Antibacterial and antifungal properties of ent-kaurenoic acid from Smallanthus sonchifolius

Chinese Journal of Natural Medicines 2012, 10(5): 0408−0414

Chinese Journal of Natural Medicines

Antibacterial and antifungal properties of ent-kaurenoic acid from Smallanthus sonchifolius Eleanor P. Padla 1, Ludivina T. Solis 1, Consolacion Y. Ragasa 2* 1

Department of Microbiology & Parasitology, College of Medicine De La Salle Health Sciences Institute, Dasmariñas, Cavite, Philippines; Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila, 1004 Philippines

2

Available online 20 Nov. 2012

[ABSTRACT] AIM: To screen for the antibacterial activity of ent-kaurenoic acid (1) from the dichloromethane extract of Smallanthus sonchifolius leaves against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, and Pseudomonas aeruginosa, and for its antifungal activity against Candida albicans, Trichophyton rubrum, and Epidermophyton floccosum. METHODS: Compound 1 was isolated by silica gel chromatography and its structure was elucidated by NMR spectroscopy. For assaying the antibacterial and antifungal activities of 1, the disk diffusion method was used, while the minimum inhibitory concentrations (MICs) were determined by the broth dilution method. RESULTS: With the disk diffusion method, 1 was found to be active against all the Gram-positive organisms tested (S. aureus, S. epidermidis, B. subtilis) at the lowest concentration of 1 000 μg·mL−1, while it was active against the fungus T. rubrum at 10 000 μg·mL−1. No inhibitory activity was observed against C. albicans, E. floccosum and all the Gram-negative test strains. The activity indices (AI) of 1 were noted to be highest against S. aureus and lowest against T. rubrum. Statistically significant differences were found between the mean inhibition zones (IZ) of 1 and the standard drugs (ofloxacin and clotrimazole). The results of the broth dilution MIC determination revealed that 1 exhibited moderate activity against S. aureus and S. epidermidis with MIC values of 125 μg·mL−1 and 250 μg·mL−1, respectively; and weak activity against B. subtilis with a MIC of 1 000 μg·mL−1. The growth of T. rubrum in the MIC assay was not inhibited at the highest tested concentration of 1 (10 000 μg·mL−1). CONCLUSION: The minimum bactericidal concentration (MBC) indicated that the bactericidal activities of 1 occurred at concentrations higher than its growth inhibitory concentrations. Furthermore, the MBC: MIC ratio of 2 : 1 clearly demonstrated the in vitro bactericidal effect of 1 against S. aureus and S. epidermidis. [KEY WORDS] Smallanthus sonchifolius; Yacon; ent-Kaurenoic acid; Antibacterial; Antifungal

[CLC Number] R284.1; R965

1

[Document code] A

Introduction

Smallanthus sonchifolius (Poepp. & Endl.) H.Rob. (Asteraceae), also known as yacon, was introduced in the Philippines in early 2000, and has since become commonly available in local markets. The growing popularity of yacon can be attributed to its nutritive, as well as medicinal, value. It has long been valued as an edible root crop. More importantly, studies have demonstrated that yacon has hypoglycemic [1-4], antioxidant [5-6], probiotic [7-9], and antimicrobial properties [10-12]. The leaves and tubers contain phenolic compounds (chlorogenic, caffeic and ferulic acids) which [Received on] 14-Nov.-2011 [*Corresponding author] Consolacion Y. Ragasa: Tel/Fax: 06325360230, E-mail: [email protected] These authors have no any conflict of interest to declare. Published by Elsevier B.V. All rights reserved

[Article ID] 1672-3651(2012)06-0408-07 exhibit antioxidant, probiotic, and hypoglycemic effects [13-15]. Moreover, the tubers are rich in non-assimilable low-calorie oligofructan and inulin, hence their use as dietary sugar and dietary fiber [5]. Although yacon has not been customarily used as an anti-infective herbal medicine, a number of sesquiterpene lactones have been identified in yacon leaves, which were shown to possess antimicrobial properties [12]. A previous study reported that ent-kaurenoic acid (1) from yacon exhibited significant anti-inflammatory and analgesic activities. Compound 1 dissolved in dimethyl sulfoxide (DMSO) also displayed potential anti-toxicity and hypoglycemic activity. It also showed low antimicrobial activities against E. coli, P. aeruginosa, S. aureus, C. albicans and T. mentagrophytes, but was found to be inactive against A. niger at 30 μg·mL−1 concentration [16]. A recent study reported the hypoglycemic potential of the aqueous extract of yacon tea and 1 [17], and an earlier study also reported 1 as a constituent of S. sonchifolius [18].

