byrsonimia cassifolia dental folk medicine

155

ANTIBACTERIAL COMPOUNDS ISOLATED FROM BYRSONIMA CRASSIFOLIA

J. Fausto Rivero-Cruza,*, Sobeida Snchez-Nietob, Guadalupe Benteza,1, Xchitl Casimiroa,1, Csar 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, -amyrin (1), betulin (2), betulinic acid (3), oleanolic acid (4), quercetin (5), (-)-epicatechin (6), gallic acid (7) and -sytosterol were isolated from a dichloromethane soluble partition of a methanol extract of Byrsonima crassifolia. All the compounds isolated were evaluated for their antimi-crobial 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 g/mL.

Keywords: Byrsonima crassifolia, nance, natural oral antimicrobials, oral patho-gens, epicatechin, terpenoids.

RESUMEN

Como parte de un proyecto conducente a la bsqueda de compuestos antimicro-bianos de plantas se logr el aislamiento de ocho compuestos conocidos, -amirina (1), betulina (2), cido betulnico (3), cido oleanolico (4), quercetina (5), (-)-epicate-quina (6), cido glico (7) y -sitosterol de la fraccin de diclorometano derivada del extracto metanlico 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 g/mL

aDepartamento de Farmacia, Facultad de Qumica, Universidad Nacional Autnoma de Mxico, Mxico D.F., C. P. 04510, MxicobDepartamento de Bioqumica, Facultad de Qumica, Universidad Nacional Autnoma de Mxico, Mxico D.F., C. P. 04510, MxicocLaboratorio de Investigacin Qumica y Farmacolgica de Productos Naturales, Facultad de Qumica, Uni-versidad Autnoma de Quertaro. Centro Universitario, Quertaro 76010, Qro, Mxico*Corresponding author. Tel: +52 55 56225281; Fax: +52 55 56225329. E-mail address:[email protected] (J. Rivero-Cruz).1Taken in part from their BS thesis.

156 J.F. RiveRo, S. Snchez, G. Bentez, X. caSimiRo, c. iBaRRa, a. RoJaS, B. RiveRo

INTRODUCTION

The fruits of a number of species of Byr-sonima 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 pa-namensis Beurl.; Malpighia crassifolia L.], which has acquired many alternate ver-nacular names: changugu, chi, nance agrio, nanche, nanchi, nancen, nan-che 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, Cura-cao, St. Martin, Dominica, Puerto Rico, Hai-ti, 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 alle-gedly 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 ul-cers (Martnez, 1989; Argueta et al., 1994; Martnez et al., 1999). A methanolic extract of the root and stem bark of B. crassifolia was found to inhibit the growth of Strep-tococcus pyogenes (Cceres et al., 1990), Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Staphylococcus epidermi-dis, Streptococcus pneumoniae y Micrococ-cus luteus (Martnez et al., 1999; Cceres et al., 1991). The methanolic extract of the leaves of B. crassifolia demonstrated weak activity against Trypanosoma cruzi (Cceres

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, (+)-epica-techin, 3-O-galloyl-(+)-epicatechin, cate-chin 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 se-lected panel of twelve bacteria as a moni-tor, led to the isolation and identification of eight known compounds namely, -amyrin (1), betulin (2), betulinic acid (3), oleanolic acid (4), quercetin (5), (-)-epicatechin (6), gallic acid (7) and -sytosterol.

METhODOLOgy

Plant material The bark of B. crassifolia was collected in Oaxaca, Oaxaca by Dr. Araceli Prez in July 2005 and identified by Dr. Robert Bye, Instituto de Biologa, UNAM.

