15]. The main benefit of producing taxanes by hazel cell cultures is that hazel is widely available, fast growing in vivo, and easier to cultivate in vitro than yew [15, 16]. Synthesis of Taxol necessitates the attachment of the N-benzoyl-3-phenylisoserine side chain to the C-13 hydroxyl group of 10-deacetyl baccatin III (10-DABIII) and the acetylation of the 10β-hydroxyl position [17]. Phenylalanine is an amino acid which is involved in the synthesis of the side chain of Taxol. Addition of Phe to the medium of Taxus calli resulted in enhanced content of Taxol [18]. Phenylalanine is also the main precursor of the phenolics metabolism pathway. Phenolic compounds are major non-enzymatic components of the antioxidant system of plants [19] and there is a relationship between phenolics content and the antioxidant activity of plant cells [20]. In the present study, the effect of phenylalanine at different concentrations on production of Taxol by suspension-cultured hazel cells and cytotoxicity of cell extracts on human cancer cells were investigated. Moreover, the phenolics content of phenylalanine-treated cells and its relationship with antioxidant activity of the extracts were elucidated.

Materials and methods

Chemicals, viz. diphenyl picrylhydrazyl (DPPH), Paclitaxel, Folin–Ciocalteu regent and methyl thiazol tetrazoliom (MTT), were purchased from Sigma (St. Louis, MO, USA). RPMI 1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were obtained from Gibco (Grand Island, NY, USA). All other reagents were obtained from Merck (Germany).

Cell culture and treatment

Calli of Corylus avellana were established from seed fragments on modified B5 medium [21] supplemented with 0.2 mg/l benzyladenine and 1.86 mg/l naphthalene acetic acid and solidified with 8 g/l plant agar. The calli emerged after 10 days in darkness at 25 °C and were sub-cultured every 2 weeks. Suspension cultures were established by transferring 1 g callus in 30 ml B5 medium and were kept at 25 °C in darkness, on rotary shakers at 120 rpm. Suspensions were also sub-cultured every 2 weeks. Treatment with phenylalanine was conducted after frequent subcultures when cells reached genetic stability. Phenylalanine was dissolved in distilled water sterilized by filtration (0.2 μm, Millipore). The filtrate was added to suspension cultures of cells on day 7 (start of logarithmic growth phase) to final concentrations of 0, 0.15, 1.5, 3, and 6 mM. Concentrations of Phe were selected according to preliminary studies as well as the available literature [4, 22]. The cells were harvested on day 14.

LC–MS detection and HPLC quantification of Taxol in hazel cells

Taxol was extracted from medium and powdered dried cells by methods previously described by Wu and Lin [23] with some modifications. In brief, the dried cells were pulverized and suspended in 10 ml methanol, filtered, and the filtrate was air-dried then re-dissolved in dichloromethane:water (1:1, v/v) followed by centrifugation at 2,400 g. The dichloromethane phase was collected, air–dried, and re-dissolved in 100 μl of methanol (HPLC grade) and filtered passing through a 0.45-μm syringe filter, before being injected into HPLC. For identification of Taxol, samples were analyzed by an Agilent 6410 HPLC system coupled to a Triple Quad ion trap mass spectrometer, equipped with an electro spray ion source. The column was an Eclipse C18 (3.5 μm particle size, 100 mm length, and 4.6 mm width). The flow rates of solvent and injection volume were 350 μl/min and 50 μl/min, respectively. Mobile phase A consisted of 0.1 % formic acid in distilled water:methanol 70:30 (v/v), and mobile phase B consisted of 0.1 % formic acid in methanol. An isocratic flow (1 ml/min) was used for 5 min. The gradient then started at 100 % A and linearly increased to 100 % B over the course of 25 min, followed by an isocratic gradient of 100 % B for 10 min, (total run time 40 min). Mass spectra were acquired in product ion mode. The presence of Taxol in the samples was verified by ESI/MS in product ion mode according to structurally diagnostic ions in the LC–MS spectra. The Taxol content in the extracts was quantified by a HPLC system (Knauer, Germany), equipped with a C-18 column (Perfectsil Target ODS3, 5 μm particle size, 250 mm length, and 4.6 mm width, MZ-Analysentechnik, Mainz, Germany). Taxol was eluted with a linear gradient of acetonitrile and water (45:55) at a flow rate of 1 ml/min and was detected at 227 nm using a UV detector (PDA, Germany). Identification of Taxol was accomplished by comparison of retention times with authentic standard [6].

