Antimalarial Activity From Bowdichia Virgiloides

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VOLUMEN 19, No.1 - 2002

ANTIMALARIAL ACTIVITY OF ORMOSANINE AND HOMOORMOSANINE FROM BOWDICHIA VIRGILOIDES José Antonio Bravo;a* Catherine Lavaud;b Geneviève Bourdy;c Eric Deharo; c Alberto Giménez;d Michel Sauvain c a Laboratorio de Química de Productos Naturales, Instituto de Investigaciones Químicas-IRD, Universidad Mayor de San Andrés, CP 303, La Paz Bolivia; bLaboratoire de Pharmacognosie UMR 6013 CNRS Bâtiment 18, BP 1039, 51097 Reims, Cedes 2, France; cInstitut de Recherche pour le Développement (ex-ORSTOM), 213 rue Lafayette, 75480 Paris cedex 10, France; dInstituto de Investigaciones Fármaco Bioquímicas, Universidad Mayor de San Andrés, CP 20606, La Paz, Bolivia *Corresponding author: [email protected]

Key Word Index: Bowdichia virgiloides; homoormosanine; Plasmodium falciparum

Leguminosae;

stem-bark;

ormosia

alkaloids;

ormosanine;

American tropical forests from Venezuela to Brazil. In Bolivia the species B. virgiloides has been reported in the departments of Beni and Santa Cruz. Ethomedical uses of Bowdichia species,2, 3 include malaria high fever relief, rheumatism as well as dismenorrhoea and hemorrhage treatment with pain relief.4 The Tacana people, an ethnic group of northern western Bolivia, use stem bark of B. virgiloides in decoction for dysentery as well as for malaria high fever relief, also the crushed stem bark is applied as cataplasms against wounds of Leishmaniasis cutaneous form.5 Some biological activities for Bowdichia species extracts have been reported, mainly dermatitis provoked by topical application of the ether extract of B. nitida,6 probably due to the presence of 2,6-dimethoxy-pbenzoquinone causing allergy on Guinea’s pig skin.7 This compound is also thought to be the responsible of the resistance of Bowdichia woods to termites. B. virgiloides has been previously tested against some bacteria and fungi8 namely Eschericha coli, Bacillus subtilis, Staphylococcus aureus and Streptococcus faecalis, Neurospora crassa, Mycobacterium smegmatis and Candida albicans, showing no activity. Two species, B. nitida and B. virgiloides have been surveyed from a chemical stand point. bowdichione, centisteine, 3’Flavonoids9 hydroxyformononetin and homopterocarpin besides 2,6-dimethoxy-p-benzoquinone7, 9 have been described in B. nitida. The existence of triterpenes like betulinic acid, betulinol10 and lupeol11 and of alkaloids homoormosanine, homo-18-epiormosanine, piptanthine, homopiptanthine, podopetaline11 have been established in B. virgiloides. The pharmacological properties described above talk about certain specificity in active principles of B. virgiloides, fact that incited us looking for antimalarial activities in the plant.

RESUMEN De la farmacopea Tacana, la corteza de tronco de Bowdichia virgiloides H.B.K., Leguminosae, ha sido estudiada mediante una separación extractiva y cromatográfica bioguiada, por sus usos contra los síntomas del paludismo. Dos alcaloides del tipo ormosia, ormosanine y homoormosanine demostraron una buena actividad in vitro contra cepas cloroquino sensibles y cloroquino resistentes de Plasmodium falciparum. Ambos compuestos en su forma sinérgica no presentaron citotoxicidad de acuerdo a pruebas sobre células KB. Las identificaciones estructurales fueron establecidas mediante técnicas de espectrometría de masas y de RMN además de un importante aporte bibliográfico. ABSTRACT From the Tacana Pharmacopoeia, the stem bark of Bowdichia virgiloides H.B.K., Leguminosae, has been studied due to properties against malaria symptoms applying bioguided extractive and chromatographic techniques. Two ormosia-like type alkaloids, ormosanine and homoormosanine showed a good in vitro activity against chloroquine sensitive and chloroquine resistant strains of Plasmodium falciparum. Acceptable low cytotoxicity was evidenced from KB cells tests. Structural identifications were carried out by application of mass and NMR techniques besides bibliographic research. INTRODUCTION Bowdichia virgiloides H. B. K.;1 is a small tree of about 4 m high used for its pharmacological properties in traditional medicine. The genus Bowdichia comports four species distributed in south

