Antibacterial activities from aqueous and ethanolic extracts of selected succulent plants

Abstract

Succulents are admired for their beauty and have been popular among plant enthusiasts during the pandemic. However, succulents can also be tapped as potential sources of antibacterial compounds. Thus, this study aimed to determine the phytochemical compounds and antibacterial activity of selected succulents, Aloe maculata, Agave potatorum, and Graptopetalum mendozae against two Gram-negative bacteria, Aeromonas hydrophila and Vibrio harveyi and a Gram-positive bacterium, Bacillus albus. Aqueous and ethanolic extracts of these succulents were prepared and screened for the presence of some phytochemical substances. Antibacterial activity was assayed using Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). The MIC was carried out based on turbidity using the microdilution method and the MBC was determined based on the ability of the substance to kill 99.9% of the target pathogen by streaking onto agar plates. The phytochemical test showed that all three succulents contained carbohydrates, phytosterols, flavonoids, phenols and tannins. A. maculata and A. potatorum showed the presence of glycosides while only G. mendozae exhibited the presence of alkaloids. MIC and MBC results for A. hydrophila showed that only the ethanolic extract of A. maculata can inhibit growth at 0.5g mL-1. Both MIC and MBC results for V. harveyi showed that all extracts are effective in inhibiting the bacterium at 0.25 g mL-1 except ethanolic extracts of G. mendozae at 0.125g mL-1 and A. potatorum at 0.5g mL-1. All extracts did not show any inhibition against B. albus. In conclusion, succulents possessed antibacterial properties against Gram-negative bacteria such as A. hydrophila and V. harveyi that could lead to the discovery of novel antibacterial medicines.

Full text

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Ordanel et al.
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2023
RESEARCH PAPER
RESEARCH PAPER RESEARCH PAPER
RESEARCH PAPER
OPEN ACCESS
OPEN ACCESSOPEN ACCESS
OPEN ACCESS
Antibacterial activities from aqueous and ethanolic extracts of
selected succulent plants
Angelo M. Ordanel
1
, Sherra Jade M. Gan
goso
1
, Jedyza L. Andallon
1
,
Rhea Mae L. Pacifico
1
, Julie Ann T. Sarabia
1
, Edda Brenda S. Yerro
1
,
Mary Lou C. Arabaca
1
, Emily M. Cataluña
1
, Christopher Marlowe A. Caipang
*1,2
1
Department of Biology, College of Liberal Arts, Sciences, and Education,
University of San Agustin, Iloilo City, Philippines
2
Division of Biological Sciences, College of Arts and Sciences, University of the Philippines Visayas,
Miag-ao, Iloilo, Philippines
Key words:
Antimicrobial, Drug discovery, Natural products, Ornament
al plants, Plant extracts
http://dx.doi.org/10.12692/ijb/22.2.111
-
119
Article published on
February
0
7
,
20
2
3
Abstract
Succulents are admired for their beauty and have been popular among
plant enthusiasts during the pandemic.
However, succulents can also be tapped as potential sources of antibacterial compounds. Thus, this study aimed
to determine the phytochemical compounds and antibacterial activity of selected succulents, Aloe maculata,
Agave potatorum, and Graptopetalum mendozae against two Gram-negative bacteria, Aeromonas hydrophila
and Vibrio harveyi and a Gram-positive bacterium, Bacillus albus. Aqueous and ethanolic extracts of these
succulents were prepared and screened for the presence of some phytochemical substances. Antibacterial activity
was assayed using Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
The MIC was carried out based on turbidity using the microdilution method and the MBC was determined based
on the ability of the substance to kill 99.9% of the target pathogen by streaking onto agar plates. The
phytochemical test showed that all three succulents contained carbohydrates, phytosterols, flavonoids, phenols
and tannins. A. maculata and A. potatorum showed the presence of glycosides while only G. mendozae exhibited
the presence of alkaloids. MIC and MBC results for A. hydrophila showed that only the ethanolic extract of A.
maculata can inhibit growth at 0.5g mL
-1
. Both MIC and MBC results for V. harveyi showed that all extracts are
effective in inhibiting the bacterium at 0.25 g mL
-1
except ethanolic extracts of G. mendozae at 0.125g mL
-1
and
A. potatorum at 0.5g mL
-1
. All extracts did not show any inhibition against B. albus. In conclusion, succulents
possessed antibacterial properties against Gram-negative bacteria such as A. hydrophila and V. harveyi that
could lead to the discovery of novel antibacterial medicines.
*
Corresponding Author: Christopher Marlowe A. Caipang  cmacaipang@yahoo.com
International Journal of Biosciences | IJB |
ISSN: 2220-6655 (Print), 2222-5234 (Online)
http://www.innspub.net
Vol. 22, No. 2, p. 111-119, 2023

