Antimicrobial Activity of Alternanthera caracasana.

ABSTRACT

The antimicrobial activity of different extracts of Alternanthera caracasana. HBK against 11 bacterial strains and 1 yeast strain was evaluated. The ethyl acetate extract showed antimicrobial activity against Staphylococcus aureus., Staphylococcus epidermidis., Bacillus subtilis., Sarcina lutea., and one strain of Vibrio cholerae.. There was no antimicrobial activity against Candida albicans.. As a bioactive compound, 7-methoxycoumarin was identified.

Keywords:

• Alternanthera caracasana
• antimicrobial activity
• coumarin
• 7-methoxycoumarin

Introduction

In México, Alternanthera caracasana. HBK (Amaranthaceae) (known as tianguis., tianguistumina., tianguispepetla., tlalpetate.) is widely used in traditional medicine. Stems, leaves, flowers, and roots are used against different kinds of ailments, such as dysentery, diarrhea, fever, and so forth (Aguilar et al., 1994ab; Argueta & Cano, 1994; Argueta & Zolla, 1994). Alternanthera caracasana. known as tianguis., is a creeping perennial herb with branching pubescent stems 10 to 50 cm long, ovoid or shortly cylindrical inflorescenses capituliform 0.5 to 1.5 cm long, axillar sessile, whitish, with numerous flowers. It is a common undergrowth in the Valley of México, on the sidewalks, on the village streets, and in some planting fields (Rzedowski, 1979ab). In traditional medicine treatments, the plant is used as an infusion prepared as a lavage or beverage (Argueta & Cano, 1994).

The purpose of the current study was to evaluate the antimicrobial activity of Alternanthera caracasana. and elucidate the structure of the active compound.

Materials and Methods

Plant material

Alternanthera caracasana. HBK (syn. A. repens.) was collected in the gardens of Facultad de Estudios Superiores Iztacala in July 1997 and was identified by Ma. Edith López Villafranco. A specimen (voucher no. 12136) was deposited at Izta-Herbarium, FES Iztacala, UNAM.

Microorganism strains

Strains of Shigella boydii. (ATCC 8700), Staphylococcus aureus. (ATCC 12398), Escherichia coli. (ATCC 25922), Enterobacter agglomerans. (ATCC 27155 Authorities: Dr. Jose Guillermo Avila Acevido), Salmonella typhi. (ATCC 19430), Vibrio cholerae. CDC V12, Vibrio cholerae. INDRE 206 (isolated from polluted water), Vibrio cholerae. isolated from a clinical case (these strains belong to the type O1, enterotoxine producers, Inaba serotype, biotype “El Tor”), Vibrio cholerae. Serotype No-01 ATCC 35971 Authorities: Dr. Jose Guillermo Avila Acevido, Enterobacter aerogenes., Staphylococcus epidermidis., Bacillus subtilis., and Sarcina lutea. were obtained from the Department of Microbiology, Facultad de Estudios Superiores Cuautitlan, UNAM. Pseudomonas aeruginosa. and Candida albicans. were isolated from patients and were kindly provided by the Clinical Analysis Laboratory of the FES-Iztacala, UNAM. Bacteria were grown on Mueller-Hinton broth (Bioxon 260-1). Candida albicans. was grown in Sabouraud broth (C-222400 Bioxon).

Extract preparation

Dried and ground aerial parts of A. caracasana. (1000 g) were macerated in 5 l of ethanol (96%) during 25 days. The ethanol extract was concentrated at reduced pressure to yield 40.8 g of residue (4.08%). The residue was successively extracted with 200 ml of hexane, chloroform, ethyl acetate, acetone, and methanol. Extracts were concentrated to approximately 10 ml at 40°C under reduced pressure, and they were dried to constant mass at 43°C. After determining the yields, extracts were stored at 4°C until their use. Yield extracts were as follows: hexane 6.2 g (0.62%), chloroform 3.4 g (0.34%), ethyl acetate 3.08 g (0.308 %), acetone 0.8 g (0.08%), and methanol 27.32 g (2.732%).

Antimicrobial testing

Antimicrobial activity of the extracts was determined in the disk bioassays (2 mg/disk) in accordance with the diffusion method of Kirby-Baüer (Van der Berghe & Vlietnick, 1991). As a positive control, chloramphenicol disks (25 µg) were used; as negative controls, the disks with the solvents used in the extract process and evaporated overnight were used (in the same manner as experimental disk). Each experiment was carried out in triplicate (a variance multifactorial analysis was fulfilled). During the purification process, determinations of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated according to the technique of Koneman (1985).