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Antimalarial tests results with extracts, fractions and pure compounds from Bowdichia virgiloides

RESULTS AND DISCUSSION The in vitro assays against P. falciparum of the hydroethanolic (3:7) extract of stem bark of B. virgiloides exhibited an IC50 = 1.0 µg/ml. Dried and powdered stem bark were extracted by a classical acidbase method12 to determine the total alkaloids content (TA). The TA extract showed and antiplasmodial activity of CI50 < 1,0 µg/ml demonstrating that the initial activity of the hydroethanolic extract corresponded to the ormosia-like type alkaloids previously reported in the species.11 The TA extract was submitted for separation to a VLC system on silica gel employing binary mixtures of CH2Cl2-MeOH of increasing eluting power. From the sixteen fractions obtained, activity concentrated on fractions 5, 6 and 7 ( 100% of inhibition of parasitaemia at 1.0 µg/ml). Fractions 5, 6 and 7 revealed by TLC to contain the same three compounds (comparable Rf’s) and being gathered were treated in an open column LC system on silica gel. The elution system to try to separate them was AcOEt-MeOH in mixtures of increasing polarity. From 262 total fractions, three alkaloids were isolated, TLC analysis put in evidence that fractions 13-48 contained the less polar compound, fractions 55-238 a more polar second alkaloid and fractions 248-262 the most polar of the three of them. Spectroscopic analysis and literature data permitted to confirm the purity of compounds and to assign structural identification to alkaloids the more polar in the tripartite mixture, namely homoormosanine 1, (fr. 55-238), and ormosanine 2, (fr. 248-262). The structure of the third alkaloid is unconfirmed by now. The activity manifested by fractions and extracts in this biologically guided pathway is resumed in the table below. H

N

H N

H

H NH

HN

[µg/ml] 1 1 1

6 (VLC)*

1

7 (VLC)*

1

33-48 (LC)§

20 2 0.2

1 (LC) §

20 2 0.2

2 (LC) §

20 2 0.2

% inhib. ¶ 100 100 100 IC50 = 0.5 (µg/ml) 100 IC50 = 0.5 (µg/ml) 100 IC50 = 0.5 (µg/ml) 45 18 0 IC50 > 20 (µg/ml) 50 27 0 IC50 > 20 (µg/ml) 89 23 0 IC50 = 5 (µg/ml)

% inhib. † 100 100 100 IC50 = 0.5 (µg/ml) 100 IC50 = 0.5 (µg/ml) 100 IC50 = 0.5 (µg/ml) 20 9 7 IC50 > 20 (µg/ml) 43 0 27 IC50 > 20 (µg/ml) 89 32 0 IC50 = 5 (µg/ml)

A next step in this pathway should conduct us to the identification of the third less polar alkaloid of the ternary mixture, to determine all the components of such a synergic composition. An HPLC analysis would provide us with the percentage composition of the synergic mixture. Anyway, the good activity found for the synergic mixture, IC50 = 0.5 (µg/ml), still remains lower than that observed for the reference molecule, chloroquine. The three alkaloids and the synergic mixture, have been separately evaluated for their toxicity levels through the test over human KB carcinoma cells and Hela. Only no toxicity was found for 5-7 VLC frs. according to KB: EC50 = 25 µg/ml and Hela: EC50 = 25 µg/ml. From a chromatographic analysis stand point and following literature data,11 we can speculate that the third and less polar alkaloid (frs. 13-48) in the synergic mixture should be either homopiptanthine or homo-18-epiormosanine, because they are among the three less polar alkaloids in B. virgiloides. The other two alkaloids in B. virgiloides, pipthantine and (-) podopetaline, are more polar than ormosanine and should be eventually gathered in VLC fractions 8-16. An increasing polarity order for the six described alkaloids in B. virgiloides, has been , reported:2, 3 10, 11 homo-18-epiormosanine (Rf 0.73), homopiptanthine (Rf 0.69), homoormosanine ( Rf 0.60), ormosanine (Rf 0.39), piptantine (Rf 0.39). According to TLC analysis made by us under the same experimental conditions than those reported in the

