Evaluation of
Cytotoxic, Anti-Inflammatory and Antibacterial Activities of Ethanol Extract of Rhinachantus nasutus Leaves
Candra
Irawan1,2, Berna Elya1*, Muhammad
Hanafi3 and Fadlina Chany Saputri1
1Faculty of Pharmacy, Universitas Indonesia, Depok, Indonesia
2Department of Food Nanotechnology,
Politeknik AKA Bogor, Bogor, Indonesia
3Research Center for Chemistry, Research Center for Chemistry, National Research
and Innovation Agency, Serpong, Indonesia
*For correspondence: berna.elya@farmasi.ui.ac.id
Received 05 April
2023; Accepted 12 April 2023; Published 28 May 2023
Abstract
Ultrasonication produced an
ethanolic extract of Rhinachantus nasutus (L.) Kurz (RX) leaves with
high total phenolic content, strong antioxidant activity and was active in
inhibiting alpha-glucosidase activity. However, its cytotoxic,
anti-inflammatory, and antibacterial properties are still unknown.
The objectives of this research were to evaluate the ethanol extract of RX leaves for its cytotoxicity using the Brine Shrimp
Lethality Test (BSLT) with Artemia salina larvae, to evaluate its
anti-inflammatory activity using the protein denaturation inhibition method,
and to evaluate its antibacterial activity against Escherichia coli and Bacillus
subtilis. Based on the results of this
study, the ethanolic extract of RX leaves was found to have a very strong
cytotoxic effect, with an LC50 value of 10.93 ppm. In addition, this
leaf extract's anti-inflammatory action is highly effective, with an IC50 value
of 7.55 ± 0.2
mg/L. In contrast, E. coli and B. subtilis bacteria do not appear
to be significantly affected by the leaf extract. These findings suggest that
the leaves of the RX have the potential to function as agents that both reduce
inflammation and prevent cancer. © 2023 Friends Science
Publishers
Keywords: Antibacterial; Anti-inflammatory; Cytotoxicity; Rhinachantus nasutus
Introduction
A plant known as Rhinachantus nasutus (L.) Kurz (RK) is a type of
shrub that is frequently discovered growing in the shade by the side of
roadways. This plant can be found growing wild throughout much of Southeast
Asia, India and China (Farnsworth and Bunyapraphatsara 1992; Maarisit et al. 2017). The community makes frequent use of RK's leaves and roots as a
treatment for a variety of conditions, including diabetes, eczema, pulmonary
tuberculosis, herpes, hypertension, leprosy and scabies, as indicated by
empirical data (Siripong et
al. 2006). According
to findings from earlier studies, a leaf extract that was obtained using
ultrasonic waves possesses a significant level of antioxidant activity (Irawan
et al. 2022a). Antioxidants found in plants are an essential component
in the fight against degenerative conditions brought on by oxidative stress
(Akinmoladun et al. 2010; Özen et al. 2010). Such as cancer and
inflammatory diseases (Joon and Takayuki 2009; Badarinath
et al. 2010).
Inflammation is a complex
biological response that is triggered when vascular tissue is exposed to
noxious stimuli such as pathogens, damaged body cells, or irritants (Egesie et al. 2011). Chronic
sickness, which is characterized by discomfort, organ and tissue dysfunction,
edema, and redness, is also associated with inflammation as a marker of the
condition (Manson et al. 2009; Ehlers
and Kaufmann 2010). There are many different treatments available to alleviate
the discomfort brought on by inflammation, but traditional medicine has not
proven to be effective enough or safe enough to be considered an acceptable
option. Because of this, there is a pressing need for a powerful and efficient
anti-inflammatory medication, in particular for the treatment of chronic
disorders (Cruz et al. 2016).
Cancer is a major killer in
developed countries and is one of the leading causes of mortality worldwide.
Cytotoxicity test carried out utilizing the Brine Shrimp Lethality Test (BSLT)
protocol with Artemia salina as the test organism. The use of leach larvae
as a preliminary test for determining anti-tumor and anti-cancer efficacy is
common practice (Janakiraman and Johnson 2016). A
connection between bacteria and particular cancers has been demonstrated by a
number of research (Cummins and Tangney 2013). Antibiotics are the medications that are prescribed for
the treatment of bacterial illnesses. The increased use of antibiotics as a
treatment for disease, the administration of doses that are lower than the prescribed
amount of medication and the consequences of discontinuing
antibiotic treatment before bacteria have been completely eradicated
by the medication all contribute to the development of antibiotic resistance in
bacteria (Dwidjoeseputro 2003; Mardiastuti
2007). Because of this, there is a
pressing need for alternative therapeutic compounds that can not only combat
the issue of bacterial resistance but also have properties that make them
effective against inflammation or cancer.
