Antibacterial and Antiparasitic Activity of Oleanolic ...

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Editor-in-Chief

Original Paper

1709

Antibacterial and Antiparasitic Activity of Oleanolic Acid and its Glycosides isolated from Marigold (Calendula officinalis)

Author

Anna Szakiel1, Dariusz Ruszkowski1, Anna Grudniak2, Anna Kurek2, Krystyna I. Wolska2, Maria Doligalska3, Wirginia Janiszowska1

Affiliation

1

3

Key words " Calendula officinalis L. ● " Asteraceae ● " triterpenoids ● " oleanolic acid glycosides ● " antibacterial activity ● " parasitic nematodes ●

received March 26, 2008 revised July 18, 2008 accepted July 30, 2008 Bibliography DOI 10.1055/s-0028-1088315 Planta Med 2008; 74: 1709– 1715 © Georg Thieme Verlag KG Stuttgart · New York Published online October 24, 2008 ISSN 0032-0943 Correspondence Dr. Anna Szakiel Institute of Biochemistry Department of Plant Biochemistry University of Warsaw ul. Miecznikowa 1 02–096 Warszawa Poland Tel.: +48-22-554 3316 Fax: +48-22-554 3221 [email protected]

Institute of Biochemistry, University of Warsaw, Warsaw, Poland Institute of Microbiology, University of Warsaw, Warsaw, Poland Institute of Zoology, University of Warsaw, Warsaw, Poland

Abstract !

The antibacterial and antiparasitic activities of free oleanolic acid and its glucosides and glucuronides isolated from marigold (Calendula officinalis) were investigated. The MIC of oleanolic acid and the effect on bacterial growth were estimated by A600 measurements. Oleanolic acid's influence on bacterial survival and the ability to induce autolysis were measured by counting the number of cfu. Cell morphology and the presence of endospores were observed under electron and light microscopy, respectively. Oleanolic acid inhibited bacterial growth and survival, influenced cell morphology and enhanced the autolysis of Gram-positive bacteria suggesting that bacterial envelopes are the target of its activity. On the other hand, glycosides of oleanolic acid inhibited the development of L3 Heligmosomoides polygyrus larvae, the infective stage of this intestinal parasitic nematode. In addition, both oleanolic acid and

Introduction !

Marigold, Calendula officinalis L. (Asteraceae) is well known for its medicinal properties and also its pharmaceutical and cosmetic uses. Aqueous extracts from flowers have diuretic, sudorific and detergent properties. In addition, there are many medical products derived from the marigold plant that have been applied in the treatment of various skin tumours, dermatological lesions, ulcers and swellings, as well as almost 200 cosmetic formulations, i. e., creams, lotions, shampoos [1], [2]. As previously shown [3], C. officinalis contains two series of triterpenic pentacyclic oleanolic acid (OA, oleanane-type triterpene) glycosides: glucosides (derivatives of 3-O-monoglucoside) and glucuronides (derivatives of 3-O-monoglucuronide), differing in the first sugar moiety

its glycosides reduced the rate of L3 survival during prolonged storage, but only oleanolic acid glucuronides affected nematode infectivity. The presented results suggest that oleanolic acid and its glycosides can be considered as potential therapeutic agents.

Abbreviations !

A600: cfu: Gal: Glc: Glcl: GlcOA: GlcUA: GlcUAOA: L3: OA:

absorbance at wavelength 600 nm colony forming unit galactose glucose 3-O-monoglucoside of oleanolic acid other glucosides of oleanolic acid glucuronic acid oleanolic acid glucuronides infective larval stage oleanolic acid

linked to the C-3 hydroxy group of the aglycone. In the various glycosides of both series, either glucose or galactose moieties form the sugar chain, which contains up to five monosaccharide units. In some representatives of both series, a single glucose molecule is also attached to the C" Fig. 1). 28 carboxyl group of oleanolic acid (● Both glucosides and glucuronides are synthesized in leaves and in young plant roots, but they differ markedly in the site of final accumulation: the latter are found in large amounts (up to 7 % of dry mass) in flowers, while the former accumulate in roots where they undergo gradual deglycosylation to the glucoside I and even to free oleanolic acid. Oleanolic acid and/or its derivatives are present in significant amounts in many medicinal plants of the class Magnoliopsida [4]. Their anti-inflammatory, anti-ulcer, immunomodulatory, cytotox-

