Antibacterial activity of Physalis

angulata L., Merremia umbellata L.,

and Cryptostegia grandiflora Roxb. Ex
R.Br. - medicinal plants of the Colombian
Northern Coast
David E. Rivera, Yanet C. Ocampo,
Jenny P. Castro, Daneiva Caro & Luis
A. Franco

Oriental Pharmacy and Experimental

Medicine

ISSN 1598-2386
Volume 15
Number 1

Orient Pharm Exp Med (2015) 15:95-102

DOI 10.1007/s13596-014-0176-0

1 23
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 Author's personal copy
Orient Pharm Exp Med (2015) 15(1):95–102
DOI 10.1007/s13596-014-0176-0

RESEARCH ARTICLE

Antibacterial activity of Physalis angulata L., Merremia umbellata

L., and Cryptostegia grandiflora Roxb. Ex R.Br. - medicinal plants
of the Colombian Northern Coast
David E. Rivera & Yanet C. Ocampo & Jenny P. Castro &
Daneiva Caro & Luis A. Franco

Received: 8 September 2014 / Accepted: 15 December 2014 / Published online: 7 January 2015
# Institute of Korean Medicine, Kyung Hee University and Springer Science+Business Media Dordrecht 2015

Abstract Among the great Colombian biodiversity are sev- Keywords Physalis angulata . Merremia umbellata .
eral vegetal species that represent a wide source of potentially Cryptostegia grandiflora . Antibacterial activity . Folk
bioactive substances; these are commonly used in the folk medicine
medicine for the treatment of various infectious diseases. In
this work we evaluated the antibacterial activity of the total
ethanolic extracts obtained from the calyces of Physalis
angulata L. and the leaves of Merremia umbellata L. and
Cryptostegia grandiflora Roxb. ex R. Br., against ATCC
strains of Staphylococcus aureus (25923), Klebsiella
Introduction
pneumoniae (13883) and Pseudomonas aeruginosa (27853).
Bacterial sensitivity tests were performed using the broth
Infectious diseases have been a major health issue for the
microdilution method. Based on the results, the most active
human population for many years, and according with the
extract was fractionated and its bacterial sensitivity and min-
World Health Organization (WHO), one of the first causes
imum inhibitory concentration was calculated. All extracts
of mortality in the world (Bibi et al. 2011; Kuete et al. 2011).
proved to be active against the bacterial strains used.
Even in the non-lethal cases, infections bring a set of burdens
Physalis angulata L. was the most active extract, showing
and complications that patients must carry on themselves
total inhibition of the bacterial growth of all microorganisms
while being treated, which is a difficulty, especially in scarce
used at 1000 μg/mL. This activity was maintained by its
resources populations (Harris and Reza 2012). Most of the
dichloromethane fraction, which showed a potent antibacterial
infectious diseases are now treatable with modern day medi-
activity with a minimum inhibitory concentration ≤128 μg/
cine; nevertheless these treatments brought along a significant
mL against all the strains, without cytotoxic activity against
increase in the incidence of side effects and the resistance that
normal human dermal fibroblasts. In conclusion, the total
pathogenic microorganisms build against several antibiotics
ethanolic extracts of the leaves of M. umbellata,
(Bazzaz et al. 2010).
C. grandiflora and the calyces of P. angulata were active
Bacterial resistance is a problem that has been on a raise
against known pathogenic bacteria. The dichloromethane
due to a general, indiscriminate and thoughtless usage of the
fraction of P. angulata showed a potent activity at low con-
new generation of drugs developed since the discovery of
centrations, without toxic effects on human cells, becoming a
penicillin (Muñoz et al. 2009). Additionally, in developing
promissory source in the search for new therapeutic
countries the treatment of infectious diseases is complicated,
alternatives.
not only because of resistant microorganisms, but also be-
cause of the low income of the population, which drastically
D. E. Rivera : Y. C. Ocampo : J. P. Castro : D. Caro : reduces the accessibility to the right medications (Kuete et al.
L. A. Franco (*) 2011). Given these reasons, scientists all over the world have
Biological Evaluation of Promissory Substances Group,
started off the search for new, safer and more affordable
Faculty of Pharmaceutical Sciences, University of Cartagena,
130015 Cartagena, Colombia therapeutic alternatives, such as natural products, to face in-
e-mail: lfrancoo@unicartagena.edu.co fections (Rangel et al. 2001).
 Author's personal copy
96
The ethnopharmacological screening of vegetal species
commonly used in the folk medicine is a valuable tool for
the discovery of new bioactive molecules with different mech-
anisms of action and less secondary effects, which in a near
future could be included in the current therapeutic arsenal
(Uzun et al. 2004) to overcome worldwide health problems
(Gibbons 2004). Our attention as a research group has been
