Carotenoids with anti-Helicobacter pylori activity from Golden delicious apple

Carotenoids with anti-Helicobacter pylori Activity from Golden Delicious Apple

Péter Molnár,1 József Deli,2 Toru Tanaka,3 Yoshiyuki Kann,3 Satoru Tani,3 Nóra Gyémánt,4 Joseph Molnár4 and Masami Kawase5*1Department of Pharmacognosy, University of Pécs Medical School, Pécs, H-7624 Hungary2Department of Biochemistry and Medical Chemistry, University of Pécs Medical School, Pécs, H-7624 Hungary3Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama 350-0295, Japan4Institute of Medical Microbiology and Immunology, University of Szeged, Szeged H-7620 Hungary5Faculty of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan

Previously it was reported that hypophasic carotenoids of Golden delicious apple peel showed potent anti-H. pylori activity (MIC50 = 36 mg/mL), comparable to metronidazole (MIC50 = 45 mg/mL). To further investigate the involved active carotenoids of the apple peel extracts, seven carotenoids were isolated for the current study: (all-E)-luteoxanthin, (all-E)-neoxanthin, (9¢Z)-neoxanthin, (all-E)-antheraxanthin, (all-E)-violaxanthin, (9Z)-violaxanthin and (all-E)-lutein. The MIC50 values of (all-E)-luteoxanthin, (all-E)-neoxanthin and (9¢Z)- neoxanthin were 7.9, 11 and 27 mg/mL, respectively. Other carotenoids and b,b-carotene did not exhibit potent anti-H. pylori activity (MIC50 > 100 μg/mL). An examination of structure and function suggested that active carotenoids contained a monofuranoid ring or an allenic bond in addition to an epoxy group and an additional two or three hydroxyl substituents on the side group. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: carotenoids; anti-H. pylori; luteoxanthin; neoxanthin; Golden delicious apple.

INTRODUCTION

Helicobacter pylori (H. pylori) is a micro-aerophilic Gram-negative, spiral-shaped, motile bacterium that colonizes the gastric mucosa of humans and is estimated to inhabit at least half of the world’s human population (Kusters et al., 2006; Sachs et al., 2003; Dunn et al., 1997). H. pylori is the root cause of chronic gastritis, peptic ulcers and gastric cancer (Di Mario et al., 2006). Currently, the most effective treatment regimens include a combination of antibiotics (β-lactams, macro-lides and quinolones), bactericidal agents (bismuth salts) and antiprotozoal agents (metronidazole) (Graham, 1997). However, the multi-drug application is associated with remarkable side effects and it is not uncommon not to be able to complete a treatment course. Thus, treatment failure is associated with the emergence of H. pylori strains that are resistant to the commonly used antibiotics clarithromycin and metroni-dazole (Matsumoto et al., 1997).

The ethnopharmacological approach has been an important source of fi nding new anti-H. pylori agents (Kawase and Motohashi, 2004). Crude extracts and iso-lated compounds from plants used in herbal traditional medicines have been tested for anti-H. pylori activity in vitro, in animal models, as well as in clinical studies, as possible sources for alternative eradication therapies

(Mahady, 2005; Ustun et al., 2006; Castillo-Juarez et al., 2007). It is important to isolate and defi ne the active principles in those experimentally active plant extracts.

Previously it was also reported that hypophasic carot-enoids of Golden delicious apple peel showed anti-H. pylori activity (Molnár et al., 2005). Of the fruits extracts and β,β-carotene, apple peel extracts displayed potent anti-H. pylori activity (MIC50 = 36 μg/mL), comparable to metronidazole (MIC50 = 45 μg/mL). Other fruits extracts and β,β-carotene did not exhibit potent anti-H. pylori activity. Thus, apple peel extracts were shown to be the most effective in anti-H. pylori potency when different fractions were compared.

To further investigate the involved active carotenoids of the apple peel extracts, seven carotenoids were iso-lated for the current study: (all-E)-luteoxanthin, (all-E)-neoxanthin, (9′Z)-neoxanthin, (all-E)-antheraxanthin, (all-E)-violaxanthin, (9Z)-violaxanthin and (all-E)-lutein. The aim of this work was, hence, to evaluate the anti-H. pylori activity of some carotenoids of the apple peel extracts and to examine the relationship between carotenoid structure and anti-H. pylori activity.

