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International Journal of Pharmacognosy and Phytochemical Research 2017; 9(10); 1344-1352
ISSN: 0975-4873
Research Article
*Author for Correspondence:erwin_martinez@msu.edu.my
In Vitro Antibacterial Activity of Spanish Moss (Tillandsia usneoides)
Crude Extract Against Skin Infection in Wound Healing
Faller E M1*, Kanes S N2, Zajmi A2, Ramli M D2
1Pharmaceutical Chemistry Unit, School of Pharmacy, Management & Science University, Shah Alam, Selangor,
Malaysia
2Department of Diagnostic and Allied Health Sciences, Faculty of Health and Life Sciences, Management & Science
University, Shah Alam, Selangor, Malaysia
Available Online:25th October, 2017
ABSTRACT
Wound healing is a dynamic phenomenon that results in the restoration of anatomic continuity and function on which can
be delayed by pathogenic bacteria. This research was designed to explore the antimicrobial efficacy of Tillandsia
usneoides against skin infections in wound healing. Physical and chemical evaluations were done through phytochemical
screening and thin layer chromatography. The effect of methanolic, ethanolic and aqueous extracts of Tillandsia
usneoides against pathogenic bacteria were evaluated on antimicrobial activities using disc diffusion and broth dilution
susceptibility assay. The excision bioassay analysis was used in examining the wound healing process in mice. The result
of the qualitative phytochemical screening showed the presence of flavonoids and alkaloids. Thin layer chromatography
revealed a high Rf value for flavonoids (0.75mm) and alkaloids (0.60mm). The antibacterial assay showed a high zone of
inhibition (ZI) for both methanolic extract (>23mm, >22mm and >20mm) and ethanolic extract (>22mm, >22mm and
>17mm) for P.aeruginosa, S.aureus and S.epidermis respectively. Resistance against aqueous extract was observed based
on the lowest zone of inhibition (<4mm). Gentamicin was used as the positive control (>28mm) and DMSO as the
negative control. The minimum inhibitory concentration and minimum bactericidal concentration results confirmed that
methanolic and ethanolic extracts restrained the growth of tested bacteria in the range of 125 to 500mg/mL and showed
bactericidal efficacy. Wound healing assay indicated that methanolic extract had a higher potency of wound closure (12
days; <1mm) compared to ethanolic and aqueous extracts (13days; >1.5mm and 13days: >1.8mm respectively).
Povidone-Iodine was used as the gold standard (15days; <2mm) in the study. In conclusion, methanolic extract of T.
usneoides has a great potential with regard to its antimicrobial and wound healing activity to be developed as a novel
drug in the future.
Keywords: Antimicrobial, Spanish moss, Tillandsia usneoides, Wound Healing, Skin Infection
INTRODUCTION
Human skin is the largest organ colonized by a diverse
milieu of microorganisms in which most of the
microorganisms are harmless or even beneficial to their
host1. The biology of the skin surface is highly variable
depending on topographical location as well as several
endogenous host factors and exogenous environmental
factors. Hence, human skin is also the natural host for
many bacterial species that colonize the skin as normal
flora. Staphylococcus aureus and Staphylococcus
epidermis are intermittent resident flora, yet they are
accountable for a wide variety of bacterial pyodermas2.
Wounds are a significant cause of morbidity worldwide.
Previous studies have shown that for every one million
patients with wound infection, at least 10,000 people die
from microbial infections3. In developing countries,
approximately 10% of the population will experience a
wound during their lifetime3. According to the latest
WHO data published in May 2014, skin disease in
Malaysia makes up 0.49% of total skin diseases globally4.
According to the Wound Healing Society, wounds are
physical injuries which result in an opening or breaking
of the skin which may cause disturbance to the normal
skin as well as its functions5. Based on current estimates,
almost 6 million people worldwide are suffering from
chronic wounds. An unhealed wound produces
inflammatory mediator that may cause pain and swelling
at the wound site.