Activity guided compounds isolationThe 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, dichlo-romethane and ethyl acetate. Water was added to the MeOH extract to afford a 50 % aqueous MeOH solution before partitio-ning with dichloromethane. The resulting four soluble partitions (petroleum ether, dichloromethane, ethyl acetate and aque-ous) were evaluated against twelve bacteria and the yeast C. albicans (BSME, Table 1). The crude dichloromethane soluble parti-tion (25.7 g) was found to exhibit strong

Antibacterial compounds isolated from Byrsonima crassifolia Rev. Latinoamer. Qum. 37/2 (2009) 157

antibacterial activity (DP, Table 1). Hence this partition was subjected to silica gel column chromatography and eluted with gradient mixtures of hexane-dichlorometh-ane, 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 subfracc-tions (F3I F3X). Compounds 1-4 and -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 CH2Cl2-MeOH to yield 10 subfractions. Fraction F8VII afforded pure compound 6 and frac-tion F8IX afforded compound 7.

INSTRUMENTATION

TLC for monitoring the fractions obtained by CC was performed on Merck silica gel 60 F254 aluminum sheets. Prepara-tive 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% vani-llin/sulphuric acid and heated (110C). 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 m, 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-

tor. Melting points of the isolates were determined using a Fisher-Johns melting point apparatus, and are uncorrected. 1H-NMR, 13C-NMR, HMQC, HMBC, and 1H-1H-COSY spectra were measured on a Varian VNMRS instrument operating at 400 and 100 MHz, respectively. Com-pounds were analyzed in CDCI3, MeOH-d4 or DMSO-d6 with tetramethylsilane (TMS) as internal standard. 13C-NMR multiplicities were determined using APT and DEPT experiments. EIMS were re-corded on a Thermo-electron DFS mass spectrometer.

Antimicrobial assaysMIC determination on Streptococcus mutans and Porphyromonas gingivalisStreptococcus 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 Medi-cine, UNAM. Brain-heart infusion broth (Difco) and trypticase soy broth-yeast ex-tract medium supplemented with cysteine hydrochloride (0.05 %), menadione (0.02 g/mL), hemin (5 g/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 form-ing units (CFU)/mL] or P. gingivalis (5 106 CFU/mL), serially diluted test compound, and the appropriate growth medium. Trip-licate samples were performed for each test concentration. The controls included inoculated growth medium without test compounds. Sample blanks contained uninoculated growth medium only. All


Tabl

e 1.

Min

imu

m in

hib

itor

y co

nce

ntr

atio

ns

in

g/m

L of

th

e co

mpo

un

ds is

olat

ed fr

om t

he

bark

of B

. cra

ssifo

lia a

nd

refe

ren

ce a

nti

biot

ics.

Mic

roor

gan

ism

sTe

sted

sam

ples

a

BSM

E

DP

12

34

56

7PG

dC

HX

d

Stap

hylo

cocc

us a

ureu

s 37

5b26

625

051

210

3010

8821

250

613

025

60.

06S.

aur

eus

310

(MR

)b13

312

551

210

3010

8821

225

326

025

66.

4n

tS.

aure

us A

TCC

259

23b

6463

256

1030

1088

212

506

520

256

0.06

nt

Ent

eroc

occu

s fa

eciu

m 3

79

(V

R)b

6463

512

1030

1088

106

506

260

256

3.2

nt

Bac

illus

sub

tilis

327

b64

6351

210

3010

8884

850

652

025

632

nt

Esc

heri

chia

col

i ipm

389

b10

6450

051

210

3010

8884

810

1252

051

22.

0n

tE

. col

i 442

b>1

064

1000

1024

1030

1088

848

1012

130

1024

3.2

nt

E. c

oli A

TCC

259

22b

>106

410

0010

2410

3010

8884

810

1252

010

2412

.8n

tK

lebs

iella

pne

umon

ia 4

25b

>106

410

0010

2410

30>1

088

>848

>101

212

4010

2412

.8n

t

Pseu

dom

onas

aer

ugin

osa

339b

>106

410

0010

24>1

030

1088

>848

>101

212

4010

24>1

2.8

nt

Can

dida

alb

ican

s 54

b>1

064

1000

>102

4>1

030

>108

8>8

48>1

012

>124

0>1

024

>12.

