Phytochemical and Antibacterial Screening of The Pseudostem of Musa (BBB Group) Saba' Against Canine Skin Pathogens

TILAMSIK: The Southern Luzon Journal of Arts and Sciences
Volume 10 | August 2018 | ISSN: 1656-1953 (Print); 2467-6209 (Online)

Published by Southern Luzon State University-College of Arts and Sciences
Phytochemical and antibacterial screening of the

pseudostem of Musa (BBB group) ‘Saba’
against canine skin pathogens
Beulah T. Grevialde & Anne Julian S. Ladiana

Bachelor of Science in Biology, College of Arts and Sciences, Southern Luzon State University
Abstract
This study was conducted to determine the presence of phytochemicals

and the antibacterial activity of the pseudostem of Musa (BBB Group)
‘Saba’ against canine skin pathogens. The fresh and rotten pseudostem
methanolic extracts were tested for the presence of different phytochemicals
such as flavonoids, alkaloids, saponins, tannins and terpenoids through
test tube reaction and thin layer chromatography. Antibacterial activity
of the two pseudostem extracts were tested through agar well diffusion
method against the test microorganisms including Staphylococcus aureus,
Escherichia coli, Klebsiella pneumoniae and the isolated pathogen
Staphylococcus pseudintermedius from the canine model organism.
Both the pseudostem extracts were mixed with petroleum jelly to prepare
the ointment, which was tested on the canine for 10 days. The results
reveal that the fresh and rotten extracts are potential sources of alkaloids,
saponins, tannins and terpenoids. For the antibacterial activity, the rotten
pseudostem extract showed partially active inhibition against S. areus and
S. pseudintermedius only. The fresh pseudostem extract showed inactive
inhibition against all the bacterial strains used. The wound healing activity
of the rotten pseudostem ointment was observed effective as early as the
5th day of application, while the fresh pseudostem ointment was observed
to take effect at the 10th day of application.
Keywords: antibacterial activity, canine skin pathogens, Musa (BBB Group)

‘Saba’, phytochemicals, wound healing activity
Research Advisers: Fides Marie Villenas & Prof. Leonisa O. Bernardo, PhD
Editor’s Note: The researchers won second place for this study in the 2017
CAS Inter-Disciplinary Research Conference.
Grevialde, Beulah T. & Ladiana, Anne Julian S. (2018). Phytochemical and

Volume 10 antibacterial screening of the pseudostem of Musa (BBB Group) ‘Saba’ against
Student Research canine skin pathogens.Tilamsik: The Southern Luzon Journal of Arts and Sciences, 125
August 2018
10. 125-142.
Phytochemical and antibacterial screening of... Musa (BBB Group)...
Student Research
Grevialde, Beulah T. & Ladiana, Anne Julian S. (2018)
1. Introduction
About 85% of all the medications for different diseases worldwide are
derived from plants (Lakshmi et al., 2015). Plant extracts are potential
sources for microbial inhibitory activity because they represent a reservoir
of effective chemotherapeutics. Plants naturally produce substances called
phytochemicals, which are used to protect themselves against bacteria,
pharmacologic effects, and viruses.
In developing countries, banana (Musa sp.) is the fourth most important

crop after rice, wheat, and maize (Dixit et al. 2014). It has many beneficial
nutritional and curative properties that prevent many kinds of illnesses (Jahan
et al. 2010). Presence of active components varies in different banana cultivars
due to its differences in growth environment, species and cultivars (Jain et al.
2011). Active components essential for the treatment of different diseases
are mostly found on the different parts of the plant such as pseudostem,
flowers, fruits, leaves and roots (Lakshmi et al., 2015). As mentioned by
Zafar, Saleha, Hoque, and Sohel (2011) in their study, Musa sapientum fruit
extract showed wound healing and antibacterial activity. Additionally, Ray and
Ghatak (2013) documented that banana pseudostem juice has antienzymatic,
astringent, and antibacterial properties that inhibit the growth of many
microorganisms. Furthermore, Musa sp. pseudostem juice has the potential
to be incorporated in antibacterial formulations in pharmaceutical industries
(Kumar, Srivastavab, Singhe, Mathdac, and Thindc, 2014) and it can be a
good source of antidermatophytic agents (Ige Mebude, Adeniyi, and Adewoye,
2015). Compared to synthetic drugs, plant-derived medications have lesser
toxicity and fewer side effects (Jain et al. 2011).
Since dogs are considered as man’s best friend, pet owners are
concerned about the health and well-being of their pets so the affected dogs with
dermatitis are brought to veterinary clinics for treatment (Sindha, Trangadia,
Vihol, Parmar, and Patel, 2014). The most common dermatologic problem
encountered in dogs is pyoderma, which is a bacterial infection (Harihan et al.,
2014). The treatment for canine pyoderma involves antimicrobial drug therapy
and the most commonly used drugs for empirical systems’ treatment are
amoxicillin-clavulonic and cephalosphorins. But S. pseudintermidius isolates
exhibits resistance to commonly prescribed drugs for canine pyoderma. Due
to the emergence of methicillin-resistant S. pseudintermidius in dogs caused
by broad use of antimicrobial agents, antibiotic resistance has increased (Han
et al., 2015). Increased resistance of pathogenic organisms to most available
antimicrobial agents demands the search for alternative medicines. Medicinal
plants are one of the best resources of bioactive compounds which can be
utilized effectively (Jain et al. 2011)
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The use of the extract of rotten banana pseudostem in curing dog’s