Antibiotic-resistant bacteria are typical bacteria that are
not controlled or killed by antibiotics6. This type of
bacteria can survive and even multiply in the presence of
an antibiotic7. Most infections are prone to resistance to
some antibacterial agents. Furthermore, it may result in
severe diseases that may stem from a single wound. One
example of such bacteria is Staphylococcus aureus which
is currently almost resistant to the penicillin antibiotic8.
This can inhibit the wound healing process.
Spanish Moss
Spanish moss (Tillandsia usneoides) is a tiny epiphyte
flowering plant that grows on larger trees, native to West
Faller et al. / In Vitro Antibacterial Activity…
IJPPR, Volume 9, Issue 10: October 2017 Page 2
India. The name of the plant species is usneoides and it
belongs to the Bromeliaceae family9 as shown in Figure
1. The Spanish moss absorbs nutrients, namely calcium
and water, from the air and rainfall. Moreover, the plant
gains its nutrients directly from the air instead of from the
soil, a host plant or trees10. This plant is colloquially
known as the ‘air plant’11. This plant hangs from trees in
long, thick masses that may reach 20 inches in length. It
is greyish-green in colour, narrowly linear, and measures
up to two inches long. One of the functions of T.
usneoides is for blood regulation12. In 2000, researchers
at Northeast Louisiana University discovered the benefits
of T.usneoides in controlling blood glucose level and for
treating diabetes13.
The Native Americans used Spanish moss as medication
for a range of purposes. In addition, it is also used in
contemporary herbal medicine among the Latin
Americans14. The plant has also been used to treat
hemorrhoids, abscesses and tumors as well as taken orally
for heart, liver and lung ailments. Research has shown
that T. usneoides exerts anti-viral, anti-inflammatory and
analgesic activities15. Besides that, this plant makes up for
a good herbal remedy. Furthermore, T. usneoides also
contains a bio-active compound that may help control
blood glucose levels. The compound is known as 3-
hydroxy-methylglutaric acid or HMG, commonly used
among diabetics as supplements16. Research has found
that this plant can boost the strength of small blood
vessels or capillaries in the skin as well as to protect the
skin from damage T. usneoides can treat the effects of
aging on the skin and the extract can inhibit the
breakdown of skin cells17.
Bacteria
S. aureus and S.epidermidis are gram-positive
microorganisms commonly found in the skin which
frequently cause surgical implant-related infections18. In
addition, Staphylococcal wound toxicities can cause
severe local and systemic complications including
bacteraemia, metastatic infection and hypotension or
organ failure19. Chronic wounds and burns are infected by
Staphylococci as well as the gram-negative opportunistic
pathogen P. aeruginosa in which the wound infection is
related to ulcer enlargement or a delay in healing20.
Wound Healing
The wound is a breakdown of the protective function in
the skin which loses its continuity of epithelium or
without loss of underlying connective tissues, for
example the muscle, bone, and nerves21. A wound may
heal by primary intention in order for the wound to be
closed with sutures, or secondary intention where the
injured tissues are restored by the development of
connective tissue and re-growth of epithelium cells where
the healing is a natural reparative process and are
typically allowed to granulate.
Skin repair and regeneration
Skin repair is an instant physiological response of the
injured tissue to restore normal functionality without any
replacement of damaged tissue22. Dermal substitute is a
wound healing which is followed by a regenerative
pathway by reducing the wound contraction.
Furthermore, in superficial layer injuries or burns, the
wound healing process will develop as a regenerative
paradigm23.
Antimicrobial assay
Antimicrobial is an agent which kills microorganisms and
inhibits the growth of bacteria. It is classified according
to the mechanism of action of antibiotics based on its
property24. Microbicidal are known as agents that kill
microbes, whereas those that inhibit the growth of
bacteria are called bacteriostatic. The application of
antimicrobial agents is also known as antimicrobial
chemotherapy25. MIC is commonly used in diagnostic
laboratories, mainly to confirm resistance. It is also used
as a research tool to determine the in vitro activity of a
new antimicrobial and studies have been conducted to
determine the MIC breakpoint26. This method can be
determined by agar dilution or broth microdilution. In a
clinical setting, the MIC is used to determine the amount
of antibiotic that the patient will use27. Meanwhile, MBC
is a determination of the MIC concentration which shows
no growth of bacteria.
