Cytotoxic Properties and Flavonoid Composition of Grapefruit Varietes

In vitro antiproliferative properties and flavanones profiles of Grapefruit (Citrus paradisi Macf.) cultivars

Lorenzo Camarda*, Vita Di Stefano, Rosa Pitonzo and Domenico Schillaci

Dipartimento di Chimica e Tecnologie Farmaceutiche,

via Archirafi n°32, 90123 Palermo, Italy

*e-mail address:

Abstract

Recent studies suggest an inverse relationship between the intake of Citrus fruits and the incidence of cardiovascular diseases, stroke and different cancer types. Protective effects shown could be ascribed to the flavonoids content of the Citrus fruits.

A reversed-phase high-performance liquid chromatographic method was developed to determine the content of flavanone glycosides, that are the main flavonoids presents in Citrus fruits. To this purpose, we analysed fresh squeezed juices of six different Grapefruit cultivars (Citrus paradisi Macf.). In all grapefruits juices tested, we found total flavanone glycosides content in the range from 33.93 mg/100 ml to 44.97 mg/100 ml of juice, and naringin was the main flavanone component (16.37 - 26.14 mg/100 ml of juice).

To evaluate pharmacological activity and, in particular, inhibitory effects in vitro on proliferation and growth of cancer cells, the six freshly squeezed grapefruit juices were tested against K562 (human chronic myelogenous leukemia), NCI-H460 (human lung cancer) and MCF-7 (human breast adenocarcinoma) cell lines.

Most all the tested grapefruits juices shown evident antiproliferative activity against above mentioned cancer cell lines; in particular Ruby Red and Foster grapefruits juices had the best activity in inhibiting the growth of NCI-H460 and MCF-7 cell lines, at 3% v/v (fresh juice volume diluted in cell culture medium).

Keywords: Grapefruits cultivars, Flavanones glycosides; HPLC; Antiproliferative activity

Introduction

Recent epidemiological studies, in vitro and in vivo, have shown that citrus fruits intake has protective effect against cancer and cardiovascular diseases (Borradaile et al., 1999 Tanaka et al., 2000 , Tanaka et al., 2001; Tian et al., 2001; Poulose et al., 2005).

Citrus plants are rich source of health promoting flavonoids, especially flavanones glycosides, that show several physiological properties (Kuo, 1996), (Yu et al., 2005; Robak and Gryglewski, 1988). Citrus fruits show different quantitative and qualitative flavanones glycosides content as a function of cultivars, growing season, location environmental conditions of growing and ripeness degree (Ross et al., 2000, Albach et al., 1981, Rouseff, R. L., Martin, S. F., & Youtsey, C. O. (1987).

Grapefruit was originally believed to be a spontaneous hybrid of the pomelo (Citrus maxima). Botanist James Macfayden was the first that named grapefruit with botanical name Citrus paradisi Macf. The varieties are different in the flesh colour of the fruits due to lycopene presence (pink/red) or absence (blond).

Our aim was to investigate freshly squeezed juices of six different grapefruit cultivars: Duncan, Imperial, Foster, Thompson with blond pulp; Red Blush and Ruby Red with pigmented pulp, to evaluate flavanone glycosides content.

To evaluate pharmacological activity and, in particular, inhibitory effects in vitro on proliferation and growth of cancer cells, the six freshly squeezed grapefruit juices were tested against K562 (human chronic myelogenous leukemia), NCI-H460 (human lung cancer) and MCF-7 (human breast adenocarcinoma) cell lines.

Materials and methods

Chemicals

Naringenin, Hesperitin, Eriocitrin (eriodictyol 7-rutinoside), Neoeriocitrin (eriodictyol 7-neohesperidoside), Narirutin (naringenin 7-rutinoside), Naringin (naringenin-7-neohesperidoside), Hesperidin (hesperetin-7-rutinoside), Neohesperidin (nesperitin 7-neohesperidoside), Didymin (isosakuranetin 7-rutinoside), Poncirin (isosakuranetin 7-neohesperidoside) were obtained from Apin Chemicals LTD UK; Quercetrin (quercetin 3-rhamnoside) was obtained from Sigma (Sigma-Aldrich Co. Germany). Methanol and water were HPLC grade (Lab-Scan, Ireland). All other chemicals used were analytical grade.

Fruit Samples Preparation

All samples of Citrus paradisi Macf. cvs. Duncan, Imperial, Foster, Thompson (blond pulp) and cvs. Red Blush and Ruby Red (pigmented pulp) were collected in an experimental orchard in February 2006, that is the optimum ripeness period.