oriented to the discovery of new antimicrobial agents
harnessing the great biodiversity of our country. In this work,
we evaluated the in vitro antibacterial effect of extracts from
the calyces of Physalis angulata and the leaves of Merremia
umbellata and Cryptostegia grandiflora, plants widely used
in the traditional medicine of the Colombian Northern Coast
due to its antibacterial, antifungal, anti-inflammatory, antipy-
retic, diuretic and immunomodulatory properties (Ashok et al.
2011; Castro-Guerrero et al. 2013; Grosvenor et al. 1995a, b;
Hwang et al. 2004; Jin et al. 2012; Lin et al. 1992; Mukherjee
et al. 1999; Singh et al. 2011). To perform the biological
evaluation of these plants we determined its values of
Minimum Inhibitory Concentrations (MIC) against ATCC
strains of Staphylococcus aureus, Klebsiella pneumoniae
and Pseudomonas aeruginosa using the broth microdilution
method (CLSI). The extract of P. angulata was selected to be
fractionated due to its potent activity. The antibacterial activity
of P. angulata primary fractions was further evaluated, and
found that dichloromethane fraction was the most active, with
special effect on S. aureus and K. pneumoniae growth. In
addition, we assessed its effects on cell viability of fibroblasts
in order to predict potential side effects.
Materials and methods
Chemical reagents and materials
Petroleum ether, dichloromethane, ethanol, methanol were an-
alytical grade and obtained from Merck (Darmstadt, Germany).
Mueller-Hinton Agar and Broth were also acquired from
Merck. Gentamicin Sulfate from Biopex SAC (Secondary
standard, Lot: 10C256). Dulbecco’s Modified Eagle Medium
(DMEM), L-glutamine, Antibiotics (Penicillin-Streptomycin),
Dimethyl sulfoxide (DMSO), Trypan blue, Phosphate Buffer
Saline (PBS) tablets were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Fetal bovine serum (FBS) was obtained
from GIBCO (Gaithersburg, MD, USA). 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl-tertazolium bromide (MTT) from
Calbiochem® (San Diego, CA, USA). All other chemicals
and reagents used were of analytical grade.
Plant material
The calyces of Physalis angulata (Solanaceae), along with the
leaves of Merremia umbellata (Convolvulaceae) and
D.E. Rivera et al.
Cryptostegia grandiflora (Apocynaceae), were collected in
Pueblo Nuevo, Bolívar (10° 44′ North-75° 15′ West,
3 m.a.s.l.). The plants were identified in the Herbarium of
the University of Antioquia; voucher specimens were ar-
chived with the identification numbers: HUA175331,
HUA175330 and HUA175328, respectively.
Extracts and fractions preparation
The plant material was dried in an oven with controlled
temperature (35±5 °C). Then it was grounded and macerated
with ethanol 96 % until exhausting. The total extracts obtained
were concentrated with a rotary evaporator and its antibacte-
rial activity was evaluated. The most active extract was frac-
tionated through liquid-liquid partition with petroleum ether,
dichloromethane and methanol. All the extracts and fractions
were dissolved in ethanol or DMSO to obtain stock solutions
that were stored at −20 °C, for its subsequent evaluation. The
final concentration of the solvents for assays was not higher
than 1 % to avoid interferences.
Phytochemical screening
The preliminary phytochemical screening of the total
ethanolic extracts was performed following standard protocols
by (Sanabria-Galindo et al. 1997) for the qualitative identifi-
cation of flavonoids, leucoanthocyanidins, phenolic com-
pounds, triterpenes, steroid nucleus, steroids, quinones, car-
diotonic glycosides, alkaloids, coumarins, tannins, saponins
and reducing sugars.
In vitro antibacterial activity
Staphylococcus aureus (ATCC 25923), Klebsiella
pneumoniae (ATCC 13883) and Pseudomonas aeruginosa
(ATCC 27853) bacterial strains were obtained from the
American Type Culture Collection (ATCC). For the evalua-
tion of the extracts the CLSI guidelines were followed, with
some modifications (CLSI 2001). Suspensions of 1×108
UFC/mL of bacteria were prepared, correspondent to a tur-
bidity of 0.5 in the McFarland scale, which was diluted to
obtain a working suspension of 5×105 UFC/mL. 50 μL of the
total extracts (1000 μg/mL), were incubated, with the same
volume of the bacterial suspensions, during 12 h at 35±2 °C.
Gentamicin (16 μg/mL) was employed as positive control.
After the incubation time, the optical density at 620 nm
(OD620) was determined in a microplate reader (Multiskan
EX Thermo®) and the bacterial proliferation was estimated
according to the maximum growth control. Given the results,
the extracts that inhibited more than 90 % of bacterial growth
were fractionated (Cockerill 2011). The antibacterial potential
of these fractions was evaluated following the same protocol,
but employing a top concentration of 256 μg/mL.
 Author's personal copy
Colombian Caribbean plants with antibacterial activity 97