Gentamicin is an antibiotic commonly used to treat many
types of bacterial infection. This antibiotic belongs to the
aminoglycoside group which are bactericidal28.
Furthermore, Gentamicin works against a wide range of
bacterial infections, which mostly are gram-negative
bacteria such as Pseudomonas and Proteus, as well as
gram-positive bacteria Staphylococcus.
MATERIALS AND METHODS
Plant collection and preparation
T. usneoides was collected in Banting, Selangor. The
plant was authenticated by Universiti Putra Malaysia with
the voucher number, SK2959/16. The grass was carefully
washed and oven dried for 1 hour at 60oC29. Next, the
grass was placed in a shaded area for further drying29.
Once the grass had dried, it was grounded in the mixer
grinder until it turned into a fine powder form30.
Collection of bacteria
Bacterial culture of S. aureus, P. aeruginosa, and S.
epidermidis were obtained from the microbiology
laboratory, Management and Science University, Shah
Alam. These bacteria were cultured in peptone water and
followed by gram staining to confirm the species of
bacteria31.
Crude extraction
Dried powder of 50g crude extract was mixed with
500mL of 70% methanol, ethanol, and aqueous solvent
for 24 hours at room temperature32. After a 24-hour
immersion, the extract was evaporated in a rotary
evaporator at 40oC33. Once evaporated, the crude extract
turned into semi-solid form.
Phytochemical screening
Detection of alkaloids (Mayer's test)
The crude extract was dissolved individually in diluted
hydrochloric acid and then filtered. For the detection of
alkaloids, Mayer’s Test was employed. In this test, 1mL
of aqueous extract and 1mL of Mayer’s reagent, which
was essentially a potassium mercuric iodine solution,
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IJPPR, Volume 9, Issue 10: October 2017 Page 3
were added. Next, the reaction was observed based on the
presence of alkaloids in a whitish or cream colour34.
Detection of flavonoids (Alkaline Reagent Test)
For the detection of flavonoids, the test used was the
Alkaline Reagent Test. 1mL of aqueous extract was
treated with 1mL of sodium hydroxide solution.
Subsequently, the reaction was observed based on the
presence of flavonoids in a yellow-coloured form, which
would turn colourless upon the addition of a few drops of
acid34.
Detection of saponins (Foam Test)
In this study, Foam Test was employed to detect
saponins. 0.5g of crude extract was taken and diluted in
2mL of distilled water. The mixture was then shaken
vigorously for 10 minutes, and the reaction was observed
based on the presence of saponins through the formation
of foam34.
Detection of tannins (Gelatin Test)
The Gelatin Test was administered to detect the presence
of tannins. For this purpose, 2-3mL of aqueous extract
was used and 1mL of gelatin containing NaCl was added.
The reaction was observed based on the presence of a
white-coloured formation, indicating the presence of
tannins34.
Detection of carbohydrates (Molisch's Test)
To detect the presence of carbohydrates, Molisch’s Test
was employed. The extract was treated with two drops of
alcoholic alpha-naphthol solution in a test tube. Then, the
reaction was observed based on the formation of a violet
ring at the junction, which indicated the presence of
Carbohydrates34.
Detection of phenols (Ferric Chloride Test)
The test used to detect the presence of phenols was the
Ferric Chloride Test. In this test, the extract was treated
with three drops of ferric chloride solution. The reaction
was observed based on the formation of a bluish black
colour, indicating the presence of phenols34.
Detection of proteins and amino acids (Xanthoproteic
Test)
For the detection of proteins and amino acids, the
Xanthoproteic Test was used. The extract was treated
with three drops of Nitric acid concentration and the
presence of proteins was observed based on the resulting
yellow-coloured formation34.
Detection of steroid (Salkowski Test)
For the detection of steroid, the Salkowski test was
performed. In this test, 2mL of extract were taken and
2mL of chloroform and 2mL of H2S04 concentration were
then added to the extract. Then, the test tube was shaken
well to observe the chloroform layers which appeared in a
red coloured-form and the acid layer which appeared in
the form of a greenish yellow fluorescence34.