8n

tSt

rept

ococ

cus

mut

ansc

266

250

256

515

542

256

512

3212

8n

t1.

7Ph

orph

yrom

onas

gin

giva

lisc

266

250

256

515

542

128

256

6464

nt

0.6

a Tes

ted

sam

ples

: B

SM

E:

met

han

olic

ext

ract

fro

m t

he

bark

of

B. c

rass

ifolia

; D

P: d

ich

loro

met

han

e pa

rtit

ion

; -

amyr

in (1

), be

tulin

(2),

betu

linic

ac

id (3

), ol

ean

olic

aci

d (4

), qu

erce

tin

(5),

epic

atec

hin

(6) a

nd

galli

c ac

id (7

)b S

tan

dard

bro

th m

icro

dilu

tion

c In v

itro

grow

th in

hib

itio

n a

ssay

per

form

ed in

96-

wel

l mic

roti

ter

plat

esd R

efer

ence

an

tibi

otic

s: P

G: p

enic

illin

; CH

X: c

hlo

rhex

idin

e gl

uco

nat

e.


plates were incubated at 37C under appro-priate 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


Figure 1. Compounds isolated from Byrsonima crassifolia

ranged from 64 to 1088 g/mL (Table 1). Among these, (-)-epicatechin (6) was the most active with MIC values of 64 g/mL for P. gingivalis, 128 g/mL for Streptococ-cus mutans and 260 g/mL for S. aureus (Table 1). The remaining compounds did not demonstrate comparable level of anti-microbial activity against all test bacteria

when compared with (-)-epicatechin (6). The MIC values obtained for compounds 1-7 were higher than that of penicillin G and chlorhexidine on the corresponding microorganisms. Although chlorhexidine and penicillin G have a considerably lower minimum inhibitory concentration values than the active compounds in this study


(Table 1), its side effects have been well documented (Quirynen et al., 2000; Abey-lath and Turos, 2008).

The flavonoids quercetin (5) and (-)-epi-catechin (6) have been previously isolated from the Byrsonima genus (Bejar et al., 1993), and subsequently, their diverse bio-logical activities have also been reported. It is also known that epicatechin inhibits enzymes such as glucosyltransferases (Wu et al., 2009) and displays anti-ulcer, anti-inflammatory, astringent, cytotoxic, antimicrobial, and amoebicidal activities (Calzada et al., 2000; Lin et al., 2001; Kwon et al., 2007). -amyrin (1) modulates acute periodontal inflammation by reducing neu-trophil infiltration, oxidative stress, and the production of pro-inflammatory cytokine TNF- (Pinto et al., 2008). Oleanolic acid (4) is known as antimicrobial, anti-inflam-matory, antitumor, hepatoprotective, an-tidiabetic, antiviral against HIV virus, and inhibitor of glucosyltransferases (Sasazuka et al., 1995; Herrera et al., 2006). Oleano-lic acid sodium salt showed antibacterial activity against S. mutans and P. gingivalis (Rivero et al., 2008). It has also been re-ported that methyl gallate and gallic acid (7) inhibit growth of B. subtilis ATCC 9372 and S. aureus MRSA ATCC 33591 (Kubo

et al., 2003; Speciale et al., 2006; Kwon et al., 2007; Rivero et al., 2008). During the search for potential antibacterial com-pounds from natural sources, several com-pounds of plant origin have shown growth inhibitory activity against pathogens with minimum inhibitory values ranging from 15 to 1250 g/mL, e.g., the naphthalene glucosides (Li et al., 1998), sanguinarine (Cowan, 2003), diterpenes (Stavri et al., 2009), and some flavonoids, including kaempferol, myricetin, and rhamnocitrin (Van der Watt et al., 2001; Shetty and Lin, 2005). We believe that plant-derived anti-microbial compounds may serve as alter-natives to the commonly used chemicals to treat bacterial diseases.

