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J. Mex. Chem. Soc.

2009, 53(2), 71-75
Article © 2009, Sociedad Química de México
ISSN 1870-249X

Evaluation of Some Plant-derived Secondary Metabolites Against
Sensitive and Multidrug-resistant Mycobacterium tuberculosis
María del Rayo Camacho-Corona,a* Juan Manuel de Jesús Favela-Hernández,a Omar González-Santiago,a
Elvira Garza-González,b Gloria María Molina-Salinas,c Salvador Said-Fernández,c Guillermo Delgado,d
Julieta Luna-Herrerae
a Laboratorio de Química de Productos Naturales, DES Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León.
Guerrero y Progreso, Col. Treviño, Monterrey 64570, Nuevo León, México.
b Departamento de Microbiología, Facultad de Medicina, Universidad Autónoma de Nuevo León. Madero y
Eduardo Aguirre Pequeño, Col. Mitras Centro, Monterrey 64460, Nuevo León, México.
c Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, Nuevo León. San Luis Potosí
y dos de Abril. Col. Independencia, Monterrey 64720, Nuevo León, México.
d Instituto de Química, Universidad Nacional Autónoma de México. Circuito Exterior, Ciudad Universitaria,
Coyoacán 04510, México, D.F.
e Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional.
Prolongación de Carpio y Plan de Ayala, México 11340, D.F., México.

Received May 18, 2009; Accepted June 29, 2009
Abstract. The results on the bioevaluation of thirty five plant-derived Resumen. Se informan los resultados sobre la bioevaluación de trein-
secondary metabolites against one sensitive and three multidrug- ta y cinco metabolitos secundarios de plantas frente a una cepa sen-
resistant clinical isolates of Mycobacterium tuberculosis are reported. sible y a tres cepas resistentes a múltiples drogas de Mycobacterium
Results toward the sensitive strain showed that five products gave MIC tuberculosis. Los resultados frente a la cepa sensible mostraron que
values of 12.5 µg/mL: the alkaloids 6-methoxydihydrochelerytrine (2) cinco metabolitos secundarios poseen valores de CMI de 12.5 µg/mL:
and 6-methoxy-dihydrochelirubine (6), the flavanone pinostrobin (17), los alcaloides 6-metoxidihidroqueleritrina (2) y 6-metoxi-dihidroque-
1-hydroxy-benzoisochromanquinone (23) and 23-hydroxy-5a-lanosta- lirubina (6), la flavanona pinostrobina (17), 1-hidroxi-benzoisocro-
7,9(11),24-triene-3-one (33). These were followed by the peracetyl- manquinona (23) y el triterpeno 23-hidroxi-5a-lanosta-7,9(11),24-
strictosidine lactam (12) and the quinone aloe-emodin (24) which trien-3-ona (33). La lactama de peracetilestrictosidina (12) y la qui-
displayed MICs of 6.25 µg/mL. Finally, liriodenine (8) was the most nona aloe-emodina (24) mostraron CMIs de 6.25 µg/mL. Finalmente,
active (MIC: 3.125 µg/mL) of all secondary metabolites. Results with liriodenina (8) fue la substancia mas activa (CMI: 3.125 µg/mL) de
the multidrug-resistant clinical isolates showed that 6-methoxy-dihy- todos los metabolitos secundarios. Los resultados con los aislados
drochelirubine (6) was the most active (MIC: 12.5 µg/mL). clínicos resistentes a multidrogas mostraron que 6-metoxi-dihidro-
Keywords: Mycobacterium tuberculosis, antituberculosis activity, quelirubina (6) fue el compuesto mas activo (CMI: 12.5 µg/mL).
bioactive secondary metabolites, alkaloids, flavonoids, quinones, tri- Palabras clave: Mycobacterium tuberculosis, actividad antituber-
terpenes, diterpenes. culosis, metabolitos secundarios bioactivos, alcaloides, flavonoides,
quinonas, triterpenos, diterpenos.