Eleanor P. Padla, et al. /Chinese Journal of Natural Medicines 2012, 10(6): 408−414

This study, which screened 1 from S. sonchifolius for its antimicrobial potential, is likely to give new impetus to the scientific validation of its therapeutic and pharmacological potential, and may provide a rationale for advancing the use of local medicinal plants and herbal remedies.

2

Experimental

2.1 General Optical rotation was taken with a Jasco DIP-370 digital polarimeter. Melting point was determined using FisherJohns melting point apparatus. NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl3 at 600 MHz for 1 H NMR and 150 MHz for 13C NMR spectra. Column chromatography was performed with silica gel 60 (70–230 mesh), while the TLC was performed with plastic backed plates coated with silica gel F254. The plates were visualized with vanillin-H2SO4 and warming. 2.2 Plant material Young leaves (1–2 months old) of Smallanthus sonchifolius were collected from Nagcarlan, Laguna in February 2011. The plant was identified and authenticated at the Bureau of Animal Industry, Manila, Philippines. 2.3 Extraction and isolation of 1 The air-dried leaves (1.6 kg) of S. sonchifolius were ground in an osterizer, and then soaked in dichloromethane for three days. The crude extract was chromatographed on silica gel eluting with increasing proportions of acetone in dichloromethane at 10% increments. The 10% to 40% acetone in dichloromethane fractions were combined and rechromatographed in petroleum ether, followed by 1% acetone in petroleum ether, and finally in 2.5% ethyl acetate in petroleum ether. The 2.5% ethyl acetate in petroleum ether fractions were rechromatographed in 5% ethyl acetate in petroleum ether, followed by 7.5% ethyl acetate in petroleum ether. The 7.5% ethyl acetate in petroleum ether fractions were rechromatographed in 10% ethyl acetate in petroleum ether to afford 1 (1.2 g). 2.4 Antimicrobial assay 2.4.1 Microbial strains The microbial strains used in the study were obtained from the Natural Sciences Research Institute, National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines, College of Public Health, and from the De La Salle Health Sciences Institute. The inhibitory activity of 1 was determined against a total of 10 microbial species, 7 bacterial and 3 fungal test strains. Amongst the bacteria tested were S. aureus (ATTC