skin lesions is already an old practice being done by old folks as stated in the
review article of Lans, Harper, Georges, and Bridgewater (2000) about the
ethno veterinary practices of people in Trinidad and Tobago. They documented
the use of Musa spp. stem exudate in dog’s skin diseases and control of
ectoparasites. Even though there are numerous studies and literatures about
the phytochemical and antibacterial property of fresh banana pseudostem,
it is the rotten banana pseudostem that is used by old folks in curing dog’s
skin lesions. Thus, the researchers became interested in conducting a
research about the antibacterial activity of both fresh and rotten pseudostems,
specifically in treating dog’s skin diseases, of Musa (BBB Group) ‘Saba’. Also,
Musa (BBB Group) ‘Saba’ was considered due to its abundance in the country
and its pseudostem’s potential as a traditional medicine. On this premise,
the researchers became interested in studying the phytochemicals present in
pseudostem of Musa (BBB Group) ‘Saba’ and its potential as an alternative
treatment for canine skin infections.
The main objective of this study is to determine the presence of

phytochemicals and the antibacterial activity of the pseudostem of Musa (BBB
Group) ‘Saba’ against canine skin pathogens. Specifically, the study sought
to detect the presence of phytochemicals on the fresh and rotten pseudostem
extracts of Musa (BBB Group) ‘Saba’; to determine the pathogen present in
canine skin through bacterial isolation and identification; to determine the
antibacterial activity of the fresh and rotten pseudostem extracts of Musa
(BBB Group) ‘Saba’ against pathogens in canine skin diseases; and to test for
the efficacy of the extract by preparing an ointment and evaluating its wound
healing activity in vivo.
2. Framework of the Study
The framework of this study is based on the concept that the phytochemicals
known to possess antibacterial property against canine skin pathogens
are alkaloids, flavonoids, saponins, tannins and terpenoids. These
phytochemicals can be extracted by using methanol as solvent. Additionally,
these phytochemicals are active against the commonly isolated pathogens
from canine skin lesions which are Staphylococcus pseudintermedius,
Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli.
In this study, fresh and rotten pseudostem of Musa (BBB Group)

‘Saba’ was used as the raw materials as source of these phytochemicals and
eventually in producing an ointment that will serve as an alternative treatment
for canine skin diseases. The phytochemicals present on the pseudostem
extract was determined through phytochemical screening as well as its
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antibacterial activity against canine skin-related pathogen. This is to provide a

scientific evidence regarding the use of banana pseudostem in treating canine
skin lesions since it is an old practice being done by old folks.
Figure 1. The Input-Process-Output model in determining the phytochemicals and

antibacterial activity of Musa (BBB Group) ‘Saba’
Figure 1 shows the Input-Process-Output Model in determining the

