ANTIBACTERIAL COMPOUNDS ISOLATED FROM� BYRSONIMA CRASSIFOLIA J. Fausto Rivero-Cruza,*, Sobeida Sánchez-Nietob, Guadalupe Beníteza,1, Xóchitl Casimiroa,1, César Ibarra-Alvaradoc, Alejandra Rojas-Molinac and Blanca Rivero-Cruza (Received May 2009; Accepted August 2009) This paper is dedicated to Professor Doctor Rachel Mata for her 60th birthday
ABSTRACT As a part of a project directed toward the discovery of oral antimicrobial compounds from plants, eight known compounds, β-amyrin (1), betulin (2), betulinic acid (3), oleanolic acid (4), quercetin (5), (-)-epicatechin (6), gallic acid (7) and β-sytosterol were isolated from a dichloromethane soluble partition of a methanol extract of Byrsonima crassifolia. All the compounds isolated were evaluated for their antimicrobial activity against twelve bacteria and Candida albicans. Compounds 1 and 4-7 inhibited the growth of the bacteria with concentrations ranging from 64 to 1088 µg/mL. Keywords: Byrsonima crassifolia, nance, natural oral antimicrobials, oral pathogens, epicatechin, terpenoids. REsuMEN Como parte de un proyecto conducente a la búsqueda de compuestos antimicrobianos de plantas se logró el aislamiento de ocho compuestos conocidos, β-amirina (1), betulina (2), ácido betulínico (3), ácido oleanolico (4), quercetina (5), (-)-epicatequina (6), ácido gálico (7) y β-sitosterol de la fracción de diclorometano derivada del extracto metanólico de Byrsonima crassifolia. Todos los compuestos aislados se evaluaron para determinar su actividad antibacteriana contra un panel de doce bacterias y Candida albicans. Los compuestos aislados inhibieron el crecimiento de las bacterias a concentraciones en el rango de 64 a 1088 µg/mL
Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, México D.F., C. P. 04510, México b Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, México D.F., C. P. 04510, México c Laboratorio de Investigación Química y Farmacológica de Productos Naturales, Facultad de Química, Universidad Autónoma de Querétaro. Centro Universitario, Querétaro 76010, Qro, México *Corresponding author. Tel: +52 55 56225281; Fax: +52 55 56225329. E-mail address:
[email protected] (J. Rivero-Cruz). 1 Taken in part from their BS thesis. a
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J.F. Rivero, S. Sánchez, G. Benítez, X. Casimiro, C. Ibarra, A. Rojas, B. Rivero
Introduction The fruits of a number of species of Byrsonima have been widely consumed by the natives of Central America and northern South America. The best-known of these is the “nance”, Byrsonima crassifolia (L.) Kunth [syns. Byrsonima cumingana Juss.; Byrsonima fendleri Turcz.; Byrsonima panamensis Beurl.; Malpighia crassifolia L.], which has acquired many alternate vernacular names: “changugu”, “chi”, “nance agrio”, “nanche”, “nanchi”, “nancen”, “nanche de perro”, “nananche”, and “nantzin” in Mexico (Argueta et al., 1994). The nance is a slow-growing tree 33 ft (10 m) high, which in certain situations may reach 66 ft (20 m). This tree is native and abundant in the wild, sometimes in extensive stands, in open pine forests and grassy savannas, from southern Mexico, through the Pacific side of Central America, to Peru and Brazil. It also occurs in Trinidad, Barbados, Curacao, St. Martin, Dominica, Puerto Rico, Haiti, the Dominican Republic and throughout Cuba. As a home remedy, the bark infusion is taken to halt diarrhea; also as a febrifuge. It is considered beneficial in pulmonary complaints, cases of leucorrhea, and allegedly tightens the teeth where the gums are diseased. In Belize, it is taken orally as an antidote for snakebites. In Guyana, the pounded bark is poulticed on wounds. Mexicans apply the pulverized bark on ulcers (Martínez, 1989; Argueta et al., 1994; Martínez et al., 1999). A methanolic extract of the root and stem bark of B. crassifolia was found to inhibit the growth of Streptococcus pyogenes (Cáceres et al., 1990), Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Staphylococcus epidermidis, Streptococcus pneumoniae y Micrococcus luteus (Martínez et al., 1999; Cáceres et al., 1991). The methanolic extract of the leaves of B. crassifolia demonstrated weak activity against Trypanosoma cruzi (Cáceres