6538), S. epidermidis (ATCC 12228), E. coli (ATCC 8739), E. aerogenes (ATCC 13048), K. pneumoniae (ATCC 13883), P. aeruginosa (ATCC 9027), and B. subtilis (ATCC 6633). The fungal test strains included C. albicans (ATCC 10231) and two dermatophytic fungi, T. rubrum and E. floccosum. Bacterial and fungal stock cultures were maintained in Nutrient Agar (Difco) and Sabouraud Dextrose Agar (Difco) slants, respectively, and kept refrigerated until used. All microbial cultures were checked for purity by plate out prior to testing. Except for the dermatophytic fungi (which were incubated at 26 ± 2 °C), all other cultures were incubated at 35 ± 2 °C. 2.4.2 Antibiotic discs and standard antimicrobials Ofloxacin (30 μg) and clotrimazole (30 μg) discs were used in the disk diffusion susceptibility testing of the bacterial and fungal test strains, respectively. Standard antibacterial and antifungal agents, ofloxacin (Sigma-Aldrich) and clotrimazole (Sigma-Aldrich), were utilized in the determination of the MIC of 1. Prior to dilution with sterile distilled water to the desired concentration, ofloxacin and clotrimazole required solubilization with 0.1 mol·L−1 sodium hydroxide (NaOH) and DMSO, respectively. 2.4.3 Preparation of inoculum For each bacterial test strain, 4-5 colonies from a purity plate were grown in 5 mL Nutrient Broth (Difco). After 18-24 hours incubation, the inoculum density was adjusted with sterile normal saline solution (NSS) to match the McFarland 0.5 turbidity standard (108 CFU·mL−1). To prepare the C. albicans inoculum, 4–5 colonies from a purity plate were picked out and suspended in 5 mL sterile NSS. The suspension was vortex-mixed and the cell density was adjusted to that of McFarland 0.5 standard. On the other hand, the dermatophyte inoculum was prepared by adding sterile NSS to 5–10 day old Sabouraud Dextrose Agar (SDA) slant culture. The conidia were dislodged from the hyphal mat using sterile inoculating loop, after which, the suspended cells were carefully pipetted into sterile tubes. The cell density was subsequently adjusted with sterile NSS to a final inoculum concentration equivalent to McFarland 0.5 standard. 2.4.4 Preparation and seeding of double layer Mueller Hinton Agar (MHA) plates Double layer MHA (Difco) plates consisting of 10 mL MHA base and 5 mL upper seeded layer were used in this study. The upper seed layer was made by inoculating a tube containing 5 mL of sterile, molten MHA with 0.1 mL of a standardized inoculum (1 × 108 CFU·mL−1). After a quick mix, the seeded molten medium was poured into a MHA base plate, and was allowed to solidify before diffusion disks were applied. The evaluation of the antibacterial and antifungal activities of 1 was conducted according to Clinical and Laboratory Standards Institute (CLSI) [19] guidelines. 2.4.5 Disk diffusion susceptibility test For assaying the antibacterial and antifungal activities of

Eleanor P. Padla, et al. /Chinese Journal of Natural Medicines 2012, 10(6): 408−414

1, the disk diffusion method was used. Compound 1 was dissolved in 95% ethanol, and then diluted with sterile NSS to obtain four ten-fold dilutions: 10 000, 1 000, 100, and 10 μg·mL−1. To plates seeded with each of the test organisms, four, 6 mm antibiotic disks impregnated with 20 µL of each of the dilutions, and a solvent blank impregnated with 20 µL of the solvent, were applied. A standard antibiotic disk and a disk impregnated with 20 μL of sterile distilled water were likewise applied as positive and negative controls, respectively. After the period of incubation (18–24 h for bacteria, 48–72 h for C. albicans and 5–10 d for dermatophytic fungi), the antimicrobial activity was assessed on the basis of the inhibition zone size. The endpoint was taken as complete inhibition of growth as judged by the naked eye. The diameters of the zones of inhibition were measured in millimeters (including the diameter of the disc) using a Vernier caliper. Values were the average of three readings. Inhibition zones equal to or greater than 7 mm were considered indicative of antimicrobial activity of 1 against the test organism or of susceptibility of the test organism to 1. Negative results were recorded as zero. The activity index (AI) of 1 was obtained by dividing its zone of inhibition by that of the standard antimicrobial agent. An AI > 0.5 was considered as significant antimicrobial activity. 2.4.6 Minimum inhibitory concentration (MIC) determination The MICs of 1 were determined by the broth dilution method for all test strains which produced inhibition zones > 7 mm. Mueller Hinton Broth (Difco) and Sabouraud Dextrose Broth (Difco) were used to prepare the dilutions for the bacterial and fungal test strains, respectively. Five, two-fold serial dilutions of 1 were set up with respective upper and lower concentrations of 2 000 μg·mL−1 and 125 μg·mL−1 for bacteria and 20 000 μg·mL−1 and 1 250 μg·mL−1 for fungi. The total volume of the concentrations prepared was adjusted to the number of organisms to be tested. Standard antimicrobials (ofloxacin and clotrimazole) were set up in similar manner. The amount of antimicrobial agent needed in preparing the dilutions was based on their specific biological activity. To each of the dilution and control tubes containing broth only, an equal volume of standardized inoculum (1 × 108 CFU·mL−1) was added, to give respective final upper and lower concentrations of 1 000 and 62.5 μg·mL−1 for bacteria and 10 000 and 625 μg·mL−1 for fungi. At the end of the incubation period (18–24 h for bacteria, 5–10 d for dermatophytic fungi), the tube containing the least concentration of 1 showing no visible sign of growth was considered as the MIC against the test organism. All MIC determinations were the average of two readings. In this study, MIC < 100 μg·mL−1 were considered with good antimicrobial activity; MICs of 100–500 μg·mL−1 with