ACkNOwLEDgMENTS

This research was supported by PAPIIT IN 208207 and PAPIIT IN 205709 and PAIP 6390-09. The authors thank the USAI, Facultad de Qumica, UNAM for their as-sistance in mass spectral data and the Nuclear Magnetic Resonance. The authors also thank Q.F.B. Alejandro Camacho, Departamento de Biologa, Facultad de Qumica, UNAM for his help in the anti-bacterial assays.

REFERENCES

Abeylath, S.C., Turos, E. (2008) Drug delivery approaches to overcome bacterial resistance to beta-lactam antibiotics. Expert Opinion Drug Delivery 9: 931-49.

Amarquaye, A., Che, C., Bejar, E., Malone, M.H., Fong, H.H.S. (1994) A new glycolipid from Byrsonima crassifolia. Planta Medica 60: 85-86.

Argueta, V., Cano, A., Rodarte, M.E. (1994) Atlas de las plantas medicinales de la medicina tradicional mexicana, pp 772, Mxico, D. F., Instituto Nacional Indigenista.


Bejar, E., Malone, M.H. (1993) Pharmacological and chemical screening of Byrsonima crassifolia, a medicinal tree from Mexico. Part I. Journal of Ethnopharmacology 39: 141-58.

Cceres, A., Cano, O., Samayoa, B., Aguilar, L. (1990) Plants Used in Guatemala for the Treat-ment of de Gastrointestinal Disorders.1. Screening of 84 Plants against Enterobacteria. Journal of Ethnopharmacology 30: 55-73.

Cceres, A., Lpez, B., Giron, M., Logemann, H. (1991) Plants used in Guatemala for the treat-ment of Dermatophytic infeccions. 2. Evaluation of antifungal activity of seven American plants. Journal of Ethnopharmacology 40: 207-213.

Cceres, A., Lpez, B., Juarez, X., Del guila, J., Garca, S. (1993) Plants used in Guatemala for the treatment of Dermatophytic infeccions. 1. Screening for antimycotic activity of 44 plant extracts. Journal of Ethnopharmacology 31: 263-276

Calzada, F. (2000) Proantocianidinas del tipo A y flavonoles con actividad antiprotozoaria de

Geranium niveum S. Watson (Geraniaceae) y Coniza filaginoides (DC) Hieron (Asteraceae). UNAM, Facultad de Qumica, Mxico, D.F. Tesis de Doctorado.

Cowan, M.M. 2003. Plant products as antimicrobial agents. Clinical Microbioliology Reviews 12: 564582.

Herrera, M.D., Rodriguez, R., Ruiz, V. (2006) Functional Properties of Pentacyclic Triterpenes contained in Orujo olive oil. Current Nutrition and Food Science 2: 45-49.

Hewitt, W., Vincent, S. (1988) Theory and application of microbiological assay, Academic Press: London, pp 38-79.

Kubo, I., Fujita, K., Nihei, K. (2003) Molecular design of multifunctional antibacterial agents against methicillin resistant Staphylococcus aureus (MRSA). Bioorganic and Medicinal Chemistry 11: 4255-4262.

Kwon, Y.I., Apostolidis, E., Labbe, R.G., Shetty, K. (2007) Inhibition of Staphylococcus aureus by phenolic phytochemicals of selected clonal herbs species of Lamiaceae family and likely mode of action through proline oxidation. Food Biotechnology 21: 71-89.

Li, X.-C., van der Bijl, P., Wu, C.D., 1998. Binaphthalenone glycosides from African chewing sticks, Diospyros lycioides. Journal Natural Products 61: 817820.

Lin, J.K., Tsai, S.H., Lin-Shiau, S.Y. (2001) Antiinflammatory and antitumor effects of flavo-noids and flavanoids. Drugs of Future 26: 145-152.