Introduction thus the screening of natural products from higher plants con-
stitutes one avenue in the search for new lead antitubercular
Tuberculosis remains one of the major deadliest infectious dis- agents. In this study we screened thirty five phytochemicals
eases for humans. Approximately 9.2 million people develop against one drug sensitive and three multidrug resistant clinical
the active disease each year, while 1.7 million cases of active isolates of M. tuberculosis using the Alamar Blue assay. The
disease result in death in the same period. The situation is tested secondary metabolites included alkaloids (1-13, scheme
worsening primarily because the association between tubercu- 1), flavonoids (14-22, scheme 2), quinones (23, 24, scheme 3),
losis and epidemic HIV/AIDS as well as the growing preva- triterpenes (25-33, scheme 4) and diterpenes (34-35, scheme
lence of multidrug-resistant (MDR) Mycobacterium tubercu- 4). The isolation and characterization of the different phyto-
losis strains [1]. These acute problems have led to search for chemicals were described previously [5-14].
structurally effective new drugs against this bacterium.
Part of our area of interest involves the search for struc-
turally novel anti-tuberculosis natural products from higher Results and Discussion
plants. Plants have been used worldwide in traditional medi-
cines for the treatment of various diseases and it is estimated Plant derived secondary metabolites 1-35 were evaluated
that even today approximately 65-75% of the world’s popu- against M. tuberculosis H37Rv and three MDR isolates (345,
lation rely only on medicinal plants as their primary source M-12 and M-20) and the compounds which displayed activ-
of medicines [2]. The phytochemical study of some of these ity are listed in Table 1. Compounds 4, 7, 11, 16, 18-22, 27,
plants has yielded a number of active natural products [3,4], 30 and 31 were considered inactive (MIC > 50 µg/mL for

Among those metabolites with moderate activity OH O OH (MIC: 12.24-triene-3-one (33) isolated from Guarea R1 R2 rhopalocarpa.24Z-dien-tirucalla-26-oic acid =O H H O R H3 COOC R 10 Strictosidine 11 Strictosidine lactam Glcp 12 Peracetylstrictosidine lactam Glcp(Ac)4 O O O H OCH3 H COOH N OCH3 OCH 3 O 13 Phaeanthine AcO AcO H 3 CO O 30 Acetyl-11A.24-triene-3-one =O pimaradiene-2B . the only secondary metabolites that showed show that seven secondary metabolites exhibited MIC values good activity against the three MDR M.18-diol BOH 14 5. Liriodenine (8) isolated from S. Structures of tested triterpenes and diterpenes.25 µg/mL were 20 Podocarpusf lavone A H CH3 22 Quercetin 21 Podocarpusf lavone B OCH3 OCH 3 the alkaloid peracetylstrictosidine lactam (12) from Cephaelis Scheme 2.25 µg/mL and only one MIC 25-50 µg/ml).5 µg/mL and two of 6. Structures of tested flavonoids. three presented isolates were the alkaloids 6-methoxydihydrocheleritrine (4.15-sandaraco- R1 16 Pinocembrin H H 7. 6-methoxydihydrosanguinarine (5.7-Dihydroxy-6-methyl-8-prenyl-flavanone CH 3 18 Aceylpinocembrin Ac Ac 19 Chrysin H H 2.3-dehydro Scheme 4. 34 Ent-8(14). Structures of tested alkaloids. 