Preparation of methanolic cell extract

Dried cells (ca. 3 g) from each treatment were pulverized and suspended in 50 ml of methanol followed by centrifugation at 1,600 g. The supernatant was air-dried. The dried material (50 mg) was re-dissolved in 5 ml of methanol and used for determination of total phenolics content, DPPH free radical scavenging capacity and ferric ion reducing/antioxidant power (FRAP) assay.

Determination of total phenolics

In order to determine total phenolics contents of the cells, the Folin–Ciocalteu method was used [24]. In brief, 0.5 ml of methanolic extract was mixed with 0.5 ml of 0.1 N Folin–Ciocalteu reagent. The mixture was kept for 2–5 min, followed by the addition of 1.0 ml of 20 % Na2CO3. After 10 min of incubation at ambient temperature, the mixture was centrifuged for 8 min (12,000 g). The absorbance of the supernatant was measured at 730 nm. The results were expressed as gallic acid equivalents in milligram per milliliter of extracts.

DPPH free radical scavenging activity

The antioxidant activity of the methanolic extract was measured by the method described by Maikai and coworkers [25] with some modifications. In brief, 0.002 % DPPH was dissolved in methanol and 1.5 ml of this solution was added to 0.5 ml of methanolic cell extract. The solution mixture was kept in the dark for 30 min and its optical density was measured at 517 nm by spectrophotometry (Cintra 6, GBC, Australia). The blank contained 1.5 ml of 0.002 % DPPH solution. The capacity of the cell extract to scavenge free radicals was calculated according to the following formula:
$$ {\text{\% }}\;{\text{of}}\;{\text{inhibition}}\;{\text{of}}\;{\text{DPPH}}\;{\text{activity}} = \left[ {{{\left( {A - B} \right)} \mathord{\left/ {\vphantom {{\left( {A - B} \right)} A}} \right. \kern-\nulldelimiterspace} A}} \right] \times 100 $$
where A is optical density of blank and B is optical density of sample. The positive control was prepared using ascorbic acid at a concentration of 10 mg/ml.

Ferric ion reducing/antioxidant power assay

Antioxidant activity was determined by FRAP assay as described by Oyaizu [26]. The methanolic cell extract (1 ml) was mixed with 1.25 ml of 1 % potassium ferricyanide. The mixture was incubated at 50 °C for 20 min and then 1.25 ml of 10 % trichloroacetic acid was added to the mixture followed by centrifugation at 1,000 g for 10 min. The supernatant (1.25 ml) was mixed with distilled water (1.25 ml) and ferric chloride solution (0.25 ml, 0.1 %). The absorbance was measured at 700 nm. Ascorbic acid at a concentration of 10 mg/ml was used as positive control.

Cancer cell culture

The breast cancer cell line (MCF-7) was obtained from the Pasteur Institute of Iran and was cultured in RPMI 1640 medium, supplemented with 10 % FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were incubated at 37 °C, 5 % CO2 and sub-cultured every 4 days.