N

H

Fraction EtOH TA 5 (VLC)*

*VLC: Vacuum Liquid Chromat. Silica 60H §LC: Liquid Chromat. In open column Silica 60 ¶: Chloroquine sensitive strain, radio isotopic method †: Chloroquine resistant strain, radio isotopic method

H N

H

VOLUMEN 19, No.1 - 2002

H

Compound 1 Compound 2 Neither compound 1 nor compound contained in fractions 13-48, manifested antiplasmodial activity like that showed by compound 2. Regarding the quest of the antimalarial activity manifested by B. virgiloides, we can conclude that the most active fractions have been obtained from the TA active extract. Only pure ormosanine (2), possesses an important antimalarial activity, however this activity appears weaker (IC50 5 µg/ml) than the activity observed in assays with the most active fractions from the TA extract, namely VLC frs. 5, 6 and 7 (100% of inhibition of parasitaemia at µg/ml). These fractions represent a mixture of three alkaloids (1, 2 and frs. 13-48 from LC).

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literature over the TA extract, the six alkaloids have been identified based on a comparison of experimental and reported Rf values. Considering these results we can deduce that the synergic mixture is composed by homopiptanthine (frs. 13-48, for which a single revealed spot appears on TLC with a purity stated by the 20 discrete peaks on the 13CNMR spectrum), compound 1, homoormosanine and compound 2, ormosanine.

H

H

H

H

N

H

homo-18-epiormosanine

N

H

N

homopiptanthine

N

H

NH

HN

H

piptanthine

NH

HN

(-)-podopetaline

H

homoormosanine

13

CNMR data for compounds 1, 2 and literature data for homoormosanine and ormosanine δ Atom

Homoormosanine*

H

The structural identification confirmation for 1 and 2, came out from NMR and EIMS analysis. Alkaloids 1 and 2, were identified after comparing 13CNMR experimental signals with literature as homoormosanine 3 and ormosanine13, respectively (see table below).

H

N

HN

N H

H

H

ormosanine

18

N

N

H

NH

N

N

N H

N H

H

H

H

H

H

H

VOLUMEN 19, No.1 - 2002

δ

δ

δ

Compound 1†

Ormosanine¶

Compound 2§

∆‡

2

55.5

55.5

55.2

53.4

+1.8

3

25.6

25.3

26.8

25.1

+1.7

4

25.0

24.6

25.3

24.6

+0.7

5

32.1

31.9

32.0

30.0

+2.0

6

64.9

65.3

63.8

62.2

+1.6

7

34.5

34.9

35.4

33.8

+1.6

8

30.0

29.5

30.7

28.6

+2.1

9

36.5

36.4

39.3

38.3

+1.0

10

63.3

62.4

56.1

54.2

+1.9

11

66.7

66.2

66.1

65.0

+1.1

13

53.2

53.1

47.0

45.4

+1.6

14

25.5

24.9

26.6

24.6

+2.0

15

-

33.5

33.2

31.6

+1.6

16

31.6

31.7

34.1

33.4

+0.7

17

37.9

37.5

38.0

37.1

+0.9

18

68.2

67.8

66.6

65.6

+1.0

19

19.8

19.7

26.8

24.6

+2.2

20

16.5

16.4

24.0

21.4

+2.6

21

25.0

24.7

25.6

23.9

+1.7

22

53.4

53.2

48.1

46.1

+2.0

24

69.8

69.7

*In CDCl3 at 22.5 MHz, δ in ppm from TMS; † In CDCl3 at 75 MHz, δ in ppm from CHCl3 (77 ppm); ¶ In CDCl3 at 50 MHz, δ in ppm from TMS; § In CDCl3-CD3OD in an equimolar proportion in deuterium, at 75 MHz, δ in ppm from CHCl3 (77 ppm);‡ Positiv value of the difference between δ values of ormosanine and 2 due to the mixture CDCl3-CD3OD used with 2 instead of pure CDCl3 reported in literature data for ormosanine