35 Ent-8(14). R HO R 2O O 2 R HO O HOH2 C R R R1 OR1 O 32 5A-Lanosta-7.24Z-dien-tirucalla-26-oic acid AOH N H N H H H 27 Acetyl-oleanolic acid BOAc 29 3-Oxo-7. tuberculosis clinical of 50 µg/mL. Chem. 2009.125 µg/mL towards the sensitive strain 12. dinklagei showed the best activity (MIC: value of 3.9(11). OR2 OH HO O H37Rv. Those with MIC values of 6. Results However.24-diol AOH pimaradiene-2A.7-Dihydroxy-8-prenyl-flavanone H 17 Pinostrobin H CH 3 15 5.18-diol AOH R1 R2 33 23-Hydroxy-5A lanosta.24. 1-hydroxy-benzoisochromanquinone from OH O Psychotria camponutans (23) and the triterpene 23-hydroxy- HO OH OH O 5-lanosta-7.13B -olide CH 3 Scheme 1. Structures of tested quinones. six had MIC values at 25 µg/mL.12-Dimethoxydihydrocheleritrine OMe OMe 6 6-Methoxydihydrochelirubine OMe Scheme 3. Soc. MIC had an MIC value of 3. Mex. 53(2) María del Rayo Camacho-Corona et al.5-50 µg/ml) and 6-methoxy-dihydrochelirubine (6.15-sandaraco- OH O triene-3A.9(11). dichroa and the quinone aloe-emodin (24) from Stephania din- klagei.125 µg/mL). M-12 and M-20).9(11). 4 6-Acetonyldihydrocheleritrine CH2COCH3 H O O N O O R1 R2 8 Liriodenin H H N O 9 Dicentrinone OMe OMe H3 CO OH CHO CO2H CH3 H COOH 1 OCH3 R H 7 Arnotianamide R2 R O R O Glcp NH H N R R 25 Oleanolic acid BOH O O 26 Epi-oleanolic acid AOH 28 3A-Hydroxy-7. values of 12.72    J. H37Rv and > 200 µg/mL for 345.12A -epoxy- N 31 Betulinic acid oleanan-28. R2 O OH OH O OH R O O O O O OH N N O H 3CO O O O OCH 3 OCH 3 R 1 R1 R2 1 Dihydrocheleritrine H H R 23 1-Hydroxy-benzoisochromanquinone 24 Aloe-emodin 2 6-Methoxydihydrocheleritrine OMe H 5 6-Methoxydihydrosanguinarine H 3 6. MIC: .5 µg/mL) were the flavonoid pinostrobin (17) from R 1O O O OH Teloxys graveolens.

5 6 (A) 12.50 200 >200 100 (Rubiaceae) Stephania dinklagei 24 (Q) 7 6. 11.5 12. Phaeanthine (13) and 1-hydroxy-ben. 12. M-12 and M-20).0 12. 18-22. platycarpa 25 (T) 14 50 >200 >200 >200 (Leguminosae) 26 (T) 25 NTb NTb NTb Celaenodendron mexicanum 28 (T) 6 50 >200 >200 >200 (Euphorbiaceae) 29 (T) 50 200 >200 >200 32 (T) 12.5 50 50 25 (Papaveraceae) 3 (A) 13 50 >200 >200 >200 5 (A) 50 25 50 12. the most active metabolite against the while 6-methoxy-dihydrochelirubine showed the same activity sensitive strain showed only moderate activity (MIC 100 µg/ (MIC 12. tuberculosis Reference (Family) (Type)a MIC (µg/mL) H37Rv 345 M-12 M-20 1 (A) 25 >200 >200 >200 Bocconia arborea 2 (A) 12. mL) against one MDR isolate. zoisochromanquinone (23) showed moderate activity against as well as 5. Liriodenine (8).25 >200 >200 >200 (Menispermaceae) E. 3-oxo-7.5 12. isolated from Bocconia arborea. the other two alkaloids were more active against tylstrictosidine lactam (12). mL) against only one of the MDR isolates.24-triene-3a. Compounds 4. 16. 7. Interestingly.5 12. 27.50 >200 >200 >200 Guarea rhopalocarpa 33 (T) 8 25 >200 >200 200 (Meliaceae) 34 (D) 25 >200 >200 >200 35 (D) 50 >200 >200 >200 Standard drugs Isoniazid 0.50 12. D: Diterpene.50 3.125 >200 >200 100 (Menispermaceae) 9 (A) 50 >200 >200 >200 Cephaelis dichroa 10 (A) 11 >50 100 >200 >200 (Rubiaceae) 12 (A) 6. Similarly. acid (29) and 5a-lanosta-7.9(11). tuberculo- sis. 30 and 31 were considered inactive (MIC > 50 μg/mL for H37Rv and > 200 μg/mL for 345.23-diol (32).5 µg/ml). Plant species Secondary metabolite M. Q: Quinone.50 12. tuberculosis H37Rv and MDR clinical isolates of M. sensitive strain and showed only weak activity (MIC: 200 µg/ respectively.125 12. b NT: not tested.06 <50 <50 <50 Ethambutol 