moderate activity; MICs of 500–1 000 μg·mL−1 with weak activity; and MICs > 1 000 μg·mL−1 with no activity. 2.4.7 Minimum bactericidal concentration (MBC)/ minimum fungicidal concentration (MFC) determination For the MBC/MFC determination, 0.1 mL from MIC tubes which did not show any sign of growth was inoculated onto MHA for bacteria and SDA for fungi by the spread plate method. After incubation (18–24 h for bacteria and 5–10 d for dermatophytic fungi), the least concentration of 1 with no visible growth on subculture was taken as its MBC/MFC against the test organisms. MBC: MIC or MFC: MIC ratios were calculated to determine the antibacterial or antifungal effect of 1 against the test strains. If the ratio is between 1 : 2 to 2 : 1, the compound is considered as bactericidal or fungicidal against the test organism and it is likely to be bacteriostatic or fungistatic if the ratio is > 2 : 1. 2.4.8 Statistical analysis Means and standard deviations of the inhibition zones (IZ) of 1 and standard antimicrobials against the test strains were computed. ANOVA was used (for S. aureus, S. epidermidis, and B. subtilis) to determine if there are statistically significant differences in mean IZs among the inhibitory concentrations (10 000 and 1 000 μg·mL−1) of 1 and ofloxacin while t-test was used for T. rubrum to determine if there is statistically significant difference between the 10 000 μg·mL−1 concentration of 1 and clotrimazole. ANOVA and t-test P-values less than 0.05 were considered as statistically significant. Furthermore, if the P-value of the F-test is < 0.05 level of significance, the Dunnet’s test was used to determine which inhibitory concentration (10 000 or 1 000 μg·mL−1) of 1 is significantly different from the standard antimicrobial. Likewise, the means and standard deviations of the AI of 1 against the test strains were computed.

3

Structural Identification

ent-Kaurenoic acid: colorless solid. [α]D –88.0 (c 0.75, CHCl3); mp: 169–170 °C; 1H NMR (600 MHz, CDCl3) δ: 0.80 (H-1, dt, J = 4.0, 13.5 Hz), 1.87 (H-1, m), 1.42 (H-2, m), 1.87 (H-2, m), 1.00 (H-3, dt, J = 4.5, 13.5 Hz), 2.15 (H-3, d, J = 14.5 Hz), 1.06 (H-5, m), 1.82 (H2-6, m), 1.44 (H-7, m), 1.52 (H-7, dt, J = 3.5, 13.0 Hz), 1.04 (H-9, d, J = 7.0 Hz), 1.55, 1.59 (H2-11, m), 1.46, 1.59 (H2-12, m), 2.62 (H-13, br s), 1.13 (H-14, dd, J = 5.0, 11.5 Hz), 1.98 (H-14, dd, J = 2.0, 11.5 Hz), 2.04 (H2-16, br s), 4.72, 4.78 (H2-17, br s), 0.93 (H3-18, s), 1.22 (H3-19, s); 13C NMR (150 MHz, CDCl3) δ: 40.68 (C-1), 19.07 (C-2), 37.80 (C-3), 43.70 (C-4), 57.03 (C-5), 21.81 (C-6), 41.26 (C-7), 44.22 (C-8), 55.08 (C-9), 39.64 (C-10), 18.42 (C-11), 33.10 (C-12), 43.84 (C-13), 39.68 (C-14), 155.90 (C-15), 48.94 (C-16), 102.98 (C-17), 15.58 (C-18), 28.95 (C-19), 183.97 (C-20) [20].