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VOLUMEN 19, No.1 - 2002

3

4

2

3

5

H

6

7 17 H 15

8 9

16

13

18

12

14

1

10 19

15 13

24

23

N1

3

5

H

7

9 11 24

H

21 22

6

7

17

N

12

H

N

4

5

6

10

16

20

11

N

14

2

N

H

H H

17

22

H

8

N

23

3

2

4

H

18

15

9 16

14

19

13

21

N 8

H

18

14

1

10 19

15 13

20

11

NH 12

H H

2

16 12

21 22

20

N1

7

9 11

H HN 23

17

N H

5

6

10

HH

8

23N 22

H

18 19

21 20

Compound 1 : homoormosanine

Compound 2 : ormosanine

EIMS spectral data obtained for 1 and 2 permits to corroborate the structural assignments according to literature data for homoormosanine3 and ormosanine.14 This survey of Bowdichia virgiloides conducted us to the qualitative characterization of a three-alkaloid-sinergicmixture, namely homopiptanthine, homoormosanine and ormosanine with an IC50 = 0.5 µg/ml over Plasmodium falciparum in vitro. The mixture represents the antiplasmodial fraction coming up from the TA extract. Also the antiplasmodial in vitro activity indexes for each of the three alkaloids have been established. A quantitative analysis of the synergic mixture should be carried out in another research in order to define the proportional composition of it. The fact of the disappearance of the methylene group at C-24 forming an open space for a double pole for interactions between N12 and N-23 and any substrate in compound 2, could be the reason for the activity loss in compound 1 which possesses in contrast such a 24-methylene group. EXPERIMENTAL

Bowdichia virgiloides H.B.K. (Leguminosae). Illustration by C. Maldonado, LPB.

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General- NMR spectra were run in a AC 300 BRUKER spectrometer. Mass spectra acquired under electronic impact at 70 eV, Optical activity measurements were measured on a PERKIN ELMER 241. VLC Silica gel : 60H MERCK (5-40 µm). LC open column silicagel :MERCK 60, 0.063-0.200 mm (70-230 mesh ASTM). TLC plates: WHATMAN (250 and 500 µm, PK 6F, gel 60A). Plant material - B. virgiloides H.B.K., Leguminosae was collected during ethnobotanical field work in the Tacana ethnic group territories, Apolo province of the La Paz department at 600 meters over sea level. A voucher specimen is deposited at the National Herbarium of the San Andrés Major University of La Paz Bolivia under the code GB 1734. Extraction and isolation of compounds – Obtention of the TA extract: Dried and powdered stem-bark (280.1 g) was moisturized in a NH4OH (NH3-H2O, 1:1, 10 ml) solution. Alkaloids salt pass to their CH2Cl2 soluble free base form. Alkalinized stem bark is extracted during 24 hr. under CH2Cl2 in a Soxhlet apparatus. The organic layer is extracted with a series of acidic aqueous fractions (5% v/v de HCl, 100 ml each). Alkaloids pass from their free base form to water soluble salts. The operation is repeated until the transfer of alkaloids from the organic layer is exhausted. The aqueous layer is alkalinized under NH4OH (NH3-H2O, 1:1, 10 ml) until pH 9 to get free alkaloids. The aqueous layer is extracted under various fractions of CH2Cl2, 100 ml each, until the transfer of alkaloids from the aqueous layer is exhausted. The organic layer is dried with anhydrous sodium sulfate. Solvent evaporation in vacuo affords the TA extract (4.64g, 1.7 % of stem bark). Alkaloids isolation: 2 g of TA extract were chromatograped in a VLC system (silica gel 60 H until 5 cm high in a fritted base funnel of 7 cm of internal diameter) eluted with mixtures of CH2Cl2-MeOH of increasing polarity, to afford 16 fractions: [100:0 (200 ml), 4.3 mg; 95:5 (200 ml), 1 mg; 90:10 (200 ml)X3, 346.9 mg, 596 mg et 84.8 mg; 87:13 (200 ml)X2, 30.4 mg and 29.6 mg; 85:5 (200 ml)X2, 14.7 mg and 16.2 mg; 83:17 (200 ml)X2, 14.6 mg and 23.7 mg; 80:20 (200 ml)X3, 43.4 mg, 51.2 mg and 64.4 mg; 0:100 (200 ml)X2, 100.5 mg and 280.0