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Introduction
One of the concerns of global health is the evolution
of various strains of microbes and our ability to treat
these diseases depends on the discovery of new drugs
through some traditional medicine (Frey and Meyers,
2010). According to Walsh (2003) and Roberts and
Zembower (2021), there are 14 million deaths
worldwide annually caused by these different disease-
causing microbes and there is a phenomenon of
antimicrobial resistance because antibiotics are
overused and misused. These problems can be solved
by the discovery of new antimicrobials through plants
being screened for their antibacterial properties
(Cowan, 1999).
There has been a surge of interest in the search for
antimicrobial drugs derived from natural products
over the past few decades, owing to the belief that
drugs derived from plants are safer and more
dependable than synthetic drugs, which may have
adverse effects on the host in addition to their high
cost (Debalke et al., 2018). Thus, it is an urgent need
to explore and discover new antimicrobials.
For thousands of years, nature has provided medical
substances, and a remarkable number of modern
medications have been identified from natural
sources. Plant-based drugs have long dominated
pharmaceutical medicine for the treatment of a
variety of diseases (Arullappan et al., 2009; Caipang
et al., 2019). The chemical and medicinal contents
found in their natural form and secondary
metabolites are a large reservoir of structural moieties
that collaborate to exhibit a wide range of biological
activities (Mangaiyarkarasi and Muhammad, 2015).
Succulents are known for their elegant beauty and are
admired by most plant enthusiasts as it is poor in
maintenance and flexes as an aesthetic. It became a
trend as a hobby during the pandemic when
lockdowns were implemented, and people tried to
shift their minds to alleviate their anxiety about the
effects of COVID-19 by taking care of plants. The
rising demand for cultivation was a big hit during the
season up until now when the disease was no longer
rampant as before. In the study of Griffiths and Males
(2017), succulent plants are known for their ability to
store water and survive drought for long periods, on
which 90-95% of the cell contents of the plant are
water. Succulents are natively grown or cultivated,
and have fleshy, thick leaves or stems; curiosity is
about the shape and form of the plant and not about
the flowers (Aileen, 2020). One of the succulent’s
most potent medicinal uses is its antimicrobial
properties (Berniyanti and Mahmiyah, 2015).
Additionally, it contains various compounds,
including saponin, aluin, ligin, antraquinones,
vitamins, and minerals. The biological capabilities of
crude extracts of plant components and
phytochemicals are well recognized, and they can be
beneficial in medicinal treatments (Khan et al.,
2013).
The present study established the medical
significance of succulent plants, namely the Agave
potatorum, Aloe maculata and Graptopetalum
mendozae from a collectors’ perspective. It aimed to
evaluate the phytochemical properties and
antibacterial activities of extracts obtained from
selected succulent plants against Gram-positive and
Gram-negative bacteria. The findings will make it
possible to discover some medically important and
pharmacologically active constituents from these
succulent plants.
Materials and methods
Study site
This study was conducted at the Biological Laboratory
of the University of San Agustin, located at Gen. Luna
St., Iloilo City, Philippines.
Collection of samples
The following succulent plants, namely, Aloe
maculata, Agave potatorum, and Graptopetalum
mendozae were utilized for this study. The identity of
the succulents was confirmed by the staff at the
Regional Office, Department of Agriculture, Iloilo
City, Philippines. Leaf samples of succulent plants
were collected, then thoroughly washed with tap
water and distilled water to remove all the impurities