Szakiel A et al. Antibacterial and Antiparasitic … Planta Med 2008; 74: 1709 – 1715


2

Original Paper

which could reduce the reproductive rate of the cereal rootknot nematode Meloidogyne artiellia. It is likely that bioactive components of C. officinalis might impede or affect free-living stages in nematode development, although the activity of isolated compounds has yet to be investigated. Heligmosomoides polygyrus is an intestinal parasite of mice [16]. The free living stages of this nematode can be grown in culture and may represent an experimental model to study the effects of plant bioactive compounds. The aim of the present study was to investigate the antibacterial activity of oleanolic acid and its glycosides from marigold and identify the cellular target of their action. The effect of these triterpenoids on the survival and infectivity of the H. polygyrus L3 stage larvae was also examined.

Materials and Methods !

Plant material Calendula officinalis L. plants (identified by H. Werblan-Jakubiec, University of Warsaw) were grown on an artificial soil substrate (glass pearls – “Perlit” Interminglass) in a greenhouse with a photoperiod of 16 h light at 21/17 °C day/night and they were fed every 7 days with 10-fold diluted Hoagland medium [17]. Voucher specimens were deposited in the herbarium of the Botanical Garden of the Institute of Botany, University of Warsaw, sample No. 000 000 9031.

Extraction and isolation of oleanolic acid and its glycosides Fig. 1 nalis.

Structure of oleanolic acid and its glycosides from Calendula offici-

ic, antimutagenic, antihepatotoxic, antidiabetic, hemolytic and antiviral activities have been described in many reports [5], [6], [7], [8]. However, few studies have examined their antibacterial activity and there is a complete lack of information on the antinematicidal activity of plant pentacyclic oleanane-type triterpenoids. Due to the increasing resistance of bacteria to antibiotics, there is a great and urgent need for alternative antimicrobial agents. Pentacyclic triterpenoids of plant origin represent potential therapeutics. Data concerning their antibacterial activity published mainly in the last three decades have demonstrated the potent growth inhibition of various bacterial genera produced by medicinal plant extracts or isolated compounds [9], [10], including effectiveness against multidrug resistant mycobacteria [11]. Information concerning the basis of this antibacterial activity is sparse, although it was demonstrated that OA is an inhibitor of insoluble glucan (ISG) synthesis by Streptococcus mutans and may therefore be considered as a potential anticaries agent [12]. It was also shown that ursolic acid negatively influences biofilm development by Escherichia coli, Pseudomonas aeruginosa and Vibrio harveyi. This compound also induced the expression of stress response genes involved in chemotaxis and mobility of E. coli [13]. Incorrect use of the available treatments for parasitic diseases has led to the development of high levels of multiple anthelmintic resistance [14], and so there is an increasingly urgent need to develop alternative methods for nematode control. Perez et al. [15] identified C. officinalis flowers as a source of compounds


Air-dried marigold leaves (1 kg) and flowers (500 g) were powdered and extracted with boiling methanol (5 × 3 L MeOH) for 30 min. Equal volumes of water were then added to each extract, the MeOH was evaporated and the remaining aqueous solutions were extracted with n-butanol (4 × 1 L n-BuOH). Subsequently, the n-BuOH extracts were evaporated to dryness (4.82 and 10.78 g, respectively) and used directly for chromatography on a DEAE Sephadex A-25 (Sigma-Aldrich) column (3 × 90 cm, 300 g) using a step gradient of ammonium acetate (0.05; 0.1; 0.2 M) in MeOH (3 fractions, 3 L each). Fractions containing the 3-O-monoglucoside of oleanolic acid (GlcI) (fractions 1 and 2), its derivatives (GlcOA) (fraction 2) and glucuronides of oleanolic acid (GlcUAOA) (fraction 3) were collected. Glucoside I was purified up to 98 % by preparative TLC on silica gel (Merck) in chloroform/methanol/water, 61/32/7, v/v/v; and ethyl acetate/acetic acid/water, 3/3/1, v/v/v. GlcOA (3.5 g) were rechromatographed on a silica gel column (4 × 40 cm, 250 g) eluting with a mixture of CHCl3/MeOH/H2O with MeOH increasing from 0 to 35 %. Separate fractions (250 mL) containing oleanolic acid glucosides were combined. GlcUAOA (3.5 g) were rechromatographed on a silica gel (MN-Kieselgel 60, 0.2 – 0.5 mm; Macherey-Nagel) column (4 × 40 cm, 250 g) eluting with a mixture of CHCl3/MeOH (95 : 5, 90 : 10) and CHCl3/MeOH/H2O (30 : 10 : 1). The fractions (250 mL) containing oleanolic acid glucuronides were combined. Purities of GlcOA (96 %) and GlcUAOA (95 %) were assessed by TLC and HPLC. Dried powdered roots (500 g) at the post-flowering stage were extracted in a Soxhlet apparatus (Carl Roth) with 3 L ethyl ether for 16 h. Oleanolic acid was separated from the extract as the insoluble Na salt. After decomposition with HCl and crystallization from MeOH, pure (99 %) oleanolic acid (1.2 g; m. p. 308 – 309 °C) was obtained. All solvents were from POCh.