Active compound isolation

In the separation process of an active compound, column chromatography (silica gel mesh 70-230 Sigma 5-2509, St. Louis, MO-USA) and preparative thin-layer chromatography (silica gel Merck Kieselgel 60-5553, Düren-Germany) were used. The ethyl acetate extract (3.08 g) was applied to silica gel column and eluted with hexane-chloroform-ethyl acetate (1:1:1); nine fractions were obtained and evaluated with 1 mg ml⁻¹ by the Koneman (1985) technique. Antimicrobial activity was observed in fraction 4. This fraction was newly chromatographed in a column and eluted with hexane-chloroform (1:1.5), chloroform-acetone (1:1), and chloroform-methanol (2:1); seven fractions were obtained and tested. Activity was observed in fraction 5. To purify the active compound, preparative thin-layer plates were used (silica gel 60 F 254 µ), using chloroform as a mobile phase; seven bands were obtained and tested. Activity was observed in the band of RF 0.55. The final yield obtained at the end of the process was 12.4 mg.

Structure identification

Identification of the active compound was determined by spectroscopic studies: melting point 115–119°C; ultraviolet absorption (Perkin Elmer UV/VIS Lambda II, Überlingen-Germany), UVλ_max MeOH: 320.42 nm. ¹H NMR (Varian Geminis 200 A, Palo Alto, Ca-USA), δ ppm: 3.88 (3H all s. 3 × OCH₃); 6.23 d. (2H J. = 9.6 Hz); 7.87 d. (2H J. = 9.6 Hz); 6.91 m., 7.52 d. (3H J. = 8.4). ¹³C NMR presents chemical signs that were assigned to the carbons of the proposed structure following the pattern of Harbone, 1989 [C-2 164.686, C-3 113.801, C-4 145.710, C-5 130.374, C-6 113.321, C-7 163.354, C-8 101.733, C-9 157.145, C-10 11.938, and C-11 56.410 ppm]. The molecular weight of the compound was 176 Da determined by EI-MS (Finningan Mat GCQ, Austin, TX-USA). Data confirm that the compound responsible for antimicrobial activity was 7-methoxycoumarin.

Results and Discussion

Antimicrobial assay

Antimicrobial susceptibility of each extract is shown in Table 1. Acetone and ethyl acetate extracts inhibited the growth of five bacterial strains: Staphylococcus aureus., Staphylococcus epidermidis., Bacillus subtilis., Sarcina lutea., and Vibrio cholerae. (No-01). The methanol extract showed activity against S. aureus. and S. epidermidis.. The hexane and chloroform extracts did not show any activity against the tested microorganisms.

Table 1. Antimicrobial susceptibility of the extracts of Alternanthera caracasana. (diameter inhibition in millimeters).

Microorganism	Kanamicin control	Methanol extract	Acetone extract	Ethyl acetate extract
S. aureus.	16.67±0.58	10.07±0.57	12.67±1.15	13.67±1.15
S. epidermidis.	16.00±0.50	12.00±1.0	12.67±0.58	13.67±0.58
B. subtilis.	21.00±0.50	—	8.00±0.50	9.00±1.00
S. lutea.	9.00±0.50	—	9.33±1.15	11.00±0.50
V. ch.. No-01	19.67±0.58	—	8.00±0.50	8.00±0.50

Determination of MIC and MBC

Minimum inhibitory and minimum bactericidal concentrations of 7-methoxycoumarin were evaluated. Results are shown in Table 2. It can be observed that V. cholerae. No-01 was more sensitive than other bacterial strains.

Table 2. Minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations of 7-methoxycoumarin.

Bacteria	MIC (mg ml^−1)	MBC (mg ml^−1)
S. aureus.	0.5	> 1.0
S. epidermidis.	0.75	> 1.0
B. subtilis.	0.75	> 1.0
S. lutea.	0.75	> 1.0
V. cholera. No-01	0.5	1.0

The ethyl acetate extract showed the largest inhibition zones. Variance analysis (p < 0.0008) was carried out to verify significant differences in susceptibility.

In accordance with our results, Gram-positive bacterial and one strain of Gram-negative bacteria were susceptible to the ethyl acetate and acetone extracts. Only two Gram-positive bacteria were susceptible to the methanol extract. C. albicans. did not show susceptibility with any extract.

Because the ethyl acetate extract was active against bacteria, we proceeded to the isolation of the active compound. According to spectroscopic studies, the active compound was 7-methoxycoumarin (Fig. 1). Both physical and spectral parameters of this compound coincided with those reported by Murray (1978), Harborne (1989), and Pouchert (1993). Coumarins have been reported to have different biological effects such as antibiotic and phytotoxic activity; they can produce hepatic damage, and so forth (Harborne, 1989). This is in concordance with the activity observed in 7-methoxycoumarin against five bacterial strains. It is understood that coumarins can impede the genetic material of bacteria and mediate antibiotic activity. V. cholerae. No-01 was more sensitive (MIC 0.5 mg/ml and MBC 1.0 mg/ml) (Table 2) than the other microorganisms.

Figure 1 7-Methoxycoumarin (C₁₀H₈O₃).

Conclusions

Among different extracts of Alternanthera caracasana., the ethyl acetate extract showed an antibacterial activity against both Gram-positive and Gram-negative bacteria. It was proved that 7-methoxycoumarin (C₁₀H₈O₃), isolated from the ethyl acetate extract, is responsible for such activity.

Acknowledgments

We are extremely grateful to Marco A. Rodríguez Monroy for the translation of the manuscript.