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Journal of Food Biochemistry ISSN 1745-4514

POTENTIAL ANTIOXIDANT, ANTIPROLIFERATIVE AND HEPATOPROTECTIVE EFFECTS OF CRATAEGUS MEYERI CENNET OZAY1,3, RAMAZAN MAMMADOV1, GULTEN TASDELEN1, EGE RIZA KARAGUR1 and HAKAN AKCA2 1 2

Department of Biology, Faculty of Science and Literature, Pamukkale University, Denizli, Turkey Department of Medical Biology, School of Medicine, Pamukkale University, Denizli 20070, Turkey

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Corresponding author: TEL: +905362827667; FAX: +902582963535; EMAIL: [email protected] Received for Publication February 10, 2015 Accepted for Publication May 12, 2015 doi:10.1111/jfbc.12161

ABSTRACT In this study, the potential antioxidant, antiproliferative and hepatoprotective effects of Crataegus meyeri Pojark. were investigated. The antioxidant activity of the ethanolic flower extracts was evaluated by using DPPH (2,2-diphenyl-1picrylhydrazyl) and β-carotene–linoleic acid assays. Total phenolic contents were also measured. The results obtained showed that C. meyeri can act as a high radical scavenger reaching 88.67%. In vitro antiproliferative activity for the same extracts was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] assay against PC3 and PC14 cells. The extracts of the plant at concentrations of 0.5, 1 and 1.25% were administered orally to the three experimental groups, including partially hepatectomized rats for 42 days. At the end of the experimental period, animals were sacrificed, and blood was collected for the assessment of serum levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase) and GGT (gamma-glutamyltransferase). In biochemical assay, a significant decrease in the levels of serum ALT and AST was found in the experimental groups.

PRACTICAL APPLICATIONS The antioxidant activity studies on Crataegus species have exhibited that these species possess considerable antioxidant potential because of their polyphenolic compounds such as flavonoids and procyanidines. In this study, the findings are consistent with these observations. However, our results also demonstrated that C. meyeri exerts a protective effect against partial hepatectomy-induced liver injury in rats and could provide a new potential approach to inhibit the proliferation of human non-small cell lung cancer cells.

INTRODUCTION Hawthorn (Crataegus spp.), belonging to the Rosaceae family, consists of small trees and shrubs, native to Northern temperate region of the world with approximately 280 species (Donmez 2004). Crataegus species are medicinal plants, which have flavonoids, triterpene acids, proanthocyanidins and organic acids as the main components. The medicinal use of extracts or tinctures prepared from the leaves, flowers and/or fruits of the plant species from the genus Crataegus dates back to the ancient times (Bahorun et al. 2003). The drug from flowers has antispasmodic, hypotensive, cardiotonic, diuretic and nervinesedative properties. It dilates coronary vessels and improves 548

blood supply to the heart (Verma et al. 2007). In the past decade, there has been a great deal of interest in the use of medicinal plants for the treatment of diseases. Many studies have focused on the therapeutic properties of medicinal plants, such as hepatoprotective, antioxidative and antimicrobial activities (Janovská et al. 2003; Rusu et al. 2005). Furthermore, many scientific studies have been extensively carried out by using medicinal plants in liver damages (Dahiru et al. 2005; Rusu et al. 2005; Aliyu et al. 2007). Liver can rapidly regenerate itself after acute liver injury, chronic hepatic diseases, liver transplantation and partial hepatectomy. Some studies have reported on different effectiveness of Crataegus spp., such as antioxidant, antiinflammatory and anticarcinogenic effects (Ahumada et al. 1997; Journal of Food Biochemistry 39 (2015) 548–553 © 2015 Wiley Periodicals, Inc.

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Kao et al. 2007; Bahri-Sahloul et al. 2009). Independent studies have established extracts of Crataegus are rich in proanthocyanidins and flavonoids (Bahorun et al. 1996; Barnes et al. 2002) and many of these phenolic compounds have been shown to be cytoprotective by reducing oxidative stress (Hertog et al. 1993; Zhang et al. 2001). Antioxidants are defined as compounds that can delay, inhibit or prevent the oxidation of oxidizable materials by scavenging free radicals and diminishing oxidative stress. Oxidative stress is an imbalanced state where excessive quantities of reactive oxygen and/or nitrogen species overcome endogenous antioxidant capacity, leading to oxidation of a variety of biomacromolecules, such as enzymes, proteins, DNA and lipids. Oxidative stress is important in the development of chronic degenerative diseases, including coronary heart disease, cancer and aging (Ames et al. 1993). Over the past few years, cancer has remained a major cause of death and the number of individuals affected with cancer is continuing to expand (Chaouki et al. 2010). Because of the enormous propensity of plants, which synthesize a variety of structurally diverse bioactive compounds, the plant kingdom is a potential source of chemical constituents with antitumor and cytotoxic activities (Kim et al. 2005; Chin et al. 2006). The aim of this study was to evaluate the in vitro antioxidant and antiproliferative activities and the hepatoprotective effects of Crataegus meyeri Pojark. extracts in the liver of partially hepatectomized rats using biochemical assays.