The BSLT approach was utilized in
the present work to carry out an analysis of the cytotoxic potential of an
ethanolic extract of RK leaves. In addition to this, the possible
anti-inflammatory and antibacterial activities of an ethanolic extract of RK
leaves were studied. Ultrasonic extraction with 70% ethanol was used to get the
RK extract that was used in this study.
Materials and Methods
Preparation and extraction of simplicia
In this study, the RK leaves
samples used were prepared by ultrasonic extraction using 70% ethanol as the solvent,
as reported in previous studies (Irawan et al.
2022a).
Cytotoxicity test
Cytotoxicity test carried out utilizing the Brine
Shrimp Lethality Test (BSLT) protocol with Artemia salina as the test
organism (Janakiraman and Johnson 2016).
The eggs of the brine
shrimp Artemia salina, amounting to 30 mg, are added to the saltwater in
a sealed container. The hatching process is aided by inserting an air hose into
the container's base. After 24 h, A.
salina eggs will hatch and become larvae. Next, 10 larvae of each species
were collected and placed into a container containing a sample solution with a
concentration of 0; 10; 100 and
250 mg/L. After 24 hours, it was clear that the A. salina larvae in
the samples and controls had died. The criteria for determining whether or not A.
salina larvae have passed away is when the larvae do not display movement
for a few seconds while being observed. Once the percentage of A. salina larvae
that died was determined, the probit value was found by searching through the
probit table, and the data was
then linearly regressed.
Anti-inflammatory test
The method of protein denaturation inhibition was used for the testing
of anti-inflammatory properties, and bovine serum albumin (BSA) served as the
substance that was put to the test (Irawan et al. 2022b).
After pipetting samples of an extract solution
containing 1000 mg/L into 80, 120 and 160 µL, each sample was placed in a volumetric flask
containing 5 mL. After that, it was dissolved using a 0.2% BSA solution that
was in tris buffered saline (TBS). After incubating the
solution at room temperature for 30 min, it was heated
in a water bath to 72°C for 5 min. For 25 min, the solution sat at room
temperature. Absorption was determined using a visible spectrophotometer with a
660 nm setting. The experiment was run five times with blank controls and three
times with sodium diclofenac (0.5, 0.75 and 1.0 mg/L) as the positive control.
Antibacterial
test
The antibacterial activity of Escherichia coli
and Bacillus subtilis was tested using the disc diffusion method, with
paper discs having a diameter of about 6 mm. The testing of the antibacterial
agents was carried out twice. After being submerged in samples
with a concentration of 100 mg/L, the paper discs were then placed on a culture
medium (Mueller Hinton Agar) that had already been injected with a pathogen
suspension. Two cycles of 24 h each were conducted at a temperature of 37°C
during the incubation process. On the paper disc, the diameter of the
inhibitory zone was seen and measured (Banjara et al. 2012; Chusri et al. 2012).
Results
Cytotoxicity activity
The BSLT method was used to
assess the cytotoxicity of RX leaves ethanol extract at concentrations of 0;
10; 100 and 500 mg/L. Table 1 displays the number and percentage of A.
salina larvae that perished at each test concentration. According to Table
1, all A. salina larvae perished at a concentration of 10 mg/L. After
obtaining these data, the statistical probit analysis is carried on, which
results in a graph depicting the relationship between the probit value and the
concentration log (Fig. 1). The probit analysis also yielded the line equation y
= 2.7675x + 2.1246 from which the LC50 value of the RK leaves
ethanol extract was calculated to be 10.93 ppm.
Anti-inflammatory activity
Inhibition of protein
denaturation was utilized as an anti-inflammatory test in this investigation.
Percent inhibition was found to be 10.20 ± 0.5, 40.82 ± 1.8 and 62.24 ± 1.4 when 4, 6 and 8 mg/L of ethanol extract of RX
leaves were applied, respectively. When compared, the percent inhibition
provided by diclofenac sodium solution at dosages of 0.5, 0.75 and 1 mg/L was
respectively 30.61 ± 1.4, 40.82 ± 1.8 and 57.14 ± 1.3 (Table 2). The linear association between
sodium diclofenac or leaves extract concentration and percent inhibition is
shown graphically in Fig. 2 (y = 53.061x + 3.0612 and y = 10.969x - 32.823,
respectively). The 50% effective dose (IC50) of sodium diclofenac
was calculated to be 0.88 ± 0.02 mg/L, while that of the leaf extract was
calculated to be 7.55 ± 0.2 mg/L.
