Communication Vol. 59, No 1/2012

The aim of the present study was to compare carotenoid extracts of Rose hips (Rosa canina L.) with regard to their phytochemical profiles and their in vitro anti-Helicobacter pylori (H. pylori), cytotoxic, multidrug resistance (MDR) reversal and radical scavenging activity. Carotenoid composition was investigated in the different fractiona-tion of rose hips, using extraction methods. Six main ca-rotenoids — epimers of neochrome, lutein, zeaxanthin, rubixanthin, lycopene, β,β-carotene — were identified from Rose hips by their chromatographic behavior and UV-visible spectra, which is in accordance with other studies on carotenoids in this plant material. The active principles in the carotenoid extract might differ, depending upon the extraction procedures.


INTRODUCTION
Carotenoids play an important role in preventing oxidative damage which are caused by free radicals in agerelated diseases such as cancer and ageing (Britton et al., 2005;Motohashi et al., 2003;Krinsky & Johnson, 2005).Most current research is focused on a proposed role of carotenoids as lipid antioxidants which can protect the cells or hosts from the oxidation and other destructive processes mediated by singlet oxygen and free radicals, though more specific effects on the immune system are now under investigation (Britton et al., 2005;Krinsky & Johnson, 2005).In this study, we present qualitative and quantitative data on the carotenoid content of Rose hips.We investigated anti-Helicobacter pylori (H.pylori), cytotoxic, multidrug resistance (MDR) reversal and radical scavenging activity by the carotenoid extracts of Rose hips.
Isolation of phytoxanthins from Rose hips (Rosa canina L.). Rose hips (1.2 kg fresh weight) have been collected in the surroundings of Pécs (Hungary).Isolation of PM2 and PM9-PM12 in crystalline state was carried out in our laboratory according to reference of Molnár et al. (2005a).PM2: Phytoxanthins of Rose hips, 680 mg crystalline sample; PM9: Hypophasic carotenoids of the ethereal extract of Rose hips, 176 mg crystalline sample; PM10: Epiphasic carotenoids of the ethereal extract of Rose hips, 350 mg crystalline sample; PM11: Hypophasic carotenoids of the MeOH extract of Rose hips, 96 mg crystalline sample; PM12: Epiphasic carotenoids of the MeOH extract of Rose hips, 180 mg crystalline sample.
Measurement of anti-H.pylori activity.The microdilution broth method was used to determine the minimum inhibitory concentration (MIC).Brain Heart Infusion (BHI) broth containing 10% fetal bovibe serum (FBS) and 0.1% glucose was used as the medium and was cultured in a jar conditioned with AnaeroPack (Campylo, Mitsubishi Gas Chemical Co., Inc.).H. pylori was inoculated in the medium and cultured at 37°C for 2 days.The collected bacterial colonies were diluted to about 10 7 colony forming unit (CFU)/ml with the medium.The fractions were dissolved in DMSO, and then diluted with the medium.To the solution of the fractions, a suspension of bacteria was added to make 10 6 CFU/150 mL/well.The mixture was incubated at 37°C for 5 days.The MIC values of the fractions were determined by observation (Kawase et al., 2003).
Cell culture.Human oral squamous cell carcinoma (HSC-2) cells (provided from Prof. Nagumo, Showa University) and human submandibular gland tumor (HSG) cells (provided from Dr. Atsumi, Meikai University) were maintained as monolayer cultures at 37°C in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-activated FBS in a humidified 5% CO 2 atmosphere, and subcultured by trypsinization.Human gingival fibroblasts (HGF) were isolated from the periodontal tissue of healthy gingival biopsy of a 10-year-old female, as described previously (Sakagami et al., 2000), according to the guideline of Meikai University Ethic Committee, after obtainig the informed consent from the patients.Cells between the fifth and seventh passages were used.
Measurement of cytotoxic activity.Near confluent HSC-2 and HSG cells were incubated for 24 h with the indicated concentrations of test samples in culture medium in 96-microwell plates (Becton Dickinson and Company, Franklin Lakes, NJ, USA), and the viable cell number was determined by MTT method (Sakagami et al., 2000).The cells were washed with PBS, and incubated for 4 h with fresh culture medium containing 0.2 mg/ mL MTT.After removing the medium, cells were lysed with 100 mL DMSO and the absorbance at 540 nm of the cell lysate was measured with Labsystems Mutiskan R with Star/DOT Matrix printer JL-10.The A 540 values of control HSC-2, HSG and HGF cells were 1.519, 0.884, and 0.283, respectively.We found that the linearity of the absorbance value was maintained between 0-2.0.The absorbance value of 1.0 was correlated to approximately 4×10 4 cells/cm 2 .The 50% cytotoxic concentration (CC 50 ) was determined from the dose-response curve.
Measurement of the MDR reversal activity.The MDR1/A expressing cells were selected by culturing the infected cells with 60 ng/mL colchicine to maintain the expression of the MDR phenotype (Kessel, 1989).The L5178 MDR cell and the L5178 Y parent cell were grown in Mc-Coy's 5A medium supplemented with 10% heat-inactivated horse serum, L-glutamine and antibiotics.The cells were adjusted to a concentration of 2×10 6 /ml and resuspended in serum-free McCoy's 5A medium, and 0.5 ml aliquot of the cell suspension were distributed into each Eppendorf centrifuge tube.Then 10 µl of 2 mg/ml test compounds (dissolved in DMSO) were added and incubated for 10 min at room temperature.Then 10 µl rhodamine 123 as indicator of drug accumulation was added to the extracts (5.2 µM final concentration) and the cells were incubated for a further 20 min at 37°C, washed twice and resuspended in 0.5 ml PBS (pH 7.4) for analysis.The fluorescence intensity of cell population was measured by flow cytometry (instrument: Beckton Dickinson FACScan).(±)-Verapamil was used as the positive control in R123 accumulation experiments (Weaver et al., 1993).The R123 accumulation was calculated from fluorescence of one height values.Then, the percentage of mean fluorescence intensity was calculated in treated MDR1 and parental cells, compared to untreated cells.The fluorescence activity ratio was calculated by following equation (Weaver et al., 1993): MDR1 reversal activity = (MDR1 treated/MDR1 control)/(parental treated/parental control) Measurement of the radical scavenging activity DPPH scavenging assay.For the determination of DPPH radical, 30 µL of 300 µM DPPH in EtOH were added to 60 µL of sample in 40% DMSO solution and analyzed 1 min after mixing.The microwave power, gain and time constant were changed to 5, 400 and 0.1 s, respectively (Molnár et al., 2005b).
1 O 2 scavenging assay.The sample was added to the reaction mixture of 20 mM 4-OH TEMP and 20 µM Rose Bengal in 20% DMSO solution and the mixture in flat cell was irradiated for 5 min at 550 nm and analyzed by using ESR spectrometer.The microwave power, modulation width, the gain and time constant were changed to 5, 0.2, 200 and 0.1 s.Singlet oxygen was measured by oxidation of 4-OH TEMP to 2,2,6,6-tetramethyl-4-hydroxy-1-piperidinyloxy (4-OH TEMPO) radical (Molnár et al., 2005b).