Determination of MIC Table 1 Phytochemical screening of Physalis angulata, Merremia

umbellata and Cryptostegia grandiflora extracts

MIC of the fractions that presented a complete bacterial Metabolite Plant

growth inhibition, at 256 μg/mL, was determined. Briefly,
50 μL of the bacterial suspensions were incubated in 96- P.angulata M.umbellata C.grandiflora
well plates, with 50 μL of the fractions at serial concentrations
Flavonoids +a – –
between 256 and 0.03 μg/mL at 35±2 °C for 12 h. OD620 was
Leucoanthocyanidins + + +
measured using a microplate reader (Multiskan EX
Phenolic Compounds + – –
Thermo®). MIC was reported as the lowest concentration, at
Terpenes and/or Steroids – ++ ++
which no apparent bacterial growth was shown.
Quinones – – –
Cardiotonic Glycosides ++ – +
Fibroblasts cell culture
Steroidal Core – – ++
Human normal dermal fibroblasts (PCS-201-012 ™ ; Alkaloids + – –
Rockville, MD, USA) were obtained from ATCC and routine- Cumarines – – –
ly cultured with DMEM supplemented with FBS (10 %), L- Tannins + ++ ++
glutamine (2 mM) and Penicillin streptomycin (100 IU/mL Saponins – – –
and 100 μg/mL, respectively), at 37 °C in a humidified Reducing Sugars – – –
atmosphere containing 5 % CO2. a
Test results are classified as strongly positive (+++), positive (++),
weakly positive (+), and non-detected (−)
MTT assay