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Escherichia coli and Bacillus megaterium were obtained from the collection of the Institute of Microbiology, University of Warsaw. Pathogenic bacteria – Yersinia enterocolitica O:8, Klebsiella pneumoniae, Klebsiella oxytoca, Shigella flexneri, Shigella sonnei, Listeria monocytogenes EGD and Staphylococcus epidermidis – were purchased from the National Institute of Hygiene (Warsaw, Poland). All strains except L. monocytogenes were grown with shaking at 37 °C in Luria-Bertani medium [18]. L. monocytogenes was grown in TSYEB medium. When required, media were solidified with 15 g.L–1 bactoagar. All constituents of media and buffers, media supplements and the reagents in special procedures were from Sigma Lab. except for TSYEB medium which was supplied by BTL.

Determination of MIC and estimation of bacterial growth, survival and cell morphology Overnight bacterial cultures were adjusted to 104 viable cells per mL before inoculating LB medium containing various amounts of OA. The cultures were then incubated for 24 h. The lowest OA concentration that resulted in no visible turbidity was taken as the minimal inhibitory concentration (MIC). As the contol, the MIC of chloramphenicol was determined (Sigma Lab.). To estimate the effect of OA on bacterial growth and survival, bacterial cultures were diluted to A600 0.05 and then incubated further until they reached A600 0.2. A600 measurements were performed using an Ultrospec III spectrophotometer (Pharmacia LKB Biochrom). OA was then added at a concentration of 2 × MIC and incubation was continued for 4 h. Control cultures without added OA were grown in parallel. Samples were taken every 30 min, the A600 was measured and, after appropriate dilution in saline, 0.1 mL aliquots were plated on solid media in order to count the number of colonies after 24 h incubation. Bacteria from overnight control cultures and from cultures grown with OA at a concentration of 0.7 × MIC were examined using a scanning electron microscope (LEO 1430VP; Carl Zeiss) at 2500 × (E. coli) and 1500 × (B. megaterium) magnification. After staining the vegetative cells with crystal violet, B. megaterium endospores were observed under a light microscope (IX 70 – S8F2; Olympus) at 1000 × magnification.

Induction of bacterial autolysis Overnight bacterial cultures were diluted to A600 0.05 and incubated until A600 0.6 was attained. The cells were then harvested by centrifugation (RC-5B Sorvall), at 20 °C, 10 min, 8000 g, and washed twice with saline. The final cell pellet was suspended in Tris-HCl (pH 8.0) to A600 0.6 and Triton X 100 was added to a concentration inducing the lysis of 80 % of bacteria in 1 h. Each cell suspension was then split in half, OA was added to a concentration equal to 0.7 × MIC to one half, while the other served as a control. Incubation at 37 °C was continued, and every 20 min samples were taken, diluted in saline, then 0.1 mL aliquots were plated on solid media in order to count the number of colonies after 24 h incubation.