Anti-H. pylori activity
Helicobacter pylori is an important human pathogen that infects up to 50% of the human population.As the leading cause of peptic ulcers, gastritis and gastric cancer worldwide, the organism has been the subject of intensive research to unravel the mysteries of its genetics and cellular biology.Antibacterial agents from a diet rich in fruit and vegetable may be superior as H. pylori control agents, when compared to many non-natural products (Kawase & Motohashi, 2004).Carotenoids, as free radicals, play an important role in the pathogenesis of gastroduodenal mucosal inflammation, peptic ulcer disease, various micronutrients are considered to protect the gastric mucosa by scavenging the free radicals.Among the tested PM2 and PM9-12 extracts only the PM10 and 11 displayed some anti-H.pylori effects (MIC 50 = 8.2 µg/ml and 11 µg/ml) comparable to metronidazole (MIC 50 = 7.0 µg/ml) (Table 1).The other compounds PM2, 9 and 12 had moderate activity.

Cytotoxic activity
Carotenoids showed potent in vitro anti-tumor promoting activity (Garewal, 1993).A higher intake of carotenoids-rich foods is associated with a reduced risk of  the development of cancer formation.Capsanthin and related carotenoids isolated from the fruits of red paprika exhibited potent antitumor-promoting activity (Maoka et al., 2001).Cytotoxic activities of PM2, PM9-12 was compared to β,β-carotene and gallic acid against six human tumor cell cultures and three normal cell cultures (Table 2).The cytotoxic effect of PM compounds were in the same range but somewhat lower than that of gallic acid as control.The tumor-specific cytotoxicity as indicated by SI selective toxicity was only slightly observed in case of PM10 and PM11.The safety index (SI) values of PM10 and PM11 were 1.9 and 2.5, respectively (Table 2).

MDR reversal on tumor cells
MDR is a major problem in cancer treatment.The overexpression of drug transport proteins is a major mechanism for the MDR.In tumor cells overexpressing P-glycoprotein (P-gp), this results in reduced intracellular drug concentrations which in turn decrease the efficacy of a broad spectrum of some antitumor drugs.Molnár and coworkers have recently examined MDR modulating activity of several carotenoids and shown that capsanthin and capsorubin enhanced the drug accumulation most potently (Molnár et al., 2004).We measured the ability of PM9-10 and PM12 extracts to inhibit the P-gp mediated R-123 efflux in the MDR mouse T cell lymphoma L5178 transfected by human MDR1 gene, which was cultured in colchicine-containing medium.The ability of PM9, 10 and 12 was studied on the drug accumulation of MDR tumor cells.These extracts showed dose dependent reversal of ABC transporter.Interestingly the β,β-carotene was more effective the same concentrations on the MDR cells, however these concentrations were toxic for the tested cells as the SSC and FSC values shows (Table 3).

Radical scavenging activity
Carotenoids are involved in the scavenging of two of reactive oxigen species (ROS), singlet molecular oxygen and peroxy radicals generated in the process of lipid peroxidation (Sies et al., 1993).At 400 µg/ml concentration PM9-10 and PM12 extracts showed lower singlet oxygen scavenging activity in comparison with the control β,β-carotene.In DPPH assay, the electrondonating capacity was strongest in the following order: PM10>PM9>PM12>β,β-carotene (Table 4).

CONCLUSION
Carotenoids are important dietary nutrients having antioxidant potential and are consequently being considered as important preventive strategic molecules.Antioxidants may protect against oxidative damages both by directly neutralizing reactive oxidants and by modulating gene expression contributing to oxidative stress (Silvaas et al., 2004).A mixture of carotenoids with different chemical structures and different antioxidants might be a promising agent for the medicinal use than individual carotenoids.

Table 3 . Effect of Rose hips carotenoids on the multidrug resistance of L-5178 cells
a FSC: Forward scatter count; SSC: Side scatter count; FL-1: Fluorescence intensity; b Par: a parenteral cell without MDR gene; c MDR: a parenteral cell transfected with MDR gene.

Table 4 . Radical scavenging activity of Rose hips carotenoids
Radical intensity of DMPO-OOH in control was 1.105.b Radical intensity of DPPH in control was 1.977.Each value represents mean of triplicate assays.S.D.<10%. a