phytochemicals and antibacterial activity of the pseudostem of Musa (BBB
Group) ‘Saba’. The input includes the fresh and rotten pseudostem of Musa
(BBB Group) ‘Saba’, equipment, apparatus, materials, chemicals and chemical
reagents for phytochemical screening, antimicrobial assay and ointment
preparation; and test organisms which are canine skin-related pathogens for
antimicrobial activity. The process conducted were extraction of pseudostem,
phytochemical screening, antibacterial assay, ointment preparation and
evaluation of wound healing activity. The fresh and rotten pseudostem
of Musa (BBB Group) ‘Saba’ was subjected to solvent extraction. The
pseudostem crude extract underwent phytochemical screening to determine
the bioactive compounds present. Antibacterial activity of the pseudostem
extracts from Musa (BBB Group) ‘Saba’ against canine skin-related pathogens
was determined through antibacterial assay. The pseudostem extract was
incorporated to yellow soft paraffin (petroleum jelly). The efficacy of the
ointment as an alternative treatment for canine skin diseases was tested
through topical application. The decrease in size of the wound during the
wound healing process was documented. The outputs of the study were the
identified phytochemicals present in the fresh and rotten pseudostem and its
antibacterial activity against canine skin-related pathogens. The final product
was the tested ointment, which can be used as an alternative treatment for
canine skin diseases.
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3.0. Materials and Methods
The research was done through experimentation. Purposive sampling was used
to collect the plant sample from a banana plantation with the needed banana
species, Musa (BBB Group) ‘Saba’. In vitro procedures were performed for
the extraction, phytochemical screening, and antibacterial screening, while in-
vivo procedure was performed for the bioassay. The research was subdivided
into five main parts: identification, collection and extraction of plant samples,
phytochemical screening, bacterial isolation and identification, antimicrobial
screening, and evaluation of wound healing activity.
The procedure by Sasidharan, et al. (2010), Banu and Cathrine

(2015), Ramu et al. (2015) were employed for phytochemical screening,
Quinto and Santos (2005) for antimicrobial screening, and James and Friday
(2010) for evaluation of wound healing activity was followed all throughout the
laboratory components of the study, with some modifications in underlined
form based on other studies. The sterile swab was gently passed through the
infected area on the dog’s skin. The swab was placed into a tube containing a
culture medium to promote organism’s growth. The test microorganisms used
are Staphylococcus pseudintermedius, Staphylococcus aureus, Klebsiella
pneumoniae, and Escherichia coli. Staphylococcus pseudintermedius was
isolated from the canine skin sample submitted at the UPLB-Veterinary
Teaching Hospital and the other microorganisms were purchased at UPLB
Biotech and SLSU Microbiology Laboratory. The microorganisms were sub
cultured in duplicate tubes of nutrient agar slant and were incubated for 18-24
hours at 35C.
Ten mL of the nutrient broth was then prepared. The bacterial

suspensions were shaken and immediately compared against the 0.5
McFarland. The turbidity was adjusted if the bacterial and fungal suspension
did not appear with the same density as the McFarland standard. To adjust
the turbidity, sterile broth was added and the resulting turbidity was again
compared to the standard.
The test organisms were aseptically inoculated onto a Mueller-

Hinton agar plate. This was performed by swabbing six times over the entire
surface of the agar plate, for even distribution on the inoculum of the surface
of the medium. The plate was rotated approximately 60 degrees after each
application. Then, the swabbed plates were left to stand for five minutes.
Three sets of bioassay plates were prepared for each test organism
and the screening was done in triplicates. Four wells in the first set of Mueller
Hinton plates and three wells in each of the second and third sets of Mueller
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Hinton plates were made using a 5.00 mm diameter flame-sterilized cork

borer. The two wells in the first plate were labelled as FM (fresh methanol) and
RM (rotten methanol), for the second plate wells were labelled as D (distilled
water), and M (methanol) and for the last plate the wells were labelled as DA
(clindamycin), AMX (amoxicillin) and CF (cephalexin). A volume of 0.055mL
of the fresh and rotten methanolic Musa (BBB Group) “Saba” pseudostem
extracts were delivered on the first plate. Distilled water and methanol were
delivered on the second plate. Also, 500 mg/L amoxicillin, clindamycin and
cephalexin were delivered on its corresponding wells on the third plate. The
plates were incubated at 35˚C for 15 to 18 hours. After the incubation period,
the extracts in the well was completely absorbed in the agar medium.
Figure 2. Agar well diffusion set up for each test microorganism
The antimicrobial activity was determined by looking for a clear round

zone, which was known as the zone of inhibition. The plates were inverted
and with the use of vernier caliper, the zone of inhibition was measured in
millimeter. The size of the agar well was also noted (Quinto and Santos, 2005).
Decontamination of plates and used cultures was done using a pressure
cooker at 15 psi for 30 minutes. After decontamination, all the agar and broth
were placed in a separate plastic bag for disposal. The used glass wares were
washed using antibacterial liquid soap, dried and stacked in a clean PP bag.
The presence of phytochemicals was determined by a series of different