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2023
on the surface. The leaves were sundried for 48 h to
reduce the moisture content of the leaves by <10%.
Phytochemical screening
Standard techniques in the phytochemical analysis
were utilized to test the leaf extracts for the presence
of bioactive compounds: alkaloids, carbohydrates,
glycosides, phenols and tannins, phytosterols, and
flavonoids as described in Khalid et al. (2018), and
Shaikh and Patil (2020).
Preparation of extracts
Aqueous extract
Sun-dried leaves were weighed, cut into small pieces
and added to distilled water at a ratio of 1:5 (w/v).
The samples were then placed inside a blender to
thoroughly grind the leaves. The ground mixture was
transferred into a sterile amber bottle and placed at
4
o
C for 24 hrs. After 24 hours, the extract was filtered
using a sterile filter paper. The filtrate was transferred
into a beaker, placed into a water bath and
evaporated to dryness. The residue of the aqueous
extracts of each plant was weighed, added with
normal saline solution to obtain a stock solution of 2 g
mL
-1
, transferred to a 10 ml centrifuge tube and
placed at -20
o
C for subsequent analyses. A working
solution containing 1 g mL
-1
of each extract was
prepared using a normal saline solution as a diluent
and two-fold serial dilutions were done for the
antibacterial assays.
Ethanol extract
The ethanolic extracts from the leaves were obtained
following the procedures used in the preparation of
the aqueous extracts. However, instead of water, 80%
ethanol was used for this purpose at a ratio of 1:4
(w/v).
Antimicrobial assays
Bacteria and culture media
The antimicrobial properties of plant extracts were
tested against Gram-negative bacteria, Aeromonas
hydrophila (Pakingking et al., 2020) and Vibrio
harveyi (Pakingking et al., 2018) and Gram-positive
bacterium, Bacillus albus, which was provided by the
Biological Laboratory of the University of San
Agustin. All the bacteria were grown in nutrient broth
with an additional 1% NaCl for V. harveyi and
incubated for 24 h. After incubation, the plate count
(CFU mL
-1
) was determined. Ten-fold serial dilutions
were prepared using normal saline solution to obtain
a final concentration of 1 x 10
4
CFU mL
-1
. This
bacterial concentration was used in all antibacterial
assays.
Minimum inhibitory concentration (MIC)
The standard broth dilution method was used to
determine the antibacterial activity from the aqueous
and ethanolic extracts of the selected succulent plants
by evaluating the visible growth of microorganisms in
the microplate. Serial two-fold dilutions of the
aqueous and ethanolic extracts in concentrations
ranging from 0.125 g mL
-1
to 1 g mL
-1
were used to
determine the MIC. Each concentration was done in
triplicate and added with a similar volume of the
bacteria at a concentration of 1 x 10
4
CFU mL
-1
. The
control contained only inoculated nutrient broth with
bacteria and incubated for 24 h at 32°C for A.
hydrophila, 28°C for V. harveyi and 35°C for B.
albus. The visual turbidity of the tubes was observed
before and after incubation to determine the MIC
values.
Minimum bactericidal concentration (MBC)
Minimum Bactericidal Concentration (MBC) is the
measure to check the growth of bacteria for 24 h of
incubation on the lowest concentration of an
antibacterial agent (Parvekar et al., 2020). MBC
values for the different extracts of succulents were
determined using growth inhibition. Using the streak
plate method, a sterile loop was dipped in the various
concentrations (1g mL
-1
, 0.5g mL
-1
, 0.25g mL
-1
, and
0.125g mL
-1
) from the microplate and streaked onto
the agar plates. Nutrient agar was used for testing
bacterial inhibition of A. hydrophila and B. albus,
while the Thiosulfate-Citrate-Bile Salts-Sucrose
(TCBS) agar was used for V. harveyi.
The plates were incubated at 32°C for 18–24 h. The
MBC was determined as the lowest concentration