2. Antimycobacterial activity de some secondary metabolites against M. F: Flavonoid. T: Triterpene.50 25 a A: Alkaloid.7-dihydroxy-6-methyl-8-prenyl-flavone (15) and the sensitive bacteria and displayed only weak activity (MIC: pinostrobin (17) showed moderate to good activity against the 100–200 µg/ml) against three and two of the resistant bacteria. perace- cal isolates.50 Rifampicin 0.Evaluation of Some Plant-derived Secondary Metabolites Against Sensitive and Multidrug-resistant Mycobacterium tuberculosis 73 Table 1.06 <50 <50 <50 Streptomycin 0.25 200 >200 >200 Triclisia patens 13 (A) 9 25 200 200 100 (Menispermaceae) Eysenhardtia platycarpa 14 (F) 14 >50 200 200 >200 (Leguminosae) 15 (F) 25 200 >200 >200 Teloxys graveolens 17 (F) 5 12. .5 Stephania dinklagei 8 (A) 7 3.24Z-dien-tirucalla-26-oic the sensitive bacteria.5 µg/ml) against the sensitive strain and MDR clini.50 200 >200 >200 (Chenopodiaceae) Psychotria camponutans 23 (Q) 12 12.

. M. then working metabolites growth of M. Becton.23-diol (32). A comparison of immediately before use in antimycobacterial test. and pyrazinamide-sensitive. (MIC) was defined as the lowest concentration of sample that ylsulfoxide at a concentration of 20 mg/mL and stored at -70 prevents a color change to pink. Each concentration was assayed in duplicate. Company. eth- oC until use. [14]. solutions in the same way and were incubated for an addi- Narváez-Mastache et al. The purity of the natural prod. ity (MIC: 12. Westlake OH) and 12 Preparations of samples for testing mL of sterile 10% Tween 80. Streptomycin. streptomycin. MDR M.24-triene-3-one culture was mixed with a sufficient volume of sterile supple- (33) and 5a-lanosta-7. dihydrochelirubine (6) and liriodenine (8) represent useful The final concentrations of metabolites tested ranged from 200 templates for the development of new anti-tuberculosis drugs. that of dihidrochelerythine (1) might be explained by a higher lipophilicity of the former. 1 standard. the same time. 16].12]. tional 24 h. tuberculosis H37Rv American Type Culture Collection Dr.15-sandaracopimaradiene-2a. 10:100 and 1:100 diluted controls The results obtained in this evaluation indicate that the were prepared from the bacterial suspension. if the well turned pink. 100:100. the higher Tamaulipas. PAICYT SA993- clinical isolates (M-12. [11. [13].5 µg/mL) against the sensitive and multidrug. Virgilio Bocanegra-García provided multidrug resistant (ATCC) 27294. After incubation time. The local ethics committee approved all activity of 6-methoxy-dihydrochelerythrine (2). compared to protocols used in this study. Stock solutions as positive for growth. Solis et al. to that of McFarland’s nephelometer No. to 0. After incubation time. stock solutions were four-fold ambutol and rifampicin were included as standard drugs. tuberculosis lowing grants: PROMEP/103. to all testing wells. The plates were re-incubated at 37 oC for 24 h. Mex. (oleic acid albumin dextrose catalase. Phytochemicals were isolated and identified previously by all the wells received the mixture of Alamar blue and Tween Camacho et al. tuberculosis strains Acknowledgments For the present study. growth of 100.7-dihydroxy-6-methyl-8-prenyl-flavanone (15) and 5. The final concentration of DMSO in wells was < 1% v/v.18-diol (34) was twice as active as