MATERIALS AND METHODS Plant Material and Extraction Procedure The flowers of the plant were collected in May 2010 from the Big Caucasus mountain range in Azerbaijan, near Quba province, and dried in the shadow for extraction. Dried flowers were pulverized and extracted with 100 mL of ethanol in a shaker water bath at 55C for 6 h. The extraction was repeated twice at the same condition. The extract was filtered and the ethanol was removed in vacuum by a rotary evaporator (IKA RV10D, Staufen, Germany) at 42–49C. The residual part of the extract was dissolved with ultrapure water and the water in the extract was lyophilized (Labconco FreeZone, Kansan City, MO). Anhydrous extract was stored at −20C until analysis for antioxidant and antiproliferative activity assays, while three different concentrations of the extract were prepared for animal assay: 0.5, 1 and 1.25%.

DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Free Radical-Scavenging Activity Free radical-scavenging activity of the extracts was determined using the free radical DPPH (Wu et al. 2006). About Journal of Food Biochemistry 39 (2015) 548–553 © 2015 Wiley Periodicals, Inc.

SOME BIOLOGICAL ACTIVITIES OF CRATAEGUS MEYERI

4 mL of the DPPH’s 0.004% methanolic solution was mixed with 1 mL (0.2–1.0 mg) of the extracts, and their absorbances were measured at 517 nm after incubation for 30 min at room temperature. Butylated hydroxytoluene (BHT) was used as a control.

β-Carotene–Linoleic Acid Assay The antioxidant activity (AA) of the extracts was evaluated using the β-carotene–linoleic acid test system (Amin and Tan 2002) with slight modifications. About 0.5 mg of β-carotene was dissolved in 1 mL of chloroform, and 25 μL of linoleic acid and 200 mg of Tween 20 were added. Chloroform was completely evaporated using a vacuum evaporator. Then, 100 mL of distilled water was added by vigorous shaking. About 2,500 μL of this reaction mixture was dispensed to test tubes, and 350 μL portions of the extracts, which was prepared at 2 g/L concentration, was added and the emulsion system was incubated for up to 48 h at room temperature. The same procedure was repeated with synthetic antioxidant, BHT, as positive control. After this incubation period, absorbances of the mixtures were measured at 470 nm. The AA was measured in terms of successful bleaching of β-carotene by using a slightly modified version of the formula from Jayaprakasha et al. (2001) and the absorbance was measured for 120 min.

AA = [1 − ( A0 − At A0 º − At º )] × 100 where A0 is the initial absorbance of the sample, At is the initial absorbance of the control, A0° is the sample’s absorbance after 120 min and At° is the control’s absorbance after 120 min.

Total Phenolic Contents The total phenolic contents in the extract of C. meyeri were expressed as microgram of pyrocatechol equivalents (PEs), which were determined with Folin–Ciocalteu reagent (FCR) according to the method of Slinkard and Singleton (1977). About 1 mL of the solution extracts (1 mg) was added to 46 mL of distilled water and 1 mL of FCR and was mixed thoroughly. After 3 min, the mixture was added to 3 mL of sodium carbonate (2%) and shaken intermittently for 2 h. The absorbance was read at 760 nm. The total phenolic contents were calculated using standard pyrocatechol graph.

Cell Proliferation Assay PC3 and PC14 cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum at 37C in a humidified incubator with 549


5% CO2. The medium was aspirated when the cells were grown to about 90% confluence. The cells were washed with phosphate buffered saline, trypsinized, counted with a hemocytometer and seeded into 96-well plates (2 × 104 cells/well). After a 24-h incubation at 37C in a 5% CO2 incubator, the medium was removed, and the cells were treated with plant extracts added to the medium in different concentrations (0.01, 0.0125, 0.025, 0.05, 0.1, 0.2 μg/mL) for 48 h. For the untreated control group, the cells were not treated with any extracts. At the end of the incubation periods, the medium was removed, and cytotoxicity in plant extract-treated and untreated control groups was measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] assay (Holst Hansen and Brünner 1998).