et al., 1990; 1993) and in vitro spasmogenic effects on rat fundus (Amarquaye, 1994). In a previous work, the ethanolic aqueous extract of the bark afforded procyanidin trimers, procyanidin dimmers, (+)-epicatechin, 3-O-galloyl-(+)-epicatechin, catechin and gallic acid (Geiss et al., 1992). In the present work, a phytochemical study, of the methanol (MeOH) soluble extract of B. crassifolia was performed. The crude extract exhibited significant antibacterial activity against twelve bacteria and the yeast Candida albicans. Bioassay-guided fractionation of this extract, using the selected panel of twelve bacteria as a monitor, led to the isolation and identification of eight known compounds namely, β-amyrin (1), betulin (2), betulinic acid (3), oleanolic acid (4), quercetin (5), (-)-epicatechin (6), gallic acid (7) and β-sytosterol. Methodology Plant material The bark of B. crassifolia was collected in Oaxaca, Oaxaca by Dr. Araceli Pérez in July 2005 and identified by Dr. Robert Bye, Instituto de Biología, UNAM. Activity guided compounds isolation The air dried bark (0.48 Kg) of B. crassifolia was extracted by maceration with MeOH (3 × 2 L) at room temperature (72 h each) and the combined methanolic extracts were evaporated under reduced pressure, yielding a residue (104 g), which was partitioned with petroleum ether, dichloromethane and ethyl acetate. Water was added to the MeOH extract to afford a 50 % aqueous MeOH solution before partitioning with dichloromethane. The resulting four soluble partitions (petroleum ether, dichloromethane, ethyl acetate and aqueous) were evaluated against twelve bacteria and the yeast C. albicans (BSME, Table 1). The crude dichloromethane soluble partition (25.7 g) was found to exhibit strong
Antibacterial compounds isolated from Byrsonima crassifolia
antibacterial activity (DP, Table 1). Hence this partition was subjected to silica gel column chromatography and eluted with gradient mixtures of hexane-dichloromethane, and then acetone and acetone-MeOH of increasing polarity to give 11 pooled fractions. Fractions F3, F8 and F10 were active in the antimicrobial assay. Active fraction F3 was chromatographed over a silica gel column with hexane-acetone of increasing polarity to yield 10 subfracctions (F3I – F3X). Compounds 1-4 and β-sitosterol were obtained from fractions F3I – F3III. Additional chromatographic separation of fraction F10 over Sephadex LH-20, using MeOH (isocratic), yielded 7 fractions. Active fraction F10VI was purified by semi-preparative HPLC, by elution with a gradient starting with CH3CN-H2O (30:70) to CH3CN-H2O (70:30) in 30 min, to afford pure compound 5 (tR = 15.7 min). Fraction F8 was chromatographed over a Sephadex LH-20 column (5 × 50 cm) using CH2Cl2MeOH to yield 10 subfractions. Fraction F8VII afforded pure compound 6 and fraction F8IX afforded compound 7. Instrumentation TLC for monitoring the fractions obtained by CC was performed on Merck silica gel 60 F254 aluminum sheets. Preparative TLC was carried out on Merck silica gel 60 F254 glass plates (2.0 mm layer thickness). TLC spots were visualized by inspection of the plates under UV light (254 and 366 nm) on a Cole-Parmer 9818 darkroom series UV viewing system. The TLC plates were sprayed with 1% vanillin/sulphuric acid and heated (110°C). All CC were performed with Kieselgel 60 Merck 70-230 mesh, 0.063-0.200 mm. A Merck Hibar® 150-4, 6 Purospher® STAR RP-18 endcapped column (5 µm, 15 x 2 cm i.d.) was used for analytic RPLC along with a Waters 960 pump and a Waters 996 photo-diode array detec-
Rev. Latinoamer. Quím. 37/2 (2009)