Thin Layer Chromatography (TLC)
Thin layer chromatography is a technique which is used
to separate non-volatile mixtures. It is performed on a
sheet of glass coated with a thin layer of adsorbent
material35. Silica gel is the standard material used in thin
layer chromatography as it can be used to identify
compounds which are present in a certain mixture. In this
study, 3g of dried powder was measured, and 10mL of
hexane was added to the powder. The powder was then
heated in a water bath for 5 minutes and then filtrated.
The amount of liquid was divided into three tubes
labelled A, B and C. For tube A, the solution was mixed
with 9mL of chloroform and 1mL of acetic acid. Then,
the mixture was heated in a water bath for 10 minutes.
For tube B, 4.9mL of chloroform, 4.9mL methanol, and
0.2mL acetic acid were added into the tube. This was
followed by heating the tube in water bath at 37oC for 10
minutes. Lastly, for tube C, the solution was mixed with
5mL of methanol and 5mL of distilled water and then
heated for 10 minutes36.
Developing a plate
TLC plates were developed in a beaker covered with an
aluminium foil. Next, the solvent was poured into three
different beakers labelled Solution A, Solution B and
Solution C. The lower edge of the plate was dipped into
the solvent. The solvent travelled up the matrix by
capillary, moving the components of the samples at
various rates due to the different degrees of interaction
with the matrix and solubility in the developing solvent37.
Disk diffusion assay method
Firstly, a few Muller-Hinton (MH) agar plates and sterile
cotton swabs were prepared38. Next, wells were made on
the different MH agar plates containing S. aureus, P.
aeruginosa and S. epidermis bacteria39. Gentamicin
antibiotic was applied as the positive control and T.
usneoides crude extract from ethanol, methanol and
aqueous were added to each agar plate containing the
bacteria. Lastly, all agar plates were incubated at 37oC for
24 hour38.
Minimum inhibitory concentration (mic)
To determine the minimum inhibitory concentration
(MIC), 21 tubes were used; 7 tubes were used for T.
usneoides crude extract, and a series of different
concentrations of crude extract with bacteria S. aureus, P.
aeruginosa and S. epidermis (500, 250, 125, 62.5, 31.25,
15.62, 7.81 mg/mL) were prepared40. Next, each bacterial
inoculum was adjusted to 0.5 McFarland standard. 0.5mL
of bacterial inoculum and plant crude extract were
dropped on each nutrient broth41. A positive control was
prepared, which contained 3mL of nutrient broth and
0.5mL of inoculum while 3mL of nutrient broth was used
as the negative control. Both tubes were incubated at
37oC for 24 hours and the turbidity of the tubes at each
concentration was observed40.
Minimum bactericidal concentration (MBC)
The minimum bactericidal concentration of the crude
extract on the bacterial culture was determined by
measuring 1mL of the mixture onto the Muller-Hinton
(MH) agar in order to obtain the value of MIC and
inhibition of the bacterial culture. This was followed by a
24-hour incubation period at 37oC42. The least
concentration value of crude extract which resulted in no
visible bacterial growth was taken as the minimum
bactericidal concentration.
Wound healing measurement
Animal ethics were applied in the university ethics
committee (AE/2016(1)/062). In this study, excision
method was used on 45 mice to examine the wound
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IJPPR, Volume 9, Issue 10: October 2017 Page 4
healing process. First, the mice were shaved to remove
their hair. After shaving, they were anesthetized with
1mL of intravenous ketamine hydrochloride (10mg/kg
body weight) prior to the excision of wound43. A 6mm
wound was created at the dorsal of the mice using a
surgical blade, forceps, and scissors44. Next, S. aureus, S.
epidermis and P. aeruginosa bacteria were applied on the
wound of each subject. Following bacterial application,
the crude extract of T. usneoides from 3 different types of
solvent (methanolic, ethanolic and aqueous), distilled
water (negative control) and an antibiotic (positive
control) was applied onto 9 subjects each. Each subject
was then observed for a period of 12 days44 and the
wound was measured in mm.