Evaluation of cytotoxicity of hazel cell extracts

The morphology of cancer cells before and after exposure to hazel cell extracts was studied under an inverted microscope (Nikon TE-2000, Japan). In addition, the cytotoxic effects of Taxol and hazel cell extracts were investigated by MTT colorimetric assay [27]. The dried hazel cells were pulverized and suspended in 10 ml methanol and then filtered. The filtrate was air-dried and re-dissolved in dichloromethane:water (1:1, v/v) followed by centrifugation at 2,400 g. The dichloromethane phase was collected, air-dried, and after quantification of their Taxol content with HPLC, appropriate amounts of the extracts were re-dissolved in methanol such that they contained 25 and 50 nM Taxol. Standards of Taxol solution at the same concentrations were also prepared. For cytotoxic effects of hazel cells extract and standard Taxol, 100 μl of MTT solution (5 mg/ml PBS) was added to MCF-7 cell cultures followed by incubation at 37 °C, 5 % CO2, for 4 h culture conditions, allowing formazan formation. Then the upper solutions were removed and 250 μl of dimethyl sulfoxide (DMSO) was added to each sample in order to dissolve formazan crystals. After 30 min, equivalent volumes of upper solutions were transferred to a 96-well plate of ELISA Reader (Anthuos 2020, Australia) and absorbance of samples was measured at 492 nm. Cytotoxity was determined by assessment of density of live cells according to the following formula
$$ \% \;{\text{viability}}\;{\text{of}}\;{\text{cancer}}\;{\text{cells}} = \left[ {{{A - B} \mathord{\left/ {\vphantom {{A - B} A}} \right. \kern-\nulldelimiterspace} A}} \right] \times 100 $$
where A is optical density of control cancer cells and B is optical density of sample.

Statistical analysis

All observations and experiments were repeated at least 3 times with 3 independent replicates. Statistical analysis was performed using Student’s t test, and the differences between the treatments were deemed significant at a level of p ≤ 0.05

Results

The effect of different concentrations of Phe on the growth of hazel cells is shown in Figure 1. The results showed that Phe up to 3 mM had no significant effect on the growth of hazel cells and at 6 mM adversely affected the dry weight (DW) of the cells (5.22 g/l vs. 7.7 g/l in control conditions). The effect of different concentrations of Phe on the contents of extracellular Taxol (released to the medium) and intracellular Taxol (cell-associated) are shown in Figures 2a, b, respectively. As shown, Phe at concentrations of 3 and 6 mM significantly increased Taxol production by hazel cells so that intracellular and extracellular Taxol contents of 6 mM-Phe-treated cells were approximately 10 and 3 times higher than those of the control cells (22.04 mg/kg DW and 48 μg/l, respectively). The level of phenolic compounds was also increased by high concentrations of Phe (3 and 6 mM) (Fig. 3a). Consequently, free radical scavenging and reducing capacity (FRAP) were increased by Phe at concentrations of 3 and 6 mM as shown in Figures 3b–c. Damage to MCF-7 cells and changes in their morphology after exposure to hazel cell extracts are shown in Figure 4. The results of the MTT assay showed that both standard Taxol and hazel cell extracts (pretreated with or without Phe) inhibited cancer cell growth (Fig. 5). Interestingly, the viability of cancer cells was more affected by hazel cell extracts than by pure Taxol.
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Fig. 1

Growth of Corylus avellana cells treated with different concentrations of phenylalanine. Data are presented as the mean ± SD with n = 3. Bars with different letters are significantly different at p ≤ 0.05 according to Student’s t test

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Fig. 2

Intracellular (a) and extracellular Taxol (b) of Corylus avellana cells treated with different concentrations of Phe. Data are presented as the mean ± SD with n = 3. Bars with different letters are significantly different at p ≤ 0.05 according to Student’s t test

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Fig. 3

Total phenolic content (a), free radical scavenging capacity (b), and reducing capacity (c) of the extract of Corylus avellana cells treated with different concentrations of Phe. Data are presented as the mean ± SD with n = 3. Bars with different letters are significantly different at p ≤ 0.05 according to Student’s t test

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Fig. 4

Morphology of MCF-7 cells before (a) and after (b) exposure to methanolic extract of hazel cells. Scale bar is equal to 100 μm

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Fig. 5

Viability of MCF-7 cells after exposure to standard Taxol and methanolic extracts of hazel cells in MTT assay. Ctrl: MCF-7 control cells; T25 and T50: The cells after treatment with 25 and 50 nM of standard Taxol, respectively; H25 and H50: MCF-7 cells after treatment with methanolic extract of hazel cells containing 25 and 50 nM Taxol, respectively; PH25 and PH50: viability of MCF-7 cells after treatment with methanolic extracts of Phe-treated hazel cells, containing 25 and 50 nM Taxol, respectively. Data are presented as the mean ± SD with n = 3. Bars with different letters are significantly different at p ≤ 0.05 according to Student’s t test

