Ganub and Monsales.

/ MSPH 602L Advanced Pharmacology Lab (2016) 1 – 5 1

MSPH 602L Advanced Pharmacology Lab

Antibacterial Activity of Rhoeo discolor Hance.

Ganub, Jean Balatero1 and Monsales, Janine Lou Matuod1
1
MS Pharm 1, MSPH 602L Advanced Pharmacology Lab, Department of Pharmacy, University of San Carlos, Cebu City 6000, Philippines

ABSTRACT

Objective: To investigate the antibacterial activity of Rhoeo discolor Hance. Methods: The antibacterial activity
was evaluated using Disc Diffusion Method against gram-positive bacteria (Bacillus subtilis, Staphylococcus
aureus) and gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) with the broad-spectrum
Cefotaxime (30μg) as positive control. Results: S. aureus, B. subtilis, E. coli and P. aeruginosa are resistant to the
1%, 3% and 5% concentration of R. discolor extract. Conclusions: The extracts do not possess an antibacterial
activity against representative gram-positive and gram-negative bacteria. Keywords: Rhoeo discolor, Antibacterial activity

1. Introduction inquiry had ensued to the utilization of a great number of

plants with therapeutic activity and detection of its novel
In the olden times, traditional medication sprouts secondary metabolites. Most commonly, the secondary
out from the ancients in the aim of seeking a cure for metabolites present in the plant are the main accountable
diseases. Traditional medication is the use of natural reason for its therapeutic effect.[10] During the last ten
resources, either plants or animals, in the treatment of years, pace of development of new antimicrobial drugs
diseases. Oftentimes, this traditional medication leads a has slowed down while the prevalence of resistance has
channel to the development of synthetic drugs. Plants are increased. The problem of microbial resistance is
one of nature’s abundant sources of medicinal agents. growing and the outlook for the use of antimicrobial
Previous study reveals that huge group of active drugs in the future is still uncertain. This prompted the
metabolites essential in the management of many need to evaluate plants as the source of potential
illnesses and diseases were discovered, extracted and chemotherapeutic agent, antimicrobial agent and their
isolated from plants.[4] The desire to have the latest and medicinal potential[11]
better drugs leads the society to search and to investigate Rhoeo discolor (L’Her.) Hance of the
the environment, more specifically the flora. This Commelinaceae family is a common ornamental plant
 Ganub and Monsales. / MSPH 602L Advanced Pharmacology Lab (2016) 1 – 5 2

found in the Philippines. It is also scientifically known as discolor, a plant commonly used for both medicinal and
Ephemerum bicolor Moench, Rhoeospathacea(Sw.) ornamental purposes was determined. The in vitro
Stearn, Tradescantia discolor L'Her., and activity of phenolic rich extracts against specifically
TradescantiaspathaceaSw. It is denoted by multiple chosen microorganisms of human health importance by
common names: locally in Tagalog as Bangka-bangkaan, measuring their susceptibility via flow cytometry was
and in English as Tradescantia, Boat lily, Moses-in-the- evaluated on the following: Gram-positive Listeria
cradle, Oyster plant, and Rheo. The plant is medicinally innocua and Streptococcus mutans, Gram-negative
used in Bangladesh for cough, cold, coughing phlegm Escherichia coli and Pseudomonas aeruginosa, and
with blood, whooping cough, nose bleeding, bacillary lastly a fungal pathogen Candida albicans. Ten different
dysentery and blood in the stool. But no significant ethno extracts were tested in eight different doses on all the
botanical information is known.[5]Commelinaceae is a microorganisms. Analytical data revealed a high content
family of herbaceous flowering plants with many species of phenolic compounds. Flow cytometry results
used in ethnobotany, particularly in South America. demonstrated that Pseudomonas aeruginosawas the least
However, thus far reports of their bioactivity are few and affected by extract exposure. However, low doses of
far between. The antioxidant and antibacterial activity of these extracts, in a range from 1 to 4 μg/mL, did produce
Commelinaceaemethanolic leaf extracts was quantified. a statistically significant bacteriostatic and bactericidal
The plants showed antioxidant content and activity, and effect on the rest of the microorganisms. These results
exhibited antibacterial activity against six species of suggest the addition of certain natural extracts from
Gram-positive and two species of Gram-negative Rhoeo discolor could act as antibacterial and antimycotic
bacteria in a range of 5–10 mg/mL based on the broth drugs or additives for foods and cosmetics.[2] The above
microdilution method.[13] There are many plants in mentioned literature stimulated the investigation of the
Mexico with medicinal properties, some of them used in antibacterial activity of the extract from Rhoeo discolor
alternative medicine to treat cancer, such is the case of Hance leaves against representative gram-positive and
Rhoeo discolor. Rhoeo discolor aqueous crude extract gram-negative bacteria. This study could contribute and
(ACE) against rat liver cancer using the resistant- can be a gateway to the discovery of a new antibacterial
hepatocyte model was showed protective effects. Results agent.
of this investigation justify continuing with further
studies of Rhoeo discolor components to develop 2. Materials and Methods
chemoprevention strategies as an option in the treatment
of cancer.[9] In addition, Rhoeo discolor is a legendary 2.1. Plant Materials
plant used for treatment of superficial mycoses.[3]
The phytochemical screening of the leaves revealed Rhoeo discolor Hance leaves were collected at
the presence of Alkaloids, Flavonoids, Saponins, Manga District, Tagbilaran City. The leaves were
Tannins & Phenolic compounds, Cardiac glycoside and washed with running water to remove dirt and other
Terpenoids.[6][8]Phytochemicals in Rhoeo discolor foreign particles. The washed leaves were then cut into
extracts have proven to have important antimicrobial smaller pieces using a set of scissors to prepare for
activity. The antimicrobial effects of extracts of Rhoeo comminution and extraction.
 Ganub and Monsales. / MSPH 602L Advanced Pharmacology Lab (2016) 1 – 5 3