MATERIALS AND METHODS

Ripe fruits of Golden delicious apple were purchased from the local market (Pécs, Hungary).

The following chemicals and reagents were obtained from the indicated companies: metronidazole and amoxicillin (Wako Pure Chem. Ind., Ltd, Osaka, Japan); β,β-carotene (Tokyo Kasei Co., Tokyo, Japan); clar-

* Correspondence to: Dr Masami Kawase, Faculty of Pharmaceutical Sci-ences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan.E-mail: [email protected]

Received 03 March 2009Revised 29 March 2009

Copyright © 2009 John Wiley & Sons, Ltd. Accepted 28 April 2009

PHYTOTHERAPY RESEARCHPhytother. Res. 24: 644–648 (2010)Published online 9 July 2009 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2912

CAROTENOIDS WITH ANTI-HELICOBACTER PYLORI ACTIVITY 645

Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 24: 644–648 (2010)DOI: 10.1002/ptr

ithromycin (Taisho Pharmaceutical Co., Tokyo, Japan). A strain of H. pylori (ATCC43504) was purchased from the American Type Culture Collection (Rockville, MD, USA).

UV/VIS spectra were acquired with a Beckman DU-65 spectrophotometer.

The isolation of carotenoids from the peel of the Golden delicious apple. The plant material (52.8 g) was homog-enized and extracted three times with MeOH and once with Et2O. The extracts were combined (total extract) and the solution was saponifi ed with 30% KOH/MeOH in heterogeneous phase (16 h, darkness, N2 or Ar). After the usual work-up, the carotenoid composition of the extract was determined by HPLC (Molnár and Szabolcs, 1980; Deli et al., 2000).

The partition of the saponifi ed extract between MeOH/H2O (9 : 1) and hexane resulted in hypophasic

and epiphasic fractions. These fractions were analysed separately by HPLC.

The hypophasic and epiphasic fractions were evapo-rated and crystallized; the hypophasic carotenoids from benzene:hexane (∼1 : 5), the epiphasic carotenoids from benzene:MeOH (∼1 : 5). The crystallization of the hypo-phasic fraction resulted in 62 mg of crystalline sample. The crystallization of the epiphasic fraction resulted in ∼550 mg of crystalline sample.

Then 30 mg of the phytoxanthin mixture isolated from the hypophasic fraction was purifi ed by classic column chromatography (CC) using CaCO3

(Biogal, Ph. Hg. VI. Hungary) adsorbent and toluene/hexane (∼4 : 1) mixture as eluent (the diameter of the column was 6 cm and the length was 30 cm).

The column chromatography resulted in nine yellow colored zones (the main components were highlighted; structures see Fig. 1): Zone 1: mixture of (Z)-isomers of

(all-E)-Neoxanthin

HO

O

HO OH

.

(9'Z)-Neoxanthin

HO

O

.OH

HO

(all-E)-Luteoxanthin

HO

O

O OH

Antheraxanthin

(all-E)-Violaxanthin

(9Z)-Violaxanthin

HO

O

OH

O

HO

O

OH

HO

Lutein

OH

HO

O

OH

O

Figure 1. Structures of luteoxanthin, neoxanthin and related carotenoids in apple peel extracts.


646 P. MOLNÁR ET AL.

neoxanthin, (Z)-isomers of luteoxanthin-epimers, (Z)-isomers of antheraxanthin, (Z)-isomers of neochrome- and (Z)-isomers of auroxanthin-epimers; Zone 2: mixture of (9′Z)-neoxanthin, (9Z)-violaxanthin, (9Z)-antheraxanthin and (Z)-isomers of luteoxanthin- epimers; Zone 3: (9Z)-violaxanthin (= violeoxanthin); Zone 4: mixture of neochrome-epimers, and (all-E)-neoxanthin; Zone 5: mixture of unidentifi ed carot-enoids, which were present only in small amount; Zone 6: epimers of (all-E)-luteoxanthin; Zone 7: mixture of (Z)-isomers of violaxanthin, (Z)-isomers of luteoxan-thin, (Z)-isomers of antheraxanthin, (Z)-isomers of lutein and unidentifi ed carotenoids; Zone 8: (all-E)-violaxanthin; Zone 9: mixture of antheraxanthin, lutein, zeaxanthin, β-cryptoxanthin, (Z)-isomer of β- cryptoxanthin and β,β-carotene.