Martnez, M. (1996) Las Plantas Medicinales de Mxico. Ediciones Botas, Mxico, D. F.Martnez, M., Gonzlez, A.R., Cazares, L., Moreno, M.N., Garca, A.N. (1999) Antimicrobial

activity of Byrsonima crassifolia (L.) H. B. K. Journal of Ethnopharmacology 66: 79-82.Pinto, S.A.H., Pinto, L.M.S., Cunha, G.M.A., Chaves, M.H., Santos, F.A., Rao, V.S. (2008) Anti-

inflammatory effect of , -amyrin, a pentacylcic triterpene from Protium heptaphyllum in rat model of acute periodontitis. Inflammopharmacology 16: 48-52.

Quirynen, M., Teughels, W., De Soete, M., y Van Steenberghe, D., (2000) Topical antiseptics and antibiotics in the initial therapy of chronic adult periodontitis microbiological aspects. Periodontology, 28: 72-90.

Rivero-Cruz, J. F., Zhu, M., Kinghorn, A.D., Wu, C.D. (2008) Antimicrobial constituents of Thompson seedless raisins (Vitis vinifera) against selected oral pathogens. Phytochemistry Letters 1: 151-154.

Sampson, B.A., Misra, R., Benson, S.A. (1989) Identification and Characterization of a New

Gene of Escherichia coli K-12 Involved in outer membrane permeability. Genetics 122: 491-501.

Sasazuka, T., Kameda, Y., Endo, M., Suzuki, H., Hiwatachi, K. (1995) Water-soluble oleanolic acid. Production, inhibition of insoluble glucan synthesis and antibacterial action. Seito Gijutsu Kenkyu Kaishi 43: 63-67.


Seebacher, W., Simic, N., Weis, R., Saf, R., Kunert, O. (2003) Complete assignments of 1H and 13C resonances of oleanolic acid, 18-oleanolic acid, ursolic acid and their 11-oxo deriva-tives. Magnetic Resonance in Chemistry 41: 636-638.

Shetty, K., Lin, Y.T. (2005) Phenolic antimicrobials from plants for control of bacterial patho-gens. In: Food Biotechnology, 2nd Edition [Shetty, K., Paliyath, G., Pometto, A.L. III and Levin, R.E. (eds)]. CRC Press, Boca Raton, FL, pp 1481-1505.

Sholichin, M., Yamasaki, K., Kasai, R., Tanaka, O. (1980) Carbon-13 nuclear magnetic reso-nance of lupine-type triterpenes, lupeol, betulin and betulinic acid. Chemical & Pharma-ceutical Bulletin 28: 1006-1008.

Singh, M.P., Petersen, P.J., Weiss, W.J., Kong, F., Greenstein, M. (2000) Saccharomicins, novel heptadecaglycoside antibiotics produced by Saccharothrix espanaensis: Antibacterial and mechanistic activities. Antimicrobial Agents and Chemotherapy 44: 2154-2159.

Speciale, A., Costanzo, R., Puglisi, S., Musumeci, R., Catania, M.R., Caccamo, F., Iauk, L. (2006) Antibacterial activity of propolis and its active principles alone and in combination with macrolides, beta-lactams and fluoroquinolones against microorganisms responsible

for respiratory infections. Journal of Chemotherapy 18: 164-171.Stavri, M., Paton, A., Skelton, B.W., Gibbons, S. (2009) Antibacterial diterpenes from Plectran-

thus ernstii 72: 1191-1194.Van der Watt, E., Pretorius, J.C. (2001) Purification and identification of active antibacterial

components in Carpobrotus edulis. Journal of Ethnopharmacology 76: 87-91.Walker, W.A., Martens, E.C., Sherman, P.M., Lampe, J.L., Hullar, M.A.J., Wu, C.D. (2009)

Functional foods for health promotion: microbes and health extended abstracts from the 11 th Annual conference on functional foods for health promotion, April 2008. Nutrition Reviews 67: 40-48.

byrsonimia cassifolia dental folk medicine

Documents