全 文 ： 408 Chin J Nat Med Nov. 2012 Vol. 10 No. 6 2012 年 11 月 第 10 卷 第 6 期

Chinese Journal of Natural Medicines 2012, 10(5): 0408−0414
doi: 10.3724/SP.J.1009.2012.00408
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*
1Department of Microbiology & Parasitology, College of Medicine De La Salle Health Sciences Institute, Dasmariñas, Cavite, Philippines;
2Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila, 1004 Philippines
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 aero-
genes, 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 diffu-
sion method, 1 was found to be active against all the Gram-positive organisms tested (S. aureus, S. epidermidis, B. subtilis) at the low-
est 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 high-
est 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 ex-
hibited 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 [Document code] A [Article ID] 1672-3651(2012)06-0408-07

1 Introduction
Smallanthus sonchifolius (Poepp. & Endl.) H.Rob. (As-
teraceae), also known as yacon, was introduced in the Philip-
pines in early 2000, and has since become commonly avail-
able 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 impor-
tantly, studies have demonstrated that yacon has hypoglyce-
mic [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: 0632-
5360230, E-mail: consolacion.ragasa@dlsu.edu.ph
These authors have no any conflict of interest to declare.
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 ses-
quiterpene 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 hypogly-
cemic 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
2012 年 11 月 第 10 卷 第 6 期 Chin J Nat Med Nov. 2012 Vol. 10 No. 6 409

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 Fisher-
Johns melting point apparatus. NMR spectra were recorded
on a Varian VNMRS spectrometer in CDCl3 at 600 MHz for
1H NMR and 150 MHz for 13C NMR spectra. Column chro-
matography 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 sonchi-
folius were collected from Nagcarlan, Laguna in February
2011. The plant was identified and authenticated at the Bu-
reau 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% ace-
tone in dichloromethane fractions were combined and re-
chromatographed in petroleum ether, followed by 1% acetone
in petroleum ether, and finally in 2.5% ethyl acetate in petro-
leum ether. The 2.5% ethyl acetate in petroleum ether frac-
tions were rechromatographed in 5% ethyl acetate in petro-
leum 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 Insti-
tute 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 to-
tal 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 mi-
crobial cultures were checked for purity by plate out prior to
testing. Except for the dermatophytic fungi (which were in-
cubated 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 bacte-
rial 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 suspen-
sion was vortex-mixed and the cell density was adjusted to
that of McFarland 0.5 standard. On the other hand, the der-
matophyte 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 care-
fully pipetted into sterile tubes. The cell density was subse-
quently adjusted with sterile NSS to a final inoculum concen-
tration 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 activi-
ties of 1 was conducted according to Clinical and Laboratory
Standards Institute (CLSI) [19] guidelines.
Eleanor P. Padla, et al. /Chinese Journal of Natural Medicines 2012, 10(6): 408−414
410 Chin J Nat Med Nov. 2012 Vol. 10 No. 6 2012 年 11 月 第 10 卷 第 6 期

2.4.5 Disk diffusion susceptibility test
For assaying the antibacterial and antifungal activities of
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 like-
wise 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 an-
timicrobial 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 antimicro-
bial 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 ac-
tivity.
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 Dex-
trose 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 re-
spective 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 in-
cubation period (18–24 h for bacteria, 5–10 d for dermato-
phytic 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)/ mini-
mum 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 de-
termine 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. epider-
midis, 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 oflox-
acin 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
2012 年 11 月 第 10 卷 第 6 期 Chin J Nat Med Nov. 2012 Vol. 10 No. 6 411

4 Results
4.1 Isolation and identification of 1
Silica gel chromatography of the dichloromethane ex-
tract 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 reso-
nances 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 re-
maining 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. Di-
agnostic 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 car-
boxylic acid was attributed to C-20 due to long-range corre-
lations 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. How-
ever, 1 was found to be inactive against all four
Gram-negative test bacteria (E. coli, E. aerogenes, K. pneu-
moniae 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 concen-
tration 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 ex-
hibited 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
10 000 μg·mL−1 1 000 μg·mL−1 100 μg·mL−1 10 μg·mL−1
Ofloxacin
(30 μg)
Clotrimazole
(30 μg)
IZ
(SD)
AI
(SD)
IZ
(SD)
AI
(SD) IZ AI IZ AI
IZ
(SD)
IZ
(SD)
Bacteria
S. aureus 9.70 (0.26)
0.35
(0.03)
8.93
(0.31)
0.32
(0.03) 0 0 0 0
28.00
(2.00) -
S. epidermidis 9.80 (0.20)
0.29
(0.01)
8.70
(0.26)
0.26
(0.01) 0 0 0 0
34.00
(1.00) -
B. subtilis 8.60 (0.35)
0.34
(0.00)
7.43
(0.15)
0.30
(0.02) 0 0 0
0