stirring until the solution turns clear golden brown in

2.2. Preparation of Extract color. The solution was then sterilized in an autoclave at
120ºC for about 20 minutes. After that, the solution was
The cut leaves were comminuted using a mortar and allowed to cool and was transferred in individual petri
pestle and were then subjected to soxhlation for 8 hours plates.
at 60oC with 95% ethanol as solvent. The solvent of the
resulting extract was initially removed using the rotary 2.6. Antibacterial Activity Testing
evaporator set to 60 rpm at 60oC for 30 minutes. The
resulting extract was then subjected to further removal of Disc Diffusion Method was used to study the
solvent using the vacufuge machine. antibacterial activity of different concentrations of
extract against Staphylococcus aureus, Bacillus subtilis,
2.3. Preparation of Test Solution Escherichia coli and Pseudomonas aeruginosa. Each
culture was then suspended in sodium chloride solution
The extract was dissolved in dimethyl sulfoxide (NaCl, 0.9%) to reach turbidity equivalent to that of the
(DMSO). Test solution for the crude extract was 0.5 MacFarland standard. Extracts were diluted in
prepared and various concentrations (1%, 3%, 5%) were dimethyl sulfoxide with the following concentrations:
prepared by dissolving 0.02 gram of extract in 2 1% (0.01g/mL), 3% (0.03g/mL), and 5% (0.05g/mL).
milliliters of DMSO, 0.06 gram of extract in 2 milliliters Each extract (1 drop) was loaded onto each sterile filter
of DMSO, and 0.10 gram of extract in 2 milliliters of paper disc. Sterilized swab was dipped in the bacterial
DMSO, respectively. suspension and was swabbed in the prepared petri plates.
The discs were placed on cultures and antibacterial
2.4. Test Organisms activity was estimated after incubation at 37oC for 18
hours, by measuring the diameter of clearing zone
The bacteria used for the test included gram-positive (ZOI).[7]
bacteria (Bacillus subtilis, Staphylococcus aureus) and
gram-negative bacteria (Escherichia coli, Pseudomonas 3. Results and Discussion
aeruginosa). The organisms were procured from the
Biology Department of University of San Carlos – The results of the Disc Diffusion Method showed
Talamban Campus. noticeable zone of inhibition. The zone of inhibition
(ZOI) on gram-positive bacterium, Staphylococcus
2.5. Preparation of the Culture Media aureus, subjected with the 1% Rhoeo discolor Hance
extract showed no clearing, with the 3% Rhoeo discolor
A total of 1000 milliliters of agar was prepared from Hance extract showed an average of 6.67 mm and with
a commercially available dehydrated base according to the 5% Rhoeo discolor Hance extract showed an average
the manufacturer’s instructions (38 grams of Müeller- of 7.33 mm as compared to 24 mm of the positive
Hinton agar in 1000 milliliters of distilled water). The control, Cefotaxime (30μg). Another gram-positive
agar in distilled water was heated with continuous bacterium, Bacillus subtilis, subjected with the 1%, 3%
 Ganub and Monsales. / MSPH 602L Advanced Pharmacology Lab (2016) 1 – 5 4