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tor. Melting points of the isolates were determined using a Fisher-Johns melting point apparatus, and are uncorrected. 1 H-NMR, 13C-NMR, HMQC, HMBC, and 1 H-1H-COSY spectra were measured on a Varian VNMRS instrument operating at 400 and 100 MHz, respectively. Compounds were analyzed in CDCI3, MeOHd4 or DMSO-d6 with tetramethylsilane (TMS) as internal standard. 13C-NMR multiplicities were determined using APT and DEPT experiments. EIMS were recorded on a Thermo-electron DFS mass spectrometer. Antimicrobial assays MIC determination on Streptococcus mutans and Porphyromonas gingivalis Streptococcus mutans (ATCC 10449) and Porphyromonas gingivalis (ATCC 33277), oral bacteria frequently associated with dental caries and periodontal disease, were chosen for this investigation. They were maintained at the College of Medicine, UNAM. Brain-heart infusion broth (Difco) and trypticase soy broth-yeast extract medium supplemented with cysteine hydrochloride (0.05 %), menadione (0.02 µg/mL), hemin (5 µg/mL), and potassium nitrate (0.02 %), respectively, were used to grow S. mutans and P. gingivalis. The antimicrobial activity was evaluated by an in vitro growth inhibition assay performed in 96-well microtiter plates. Overnight cultures of test bacteria were centrifuged (10,000 rpm, 10 min), washed twice with 0.05 M phosphate buffered saline (PBS, pH 6.8) and re-suspended in PBS. Each well in the microtiter plate contained S. mutans [final concentration of 5 × 105 colony forming units (CFU)/mL] or P. gingivalis (5 × 106 CFU/mL), serially diluted test compound, and the appropriate growth medium. Triplicate samples were performed for each test concentration. The controls included inoculated growth medium without test compounds. Sample blanks contained uninoculated growth medium only. All
1000
>1064
c
500
1064
250
266
>1030
1030
1030
1030
1030
1030
1030
1030
1030
1030
256
256 515
515
>1024 >1030
1024
1024
1024
1024
512
512
512
256
512
512
542
542
>1088
1088
>1088
1088
1088
1088
1088
1088
1088
1088
1088
3
128
256
>848
>848
>848
848
848
848
848
106
212
212
212
4
256
512
>1012
>1012
>1012
1012
1012
1012
506
506
506
253
506
5
64
32
>1240
1240
1240
520
130
520
520
260
520
260
130
6
64
128
>1024
1024
1024
1024
1024
512
256
256
256
256
256
7
nt
nt
>12.8
>12.8
12.8
12.8
3.2
2.0
32
3.2
0.06
6.4
0.06
PGd
0.6
1.7
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
CHXd
a
Tested samples: BSME: methanolic extract from the bark of B. crassifolia; DP: dichloromethane partition; b-amyrin (1), betulin (2), betulinic acid (3), oleanolic acid (4), quercetin (5), epicatechin (6) and gallic acid (7) b Standard broth microdilution c In vitro growth inhibition assay performed in 96-well microtiter plates d Reference antibiotics: PG: penicillin; CHX: chlorhexidine gluconate.
Phorphyromonas gingivalis
250
266
Streptococcus mutansc
1000
>1064
Candida albicans 54b
1000
1000
>1064 >1064
1000
>1064
Pseudomonas aeruginosa 339b
Klebsiella pneumonia 425
E. coli ATCC 25922b
E. coli 442
b
Escherichia coli ipm 389
b
63
64 b
Bacillus subtilis 327b
63
64
Enterococcus faecium 379 (VR)b
63
125
133 64
250
266
S.aureus ATCC 25923b
b
S. aureus 310 (MR)
Staphylococcus aureus 375b
2
Microorganisms 1
Table 1. Minimum inhibitory concentrations in mg/mL of the compounds isolated from the bark of B. crassifolia and reference antibiotics.
DP
J.F. Rivero, S. Sánchez, G. Benítez, X. Casimiro, C. Ibarra, A. Rojas, B. Rivero
Tested samplesa BSME
158
Antibacterial compounds isolated from Byrsonima crassifolia
plates were incubated at 37°C under appropriate atmospheric conditions [S. mutans was grown aerobically and P. gingivalis was incubated in an anaerobic growth chamber (Fisher Scientific) in 10 % H2, 5 % CO2 and 85 % N2], growth was estimated spectrophotometrically (A660 nm), after 24 and 48 h using a microtiter plate reader. The MIC value for each test organism was defined as the minimum concentration of test compound limiting turbidity to