Statistical analysis
Figure 1: Spanish moss(Tillandsia usneoides).
0
5
10
15
20
25
Methanol Ethanol Aqueous
Zone of Inhibition
Type of Solvent
S.aureus
S.epidermis
P.aeruginosa
Figure 2: Zone of inhibition of bacteria using crude extracts of T.usneoides.
Table 1: Phytochemical screening of T. usneoides.
Solvents
No.
Metabolites
Name of Test
Methanol
Ethanol
Aqueous
1
Alkaloids
Mayers's Test
+
+
+
2
Flavonoids
Alkaline Reagent Test
+
+
+
3
Tannins
Gelatin Test
+
+
-
4
Saponins
Foam Test
-
+
-
5
Phenols
Ferric Chloride Test
+
+
-
6
Steroids
Salkowski Reagent
-
+
+
7
Protein and Amino acids
Xanthoproteic Test
+
-
-
8
Carbohydrates
Molisch's Test
-
-
-
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IJPPR, Volume 9, Issue 10: October 2017 Page 5
Figure 3: Wound healing measurement on methanolic extract.
Figure 4: Wound healing measurement on ethanolic extract.
Figure 5: Wound healing measurement on aqueous extract.
12 13 13
15 15
0
2
4
6
8
10
12
14
16
Methanol Ethanol Aqueous Standard Control
DAYS
GROUPS
Time Taken to Heal the Wound
Figure 6: Graph of time taken to heal the wound.
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IJPPR, Volume 9, Issue 10: October 2017 Page 6
Data were collected and analysed using SPSS (Statistical
Package for Social Science) version 22.0. The Parametric
test of ANOVA (One Way Analysis of Variance) was
employed. Differences with a p value of <0.05 were
considered to be statistically significant.
RESULTS
Phytochemical screening
In the present study, primary and secondary metabolites
in T. usneoides plant were qualitatively and quantitatively
analyzed. T. usneoides plant extracts such as methanol,
ethanol and aqueous were studied. Out of these three
extracts, ethanol showed the maximum number of plant
constituents such as alkaloids, flavonoids, tannins,
saponins, phenols and steroids. These results are
exhibited in Table 1.
The test results were determined by the colour change as
well as the presence or absence of precipitate or any ring
formation at the top, bottom or between two layers in the
test tube. The results were then compared to the standard
reference to determine the presence or absence of
phytochemical constituents.
Thin layer chromatography
Results revealed that a high Rf value for flavonoid
(0.75mm) and alkaloid (0.60mm) was obtained. A
previous study on T. usneoides concluded that flavonoid
content was the highest, and alkaloid content was the
second highest45.
Disk diffusion
The disk diffusion was done using the Modified Kirby-
Bauer Method. MH agar was done in triplicate sets.
500mg/mL and 1000mg/mL of T. usneoides
concentration were used, and the result showed that the
500mg/mL concentration of T. usneoides showed the
largest zone of inhibition. Furthermore, methanolic
extract showed the largest zone of inhibition for S.
aureus, which was 22mm, whereas the zone of inhibition
for S. epidermis and P. aeruginosa was 20mm and 23mm,
respectively. Similarly, ethanolic extract also showed the
largest zone of inhibition for S. aureus with the
measurement of 22mm, whereas for S. epidermis and P.
aeruginosa, the zone of inhibition was 17mm and 22mm,
respectively. On the other hand, the aqueous extract
showed the lowest zone of inhibition for S. aureus which
was 4mm, and the zone of inhibition for S. epidermis and
P. aeruginosa was 4 mm and 3mm, respectively.
Minimum inhibition concentration and minimum
bacterial concentration
MIC and MBC results confirmed that both methanolic
and ethanolic extracts restrained the growth of tested
bacteria at a concentration range of 125 to 500mg/mL and
also demonstrated bactericidal efficacy.
Wound healing measurement
Wound healing assay indicated that methanolic extract
has a higher potency of wound closure (12 days; <1mm)
compared to ethanolic and aqueous extracts (13days;
>1.5mm and 13days: >1.8mm, respectively). Povidone-
Iodine was used as the gold standard (15days; <2mm) in
the study.