Zone 4 [contained (all-E)-neoxanthin] and Zone 9 [contained (all-E)-lutein] were purifi ed by repeated CC. After these procedures, the following carotenoids were crystallized from benzene/hexane (∼1 : 5): (9Z)- violaxanthin (6 mg), (all-E)-neoxanthin (2 mg), epimers of (all-E)-luteoxanthin (2 mg), (all-E)-violaxanthin (4 mg) and (all-E)-lutein (4 mg). The crystalline materials were characterized by spectroscopic methods (UV/VIS, 1H-NMR, CD, MS). Their purity was checked by HPLC.

HPLC analysis of apple peel carotenoids. HPLC was performed by using a Gynkotek Model 300B pump, a Gynkotek gradient former, a Hewlett-Packard 1050 detector, with HP ChemStation software and a Waters 991 photodiode array detector. Carotenoids were anal-ysed on a 250 mm × 4.6 mm i.d., 6 μm particle, end-capped Chromsyl C18 column. Gradient elution was performed with the mobile phase A (12% H2O in MeOH), B (MeOH) and C (30% CH2Cl2 in MeOH). The gradient program was 0–2 min; 100% A; 2–10 min: to 80% A + 20% B; 10–18 min: to 50% A 50% + B; 18–25 min: to 100% B; 25–27 min, 100% B; 27–34 min: to 100% C; 34–41 min: 100% C (in linear steps). The fl ow rate was 1.2 mL/min (Deli et al., 2000).

Measurement of anti-H. pylori activity. The micro-dilution broth method was used to determine the minimum inhibitory concentration (MIC). Brain heart infusion (BHI) broth containing 10% fetal bovine serum (FBS) (Biofl uid, Inc. Rockville, MD, USA) and 0.1% glucose was used as the medium and was cultured in a jar conditioned with AnaeroPack (Campylo) (Mit-subishi Gas Chemical Co., Inc.). Briefl y, H. pylori was inoculated in the medium and cultured at 37 ºC for 2 days. The collected bacterial colonies were diluted to about 107 colony forming units (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 106 CFU/150 μL/well. The mixture was incubated at 37 ºC for 5 days. The MIC values of the fractions were determined by observation (Kawase et al., 2003).

RESULTS AND DISCUSSION

Characterization of carotenoid fractions

Apple peel contains carotenoids which are esterifi ed with fatty acids, as well as unesterifi ed carotenoids

(Britton et al., 1995). Therefore, the extract was saponi-fi ed and partitioned between MeOH/H2O (9 : 1) and hexane. The partition gave two fractions, hypophasic and epiphasic fractions, respectively. Thus, it should be expected that the hypophasic fractions contained oxy-genated carotenoids, while the epiphasic fractions have less oxygenated, more lipophilic carotenoids. The HPLC analyses of hypophasic and epiphasic fractions were per-formed. Peak assignment of the carotenoids in the extract was based on comparison of HPLC retention times and UV-visible absorption spectra with those of authentic samples (Molnár and Szabolcs, 1980). The carotenoids in a hypophasic fraction of apple peel were mainly com-posed of epimers of (all-E)-luteoxanthin, (all-E)- neoxanthin, (9′Z)-neoxanthin, (all-E)- antheraxanthin, (all-E)-violaxanthin, (9Z)-violaxanthin and (all-E)-lutein (Fig. 1). These seven carotenoids were separated by repeated column chromatography using CaCO3 adsorbent and crystallized. The epiphasic fraction con-tained very small amounts of carotenoids (β-cryptoxan-thin 5,6-epoxide, β-cryptoxanthin and β,β-carotene), but a large amount of steroids and other compounds.