24.97
(1.08)
-

E. coli 0 0 0 0 0 0 0 0 39.97 (0.25) -
E. aerogenes 0 0 0 0 0 0 0 0 22.27 (2.42) -
K. pneumoniae 0 0 0 0 0 0 0 0 38.33 (1.53) -
P. aeruginosa 0 0 0 0 0 0 0 0 28.33 (1.15) -
Fungi
C. albicans 0 0 0 0 0 0 0 0 - 17.00 (1.00)
T. rubrum 7.20 (0.26)
0.21
(0.01) 0 0 0 0 0 0 -
35.00
(1.00)
E. floccosum. 0 0 0 0 0 0 0 0 - 19.83 (0.76)
IZ (inhibition zone, in millimeters) = mean of triplicate readings; AI (activity index) = IZ of 1/IZ of standard drug


Eleanor P. Padla, et al. /Chinese Journal of Natural Medicines 2012, 10(6): 408−414
412 Chin J Nat Med Nov. 2012 Vol. 10 No. 6 2012 年 11 月 第 10 卷 第 6 期

0.32 and 0.26, respectively at the lowest inhibitory concen-
tration 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 suscepti-
bility to the compound by the disk diffusion method.
The results of post hoc comparisons of the mean IZ be-
tween 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 com-
puted 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 dif-
ferences 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
Mean difference
(95 % CI)
1 – standard
Dunnet’s
Test’s
P-value
1 (10 000 μg·mL−1)
vs ofloxacin
−18.30
(−21.05, −15.55) < 0.001
S. aureus
1 (1 000 μg·mL−1)
vs ofloxacin
−19.07
(−21.82, −16.31) < 0.001
1 (10 000 μg·mL−1)
vs ofloxacin
−24.20
(−25.62, −22.78) < 0.001S. epider-
midis 1 (1 000 μg·mL−1)
vs ofloxacin
−25.30
(−26.72, −23.88) < 0.001
1 (10 000 μg·mL−1)
vs ofloxacin
−16.37
(−17.91, −14.82) < 0.001
B. subtilis
1 (1 000 μg·mL−1)
vs ofloxacin
−17.53
(−19.08, −15.99) < 0.001
T. rubrum 1 (10 000 μg·mL
−1)
vs clotrimazole
−27.80
(29.46, −26.14) < 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. sub-
tilis with a MIC value of 1 000 μg·mL−1. Although 1 pro-
duced 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, respec-
tively, and highest for B. subtilis, with values of > 1 000
μg·mL−1. The MFC of 1 for T. rubrum was no longer deter-
mined, 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 activi-
ties of 1 occur at concentrations higher than its growth in-
hibitory concentrations.
Table 3 Parameters of antimicrobial activity of
ent-kaurenoic acid (1)
Test bacteria
Minimum inhibi-
tory concentration
(MIC)/(μg·mL−1)
Minimum bactericidal
concentration (MBC)
/(μg·mL−1)
MBC : MIC
ratio
S. aureus 125 250 2 : 1
S. epider-
midis 250 500 2 : 1
B. subtilis 1 000 > 1 000 NC
Test Fungus
Minimum inhibi-
tory concentration
(MIC)/(μg·mL−1)
Minimum fungicidal
concentration (MFC)
/(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 ex-
erted a clear bactericidal action against S. aureus and S. epi-
dermidis. 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 ter-
penes 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 antimicro-
bial 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. epider-
midis. However, ancillary investigations such as time-kill
studies are needed to ascertain if the lethal activity is concen-
tration- 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
2012 年 11 月 第 10 卷 第 6 期 Chin J Nat Med Nov. 2012 Vol. 10 No. 6 413

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 con-
ditions 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 po-
tent antimicrobial activity, but possible additive or synergistic
effects with other plant constituents may confer greater effi-
cacy, particularly in preparations other than of the pure sub-
stance. 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 re-
sistance 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-kau-
renoic acid. The kind assistance of the Center for Basic Bio-
medical Research-De La Salle Health Science Institute and
the College of Science-De La Salle University is likewise
acknowledged.
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