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which inhibited the growth of the respective
microorganisms, as indicated by the absence of
bacterial growth on the agar plates. All assays were
performed in triplicate. Nutrient broth inoculated
with the bacteria served as a negative control for all
the assays.
Results and discussion
Phytochemical analysis
In the present study, results of the phytochemical
analysis show that Agave potatorum, Aloe maculata
and Graptopetalum mendozae contained
carbohydrates, phytosterols, flavonoids, phenols, and
tannins. A. maculata and A. potatorum showed the
presence of glycosides, while only G. mendozae
exhibited the presence of alkaloids, as shown in Table
1. The results of this study confirm the findings of
earlier works on the phytochemical composition of
these succulents. For example, A. potatorum
contained significant amounts of coumarins,
alkaloids, flavonoids, saponins, tannins, cardiac
glycosides and triterpenoid compounds in a
qualitative assays conducted by Soto-Castro et al.
(2021) and Almazán-Morales et al. (2022). A.
maculata, which belongs in the Aloe family, contains
tannins, saponins, flavonoids, steroids, terpenoids,
and cardiac glycosides anthroquinones (Noor et
al.,2008; Cock, 2015). Genova-Kalou et al. (2022)
observed high levels of phenolic compounds and
anthocyanins in Graptopetalum paraguayense, and
further characterization using high performance
liquid chromatography (HPLC) revealed the presence
of gallic acid, flavone, genistin, daidzin, and quercetin
(Chao et al., 2019). Both G. paraguayense and G.
mendozae belong in the same family, and it would not
be surprising of the latter will contain these
phytochemicals. The ability of the succulents to
biosynthesize phytochemicals could be tapped as
good sources of natural antioxidants (Karabourniotis
and Fasseas, 1996).
Table 1. Qualitative analyses of phytochemical substances in different extracts of selected succulent plants.
Alkaloids
Carbohydrates
Glycosides
Phytosterols
Flavonoids
Phenols and Tannins
G. mendozae
++
++
-
++
++
+
A. maculata
-
++
++
+
+
++
A. potatorum
-
++
++
++
+
++
++: intensely present, +: Present, - : Absent.
Antibacterial assays
After a 24-h incubation under aerobic conditions, the
aqueous extracts of the three succulents had MIC
(Table 2) and MBC (Table 3) of 0.125 g mL
-1
against
A. hydrophila. In comparison, the MIC and MBC of
the ethanolic extracts of the three succulents against
the bacterium were 0.125 g mL
-1
, 0.5 g mL
-1
, and
0.125 g mL
-1
for A. potatorum, A. maculata, and G.
mendozae, respectively. After a 24-h incubation
under aerobic conditions, the aqueous extracts of the
three succulents had MIC (Table 4) and MBC (Table
5) of 0.25 g mL
-1
against V. harveyi. In comparison,
the MIC and MBC of the ethanolic extracts of the
three succulents against the bacterium were 0.5 g mL
-
1
, 0.25 g mL
-1
, and 0.125 g mL
-1
for A. potatorum, A.
maculata, and G. mendozae, respectively.
Table 2. Minimum Inhibitory Concentration (MIC) of aqueous and ethanolic extracts against Aeromonas
hydrophila.
Scientific
name
Type
of extract
0.125g/mL
0.25g/mL
0.5g/mL
1g/mL
Agave
potatoru
m
Aqueous
-
-
-
-
Ethanol
-
-
-
-
Aloe
maculata
Aqueous
-
-
-
-
Ethanol
+
+
-
-
Graptopetalum mendozae
Aqueous
-
-
-
-
Ethanol
-
-
-
-
Control
Control
+
+
+
+
+ turbid; - not turbid.

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2023
After a 24-h incubation, all three plant extracts did
not inhibit B. albus as shown by the presence of
turbidity (Table 6) and bacterial growth (Table 7) in
all the concentrations tested. Succulents are plants
that are known for their ability to store water and
survive drought for extended periods (Griffiths and
Males, 2017). These plants were selected for use in
this study because these could be potential sources of
pharmacological properties due to the presence of
various phytochemical compounds that could inhibit
microorganisms (Choi et al., 2015).
Table 3. Minimum Bactericidal Concentration (MBC) of aqueous and ethanolic extracts against Aeromonas
hydrophila.
Scientific
name
Type
of extract
0.125g/mL
0.25g/mL
0.5g/mL
1g/mL
Agave
potatorum
Aqueous
-
-
-
-
Ethanol
-
-
-
-
Aloe
maculata
Aqueous
-
-
-
-
Etha
nol
+++
+++
-
-
Graptopetalum mendozae
Aqueous
-
-
-
-
Ethanol
-
-
-
-
Control
Control
+++
+++
+++
+++
*(+/++/+++) indicates bacterial growth on replicate plates; (-) no bacterial growth.
Agave potatorum is distinguished by its compact
rosettes, layered with flat sheets, small teeth, and a
spike on each leaf (García-Mendoza, 2010). In the
study of Soto-Castro et al. (2021), fresh leaves of A.
potatorum were used to test its effect on the mycelial
growth of a fungus, Pleurotus spp, as it contains
secondary metabolites that are capable of accelerating
mycelial growth, while in the study of Monterrosas-
Brisson et al. (2013), leaves of Agave tequilana,
Agave angustifolia, and Agave americana were
tested for the identification of anti-inflammatory
compounds due to these species being used as
traditional medicines in Mexico in treating various
diseases.
Table 4. Minimum Inhibitory Concentration (MIC) of aqueous and ethanolic extracts against Vibrio harveyi.
Scientific
name
Type
of extract
0.125g/mL
0.25
g/mL
0.5g/mL
1g/mL
Agave
potatorum
Aqueous
+
-
-
-
Ethanol
+
+
-
-
Aloe
maculata
Aqueous
+
-
-
-
Ethanol
+
-
-
-
Graptopetalum
mendozae
Aqueous
+
-
-
-
Ethanol
-
-
-
-
Control
Control
+
+
+
+
+ turbid; - not turbid.
Aloe species are native to South Africa, where the
warm and arid climate is favorable; it exhibits a spiky
tongue shape, the epidermis of the leaves has a thick
cuticle, and the main bulk is mostly Aloe gel wrapped
by the mesophyll tissue (Choi et al., 2015). G.
mendozae was first considered rare in the wild.
However, due to various local cultivations, the species
are now commonly grown (Cházaro-Basñez and
Acevedo-Rosas, 2009). The same study also
elucidated that aqueous extract of G. paraguayense
shows anti-inflammatory and antioxidant activity
(Chung et al., 2005) and down-regulates the
expression level of some oncoproteins of
hepatocellular carcinoma (Hsu et al., 2015).