Discussion

According to this research, production of Taxol by suspension-cultured hazel cells (0.16 mg/l) was not as high as has been reported for Taxus sp. [3, 5, 7, 11]. Nonetheless, it should be noted that the aforesaid yield of Taxol was obtained only after 14 days of subculture of hazel cells. Moreover, the cells were easily available, fast-growing, and their Taxol product could be easily enhanced by feeding with Phe. The dry weight loss of Phe-treated hazel cells may be due to advancing the cell metabolism to production of phenolic compounds and secondary metabolites such as Taxol. Our results are consistent with the finding of Edhahiro and coworkers [28] who observed a strong reduction in cell growth at a high concentration of phenylalanine. Recently, a few studies have shown that manipulation of hazel cell cultures resulted in improved Taxol production. The higher Taxol biosynthetic capacity observed in phenylalanine-treated cells could be explained by a probable metabolization of this precursor into taxoid pathway intermediates, affording conversion of phenylalanine to phenylisoserine and also to baccatin III, followed by N-benzoylation and synthesis of the side chain of Taxol [17]. Therefore the increment of Taxol content in suspension-cultured hazel cells can be attributed to increased synthesis of the side chain of Taxol. In addition, consistent with the results obtained here, there is evidence that exogenously applied phenylalanine improved production of secondary metabolites in some plant species, e.g., Taxus chinensis [29].

Phenylalanine, an aromatic amino acid, is the substrate of phenylalanine ammonia-lyase (PAL) that catalyses the reductive de-amination of L-phe into trans-cinnamic acid as the first step of the biosynthesis of plant phenolic compounds [30]. Enhanced levels of phenolic compounds in Phe-treated cells is therefore reasonable.

The antioxidant activity of hazel cell extract was determined by the DPPH method. This method has been extensively used for screening antioxidant activity, since DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become a stable diamagnetic molecule [31, 32].

To the best of our knowledge, this is the first report showing the antioxidant activity of hazel cell extract. Antioxidant activity is usually attributed to the redox potential of phenolic compounds and is proven by different studies [33]. The results presented here demonstrated that the antioxidant activity of hazel cell extract was correlated closely with its phenolics content. In this connection, providing hazel cells with higher concentrations of Phe resulted in higher phenolics content as well as higher antioxidant capacity of the cell extract.

The presence of reductants in the extracts causes the reduction of Fe3+/ferricyanide complex to the ferrous form [34]. Reduction of Fe3+ is a significant indicator for electron donation and this is an important mechanism of antioxidant activity in phenolic compounds. The results of the reducing power of the hazel cell extract were coincident with their phenolics content and DPPH radical scavenging activity.

Exposure of MCF-7 cells to methanolic extract of hazel cells resulted in their damage and significant changes in their morphology. The mitochondrial succinate dehydrogenase reduces the MTT (tetrazolium salt) to purple formazan crystals. The more viable cells that are present in a well, the more formazan dye is produced [35]. Whether the hazel cell extracts induced apoptosis rather than other kinds of death in MCF-7 cells was outside the scope of the present study, but the MTT test and decrease in formazan staining in those cells treated with hazel cell extracts provides indirect evidence showing occurrence of apoptosis and mitochondrial damage in these cells. As mentioned previously, the viability of cancer cells was more affected by the cell extracts even when their Taxol content was identical to that of pure standard of Taxol. This may be due to the existence of Taxol-like compounds in hazel cell extracts, as was noted by other researchers [15].

In conclusion, the results of the present research demonstrated that application of phenylalanine as a precursor of Taxol can be a good method to increase its production by hazel cells. Due to its high efficiency in killing cancer cells, the extract of hazel cells, in particular when treated with Phe, can be suggested as an alternative and effective probe for treating human cancer.

Copyright information

© The Japanese Society of Pharmacognosy and Springer 2012