Confluent cells were trypsinized and cultured for at 37 °C in
5 % CO 2 in 96-well microplates until confluence.
Subsequently, the culture medium was replaced with various
concentrations of test fraction. Control group was incubated
with the same amount of DMSO. Triton X-100 (2 %) was
used as positive control. After 24 h, the medium was removed
and cells incubated with MTT solution (3 mg/mL). Four hours
later medium was carefully aspirated, formazan crystals were
dissolved in DMSO, and OD550 was measured using a micro-
plate reader (Multiscan EX Thermo®). Percentages of cell
survival relative to control group were calculated, as well as
the concentration that reduces survival to 50 % (LC50).
Statistical analysis
Results are expressed as the mean±standard error of the mean
(S.E.M). MIC50 and LC50 were calculated employing non-
linear regression and expressed as mean and its 95 % confi-
dence interval. Selectivity index (SI) was calculated as the
ratio LC50/MIC50. Data were analyzed using one-way analysis
of variance (ANOVA), followed by Dunnett’s post hoc test, to
determine the differences between groups. Values of P<0.05
were considered significant.
Results
The results of preliminary phytochemical screening
are showed in Table 1. P. angulata was the species with more
metabolites found throughout the test; cardiotonic glycosides
assessment came out positive (++), while flavonoids, phenolic
compounds, leucoanthocyanidins, alcaloids and tannins signal
was weakly positive (+). On the other hand, M. umbellata
presented low variety of metabolites, with tannins (++), ter-
penes and steroids (++); and leucoanthocyanidins (+).
Between this two species, C. grandiflora extract was posi-
tioned with an intermediate diversity of metabolites showing
tannins (++), steroids and terpenes (++) as its major constitu-
ents, and leucoanthocyanidins (+) and cardiotonic glycosides
(+) were found with weaker signals. Saponins, reducing
sugars, cumarines and quinones were not detected in neither
in any of the ethanolic extracts of P. angulata, M. umbellata
and C. grandiflora.
The results of the antibacterial effect evaluation of the total
ethanolic extracts of P. angulata, M. umbellata, and
C. grandiflora, are presented in Fig. 1. DMSO, used as vehicle,
did not produce any significant alteration in cell viability, even
at the highest concentration employed (1 %), which indicates
that it did not interfere with the observed activity of extracts.
As expected, gentamicin (16 μg/mL), utilized as positive con-
trol, completely inhibited the growth of all the tested microor-
ganisms, confirming its utility in the assay. P. angulata,
M. umbellata and C. grandiflora extracts inhibited significant-
ly the growth of the bacterial strains employed, with inhibition
percentages higher than 20 %. However, the extract of the
calyces of P. angulata, managed to inhibit almost completely
the growth of all the bacteria strains.
In order to classify the antibacterial activity of the evaluated
extracts, they were considered as strongly active (+++), when
the bacterial proliferation percentage was 0–10 %, active (++)
when it was 10–50 %; weakly active (+) when around 50–
 Author's personal copy
98 D.E. Rivera et al.

Fig. 1 Effect of the total extracts

on the growth of Klebsiella
pneumonia, Staphylococcus
aureus and Pseudomonas
aeruginosa. Values represent the
mean±S.M.E. (n=12).
***P<0.001 ANOVA statistical
significance compared with
control group. Extracts were
classified as strongly active
(+++), active (++), weakly active
(+), and inactive (−) as described
in results

75 % and inactive when higher than 75 %. P. angulata extract
was selected to further continue with fractionation through
liquid-liquid partition as it was the only one that exerted a
strongly active effect, and the influence on bacterial growth of
the primary fractions obtained was investigated.
Table 2 present the results of the antibacterial activity of
primary fractions from P. angulata extract. As can be seen,
ether and methanol fraction did not produce a significant
effect, whereas dichloromethane fraction presented a strong
antibacterial effect on bacterial growth of all tested strains,
with inhibition percentages higher than 94 %. Consequently,
these results suggest that the active metabolites of P. angulata
are concentrated on the medium polarity fraction. As the
dichloromethane fraction was the most active, the MIC value
for each strain was calculated. As can be seen in Fig. 2, the
dichloromethane fraction of P. angulata showed a potent
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Colombian Caribbean plants with antibacterial activity 99

Table 2 Antibacterial activity of the fractions obtained from the calyces of Physalis angulata L. on Klebsiella pneumoniae, Staphylococcus aureus,
and Pseudomonas aeruginosa

Fractions Inhibition Percentage (%)

K. pneumoniae S. aureus P. aeruginosa

Petroleum Ether 18.28±5.49 (−)a 10.67±3.52 (−) 16.39±1.45 (−)