Inhibition of H. pylori

With regard to the carotenoid composition of apple, (all-E)-luteoxanthin, (all-E)-neoxanthin, (9′Z)-neoxanthin, (all-E)-antheraxanthin, (all-E)-violaxanthin, (9Z)- violaxanthin and (all-E)-lutein have been identifi ed and isolated as the major carotenoids in this study. Each of these seven carotenoids was tested for inhibition of H. pylori in vitro and the degree of inhibiting activity is expressed as half the minimum inhibitory concentration (MIC) value. The effect of these carotenoids and β,β-carotene in the MIC assay is shown in Table 1. The MIC50 values of (all-E)-luteoxanthin, (all-E)-neoxan-thin and (9′Z)-neoxanthin were 7.9, 11 and 27 μg/mL, respectively. The MIC50 values observed from three carotenoids are considerably higher than those exhib-ited by antibacterial agents such as amoxicillin (MIC50 = 0.16 μg/mL) and clarithromycin (MIC50 = 0.022 μg/mL), using the same assay procedure. However, these carot-enoids had stronger activity than metronidazole (MIC50 = 45 μg/mL) and displayed higher activity than the apple peel carotenoid extract (MIC50 = 36 μg/mL). Other carotenoids and β,β-carotene did not exhibit potent anti-H. pylori activity (MIC50 > 100 μg/mL) (Table 1).

Table 1. Inhibition of H. pylori by apple peel carotenoids

CompoundMIC50

(μg/mL)

(9′Z)-Neoxanthina 27(all-E)-Neoxanthinb 11Luteoxanthinc 7.9Antheraxanthin >100Lutein >100Violaxanthin >100(9Z)-Violaxanthin >100β,β-Carotene >100Metronidazole 45Clarithromycin 0.022Amoxicillin 0.16

a (9′Z)-Neoxanthin: 95% purity determined by HPLC.b (all-E)-Neoxanthin: 72% purity determined by HPLC.c Luteoxanthin: mixture of ∼1 : 1 epimers.

CAROTENOIDS WITH ANTI-HELICOBACTER PYLORI ACTIVITY 647


The active carotenoids are highly polar xanthophylls and have the characteristic structure of a monofuranoid ring or an allenic bond. Other xanthophylls, such as violaxanthin and antheraxanthin with two or one epoxy group, are inactive. Our previous study suggested that capsanthin, zeaxanthin, β-cryptoxanthin and violaxan-thin, did not show potent activity (Molnár et al., 2005). Indeed, the present results indicate that violaxanthin has no activity.

Comparisons between structural characteristics and carotenoid activity reveal that the existence of an allene bond or furanoid ring in the molecular structure may be essential for the activity. To our knowledge, this is the fi rst report of the screening of carotenoids for activity against H. pylori.

CONCLUSION

In the present study, seven carotenoids were screened for potential anti-H. pylori activity, with the aim of determining an essential structure of active carotenoids. Overall, luteoxanthin and neoxanthin showed an encouraging activity, while the other carotenoids did not show such an effect. The difference in activity is possibly associated with the different substituents in the end groups of individual carotenoids. Neoxanthin has the characteristic structure of an allenic bond and 5,6-monoepoxide. Luteoxanthin is endowed with a furanoid ring and a 5,6-monoepoxide.

Antibacterial agents from a diet rich in fruit and veg-etable may be superior as H. pylori control agents, when compared with many non-natural products (Kawase

and Motohashi, 2004). As free radicals play an impor-tant role in the pathogenesis of gastroduodenal mucosal infl ammation, peptic ulcer disease and probably even gastric cancer, various micronutrients are considered to protect the gastric mucosa by scavenging the free radi-cals (Correa et al., 2000). A high intake of carotenoids has been shown to prevent the development of H. pylori-associated diseases (Zhang et al., 2000; Nair et al., 2000). Astaxanthin, a naturally occurring carotenoid with strong antioxidant properties, was reported to be effective in reduction of refl ux gastritis in H. pylori posi-tive patients, but not in H. pylori negative patients (Kupcinskas et al., 2008).

Neoxanthin is one of the major carotenoids in green leafy vegetables such as spinach (Spinacia oleracea) (Khachik et al., 1986). Neoxanthin has been reported to suppress the proliferation of several cancer cells in vitro, and to inhibit chemically induced carcinogenesis in animal models (Terasaki et al., 2007). Moreover, recent studies have indicated potential health benefi ts of pre-venting cancer and obesity (Okada et al., 2008; Tapiero et al., 2004).

Therefore, carotenoids are important dietary nutri-ents having antioxidant potential and are consequently being considered as important preventive strategic mol-ecules in gastric diseases.

Acknowledgement

This study was supported by grants OTKA K 60121 and K 76176 from the Hungarian National Research Foundation, and from the Szeged Foundation for Anticancer Research, Hungary to the Hungarian authors.

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Carotenoids with anti-Helicobacter pylori activity from Golden delicious apple

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