Twelve fruits each variety were randomly collected. Fruits were immediately hand-squeezed and juices were centrifuged at 7000 rpm for 20 minutes, at controlled temperature of 4°C. Clarified juices samples (20 mL) were diluted with water HPLC grade (1:1 v/v), filtered through a 0.45 mm hydrophilic cellulose acetate membrane syringe filter (Sartorius) and stored at -8°C until analysis.

Preparation of stock solutions

Each flavanone stock solutions 0.1 mg/ml were prepared in methanol (HPLC grade) and stored at -8°C. Daily, well-defined volume samples were drawn from each stock solution, put together and diluted in methanol to produce four working standard solution at 0.005, 0.02, 0.1 and 0.5 mg/ml for each flavanone. Standard solutions obtained, were filtered through a 0.45 mm hydrophilic cellulose acetate filter (Sartorius), injected into the HPLC system and assayed in triplicate to generate the corresponding standard curves.

HPLC method

The separation was performed on a liquid chromatograph Agilent 1100 Series coupled to a Chemstation HP. The instrumentation included a binary pump, model G1312A and a UV-variable wavelength detector, model G1314A.

At the beginning of our study we was looking for the maximum UV absorption wavelength for each flavanone stock solution on the basis of UV spectra for everyone. We found that 280 nm was the best wavelength for all flavanones to have good sensibility and maximum signal/noise ratio. Separation was achieved using a reversed-phase Supelcosil LC-18 (Supelco, USA) analytical column (4.6 mm i.d x 250 mm, 5 mm particle size), maintained at 25 °C with a column temperature controller (Thermosphere TS-130. Phenomenex, Torrance, California).

HPLC gradient analysis were performed using the following mobile phase: water containing 0.1% formic acid (A) and methanol (B). A gradient program was used as follows: 0-15 min 10-25% B; 15-50 min 25-95% B; 50-60 min 95-10% B. Each mobile phase was prepared fresh every day and degassed ultrasonically for 20 minutes before use. The flow rate was 0.7 mL/min and the injected volume was 20 µl. The separation took about 60 min under the described conditions including column equilibration.

The HPLC conditions were optimized for separation of all flavanone glycosides investigated. The order of flavanones elution was confirmed by analyses of individual standard solutions and their mixtures; flavanones identification was performed by characterizing the sample peaks in terms of retention times, compared with those in standard solutions (Figura e tempi di ritenzione?).

Data analysis

Calibration curves were produced using linear regression of the peak area against concentration of each flavanone standard solution. All curves were linear over the concentration ranges considered for each flavanone (r2>0.99). Calculation of flavanone glycosides concentration (expressed in mg/100 ml) was carried out by an external standard method, using calibration curves.

The value of means ±RDS were calculated.

Cytotoxicity studies in vitro

Fruit juices of six different grapefruit cultivars were tested in vitro for antiproliferative activity against K562 (human chronic myelogenous leukemia), NCI-H460 (human lung cancer) and MCF-7 (human breast adenocarcinoma) cell lines. These cell lines were grown at 37 °C in a humidified atmosphere containing 5% CO2, in RPMI-1640 medium or MEM (Sigma) in the case of NCI-H460 and MCF-7, supplemented with 10% fetal calf serum and antibiotics.

K562 cells were suspended at a density of 1 X 105 cells/ml in growth medium, transferred to 24-welI plate (1 ml per well), cultured with or without (in the case of control wells) a screening volume of 50 ml of fruit juices and incubated at 37 °C for 48 h. Control wells were added with 50 ml of a citric acid solution 1% w/v whose pH was comparable to pH of frail juices.

Numbers of viable cells were determined by counting in a hematocytometer after dye exclusion with trypan blue [10], The antiproliferative activity against NCI-H460 and MCF-7 was determined by MTT (methyltetrazolium) assay [11].

The experiment was carried out as follows: cells were suspended at a density of 4 X 105 cells per ml for NCI-H460 or 2 X 105 cells for MCF-7 per ml in MEM, supplemented with 10% fetal calf serum and antibiotics, transferred (100 ml per well) to 96-well plate and incubated at 37 °C for 4 days until the formation of cellular monostrate. After this time, the original medium was replaced with RPMI without red phenol and a juice fruit screening volume of 5 ml was added to each well and incubated for three days.

The antiproliferative effects of the fruit juices were estimated in terms of percent growth inhibition comparing cell viability of treated and untreated cells (but added with 5 ml of a citric acid solution 1%) by their reduction of the tetrazolium substrate, MTT.

Moreover, methanolic standard solution of naringin, naringenin, and narirutine, were tested against the tumoral cell lines at screening concentration of 100 mg/ml.

Results and Discussion

HPLC studies of fresh squeezed juices of six different grapefruit cultivars showed the presence of naringin as the main flavanone glycoside component (Rt = 31.8 min) and narirutin, hesperidin, neohesperidin and poncirin at approximate retention times of 31.2, 32.4, 32.8 and 36.2 minutes, respectively.