The graph above shows the time taken for wound healing.
Based on the results, methanolic extract showed a faster
recovery time compared to the gold standard, which is the
Povidone-Iodine.
DISCUSSION
Antimicrobial activity in the alcoholic extracts of T.
usneoides can be primarily due to the presence of
phytochemicals such as phenols, flavonoids, tannins,
steroids and so on. These bio-active components exhibit
their actions through various mechanisms. Tannins inhibit
cell wall synthesis by forming irreversible complexes
with prolene-rich proteins46 while saponins causes
leakage of proteins and certain enzymes from the cell47.
Coincidentally, flavonoids are excellent antimicrobial
substances as they are capable of complexing with
extracellular and soluble proteins as well as bacterial cell
walls48. Steroids, on the other hand, act on membrane
lipids and leakage from bacterial liposomes become
apparent49.
In this research, T. usneoides showed antimicrobial
activity at the concentration of 500mg/mL. Previous
studies of Silva et.al (2013) on T. usneoides did not show
any antimicrobial activity at the concentration of
50mg/mL and 100mg/mL. When the concentration was
increased to 500mg/mL, the antimicrobial effect which
was represented by the diameter of the zone of inhibition
gradually increased. Hence, the earlier negative results do
not necessarily justify the absence of bio-active
compounds in the plants used in this study nor do they
indicate their inactivity.
Methanolic and ethanolic extracts have a potential in
terms of their antimicrobial activity against common skin
bacteria compared to aqueous, which showed the smallest
inhibition zone. Overall, these findings indicate that the
prominent antimicrobial activity in T. usneoides is due to
the presence of phenols in methanolic and ethanolic
extracts. The presence of phenols indicates stronger
antimicrobial properties.
The MIC for methanolic and ethanolic extracts against S.
aureus, S. epidermis, and P. aeruginosa were the same at
a concentration of 125 to 500mg/mL. Turbidity was
observed at the lowest concentration, which indicated the
presence of bacterial growth. However, at a concentration
of 125 to 500mg/mL, the extracts were not only able to
inhibit the growth of the tested bacteria but it could also
kill the bacteria, thus justifying the potency of their
inhibitory effects.
In general, the MIC for aqueous extract was fairly low
against the tested bacteria (S.aureus, S.epidermis and P.
aeruginosa) as the aqueous extract is a water-based
solvent, which makes it difficult to kill or exert effects
against these bacteria. Therefore, it can be concluded that
the aqueous extract is not bactericidal against the tested
bacteria S.aureus, S.epidermis and P. aeruginosa.
The investigation using the excision wound was
conducted by calculating the contraction in the wound
area over a period of 15 days as well as the calculation of
the percentage of wound contraction on a weekly basis
for a period of 15 days. The observed results revealed that
Faller et al. / In Vitro Antibacterial Activity…
IJPPR, Volume 9, Issue 10: October 2017 Page 7
the methanolic and ethanolic extracts of T. usneoides had
a significant wound healing effect on the excision wound
with an area measuring 1mm compared to the control
area, which was 3.5mm. The time taken for the wound to
heal upon application of each extract was 12 days; this
was faster compared to the control group which took
about 15 days to recover. The aqueous extract of T.
usneoides also exerted wound healing effect with a total
area of 2mm. It took 13 days for the wound to heal upon
application of aqueous extract, compared to the control
group which took about 15 days to recover.
CONCLUSION
Spanish moss shows a significant antimicrobial activity
against S. aureus, S. epidermis, and P. aeruginosa.
Additionally, the findings of this study also revealed that
the methanolic extract of T. usneoides exerts a significant
wound healing effect. Conclusively, it can be inferred that
the Spanish moss has the capability of being developed
into a novel drug delivery system.
ACKNOWLEDGEMENT
The author would like to thank Management and Science
University (MSU) for supporting the research by
providing financial assistance through the MSU Seed
Grant (SG-358-0915-SPH).
CONFLICT OF INTEREST
No conflict of interest was declared by the authors.
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