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(14), 151 (44), 150 (20), 149 (91), 134 (27) 122 (16), 110 (22); 13CNMR (75.0 MHz): 53.4 (t, C-2), 25.1 (t, C-3), 24.6 (t, C-4), 30.0 (t, C-5), 62.2 (d, C-6), 33.8 (d, C-7), 28.6 (t, C-8), 38.3 (s, C-9), 54.2 (t, C-10), 65.0 (s, C-11), 45.4 (t, C-13), 24.6 (t, C-14), 31.6 (t, C-15), 33.4 (d, C-16), 37.1 (t, C-17), 65.6 (d, C-18), 24.6 (t, C-19), 21.4 (t, C-20), 23.9 (t, C-21), 46.1 (t, C-22), 69.7 (t, C-24). Antimalarial in vitro assays. The culture of chloroquine resistant (Indo) and chloroquine sensitive (F32-Tanzania) Plasmodium falciparum strains is realized according to Trager and Jensen15 in a RPMI 1640 medium enriched by glucose and completed by a 10% of human serum at 37ºC. 50 µl of DMSO are added to samples of vegetal extracts solved in RPMI 1640 medium using an ultrasound box. Final concentration of DMSO never exceeds 0.1%. 150 µl of culture medium containing the dilute extract and a human hematies (“O” + group, 5% of hematocrite) with 1% of parasitaemia, all placed in 96-well-plates for micro titration. All test were executes by triplicate. After a 24-hours-incubation period at 37°C in an incubator containing a candle, the medium is renewed and incubation continues during still 48 more hours. At the third day a sample of blood is extracted from each well to measure the parasitaemia. Each assay is correlated to a solvent untreated witness and chloroquine positive witness. The parasitaemia of each receptacle as well as the inhibition percentage for each extract concentration are calculated regarding the untreated witness. The values of the IC50 were determined graphically taking into account the concentration versus the percentage.

mg]. Fractions 5, 6 and 7 [90:10 (200 ml), 84.8 mg, 87:13 (200 ml)X2, 30.4 mg and 29.6 mg] that manifested the best activity indexes, and after corroborating the same chemical composition in alkaloids through TLC analysis (SiO2, CH2Cl2 and NH3 vapor, revealing agent: Dragendorff reagent), were gathered (166 mg) and then separated in a LC open column on silica gel 60 (6.6 g) using AcOEtMeOH mixtures. 262 fractions were obtained: [98:2 (2.5 l); 97:3 (0.08 l); 96:4 (0.15 l); 95:5 (0.12 l); 94:6 (0.09 l); 93:7 (0.01 l); 92:8 (0.14 l); 90:10 (0.12 l); 85:15 (0.12 l); 80:20 (0.32 l), 0:100 (0.05 l)]. Fractions 13-48 afforded one pure alkaloid according to TLC and NMR analysis, AcOEt-MeOH (98:2). Fractions 55-238 afforded compound 1 AcOEt-MeOH (98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 90:10, 85:15). Fractions 248-262 afforded compound 2 AcOEtMeOH (80:20, 0:100). Compound 1. Homoormosanine. [α]D -5.7° (CHCl3MeOH, 1.0:0.2 c 0.30); EIMS 70 eV: m/z (rel. int.): 329 [M]+ (100), 247 (25), 246 (80), 233 (23), 232 (18), 231 (62), 217 (13), 205 (16), 164 (12), 163 (25), 162 (22), 150 (27), 149 (25), 148 (26), 136 (17), 134 (19), 123 (12), 122 (13), 110 (18); 1HNMR (300 MHz): 3.06 (1H, H-1), 2.12 (1H, H-1’), 1.86 (1H, H-3), 1.61 (1H, H-3’), 1.76 (1H, H-5), 1.01 (1H, H-5’), 1.93 (1H, H-6), 1.68 (1H, H-7), 1.81 (1H, H-8), 0.94 (1H, H-8’), 2.91 (1H, H10), 2.70 (1H, H-10’), 2.80 (1H, H-11), 2.78 (1H, H-13), 1.95 (1H, H-13’), 1.56 (1H, H-15), 1.58 (1H, H-16), 1.41 (1H, H-17), 1.10 (1H, H-17’), 1.49 (1H, H-18), 1.10 (1H, H-19), 0.85 (1H, H-19’), 1.80 (1H, H-20), 1.18 (1H, H-20’), 2.93 (1H, H-22), 3.40 (1H, H-24), 3.24 (1H, H-24’); 13CNMR (75.0 MHz): 55.5 (t, C-2), 25.3 (t, C-3), 24.6 (t, C-4), 31.9 (t, C-5), 65.3 (d, C-6), 34.9 (d, C-7), 29.5 (t, C-8), 36.4 (s, C-9), 62.4 (t, C-10), 66.2 (s, C-11), 53.1 (t, C-13), 24.9 (t, C-14), 33.5 (t, C15), 31.7 (d, C-16), 37.5 (t, C-17), 67.8 (d, C-18), 19.7 (t, C-19), 16.4 (t, C-20), 24.7 (t, C-21), 53.2 (t, C-22), 69.7 (t, C-24). Compound 2. Ormosanine. [α]D -9.1° (CHCl3-MeOH, 1.0:0.2 c 0.28); EIMS 70 eV: m/z (rel. int.): 317 [M]+ (65), 259 (7), 246 (15), 234 (43), 233 (34), 232 (34), 231 (19), 219 (100), 205 (7), 191 (13), 176 (8), 162