Neoeriocitrin (Rt = 29.1 min) was identified only in juices of Imperial cv.; didimin (Rt = 35.8 min) in Foster, Ruby Red, Red Blush and Tompson cvs.; quercetrin (Rt = 36.2 min) in Foster, Duncan, Red Blush and Tompson cvs.

Table 1 shows the flavanones glycosides quantities (mg/100 mL of juice) in filtered fresh squeezed juices of six different grapefruit cvs. Duncan, Imperial, Foster, Thompson (blond pulp) and cvs. Red Blush and Ruby Red (pigmented pulp). Significant amounts of naringin (26.14 mg/100 mL) and narirutin (10.67 mg/ 100 ml) were found in Ruby Red cv. Narirutin was the dominant flavanone glycoside in all grapefruit cvs: Red Blush (21.45 mg/100 mL), Duncan (20.67mg/100 mL), Tompson (20.38mg/100 mL), Foster (19.84mg/100 mL) and Imperial (16.37mg/100 mL).

High amount of neoesperidin was found in Imperial cv. (8.25 mg/100 mL) and neoeriocitrin was found only in Imperial cv. (3.04 mg/100 mL).

The total content of flavanone glycosides in all grapefruits juices tested ranged from 44.97 mg/100 ml in Ruby Red variety to 33.93 mg/100 ml in Duncan (grafico 1)

Table 1 : The flavonoid content (mg/100 mL of juice) in filtered fresh squeezed juices of six different grapefruit cvs. Duncan, Imperial, Foster, Thompson (blond pulp) and cvs. Red Blush and Ruby Red (pigmented pulp).

All the investigated juices exhibited evident antiproliferative activities against human chronic myelogenous keukemia K562, human breast adenocarcinoma MCF-7 and human lung cancer NCI-H460 (Table 2). Foster and Ruby Red cv. exhibited the great significant inhibitory effect against all cell lines above mentioned at screening concentration 5% v/v. As shown in Table 2, juices of Ruby Red cv. inhibited completely proliferation of human lung cancer NCI-H460, and proliferation of human breast adenocarcinoma MCF-7 for 98.36%.

K-562

Values are the mean of at least three independent determinations; coefficient of variation was less than 15%; ns = not significant i.e. below 10% inhibition.

Table 2: Growth inhibition percentages evaluated at screening concentration of 5% v/v.

Since juices of Foster and Ruby Red cvs. have shown the highest growth inhibition percentages (Table 2), we evaluated growth inhibition percentages at screening concentration of 3% v/v (Table 3).

K-562

Table 3: Growth inhibition percentages evaluated at screening concentration of 3% v/v.

Data obtained showed that juices of Ruby Red cv. had the best activities against all cell lines tested at both screening concentrations of 3% and 5% v/v. As it is possible to note in Table 1, naringin and narirutin were flavonones glycosides present in great quantity in juices of Ruby Red cv., compared to others flavonones. So, in order to evaluate the role of naringin and narirutin towards antiproliferative activity, we tested each flavanone at screenining concentration of 100 mg/mL. (che correlazione c’è con le %v/v? Si può fare un paragone?).

In Table 4 are reported growth inhibition percentages against NCI-H460 and MCF-7 cell lines. (Nella Tabella 4 metterei le % due flavononi!)

Moreover, we tested antiproliferative activity of naringenin, that is the aglycone form of narirutin and naringin. It is possible to note the significant activity against NCI-H460 and MCF-7 cell lines (Table 5).

Only narirutin, naringin and naringenin aglycone, showed antiproliferative activity expecially on NCI-H460 and MCF-7 cell lines (Table 4).

K-562

Table 5: Growth inhibition percentages evaluated at screening concentration of 100 μg/mL.

A probably explanation of observed increasing (?) growth inhibition percentages of naringenin, compared to narirutin and naringin percentages, could be related to its metabolism from glycoside form to aglycone, in vivo.

Some studies are reported about the role of bioactive flavonoid forms in vivo (Williams RJ et al, 2004. Spencer JPE et al, 2003). In particular there is strong evidence for the extensive phase I -deglycosylation- and phase II –metabolism-. During these phases, across small intestine and liver, flavonones glycoside are converted in aglycones and then conjugated as glucuronides, sulphates and O-methylated forms. Further transformations has been reported in the colon, where the enzymes of the gut microflora degrade flavonoids to simple phenolic acids (Scheline, RR 1999), then metabolized in the liver.

References

1  Borradaile et al., 1999 N.M. Borradaile, K.K. Carroll and E.M. Kurowska, Regulation of hepg2 cell apolipoprotein b metabolism by the citrus flavanones hesperetin and naringenin, Lipids 34 (1999), pp. 591–59