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Table 5. Minimum Bactericidal Concentration (MBC) of aqueous and ethanolic extracts against Vibrio harveyi.
Scie
ntific
name
Type
of extract
0.125g/mL
0.25g/mL
0.5g/mL
1g/mL
Agave
potatorum
Aqueous
++
-
-
-
Ethanol
+
+
-
-
Aloe
maculata
Aqueous
++
-
-
-
Ethanol
+
-
-
-
Graptopetalum
mendozae
Aqueous
+++
-
-
-
Ethanol
-
-
-
-
Control
Control
+++
+++
+++
++
+
*(+/++/+++) indicates number of bacterial growth on replicate plates; (-) no bacterial growth.
Table 6. Minimum Inhibitory Concentration (MIC) of aqueous and ethanolic extracts against Bacillus albus.
Scientific
name
Type
of
extract
0.125g/mL
0.25g/mL
0.5g/mL
1g/mL
Agave
potatorum
Aqueous
+
+
+
+
Ethanol
+
+
+
+
Aloe
maculata
Aqueous
+
+
+
+
Ethanol
+
+
+
+
Graptopetalum
mendozae
Aqueous
+
+
+
+
Ethanol
+
+
+
+
Control
Control
+
+
+
+
+ turbid; -not turbid.
In this study, the MIC and MBC of selected succulent
plants (A. potatorum, A. maculata, and G.
mendozae) against Gram-negative (A. hydrophila
and V. harveyi) and Gram-positive (B. albus) bacteria
were determined by microdilution method. It was
noted that different bacteria differ in growth
preferences wherein in A. hydrophila, only 0.125 g
mL
-1
and 0.25 g mL
-1
tubes containing ethanol
extracts of A. maculata have bacterial growth, and for
V. harveyi, all 0.125 g mL
-1
tubes containing ethanol
and aqueous extract have bacterial growth except for
the ethanolic extract of G. mendozae which has no
growth. Also, only ethanolic extract of A. potatorum
has bacterial growth at a concentration of 0.25g mL
-1
.
It implies that the bacteria will grow at low
concentrations of the extract regardless of the
extracting solvent that is used. On the other hand, all
the concentrations tested were ineffective against
Gram-positive bacteria as shown by the bacterial
growth on the agar plates.
Table 7. Minimum Bactericidal Concentration (MBC) of aqueous and ethanolic extracts against Bacillus albus.
Scientific
name
Type
of extract
0.125g/mL
0.25g/mL
0.5g/mL
1g/mL
Agave
potatorum
Aqueous
+++
+++
+++
+++
Ethanol
+++
+++
+++
+++
Aloe
maculata
Aqueous
+++
+++
+++
+++
Ethanol
+++
+++
+++
+++
Graptopetalum
mendozae
Aqueous
+++
+++
+++
+++
Ethanol
+++
+++
+++
+++
Control
Control
+++
+++
+++
+++
*(+/++/+++) indicates number of bacterial growth on replicate plates; (-) no bacterial growth.
In summary, all three succulent plants contained
carbohydrates, phytosterols, flavonoids, phenols, and
tannins. However, A. maculata and A. potatorum
showed the presence of glycosides, while the G.
mendozae exhibited the presence of alkaloids. Both
the aqueous and ethanolic extracts of A. potatorum,
A. maculata and G. mendozae inhibited the growth of
Gram-negative bacteria such as A. hydrophila and V.

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harveyi. In contrast, the Gram-positive bacterium, B.
albus was resistant to the extracts. Hence, this study
confirmed that succulent plants possessed
antibacterial properties against Gram-negative
bacteria. The data could be used as baseline
information in discovering antimicrobial compounds
for future therapeutic applications.
Acknowledgment
The authors greatly acknowledge the support
provided by the Friar administrators of the University
of San Agustin and the technical support extended by
the laboratory staff at the Department of Biology,
College of Liberal Arts, Sciences, and Education of the
same university.
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