ent-8(14). tuberculosis H37Rv by ≥ 94% at 12. Before the assay. etham. Each diluted in supplemented Middlebrook 7H9 media. Wells with a well-defined pink color were scored ucts and derivatives was determined by HPLC. A similar trend M. USA) until logarithmic growth was achieved. with the mented Middlebrook 7H9 broth to reach turbidity equivalent exception of acetyl oleanolic acid (25) and oleanolic acid (26). water (200 mL) was poured into outer perimeter wells of Finally.9(11). Navarro et al. CONACYT SALUD2004-C01-161and SIP/IPN. 10 and 1% of the bacterial population tested. The sus- The antimycobacterial activity detected for 25 and 26 is in pension was then diluted 1:20 with the same culture medium agreement to that previously reported [17.18]. In assessing structure-activity relationships. it has been reported that the benzo(c)phena­ the microplate. as well as to the drug-free control wells. the diterpene epimers of sandaracopimaradiene indicates that the configuration of the hydroxyl group at C-2 is important for Antimycobacterial test activity.9(11). Each brin (16). México. representing the structural skeletons of the most active alkaloids 6-methoxy. Dickinson and droxy-8-prenyl-flavanone (14). The Minimal Inhibitory Concentration were prepared by dissolving the various metabolites in dimeth. At resistant isolates. Each mycobacteria was cultured at 37 oC in Middlebrook acetylstrictosidine lactam (12) and strictosidine lactam (11). experiment was performed at least twice. This study was supported by the fol- butol. All other wells received 100 mL of supple- nthridine alkaloids chelerythrine and chelirubine inhibited the mented Middlebrook 7H9 broth.18-diol (35) against the sensitive strain.5 µg/mL solutions (100 mL) were poured into the first well of each row. The activity of all phytochemicals against the aforementioned ene-2b. isolated clinical strains. Reynosa. isoniazid. Each microplate was incubated for 5 days at 37oC in a 5% CO2 atmosphere in a sealed plastic bag. since ent-8(14). pinostrobin (17) and pinocem. [5-10]. COFAA. which may facilitate its passage Preparation of test inoculum across the cell membrane of the mycobacteria. The test inoculum (100 mL) was added whereas 6-methoxy-dihydrochelirubine (6) has the same activ. Soc.74    J. 53(2) María del Rayo Camacho-Corona et al. rifampicin. and 23-hydroxy-5a-lanosta-7. isoniazid. SNI and EDI fellow and acknowledges their support. A similar argu- ment might be made for the following pair of metabolites: per.24-triene-3á. Jurisdicción Sanitaria IV. [3]. Experimental one control growth was developed with a mixture of 20 mL of Alamar blue solution (Trek Diagnostics. tuberculosis strains was tested using the microplate Alamar was observed between oleanolic acid (25) and epi-oleanolic Blue assay described previously [15. JLH is de Salud de Tamaulipas. MIC: 25 µg/mL) from which two-fold dilution series were made through the was half as active as chelerythrine against the sensitive strain.5/04/1371. M-20 y 345) obtained from Secretaría 04.2% glycerol and 10% OADC 5. We found that dihydrochelerythrine (1. microplate column. Chem. 7H9 broth supplemented with 0.15-sandaracopimaradi.7-dihy.097 mg/ml. Sterile distilled acid (26). the following bacteria were used: M. 2009.

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