Parasites Adult stages of Heligmosomoides polygyrus (Nematoda, Trichostrongylidae) were isolated from the small intestine of BALB/c mice on the 28th day of infection. The nematodes were washed five times in 0.1 M PBS (pH 7.2) containing 100 U.mL–1 penicillin (Gibco) and 100 μg.mL–1 streptomycin (Gibco) at 37 °C, and then transferred to RPMI 1640 culture medium (Gibco Paisley) sup-

plemented with 10 μl.mL–1 L-glutamine (Gibco). Females were maintained on plates incubated in an atmosphere containing 5 % CO2 at 37 °C for 24 h. Eggs were then decanted, washed 3 times in PBS (pH 7.2), counted and split into glass plates containing 5 mL of Nematode Growth Medium (NGM) agar with E. coli strain OP50 added as a food source [19]. At least 2000 eggs were cultured on each plate.

Effect of triterpenoids on L3 development H. polygyrus eggs were split into NGM containing 70 and 350 μg.mL–1 of a mixture of OA and its glycosides or on plates of media without additions. As the compounds were dissolved in ethanol, further control plates of NGM, containing an equivalent concentration of ethanol were also prepared. After incubation, the number of L3 native larvae on the different treatment were counted and recorded as a percentage of the eggs split into those media.

Effect of triterpenoids on L3 survival H. polygyrus eggs were split into NGM supplemented with 70 μg.mL–1 OA, GlcI, GlcOA or GlcUAOA. The number of L3 larvae was counted 1 week later. The larvae were transferred to water then held at 4 °C and the number of surviving nematodes was recorded as a percentage 4 weeks later.

Infectivity of larvae developed in the presence of marigold triterpenoids L3 larvae collected from agar cultures containing 70 μg.mL–1 of OA, GlcI, GlcOA or GlcUAOA were used to infect 8 week-old male BALB/c mice via the alimentary route. 4 weeks later adult worms were isolated from the intestines of each animal and counted. The experiment was conducted in accordance with the guidelines of the Local Ethical Committee, the permit No. 11/WB/ 2005.

Statistical analysis The experiments to examine the antibacterial effect of oleanolic acid and its glycosides were performed at least three times and the mean ± standard error was calculated. The effect of these triterpenoids on free-living larvae of H. polygyrus was evaluated at least three times with 5 replicates in each experiment. Statistical significance of the differences between experimental groups was calculated using Student's t test and presented on graphs. All values are expressed as mean ± SEM and a P value < 0.05 was considered statistically significant.

Results and Discussion !

The antibacterial activity of free oleanolic acid as well as its 3-Omonoglucoside, and other glucosides and glucuronosides isolated from marigold was investigated. OA, GlcI, GlcOA and GlcUAOA are accumulated in different parts of the plant and perform different physiological functions, so their biological activity was examined separately. MICs of the tested compounds were determined for 9 bacterial species and OA showed the highest antibacterial activity. Bacterial susceptibility to this compound varied depending on the " Table 1). Gram-positive bacteria were highly suscepspecies (● tible to OA, whereas Gram-negative bacteria were markedly less so. OA did not inhibit the growth of E. coli but an effect on its survival was noted, the number of viable E. coli cells.mL–1 decreased



Bacterial strains, media and growth conditions

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Original Paper

MIC ( μg.mL –1)

Strain OA Gram-negative

Gram-positive

a

Table 1 Antibacterial activity of oleanolic acid (OA)

Chloramphenicol a

E. coli

35

Y. enterocolitica O:8

60

2.5 5

K. pneumoniae

100

2

K. oxytoca

125

1

S. flexneri

50

1

S. sonnei

55

52.5

B. megaterium

35

3

L. monocytogenes

15

4.5

S. epidermidis

10

3

Antibiotic used as a positive control in antibacterial tests. The same MIC values were obtained in three separate experiments.

2-fold after 2 h incubation with OA if compared to the control " Figs. 2A, C). The effect of OA on L. monocytogenes was more (● pronounced: growth was inhibited 5-fold, although no cell lysis was observed, and the number of viable cells decreased 20-fold " Figs. 2B, D). (●

Microscopic observations revealed that OA affected bacterial morphology. E. coli cells appeared several-fold longer after overnight incubation with OA at a concentration of 0.7 × MIC " Figs. 3A, B), indicating potential influence of OA on protein (● PBP3 (penicillin binding protein 3), which is involved in bacterial