Dichloromethane 94.05±1.94 (+++) 96.57±1.69 (+++) 96.87±0.50 (+++)
Methanol 10.25±0.97 (−) 7.97±4.26 (−) 6.11±10.21 (−)
Gentamicin 99.04±0.58 (+++) 99.17±0.44 (+++) 99.33±2.96 (+++)
a
Values represent the mean±S.M.E. (n=12). Fractions were classified as strongly active (+++), active (++), weakly active (+), and inactive (−) as
described in results

antibacterial activity inhibiting the bacterial growth of all the
evaluated strains in a concentration-dependent manner show-
ing MIC values lower than 128 μg/mL (Table 3).
Once the MIC values of dichloromethane fraction of
P. angulata were established, the effect of this fraction on
human normal dermal fibroblasts was also evaluated
employing MTT assay (Fig. 2d). Dichloromethane fraction
inhibited cell viability of normal fibroblasts in a
concentration-dependent manner. However, the results also
demonstrated that fibroblasts are viable at concentrations that
completely inhibited K. penumoniae and S. aureus bacterial
growth, presenting SI values higher than 1 (Table 3). This
experimental evidence indicates the promissory antibacterial
potential of this P. angulata fraction.

Fig. 2 Effect of dichloromethane fraction isolated from the calyces of normal dermal fibroblasts (PCS-201-012™) cell viability (d). Values
Physalis angulata L. against Klebsiella pneumoniae (a), Pseudomonas represent the mean±S.M.E. (n=12)
aeruginosa (b), Staphylococcus aureus (c) bacterial growth, and human
 Author's personal copy
100
Table 3 Effect of dichloromethane fraction from Physalis angulata L.
calyces on human normal dermal fibroblasts viability and Klebsiella
Strain IC50 (μg/mL)
K. pneumoniae 24.33 (17.64–32.84)
S. aureus 16.22 (10.69–24.69)
P. aeruginosa 43.37 (34.87–53.66)
Each value represents mean±S.E.M. (n=8)
Discussion
Microorganisms take advantage of numerous adaptability ca-
pacities to hide, defend and overcome, not only the immuno-
logical response of the host, but also the current therapeutic
approaches, that given the great number of infections and the
recurrent use of antimicrobial agents, has become inefficient
due to the antibiotic resistance development by microorgan-
isms from the Staphylococcus, Salmonella, Pseudomonas,
Escherichia and Klebsiella genus which are the main cause
of the majority of infections in human beings (Acquah et al.
2013). On the other hand, the use of antibiotics is also related
with the appearance of multiple secondary effects, pushing the
patients to discontinuation of the therapy. Additionally, the
elevated costs lessen the opportunities of some patients to get
the treatment, especially in developing countries (Barbosa
et al. 2006; Bibi et al. 2011; Zampini et al. 2007). This
evidences the global need for the development of new anti-
bacterial therapies with reduced toxicity, enhancement of bio-
availability and counteraction of resistance, with natural prod-
ucts as a promissory alternative to take on the task.
Our biological study in vitro demonstrated that the
growth of S. aureus, K. pneumoniae and P. aeruginosa,
was inhibited by the total ethanolic extracts of P. angulata,
M. umbellata and C. grandiflora. These results confirm the
popular employment of these plants for the treatment of
infections and complement the results of previous investiga-
tions (Grosvenor et al. 1995a, b; Mukherjee et al. 1999;
Osho et al. 2010; Silva et al. 2005). In addition, the results
showed that the sensitivity of the microorganisms grows
according to the number of secondary metabolites detected
on the phytochemical screening, since the bacterial growth is
always slightly lower for P. angulata (6 detected com-
pounds) and C. grandiflora (5 detected compounds) com-
pared to M. umbellata (3 detected compounds).
Although our data highlight P. angulata activity, which
exerted a remarkable antibacterial activity against both Gram-
positive and Gram-negative strains; M. umbellata and
C. grandiflora are promissory extracts that should be further
studied, since they significantly affect bacterial growth of clin-
ically relevant bacterial strains. This effect combined with their
anti-inflammatory effect in mice models of edema; reinforce
their potential as treatment of infectious diseases, which is also
D.E. Rivera et al.
pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa
bacterial growth
LC50 (μg/mL) Selectivity Index
30.71 (24.27–38.67) 1.26
1.89
0.71
correlated with inflammation by immune response to microor-