ACKNOWLEDGMENTS The authors wish to thank the National Herbarium of Bolivia. The ethnic group Tacana. The FONAMA for financial support. Miss Lic. Yvon Rojas for antimalarial ant cytotoxic assays.

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2

3 4 5

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14 15

BRANDAO, M. G. L., GRANDI, T. S. M., ROCHA, E. M. M., SAWYER, D. R. KRETTLI, A. U. J. of Ethnopharmacology, 1992, 36, 175. ACHENBACH, H., BAUEREIβ, P., TORRENEGRA, R. Arch. Pharm. Weinheim Ger., 1988, 321, 675. TORRENEGRA, R. G., ESCARRIA, S. R., BAUEREIβ, P., ACHENBACH, H. Planta Medica, 1985, 3, 276. ELISABETSKY, E., POSEY, D. A. J. of Ethnopharmacology, 1989, 26, 299. BOURDY, G. Personal communication IRD-UMSA, Mission La Paz, Bolivia. GONÇALO, S. Contact Dermatitis, 1992, 26, 205. SCHULTZ, K. H., GARBE, I., HAUSEN, B. M., SIMATUPANG, M. H. Arch. Dermatol. Res., 1979, 264, 275. CHIAPPETA, A. D. A., FRANCISCO DE MELO, J., MACIEL, G. M. Rev. Inst. Antibiot. Univ. Fed. Pernambuco Recife, 1983, 21, 43. BROWN, P. M., THOMSON, R. H., HAUSEN, B. M. Justus Liebigs Ann. Chem., 1974, 1295 MARINHO, L. C., CARNEIRO DA CUNHA, M. T. M., THOMAS, G., BARBOSA, FILHO, J. M. Fitoterapia, 1994, LXV, 475. TORRENEGRA, R., BAUEREIβ, P., ACHENBACH, H. Phytochemistry, 1989, 28, 2219. BRAVO, J. A., SAUVAIN, M., BALDERRAMA, L., MORETTI, C., RICHOMME. P. BRUNETON, J. Rev. Bol. de Quim., 1996, 13, 19 BHACCA, N. S., BALANDRIN, M. F., KINGHORN, A. D., FRENKIEL, T. A., FREEMAN, R., MORRIS, G. A. J. Am. Chem. Soc. 1983, 105, 2538 KINGHORN, D. A., BALANDRIN, M. F., LIN, L-J Phytochemistry, 1982, 21, 2269. (14) TRAGER W, JENSEN J B Human malaria in continous culture. Science, 1976, 193, 673-675.

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