1712

Fig. 2 Effect of OA on bacterial growth and survival. E. coli growth curve (A), E. coli survival curve (B), L. monocytogenes growth curve (C), and L. monocytogenes survival curve (D). Black squares – control cultures, black diamonds – cultures with added OA. Each symbol with bars indicates the mean ± S.D. of three experiments. The arrows indicate the time of OA addition.


istically enhance the activity of β-lactams against methicillin-resistant S. aureus (MRSA) [22]. It was also recently demonstrated that salvipisone and aethiopinone isolated from hairy roots of Salvia sclarea showed synergy with antibiotics of the β-lactam, glycopeptide and oxazolidone groups and caused alterations in cell surface hydrophobicity and cell wall/membrane permeability [23]. In the life cycle of many parasitic nematodes, the stages of egg embryonation, hatching and growing of L1, L2 and L3 larvae occur in the external environment. Investigations of the influence of plant active substances on these free-living stages might identify potential control strategies that could reduce the infectivity and abundance of these parasites in the external environment. In this study, unembryonated eggs of H. polygyrus were maintained in NGM agar cultures supplemented with a mixture of OA and its glycosides. Larvae reached the infective stage L3 after 7 days. The embryonation and hatching of nematode eggs progressed and after two days the motile larval stage L1 was observed in the culture. The larvae subsequently moulted and reached stage L2. In the presence of OA glycosides, fewer eggs developed and hatched and fewer larvae reached the infective " Table 2. stage L3. The results of the in vitro assays are shown in ● The mixture of all OA glycosides used at a high concentration caused a significant reduction in the number of L3 larvae, and the effect appeared to be dose-dependent. The concentration at which 50 % of hatched larvae failed to develop fully into infective stage L3 larvae was 350 μg.mL–1. A very weak side-effect of ethanol was also observed.

Fig. 3 Effect of OA on bacterial cell morphology and endospore formation. E. coli, control (A), E. coli, OA (B), B. megaterium, control (C), B. megaterium, OA (D), B. megaterium endospores, control (E), B. megaterium endospores, OA (F). The scale bars corresponds to 1 μm. Scanning electron microscope images (A, B, C, D) and light microscope images (E, F) of bacteria stained with crytal violet. E. coli cells were longer and B. megaterium cells became shorter after overnight incubation with OA at a concentration of 0.7 × MIC. OA efficiently inhibited sporulation by B. megaterium.

septation as a part of the divisome [20]. In contrast, cells of B. megaterium incubated overnight with OA (0.7 × MIC) became shorter, suggesting the potential influence of this compound on PBP2 located preferentially in the lateral wall and required for " Figs. 3C, D) [21]. OA was also a very lateral murein synthesis (● " Figs. 3E, F). efficient inhibitor of sporulation in B. megaterium (● The possibility that the cell wall constitutes the target of OA is strengthened by the demonstration that treatment with this compound greatly enhanced B. megaterium autolysis induced " Fig. 4B). As autolysis is the reby the surfactant Triton X-100 (● sult of unbalanced murein turnover, the observed effect of OA could be caused by inhibition of the murein synthesising activity of PBPs or the stimulation of murein hydrolases leading to the increased degradation of this macromolecule. The lack of any ef" Fig. 4A) may be due to the presfect of OA on E. coli autolysis (● ence of an additional surface layer – the outer membrane – in Gram-negative bacteria. There are several previous reports that cell wall/envelope structures of Staphylococcus aureus are the site of action of plant derived diterpenoids, catechins and flavones. Epigallocatechin gallate extracted from tea (Camellia siniensis) directly or indirectly affected the peptidoglycan component of the cell wall to synerg-

Fig. 4 Influence of OA on bacterial autolysis induced by Triton X-100. E. coli (A), B. megaterium (B). White bars – control cultures with Triton X-100 only, striped bars – cultures with Triton X-100 and OA. The results are the mean of three experiments and the bars indicate ± S.D.