ganisms (Castro-Guerrero et al. 2013; Castro et al. 2014).
P. angulata is widely distributed in tropical and subtropical
regions of the world. Extracts and infusions of this plant are
used in the folk medicine as antiparasitic, anti-inflammatory,
antimicrobial, antimalarial, antileishmanial, immunosuppres-
sive, anti-asthmatic, diuretic and anti-tumoral. The diversity of
activities of this plant is related to the presence of compounds
as steroids, withanolides and glycosides (Bastos et al. 2006;
Rengifo Salgado and Vargas Arana 2013a; Soares et al. 2003;
Vieira et al. 2008; Wu et al. 2004).
Despite the great number of previous studies related to
P. angulata and the great number of pharmacologically activ-
ities it metabolites possess; to our knowledge, this is the first
report of antibacterial activity from its calyces. This charac-
teristic plant structure of Physalis genus, is a set of modified
leaves or sepals in charge of covering and protecting the fruit,
which suggest that the amount of bioactive molecules useful
in the treatment of infectious diseases might be higher in the
calyces than in the rest of the plant (Kindscher et al. 2012;
Rengifo Salgado and Vargas Arana 2013b). According to our
data, glycosides were the most abundant metabolites of
P. angulata extract, suggesting that these compounds greatly
contribute in its antibacterial effect. These results are consis-
tent with the known potential of glycosides to inhibit bacterial
proliferation (Arthan et al. 2002; Gibson et al. 2012; Li and
Zhang 2014). Additionally, a previous study focused on an-
other specie of this genus also showed an important antibac-
terial activity of an ethereal extract and glycoside-enriched
fraction from calyces of Physalis peruviana L, ratifying this
hypothesis and the potential of the members of the Physalis
genus as a source of promissory compounds from a therapeu-
tic and pharmacological approach (Franco et al. 2013).
The powerful antibacterial activity showed by the ethanolic
extract of the calyces of P. angulata, was kept by its dichloro-
methane fraction, which greatly inhibited the growth of
S. aureus, K. pneumoniae and P. aeruginosa, at concentrations
four times lower than the employed with the total extract. This
activity augmentation might be related to the concentration of
the metabolites responsible for the antibacterial effect, which is
confirmed by the MIC values (MIC≤128 μg/mL) and impulse
future studies to isolate, purify and identify the active molecules
and the mechanisms of action involved (Mulaudzi et al. 2011).
 Author's personal copy
Colombian Caribbean plants with antibacterial activity
According to the IC50 calculated values the fraction was
active in all the strains used (K. pneumonia IC50 =24.33 μg/
mL and P. aeruginosa IC50 =43.37 μg/mL), but the most
susceptible bacterial strain was S. aureus (16.22 μg/mL).
This microorganism is one of the main causes of nosocomial
infections around the world, which makes it a global public
health issue, especially when the cases are associated with
methicillin-resistant S. aureus (MRSA) (Williamson et al.,
2014). For this matter, the dichloromethane fraction of the
calyces from P. angulata stands out as possible therapeutic
alternative for the treatment of infectious diseases.
In conclusion, K. pneumoniae, S aureus, and P. aeruginosa
can be effectively inhibited without significant damage to
human normal dermal fibroblasts, employing extracts from
P. angulata, M. umbellata and C grandiflora. Our results give
a scientific foundation to the employment of these plants in the
Colombian folk medicine. The dichloromethane fraction of
P. angulata calyces demonstrated a potent antibacterial effect,
suggesting a concentration of bioactive metabolites. Further
scrutiny of this fraction to isolate antibacterial compounds and
to evaluate its activity using in vivo experimental models is
thus warranted to assess its clinical potential to treat bacterial
infections.
Acknowledgments The authors thank the University of Cartagena,
Colombia, for the financial and institutional support (Project Code: 055-
2012), also to the Young researchers and innovators “Virginia Gutiérrez
de Pineda” program from Colciencias for the support given to Daneiva
Caro and Jenny Castro (Call 525 of 2011).
Conflict of Interest The authors declare that they have no conflict of
interest.
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