chemical treatments in test tube reactions and thin layer chromatography.
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Color change and precipitation was examined. Table 1 and Table 2 show the
qualities that were visualized to confirm a positive result to a specific test.
Table 1. Indication of a positive result in test tube reactions
Table 2. Indication of a positive result in TLC
Results obtained were tabulated and positive result was indicated

by a positive sign (+) and negative result was indicated by a negative sign
(-). Variation of results in the two methods used in a single phytochemical
was considered as negative since the amount of the compound might not be
sufficient to be detected in a test tube reaction. On the other hand, further
confirmatory test should be done for the presence of phytochemical where
either just TLC or test tube reactions were done. Hence, phytochemicals
present were measured qualitatively through different color reactions and
precipitations.
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In the antibacterial screening, the size of the zone of inhibition

determined the plant extracts’ antibacterial activity against canine skin
pathogens. For a 5mm diameter agar well, the diameter zones of inhibition
that was observed with corresponding inference are presented in Table 3.
Table 3. Indication of a positive result in TLC
The ointment was prepared by obtaining 5 g of the crude extract of

pseudostem of the Musa (BBB Group) and was mixed with 20g of petroleum
jelly. The tube with 20g petroleum jelly was submerged in a water bath while
adding the extract for a certain period of time until it was thoroughly mixed.
Two ointments were prepared; the first one is the mixture of the 5g of the crude
extract of fresh pseudostem of the Musa (BBB Group) and 20g of petroleum
jelly. The second one is the mixture of the 5g of the crude extract of rotten
pseudostem of the Musa (BBB Group) and 20g petroleum jelly (Sasidharan et
al., 2010).
The prepared ointment was applied on the dog’s skin lesion by

dabbing a sufficient amount of the ointment all over the infected area. There
were two experimental set-ups and two control groups, one positive and one
negative control. For experimental set-up 1, dogs’s skin lesions were treated
topically with formulated crude extract (fresh sample) and petroleum jelly. For
experimental set-up 2, dog’s skin lesions were treated topically with formulated
crude extract (rotten sample) and petroleum jelly while the control set up was
treated only with petroleum jelly and the other was left untreated for 10 days.
The dog’s skin infection was observed and documented every day for
10 days. Improvement of the infected site was documented every day and was
the basis for the efficacy of the formulated ointment (James and Friday, 2010).
4. Results and discussion
4.1. Identification and screening

The plant sample submitted to the Museum of Natural History,
UPLB was identified as Musa (BBB Group) ‘Saba’. As to phytochemical
screening, alkaloid was present in the test tube reactions and TLC analysis
in the methanolic extract of both fresh and rotten Musa (BBB Group) ‘Saba’
pseudostem. Likewise, in the study of Ramu et al. (2015) alkaloids were
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present in banana (Musa sp. var Nanjangud Rasa Bale) pseudostem. This
denotes the occurrence of basic nitrogenated compounds, comprising the
large class of secondary metabolites (Aguinaldo et al., 1998). Alkaloids are
used for medications, as recreational drugs, or in entheogenic rituals due to its
pharmacological effects. They have antimicrobial and anti-parasitic properties
and some can act as narcotics (Linne, 2007). Contrary to this are the findings
of Kumar et al., (2014) wherein alkaloids are not detected in Musa acuminate
pseudostem.
Flavonoids was existent only in the rotten pseudostem extract of

Musa (BBB Group) ‘Saba’ as confirmed by the Alkaline reagent test and TLC
test. This is in contrast with the findings of Kumar et al. (2014) and Onyema
et al. (2016), wherein flavonoids are present in the fresh pseudostem of Musa
acuminate and in Musa sp. var Nanjangud Rasa Bale according to (Ramu et
al. 2015). Flavonoids are ubiquitous in photosynthesizing cells and therefore
occur widely in the plant kingdom. They are found in fruits, vegetables, nuts,
seeds, stems and flowers, as well as tea and wine. They have been reported
to exhibit many beneficial properties, including antimicrobial activity, (Cushnie
and Lamb, 2005). According to Muhammad et al. (2013), bioactive component
identified from Moringa oleifera that was found to be beneficial in wound
healing belongs to the group of flavonoid compounds. Similarly, the component
responsible for the wound healing activity in dogs was flavonone (Moulari et
al. 2016). One of the factors affecting the presence of flavonoids on the rotten
pseudostem was mechanical or chemical injury, which may lead to rotting.
Induced production occurs wherein new compounds that are originally not
present in the plant can be produced. This response depends on the plants‘
species, developmental stage, the amount of light, and the availability of water
Figueiredo (2008).
Presence of tannins was detected in both fresh and rotten methanolic