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Original Paper

Original Paper

Table 2

Effect of different concentration of marigold fractions used as mixture: OA, GlcI, GlcOA, and GlcUAOA on H. polygyrus L3 development in NGM

Concentration

The rate of inhibition ( %)

The percentage of developed larvae OA glycosides

70 μ g.mL

–1

a

17.2

49.0 ± 6.2

350 μ g.mL –1

51.4

28.8 ± 7.7b,c

Ethanol control

NGM control

53.0 ± 2.2

59.2 ± 1.3

53.4 ± 3.4

59.2 ± 1.3

The rate of inhibition was calculated as the amount of larvae in NGM with glycoside fractions divided by the amount of larvae developed in control. Statistical significance: a

P < 0.05 OA glycosides (70 μg.mL –1) vs. NGM control; OA glycosides (70 μ g.mL –1) vs. ethanol control.

b

P < 0.005 OA glycosides (350 μ g.mL –1) vs. NGM control; OA glycosides (350 μg.mL –1) vs. ethanol control.

c

P < 0.001 OA glycosides (350 μ g.mL –1) vs. OA glycosides (70 μ g.mL –1).

The effect of the individual compounds OA, GlcI, GlcOA and GlcUAOA on the survival of H. polygyrus larvae was recorded af" Fig. 5). The number of larvae that hatched and ter 4 weeks (● developed in NGM containing 70 μg.mL–1 of OA, GlcI, GlcOA or GlcUAOA was reduced in all cases in comparison with the control NGM culture. The number of live and motile larvae dropped by more than half. GlcUAOA was identified as the most effective inhibitor of larval development. OA and its glycosides also reduced the infectivity of H. polygyrus larvae. In mice inoculated with stage L3 larvae, that had developed in NGM containing a mixture of all OA glycosides, the level of infection was significantly reduced in comparison to both control larvae, i. e., maintained in NGM medium alone or NGM with ethanol (data not shown). The antilarval activity appears to reside in the GlcUAOA fraction, which was identified as the " Fig. 6). At a concenmost potent in reducing larval infectivity (● tration of 70 μg.mL–1, this fraction reduced the number of worms recovered from mouse intestines; only 26.6 % of the infective dose persisted, while in control mice as much as 62.4 % of larvae settled and developed into the adult stages. It is interesting to note that all of the investigated glycosides: GlcI, GlcOA and GlcUAOA as well as free OA, reduced prolonged survival of L3 larvae; however, only GlcUAOA reduced the infectivity of these larvae in a statistically significant way. OA glycosides added to the agar culture medium inhibited the development of infective stage L3. The period of larval survival and nematode infectivity were also reduced. The most active compounds were again GlcUAOA. In conclusion, the results of this study indicate that marigold may be a potentially good source of compounds that can be ap-

Fig. 6 Effect of OA and its glycosides on H. polygyrus infectivity in BALB/c mice. Mice were infected with 50 L3 larvae developed in NGM containing 70 μg.mL–1 OA, GlcI, GlcOA or GlcUAOA. The number of adult worms in the intestines of each mouse was counted after 4 weeks. Five mice were examined in each group; the bar represents mean + SD. GlcUAOA vs. NGM: P < 0.001.

plied for the successful reduction of parasitic nematode infectivity. Recently, with the increased interest in organic food and natural therapies, there is a growing demand for natural and environmentally safe treatments for disease. These are frequently based on herbal medicines that have been traditionally used in developing countries for health-care in both humans and animals [24]. In our experiments, clear relationships between the chemical structure of oleanolic acid and its glycosides isolated from marigold and their antibacterial and wormicidal activities were observed. The aglycone structure appeared essential in the antibacterial activity of these compounds. However, the wormicidal activity of OA glycosides was greater than that of the aglycone and the level of activity was dependent on the nature of the sugar side-chain at the C-3 position. The first sugar molecule of glucuronides, i. e., the glucuronic acid attached to the aglycone, appeared to be vital to the antyparasitic properties of these compounds.

Acknowledgements !

Fig. 5 Effect of OA, GlcI, GlcOA and GlcUAOA on prolonged survival of H. polygyrus L3 larvae after 4 weeks of storage in 4 °C. Each bar indicates the mean ± SEM. Five samples were evaluated in two independent experiments. * P < 0.001.


This study is partially supported by the Ministry of Science and Higher Education grant N 304 117 32/4332 and grant BW1720/22 (through the Faculty of Biology, University of Warsaw). The participation of undergraduate students, A. Klicka and Ł. Samluk in this study is highly appreciated. We also thank M. Laskowska for excellent technical assistance.


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