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serum and were stored at −20C. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gammaglutamyltransferase (GGT) were measured for the determination of liver function. The biochemical results were evaluated using the Kruskal–Wallis test in SPSS 15.0 program (SPSS, Inc., Chicago, IL). The level of significance was set at P < 0.05.

RESULTS AND DISCUSSION Antioxidant Activity and Total Phenolic Contents

Male albino rats, weighing approximately 150–200 g, were obtained from the Pamukkale University, Faculty of Medicine, Experimental Research Center, Denizli, Turkey. The animals (ethical approval was obtained from the Pamukkale University Animal Ethics Committee) were allocated into four groups with five rats in each group. Before the experimental period, 50% partial hepatectomy was performed under anesthesia by removing the left lateral lobe from all the groups. Group I: Control animals received normal rat diet and water, ad libitum. Group II: The plant extract at concentration of 0.5% was given orally for 6 weeks. Group III: The plant extract at concentration of 1% was given orally for 6 weeks. Group IV: The plant extract at concentration of 1.25% was given orally for 6 weeks. After the experimental period, the animals were sacrificed under anesthesia, and blood samples were collected for the biochemical assays.

In the DPPH assay, the purple radical (picrylhydrazyl) is reduced by antioxidant compounds to the corresponding pale yellow hydrazine (picrylhydrazine). The discoloration indicates free radical-scavenging activity of the tested sample. The extract showed scavenging activity in a concentration-dependent manner. The DPPH radicalscavenging activity at 1 mg/mL concentration in the extract was found to be 88.67 ± 2.75%, also in the β-carotene– linoleic acid test system, inhibition value of C. meyeri was observed to be 76.44 ± 1.26%. (Table 1). A previous study reported that C. meyeri fruit extract had higher phenolic and flavonoid contents than Crataegus pontica and its DPPH value was 95.43 ± 0.074% (Dolatkhani and Jameie 2013). The total phenolic contents in the extract of C. meyeri flowers were expressed as μg PEs/mg of extracts as shown (60.55 ± 0.65) in Table 1. In a previous study, where 52 indigenous Crataegus species of Turkey were compared in terms of their antioxidant capacities, C. meyeri flower extract has exhibited high antioxidant activity and phenolic contents in comparison with the leaf extract (Özyürek et al.2012). A positive correlation was observed between antioxidant activity and amount of phenolic contents of the extracts. Similar results for Irish York cabbage (Jaiswal et al. 2011) and Cyclamen graecum (Metin et al. 2013) have been reported.

Biochemical Assays

Cytotoxic Activity

Blood samples were taken by cardiac venipuncture on the second, fourth and sixth weeks after the initial treatment. Then, they were centrifuged at 200 g for 10 min to collect

The effect of the crude ethanolic flower extracts of C. meyeri on the growth of PC3 and PC14 cell lines was investigated by the MTT assay. This assay detects the

Treatment of Animals

Sample

β-carotene–linoleic acid assay Antioxidant activity (%)

DPPH assay Scavenging activity (%)

Total phenolic content (μg PEs/mg extract)

C. meyeri BHT

76.44 ± 1.26 92.33 ± 0.28

88.67 ± 2.75 93.30 ± 0.15

60.55 ± 0.65 –

Values expressed are means ± standard deviation of three parallel measurements. BHT, butylated hydroxytoluene; DPPH, 2,2-diphenyl-1-picrylhydrazyl; PEs, equivalents.

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TABLE 1. ANTIOXIDANT ACTIVITY AND TOTAL PHENOLIC CONTENTS OF CRATAEGUS MEYERI

pyrocatechol

Journal of Food Biochemistry 39 (2015) 548–553 © 2015 Wiley Periodicals, Inc.

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TABLE 2. SERUM AST, ALT AND GGT LEVELS OF 2 WEEKS IN PARTIALLY HEPATECTOMIZED RATS Groups

AST (IU/L)

92.24 ± 12.56 0.5% 36.42 ± 10.01* Experimental 1% 56.02 ± 15.11* 1.25% 68.71 ± 14.06* Control

ALT (IU/L)

GGT (IU/L)

33.65 ± 9.16 20.94 ± 6.02 23.46 ± 6.13* 26.33 ± 5.08*

13.35 ± 0.12 13.41 ± 0.13 13.55 ± 0.22 13.58 ± 0.31

* P < 0.05 compared to control. ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyltransferase.