extract of Musa (BBB Group) ‘Saba’ pseudostem. Tannins are naturally
occurring plant polyphenols which combine with protein and other polymers to
form stable complexes (Lim et al. 2006). Presence of tannin was confirmed
by the ferric chloride test, gelatin test and TLC. The gelatin test, gives a jelly
precipitate that connotes the presence of tannins. Tannins are water soluble
polyphenols, which differs in most other natural phenolic compounds in their
ability to precipitale such as gelatin from solution (Scalbert, 1991). Tannins
are also present in banana (Musa paradisiaca) pseudostem based on the
findings of Ray and Ghatak (2013). However, Ramu et al. (2015) documented
that tannins are absent in banana (Musa sp. var Nanjangud Rasa Bale)
pseudostem.
In the TLC test, upon spraying the TLC plate with the corresponding
spray reagent for tannins, which is the ferric chloride and potassium
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ferrycyanide solution, appearance of blue spots was observed, which indicates

the presence of tannins.
Terpenoids were present in methanolic extract of both fresh and rotten

Musa (BBB Group) ‘Saba’ pseudostem. Both test tube reactions and TLC test
confirmed the positive result for terpenoids. Based on the findings of Ramu et
al. (2015) and Onyema et al. (2016), terpenoids are present in the fresh banana
pseudostem. This denotes the occurrence of the numerous and structurally
diverse plant natural product. They are major constituents of plant resin and
essential oils extracted from such plants. Also, they have been exploited for
their potentials and effectiveness as medicine and flavor enhancers (Zwenger
and Basu, 2008).
Both fresh and rotten Musa (BBB Group) ‘Saba’ pseudostem sample
gave positive result for the froth test for saponins. Similarly, saponins are present
in fresh Musa sp. var Nanjangud Rasa Bale (Ramu et al. 2015) pseudostem
extract. On the contrary, saponins are absent in fresh Musa paradisiaca (Ray
and Ghatak, 2013) and Musa accuminata (Kumar et al. 2014) pseudostem
extract. Saponins are bioactive compounds produced mainly by plants, but
also by some insects and marine organisms. They are able to interact with cell
membranes and decrease the surface tension of an aqueous solution because
of their lyobipolar properties (Thakur et al. 2011). From this, it can be deduced
that the existence of the persistent foam in the tube contains triterpenoid
glycosides that are characterized by their ability to froth when the aqueous
solution is agitated.
The pathogen isolated from the canine skin lesion was Staphylococcus
pseudintermedius as identified using BBL Crystal Identification. This is similar
to the findings of Hariharan et al. (2014), Bryan et al. (2012) and Han et al.
(2015), which revealed that most common isolated pathogen from canine skin
diseases was Staphylococcus pseudintermedius.
For the antibacterial activity of the fresh and rotten methanolic crude
extract and ointment, it was evaluated in vitro using agar-well diffusion method
for antibacterial assay and its antibacterial activity was measured based on the
diameter of the zone of inhibition surrounding the agar well. Table 4 shows the
antibacterial activity of the fresh and rotten extract against gram positive and
gram negative bacteria as well as their reaction to the positive and negative
controls.
The diameter of inhibition of the rotten methanolic extract in the

isolated canine pathogen, Staphylococcus pseudintermedius was considered
partially active with a diameter of 11mm. It was larger than the other
microorganisms used in the antibacterial assay. In contrast, the isolated
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Table 4. Antibacterial activity of fresh and rotten extract and ointment by Agar
well diffusion
pathogen exhibited resistance against the fresh methanolic crude extract. The
other gram positive bacterium used in the assay was Staphyloccocus aureus.
It exhibited a 10mm zone diameter of inhibition against rotten methanolic
extract of ‘Saba’ pseudostem, which was considered as partially active, while
the fresh methanolic extract exhibited a 9mm zone diameter of inhibition,
which was considered to be inactive or resistant. Meanwhile, fresh and rotten
methanolic extract showed no inhibitory activity against the two gram negative
bacteria used, E.coli and K. pneumoniae. The resistance of the gram negative
bacterial strains to the fresh and rotten methanolic extract was because of the
presence and absence of different phytochemicals. The largest zone diameter
of inhibition was made by the rotten methanolic crude extract against the gram
positive bacterial strain S. pseudintermedius that was isolated from the skin
lesion of the canine model test organism. This result shows that the rotten
extract was only inhibitory to the strain isolated from an infected canine skin.
These results provide scientific explanation to the application of rotten banana
pseudostem on canine skin infections, which was done by old folks.
Though there was an inhibition zone observed, it was only categorized