Eleanor P. Padla, et al. /Chinese Journal of Natural Medicines 2012, 10(6): 408−414

4

Results

4.1

Isolation and identification of 1 Silica gel chromatography of the dichloromethane extract of young yacon leaves (1–2 months) afforded 1. The structure of 1 was elucidated by extensive 1D and 2D NMR spectroscopy as follows. The 1H NMR data of 1 (see experimental) gave resonances for two methyl singlets at δ 0.93 and 1.22 and an exocyclic methylene at δ 4.72 (br s) and 4.78 (br s). The remaining resonances in the shielded region were assigned to methine and methylene protons in 1. The 13C, DEPT and HSQC NMR data of 1 (see experimental) confirmed the presence of twenty carbons, with a total of two methyls, ten methylenes, three methines and five quaternary carbons. Diagnostic features of the diterpene included a carboxylic acid at δ 183.97, a non-protonated olefinic carbon at δ 155.91 and an exocyclic methylene carbon at δ 102.98. Protons attached to carbons were assigned from HSQC 2D NMR data (see experimental) and the structure of 1 was elucidated by analysis of the HMBC 2D NMR data. The carboxylic acid was attributed to C-20 due to long-range correlations observed from H3-19, H2-3 and H-5 to this carbon. The exocyclic methylene was assigned to C-15 since long-range correlations were observed from H2-16, H2-17 and H-14 to this carbon. The second methyl was assigned to C-18 on the basis of correlations from H2-1, H-5 and H-9 to this carbon. All long-range correlations are consistent with the

structure of 1 which was confirmed by comparison of its 13C NMR data with those reported for ent-kaurenoic acid [20]. 4.2 Antimicrobial assay The inhibitory activities of 1 against seven bacterial and three fungal test strains as determined by the disk diffusion method are presented in Table 1. The results indicate that 1 exhibited antibacterial activity against all three Gram-positive organisms tested (S. aureus, S. epidermidis and B. subtilis) at the lowest inhibitory concentration of 1 000 μg·mL−1. However, 1 was found to be inactive against all four Gram-negative test bacteria (E. coli, E. aerogenes, K. pneumoniae and P. aeruginosa) even at the highest concentration used (10 000 μg·mL−1). Amongst the Gram-positive bacteria, S. aureus and B. subtilis were found to be the most and the least susceptible, respectively, with corresponding inhibition zones of 8.93 and 7.43 mm at the lowest inhibitory concentration of 1 (1 000 μg·mL−1). Among the three fungal species tested, only the dermatophytic fungus T. rubrum was found susceptible with a 7.20 mm inhibition zone at 10 000 μg·mL−1 concentration of 1. The data also show that the mean IZs of 1 were less than those of the standard drugs, and the test P-values (< 0.000 1) indicate that the mean differences are statistically significant. The AI of the different concentrations of 1 are likewise shown in Table 1. Relative to the activity of ofloxacin, 1 exhibited the greatest antibacterial activity against S. aureus, and the least activity against S. epidermidis with AI values of

Table 1 Disk diffusion-based inhibitory activities of ent-kaurenoic acid (1) against 10 microbial test strains 1 −1

10 000 μg·mL IZ AI (SD) (SD)

1 000 μg·mL−1 IZ AI (SD) (SD)

9.70 (0.26) 9.80 (0.20) 8.60 (0.35)

0.35 (0.03) 0.29 (0.01) 0.34 (0.00)

8.93 (0.31) 8.70 (0.26) 7.43 (0.15)

0.32 (0.03) 0.26 (0.01) 0.30 (0.02)

E. coli

0

0

0

E. aerogenes

0

0

K. pneumoniae

0

P. aeruginosa

100 μg·mL−1

10 μg·mL−1

IZ

AI

IZ

AI

0

0

0

0

0

0

0

0

Ofloxacin (30 μg)

Clotrimazole (30 μg)

IZ (SD)

IZ (SD)