and 5% Rhoeo discolor Hance extract showed no value of medicinal plants lies in the various chemical
clearing as compared to 34.67 mm of the positive constituents present. The bioactivity of plant extract is
control, Cefatoxime (30μg). The zone of inhibition (ZOI) attributed to phytochemical constituents. There are
on gram-negative bacterium, Escherichia coli, subjected reports that plants rich in tannins have antibacterial
with the 1% Rhoeo discolor Hance extract showed an potential due to their basic character that allows them to
average of 6.33 mm, with the 3% Rhoeo discolorHance react with proteins to form stable water soluble
extract showed an average of 7 mm and with the 5% compounds thereby killing bacteria by directly damaging
Rhoeo discolor extract showed an average of 7.67 mm as its cell membrane. Alkaloids are commonly found to
compared to 27 mm of the positive control, Cefatoxime have antimicrobial properties. Alkaloids, flavonoids,
(30μg). Another gram-negative bacterium, Pseudomonas terpenoids, tannins and phenols are antimicrobial
aeruginosa, subjected with the 1%, 3% and 5% Rhoeo phytochemicals according to Tiwari et al (2011) [14] as
discolor Hance extract showed no clearing as compared shown in Table 3 (Activity of Phytochemicals).
to 13.67 mm of the positive control, Cefatoxime (30μg). Staphylococcus aureus, Bacillus subtilis, Escherichia
coli is susceptible to the positive control, Cefotaxime
Table 1.Disk Diffusion Test Results (30μg), with the exception of the resistant Pseudomonas

Bacteria Concentrations Trials (mm) aeruginosa.

1 2 3 Ave
S. aureus 1% - - - -
4. Conclusion
3% 6 7 7 6.67
5% 7 8 7 7.33
Cefotaxime 25 23 24 24.00 Based on the results from the experiment, the
B. subtilis 1% - - - -
researchers concluded that the 1%, 3%, 5%
3% - - - -
5% - - - - concentration of the extract from the leaves of Rhoeo
Cefotaxime 40 32 32 34.67 discolor Hance does not possess antibacterial activity
E. coli 1% 6 6 7 6.33
3% 6 7 8 7.00 against gram-positive Bacillus subtilis and gram-
5% 7 8 8 7.67 negative Pseudomonas aeruginosa due to the absence of
Cefotaxime 28 29 24 27.00
zone of inhibition. It was also concluded that the
P. aeruginosa 1% - - - -
3% - - - - extracts does not possess an efficacious antibacterial
5% - - - - activity against gram-positive Staphylococcus aureus
Cefotaxime 20 8 13 13.67
and gram-negative Escherichia coli due to the presence
of ≤16 mm diameter of clearing zone which was
The results exhibited that the Staphylococcus
interpreted by the criteria[1] as resistant. The presence of
aureus, Bacillus subtilis, Escherichia coli and
clearing zone, however, may be due to the presence of
Pseudomonas aeruginosa are resistant[1] to the 1%, 3%
various active principles as seen in the study of Nikam et
and 5% concentration of Rhoeo discolor Hance extract.
al (2013)[6] and Parivuguna et al (2008)[8] which are said
However, the clearing zones may be due to the presence
to have antimicrobial activities according to Tiwari et al
of phytochemicals i.e. alkaloids, flavonoids, terpenoids,
(2011)[14].
tannins and phenols as shown in the study of Nikam et al
(2013)[6] and Parivuguna et al (2008)[8]. The therapeutic
 Ganub and Monsales. / MSPH 602L Advanced Pharmacology Lab (2016) 1 – 5 5

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