as partially active unlike the reaction of the canine bacterial strains to the
positive controls of antibiotics. The antibiotics that served as positive control
were amoxicillin, cephalexin and clindamycin. These antibiotics exhibited an
effective inhibitory activity against the canine bacterial strains, which was
considered to be active or susceptible, except for both of the gram negative
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bacteria, E.coli and K. pneumoniae, which was resistant to clindamycin. There

was also no inhibitory activity observed from the negative control solutions,
which was distilled water. In contrast, the negative control solution, methanol
shows partially active inhibition activity against all canine bacterial strains. The
inhibitory activity exhibited by the fresh and rotten extract against both gram
negative bacteria was not observed, while the extracting solvent methanol
shows partially active inhibition.
Methanol was not responsible for the inhibitory activity exhibited by the
fresh and rotten extract because the extracting solvent was already evaporated
when subjected to the rotary evaporator. For confirmation that there was no
trace of methanol left on the extract, which may inhibit the growth of bacteria,
the extract was smelled. Also, from a total volume of 200ml methanol added to
100g fresh and rotten pseudostem, the remaining crude extract after extraction
was 13.7ml and 17.7 ml fresh and rotten extract, respectively.
According to Onyema et al. (2016) the antibacterial activities of

banana (Musa acuminata) pseudostem extract was due to the presence of
large classes of secondary metabolites. Thus, the inhibitory activity of the
rotten and fresh pseudostem extracts was due to the presence of secondary
metabolites such as alkaloids, flavonoids, tannins, terpenoids and saponins.
Resistance and susceptibility of Staphylococcus pseudintermedius, which was
the leading cause of most canine skin diseases in fresh and rotten ‘Saba’
pseudotem extract, respectively was mainly because of the absence of
flavonoids in the fresh ‘Saba’ pseudostem extract. According to Xu and Lee
(2001), the antibacterial activity of flavonoids is in relation with its structure.
Flavonoids lacking hydroxyl groups in ring B do not possess antibacterial
activity. Presence of flavonoids in high amounts accounts for the plants high
antibacterial and lipid peroxidation activity (Bhaskar et al. 2011). Similarly, in
the study of Ramu et al. (2015) the presence of flavonoids in banana (Musa sp.
var Nanjangud Rasa Bale) accounts for its antimicrobial activity. In addition,
flavonoid is the compound responsible for the wound healing activity in dogs
(Moulari et al. 2006). Granting that terpenoids are present in both of the fresh
and rotten pseudostem extracts, fresh ‘Saba’ pseudostem extract did not
exhibit active inhibitory effect against all the bacterial strains. In contrast to
this are the findings of Mastelic et al (2005), which showed active inhibitory
activity against Staphyloccocus aureus and concluded that the antibacterial
activity of Helichrysum italicum was due to the presence of terpenoids.
The reaction of the two gram negative bacteria used, E.coli and K.
pneumoniae, against the fresh and rotten methanolic extract on the bacterial
assay was in contrast to the findings of Kumar et al. (2014), where they
observed that the fresh methanolic banana pseudostem extract was effective
against E. coli and Klebsiella pneumoniae. Also the alkaloids which are present
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in both the rotten and fresh methanolic extracts of the ‘Saba’ pseudostem were
found to have an active inhibitory activity against gram positive pathogenic
bacteria only based on the findings of Deng et al. (2011). Similarly, saponins,
which are present in both of the ‘Saba’ pseudostem extracts, did not exhibit
an inhibitory activity against gram negative bacteria (Soetan et. al, 2006). The
study of Ray and Ghatak (2013) states that the documented fresh banana
pseudostem extract was found to inhibit the growth of E. coli mainly because
of its tannin content, which was also present in the fresh and rotten ‘Saba’
pseudostem extract.
The inactive and partially active inhibition exhibited by the pseudostem