Bacteria S. aureus S. epidermidis B. subtilis

0

28.00 (2.00) 34.00 (1.00) 24.97 (1.08) 39.97 (0.25) 22.27 (2.42) 38.33 (1.53) 28.33 (1.15)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

C. albicans

0

0

0

0

0

0

0

0

-

T. rubrum

7.20 (0.26)

0.21 (0.01)

0

0

0

0

0

0

-

0

0

0

0

0

0

0

0

-

-

Fungi

E. floccosum.

IZ (inhibition zone, in millimeters) = mean of triplicate readings; AI (activity index) = IZ of 1/IZ of standard drug

17.00 (1.00) 35.00 (1.00) 19.83 (0.76)

Eleanor P. Padla, et al. /Chinese Journal of Natural Medicines 2012, 10(6): 408−414

0.32 and 0.26, respectively at the lowest inhibitory concentration of 1 (1 000 μg·mL−1). For T. rubrum, 1 gave an AI value of 0.21 at the highest concentration tested (10 000 μg·mL−1). The results further show that the AI values of 1 were highest against S. aureus and lowest against T. rubrum. Based on an AI cut-off value of > 0.5, it can be concluded that no significant inhibitory activity was exhibited by 1 against any of the four strains which demonstrated susceptibility to the compound by the disk diffusion method. The results of post hoc comparisons of the mean IZ between 1 and standard drugs are shown in Table 2. The least computed mean difference was against B. subtilis between 1 at 10 000 μg·mL−1 and ofloxacin, while the greatest computed mean difference was against T. rubrum between 1 at 10 000 μg·mL−1 and clotrimazole. All of the Dunnet’s test P-values are < 0.05 level of significance, thus all mean differences in IZ between 1 and standard drugs are statistically significant. Table 2 Inhibition zone mean differences (95% confidence interval) between ent-kaurenoic acid (1) and standard drugs Test ctrain

Post Hoc comparison −1

S. aureus

S. epidermidis

1 (10 000 μg·mL ) vs ofloxacin −1

1 (1 000 μg·mL ) vs ofloxacin 1 (10 000 μg·mL−1) vs ofloxacin −1

1 (1 000 μg·mL ) vs ofloxacin −1

B. subtilis

T. rubrum

1 (10 000 μg·mL ) vs ofloxacin 1 (1 000 μg·mL−1) vs ofloxacin −1

1 (10 000 μg·mL ) vs clotrimazole

Mean difference (95 % CI) 1 – standard −18.30 (−21.05, −15.55) −19.07 (−21.82, −16.31) −24.20 (−25.62, −22.78) −25.30 (−26.72, −23.88) −16.37 (−17.91, −14.82) −17.53 (−19.08, −15.99) −27.80 (29.46, −26.14)

Dunnet’s Test’s P-value < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001

The parameters of antimicrobial activity (MIC and MBC) of 1 are shown in Table 3. The results of the broth dilution MIC determination indicate that 1 was moderately active against S. aureus and S. epidermidis with MICs of 125 and 250 μg·mL−1, respectively, and weakly active against B. subtilis with a MIC value of 1 000 μg·mL−1. Although 1 produced inhibition zones against T. rubrum by disk diffusion, in the MIC assay, it was inactive against the fungus, with MIC of > 10 000 μg·mL−1. Similarly, the MBC of 1 was lowest for S. aureus and S. epidermidis with values of 250 and 500 μg·mL−1, respectively, and highest for B. subtilis, with values of > 1 000 μg·mL−1. The MFC of 1 for T. rubrum was no longer determined, since all of the MIC dilution tubes showed visible growth on examination. The MBC values were apparently higher than the MICs, suggesting that the bactericidal activities of 1 occur at concentrations higher than its growth inhibitory concentrations.