FIG. 1. ANTIPROLIFERATIVE EFFECTS ON PC14 AND PC3 CELL LINES OF CRATAEGUS MEYERI

As it is known, the values of ALT and AST are used as biochemical markers of liver damage (Sturgill and Lambert 1997). Tables 2–4 give the mean serum AST, ALT and GGT levels for 2, 4 and 6 weeks in all groups. There was a significant difference in serum AST and ALT levels of the experimental groups when compared to controls (P < 0.05). Administration of C. meyeri extract at different concentrations lowered ALT and AST levels when compared to controls at the end of the treatment. However, there was a significant decrease of AST level at concentrations of 0.5 and 1 compared to that of 1.25. This effect appeared to be dose-dependent. On the other hand, the extract did not affect the level of GGT. These findings showed that the extract of C. meyeri has a protective effect against partial hepatectomy-induced liver injury. It has been reported that carvacrol obtained from Origanum onites L. (Lamiaceae) plant oil increases the liver regeneration rate following hepatectomy in rats (Uyanoglu et al. 2008). A previous

reduction of MTT by mitochondrial dehydrogenase to blue formazan product, which reflects the function of mitochondria and cell viability (Holst Hansen and Brünner 1998). The antiproliferative effect of the extract was determined by comparing the optical density of the treated cells against the optical density of the untreated cells. Control containing the appropriate volumes of the blank solutions was included in the assay and the cytotoxic activity obtained from the test extracts were normalized with that of the control. NSCLC (non-small-cell lung carcinoma) accounts for 80% of all lung carcinomas and is composed of heterogeneous groups, such as adenocarcinoma, squamous cell carcinoma and large-cell carcinoma. Conventional anticancer drug discovery and development have focused on the cytotoxic agents. Figure 1 clearly indicates that C. meyeri has cytotoxic effects on the NSCLC cells. The cytotoxicity of the extract on the proliferation of PC3 and PC14 cell lines was found to be 58 and 50%, respectively. The antiproliferative activities of the flower extract suggest that it may have contained some substances responsible for the antiproliferative activities.

92.35 ± 12.23* 0.5% 34.42 ± 14.09* Experimental 1% 54.02 ± 10.51* 1.25% 67.71 ± 13.05*

Hepatoprotective Activity

* P < 0.05 compared with control. ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyltransferase.

Many studies showed that some Crataegus extracts could protect the myocardium from injury in animal models of coronary ischemia and reperfusion (Krzeminski and Chatterjee 1993; Veveris et al. 2004). The plant is also used as therapeutic agent for cancer, diabetes and sexual weakness in Arab traditional medicine and is considered to be generally safe and well-tolerated (Ljubuncic et al. 2005), while its different parts are used in Turkish traditional medicine for various diseases such as cough, flu, asthma, stomach ache, rheumatic pain, nephritis and hemorrhoids (Özyürek et al.2012). Journal of Food Biochemistry 39 (2015) 548–553 © 2015 Wiley Periodicals, Inc.


AST (IU/L)

Control

ALT (IU/L)

GGT (IU/L)

35.55 ± 9.59 21.64 ± 6.11* 23.54 ± 6.05* 26.43 ± 5.18*

13.15 ± 0.65 13.10 ± 0.63 13.26 ± 0.57 13.39 ± 0.49


AST (IU/L)

90.24 ± 12.25 0.5% 36.42 ± 10.06* Experimental 1% 56.02 ± 15.17* 1.25% 68.71 ± 14.04* Control

ALT (IU/L)

GGT (IU/L)

32.74 ± 9.62* 21.61 ± 6.16 23.95 ± 6.18* 26.22 ± 5.07*

13.17 ± 0.59 13.23 ± 0.11 13.27 ± 0.17 13.48 ± 0.15

* P < 0.05 compared to control. ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyltransferase.

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study has shown that Crataegus aronia var. dentata extract can decrease the levels of serum ALT and AST and play a role in apoptosis of hepatocytes in the liver of partially hepatectomized rats (Keskin et al. 2012). In this study, the extracts of C. meyeri may increase the regeneration rate in hepatocytes in comparison to control group in the liver of partially hepatectomized rats.

CONCLUSIONS In conclusion, C. meyeri extract may have potent antioxidant activity, as reflected by reduced serum AST and ALT levels in partially hepatectomized rats. The mechanism of AA may be explained by the phenolics present in the extract. Moreover, the plant extract may enhance the regenerative capacity of hepatocytes in the experimental groups. However, further studies are required to confirm the effects of the plant extract on hepatoprotection. Also, the extract has cytotoxic effects on non-small cell lung cancer cell lines such as PC3 and PC14. Future studies will be aimed at investigating the effects of different parts of C. meyeri upon isolating and identifying the substances responsible for the antioxidant and antiproliferative effects.

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