extracts suggests that the concentration of bioactive compounds in the crude
extracts vary between the state of the pseudostem when it was extracted,
fresh and rotten. As observed in the results, the extracting solvent methanol
showed partially active inhibition against the canine bacterial strains, which
was in contrast with the findings of (Ahmed et al. 2015) where the solvents
used to extract species of Rhododendron was methanol and ethyl acetate
had no impact on the growth of any of the tested bacterial organisms. Their
test organisms possess a broad spectrum as well as intragenus diversity.
According to the study of Wendisch and Lebmeier (2015), methanol was found
to inhibit the growth of bacteria suggesting that methanol tolerance limits the
fermentative production processes of some bacteria
4.2. Evaluation of wound healing activity
For the evaluation of the ointment prepared from mixing the fresh and
rotten extracts of ‘saba’ pseudostem with petroleum jelly, dogs’ skin infection
was treated topically with the prepared ointments and petroleum jelly. In the
first day of application, it can be noted that wound and its exudates are present
in all the four infected area in the canine skin as shown in Figure 3.
Initially, massive hair loss and manifestation of ticks was also observed.
Dry skin or white flakes were also noticeable due to the dark colored hair or
dog’s coat. After two (2) days of topical application, scabs were present in the
infected area treated with RO. The wound and its exudate became relatively
smaller compared to the first day of application. In the infected area treated
with FO, wound and its exudate also became smaller, but scabs were not
present. In the control set up, there was no apparent improvement compared
to the first day of application. On the other hand, the untreated area became
severe.
After five (5) days of application, the infected area treated with RO
was completely cured showing no marks of wound and scabs. Also, the area
regenerated. In the area treated with FO, the condition improved but exudates
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Figure 2. Infected areas treated with RO, FO and PJ and untreated area at Day 0, 5 and 10
were still present. Dryness all over the area was lessened and wound exudates
are still evident in the infected area treated with petroleum jelly. The lesions in
the untreated area became serious and gradual hair loss was observed. Since
the infected area treated with RO completely healed after five days, topical
application was stopped.
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For the FO, application was continuous until the area was completely
healed. After 10 days of application, the lesion has completely disappeared,
but scars were still present unlike in the area treated with RO. On the other
hand, at the 10th day of observation, the area treated only with petroleum jelly
still has little exudates and dryness has lessened and the area appears to be
pinkish in color. It was also observed that sloughs are present in the area.
These results could be explained by the occlusive and moisturizing action of
petroleum jelly. It prevents tissue from drying and prevents ischemia of deeper
tissues, preventing the increase of the lesion (Franco et. al, 2012). Also, after
being left untreated for ten days, severe hair loss has occurred.
Shorter time of treatment with RO was maybe due to the presence of

all the five phytochemicals, which are known to have wound healing potential,
especially flavonoids, which was absent in the fresh extract. According to
Tsuchiya (2015), this was due to the membrane-interacting properties of
flavonoids. It modifies the fluidity, order, and permeability of artificial and
cellular membranes. These characteristics of flavonoids are correlated with
its antitumor, antiplatelet, antioxidant, antimicrobial, and anti-inflammatory
effects. Presence of flavonoids in the rotten extract may be due to mechanical
or chemical injury Figueiredo (2008), which may lead to rotting of the Musa
(BBB Group) “Saba” pseudostem. Additionally, according to Berdami, 2001,
petroleum jelly is an effective base ointment vehicle for the transport of active
constituents in topical animal medications.
5. Conclusions and Recommendations
Alkaloids, saponins, tannins and terpenoids were present in both fresh

and rotten extract, while flavonoids were present only on the rotten extract.
The isolated bacterium from the wound of the canine model organism was
Staphylococcus pseudintermedius only, which is a common canine skin
pathogen. The rotten pseudostem extract exhibited partially active inhibitory
activity against Staphylococcus pseudintermedius and Staphylococcus
aureus and inactive inhibitory activity against Escherichia coli and Klebsiella
pneumonia, while the fresh pseudostem extract showed an inactive inhibitory
activity against all the bacterial strains used. The ointment with extract from
rotten sample required a shorter time (five days) in treating canine skin lesions,
while the ointment with extract from fresh sample took 10 days to completely
heal the canine skin lesion.
The researchers recommended that the rotten extract of Musa (BBB