Table 3 Parameters ent-kaurenoic acid (1)

of

antimicrobial

activity

of

Minimum inhibi- Minimum bactericidal MBC : MIC Test bacteria tory concentration concentration (MBC) ratio −1 −1 /(μg·mL ) (MIC)/(μg·mL ) S. aureus S. epidermidis B. subtilis

125

250

2:1

250

500

2:1

1 000

> 1 000

NC

Minimum inhibi- Minimum fungicidal Test Fungus tory concentration concentration (MFC) /(μg·mL−1) (MIC)/(μg·mL−1) T. rubrum

> 10 000

ND

--

MIC and MBC values represent the average of two readings; ND = not determined; NC = not calculable

As indicated by the ratios of MBCs to the MICs, 1 exerted a clear bactericidal action against S. aureus and S. epidermidis. The MBC: MIC ratio of 1 against B. subtilis was denoted as not calculable (NC), since its MBC exceeded the highest concentration tested (1 000 μg·mL−1).

5

Discussion

Major antimicrobial compounds from plants include terpenes and terpenoids, and their specific antibacterial [21-25] and antifungal [26-29] activities have been previously reported. In yacon, such antimicrobial activity has been suggested by a number of bioactive terpenes. Sesquiterpene lactones from yacon leaves have been shown to exhibit potent antibacterial [12] and antifungal activities [11]. An earlier study reported that ent-kaurenoic acid (1) from yacon exhibited low antimicrobial activities against E. coli, P. aeruginosa, S. aureus, C. albicans and T. mentagrophytes, and was found to be inactive against A. niger at 30 μg·mL−1 concentration [16]. In this study, 1 was evaluated for its antibacterial and antifungal properties against ten microorganisms at different concentrations. The results from this study revealed that 1 possesses specific antibacterial activity, being active only against Gram-positive organisms (S. aureus, S. epidermidis, and B. subtilis). This finding is consistent with other studies which demonstrated the greater susceptibility of Gram-positive organisms to plant-derived compounds than Gram-negative bacteria [30-33]. Differences in cell wall structure and cell membrane permeability have been suggested to account for this susceptibility pattern and outcome [34-36]. As to the nature of the antibacterial activity, 1 clearly exerted a bactericidal effect against S. aureus and S. epidermidis. However, ancillary investigations such as time-kill studies are needed to ascertain if the lethal activity is concentration- or time-dependent, and to gain further insight into the therapeutic and pharmacologic potential of the compound. Although capable of bactericidal activity, 1 was only moderately active against S. aureus and S. epidermidis, a finding which may reduce its utility as lead compound in drug discovery and development. Nevertheless, 1 may still be

Eleanor P. Padla, et al. /Chinese Journal of Natural Medicines 2012, 10(6): 408−414

useful in antiseptic and disinfectant formulations. In fact, yacon has recently been developed in the Philippines as an herbal soap with claims of efficacy for wound and skin conditions and for skin rejuvenation [37-38], among others. The established antibacterial [10-12] and antioxidant activities [5-6] of yacon may well account for the soap’s rejuvenating and skin/wound healing effects. Compound 1 may not have potent antimicrobial activity, but possible additive or synergistic effects with other plant constituents may confer greater efficacy, particularly in preparations other than of the pure substance. Moreover, since staphylococci are the most common cause of bacterial skin infections [39], and 1 has proven anti-staphylococcal property, it is not surprising to find yacon as an effective herbal skin remedy and a successful niche product in the future. Thus, this study not only confirmed the plant’s therapeutic utility, but has also substantiated pertinent benefit claims. This study failed to show significant inhibitory activity of 1 against the fungal species tested. The differences in cell wall composition and in mechanism(s) of action between bacteria and fungi may account for the observed greater resistance of the fungal strains to 1. The results of this study show that the in vitro activity of 1 is consistent with the plant’s traditional and contemporary use, and should therefore encourage wider use of yacon and greater recognition of its market potential.

Acknowledgments The authors gratefully acknowledge the financial support from the Heath Research Development Consortium-Region IV for the antimicrobial assays, and the College Research Fund of De La Salle University for the isolation of ent-kaurenoic acid. The kind assistance of the Center for Basic Biomedical Research-De La Salle Health Science Institute and the College of Science-De La Salle University is likewise acknowledged.

[6]

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