Group) ‘Saba’ pseudostem may be utilized in the preparation of ointment for
curing canine skin lesions. Test for the antimicrobial activity of the extract
by using a wide range of microorganisms that can be found on canine skin
Volume 10
Student Research
diseases such as bacteria and fungi may be conducted. The active components
from extract of Musa (BBB Group) ‘Saba’ may be isolated and determine its
minimum inhibitory concentration (MIC) against canine skin diseases. Different
extracting solvents such as ethanol and dichloromethane maybe used.
Removal of the remaining water from the extract before incorporation with the
base ointment (petroleum jelly) may be done. Histopathological analysis on
the wound of the model organism may be performed for further evaluation of
the healing activity of the ointment.
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About the Authors
Beulah T. Grevialde finished her Bachelor of Science in

Biology degree at Southern Luzon State University in 2017.
As a student, she was a scholar of the provincial government
of Laguna, and a member of the SLSU Haynayanon.
Anne Julian S. Ladiana finished her degree of Bachelor

of Science in Biology at Southern Luzon State University
in 2017. She was a scholar of the provincial government
of Laguna and a member of the SLSU Haynayanon during
her student days.
Corresponding author: beulahg05@gmail.com
Volume 10
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Phytochemical and Antibacterial Screening of The Pseudostem of Musa (BBB Group) Saba' Against Canine Skin Pathogens

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TILAMSIK: The Southern Luzon Journal of Arts and Sciences

Volume 10 | August 2018 | ISSN: 1656-1953 (Print); 2467-6209 (Online)

Phytochemical and antibacterial screening of the

Beulah T. Grevialde & Anne Julian S. Ladiana

This study was conducted to determine the presence of phytochemicals

Keywords: antibacterial activity, canine skin pathogens, Musa (BBB Group)

Grevialde, Beulah T. & Ladiana, Anne Julian S. (2018). Phytochemical and

In developing countries, banana (Musa sp.) is the fourth most important

The use of the extract of rotten banana pseudostem in curing dog’s

The main objective of this study is to determine the presence of

2. Framework of the Study

In this study, fresh and rotten pseudostem of Musa (BBB Group)

antibacterial activity against canine skin-related pathogen. This is to provide a

Figure 1. The Input-Process-Output model in determining the phytochemicals and

Figure 1 shows the Input-Process-Output Model in determining the

3.0. Materials and Methods

The procedure by Sasidharan, et al. (2010), Banu and Cathrine

Ten mL of the nutrient broth was then prepared. The bacterial

The test organisms were aseptically inoculated onto a Mueller-

Hinton plates were made using a 5.00 mm diameter flame-sterilized cork

Figure 2. Agar well diffusion set up for each test microorganism

The antimicrobial activity was determined by looking for a clear round

The presence of phytochemicals was determined by a series of different

Table 1. Indication of a positive result in test tube reactions

Table 2. Indication of a positive result in TLC

Results obtained were tabulated and positive result was indicated

In the antibacterial screening, the size of the zone of inhibition

Table 3. Indication of a positive result in TLC

The ointment was prepared by obtaining 5 g of the crude extract of

The prepared ointment was applied on the dog’s skin lesion by

4. Results and discussion

4.1. Identification and screening

Flavonoids was existent only in the rotten pseudostem extract of

Presence of tannins was detected in both fresh and rotten methanolic

ferrycyanide solution, appearance of blue spots was observed, which indicates

Terpenoids were present in methanolic extract of both fresh and rotten

The diameter of inhibition of the rotten methanolic extract in the

Though there was an inhibition zone observed, it was only categorized

bacteria, E.coli and K. pneumoniae, which was resistant to clindamycin. There

According to Onyema et al. (2016) the antibacterial activities of

The inactive and partially active inhibition exhibited by the pseudostem

4.2. Evaluation of wound healing activity

Shorter time of treatment with RO was maybe due to the presence of

5. Conclusions and Recommendations

Alkaloids, saponins, tannins and terpenoids were present in both fresh

The researchers recommended that the rotten extract of Musa (BBB

Ahmed, R. 2015. Phylogenetic spectrum and analysis of antibacterial activities of leaf

About the Authors

Beulah T. Grevialde finished her Bachelor of Science in

Anne Julian S. Ladiana finished her degree of Bachelor

Corresponding author: beulahg05@gmail.com

Volume 10 | August 2018 | ISSN: 1656-1953 (Print); 2467-6209 (Online)

College of Arts and Sciences

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