Fig. 1: Probit chart for RX leaves ethanol extract
Fig. 2: A graph that shows how the IC50 values for
sodium diclofenac (A) and ethanol leaves extract RX (B) were found
Antibacterial
activity
The results of an antibacterial activity test performed
on an ethanol extract of the RnLK leaves are shown in Table 3. The bacteria
strains E. coli and B. subtilis were used in the Table 1: RX leaves ethanol extract cytotoxicity test results
Concentration (mg/L) |
Log
Concentration |
Total Larva Test |
Mortality |
% Mortality |
Probit Value |
0 |
0 |
10 |
0 |
0% |
0 |
10 |
1 |
10 |
10 |
100% |
8,09 |
100 |
2 |
10 |
10 |
100% |
8,09 |
500 |
2,699 |
10 |
10 |
100% |
8,09 |
Table 2: Observations
from anti-inflammatory drug trials
Sample |
Concentration (mg/L) |
% Inhibition |
IC50 (mg/L) |
Sodium
Diclofenac |
0.50 |
30.61 ± 1.4 |
0.88 ± 0.02 |
0.75 |
40.82 ± 1.8 |
||
1.00 |
57.14 ± 1.3 |
||
Ethanolic Extract |
4 |
10.20 ± 0.5 |
7.55 ± 0.2 |
6 |
31.63 ± 3.0 |
||
8 |
57.14 ± 2.4 |
Table 3: The findings of the antibacterial test conducted on RnLK
leaves ethanol extract against E. coli
and B. subtilis
Bacteria |
Average (mm) |
The activity of the antimicrobial
inhibition zone |
E. coli |
1.30 |
Weak |
B. subtilis |
4.70 |
Weak |
experiment. By
observing the zone of inhibition around the paper disc, antibacterial activity may be gauged. In order to observe
the zone of inhibition, the agar diffusion technique was performed. The test material extract was tested for its activity
against E. coli and B. subtilis at a concentration of 0.11 mg/L
and the results are shown in Fig. 3. The resulting inhibition zones were 1.30
and 4.70 mm, respectively.
Discussion
In this investigation, the ethanol extract of RX
leaves exhibited a cytotoxicity level of 10.93 ppm. This indicates that the
extract is poisonous, as it caused the death of 50 percent of the test animals
at concentrations below 1000 ppm (Meyer et
al. 1982). It has been found that there is a positive link between the
cytotoxicity of the extract and the antiproliferative power of the extract
against cancer cells. Cytotoxicity is the ability of a substance to kill cancer
cells. Antiproliferative power stops the growth of cancer cells. According to
the BSLT test, the ethanol extract from the leaves of the RX plant, which is
poisonous, has the potential to have an antiproliferative effect on cancer
cells (Elsyana et al. 2016; Bouothmany et al. 2022).
Previous
research has demonstrated that the ethanolic extract of nasustus leaves
contains a significant quantity of total phenol (Irawan et al. 2022a).
The category of chemicals known as polyphenols is the one that is found in the
widest variety of plant species. Polyphenolic substances can be subdivided into
groups of phenolic acids, flavonoids, polyphenol amides and other polyphenols
based on the structure of the aglycoside. Each of these classes of polyphenols
possesses their own unique features (Tsao 2010). Research has been done on phenolic compounds to
investigate the antioxidant, anti-carcinogenic, alpha-glucosidase activity
inhibiting, anti-inflammatory, and free radical scavenging characteristics they
possess (Perez et al. 1989; Yin et
al. 2014; Khan et al. 2019).
As seen
in Williams' research, blocking BSA protein denaturation can serve as a
preliminary screening for anti-inflammatory action before anti-inflammatory
tests are conducted on experimental animals (Williams et al. 2008). BSA is denatured when heated so that the secondary and
tertiary structures change. This suggests that heat damages albumin, leading
the organism to react negatively to itself. Because of this, the body has
trouble controlling its inflammatory responses (Leelaprakash and Mohan 2010; Ingle and Patel 2011). The body's proteins are vulnerable
to denaturation due to the production of free radicals, which results in the
release of inflammatory mediators and subsequently causes inflammation (Chick
and Martin 1990).
The fact
that RnK leaf extract has a high anti-inflammatory effect is in line with the
fact that it has a high total phenolic content and a high antioxidant effect
(Irawan et al. 2022a). Natural ingredients with antioxidant properties have phenolic chemicals in them. Denatured albumin
acts as an antigen in immune reactions
like type III hypersensitivity, serum sickness and glomerulonephritis, which is
an inflammatory-based autoimmune
disease. Then, an agent that stops albumin from breaking down or stabilizes it
by more than 20% can be thought to have anti-inflammatory properties and may be
put through more anti-inflammatory testing (Williams et al. 2008; Tatti et al. 2012; Bailey-Shaw et al. 2017).
Fig. 3: The diameter of the inhibitory zone created by the ethanolic extract of RK
leaves against E. coli (X)
and B. subtilis (Y)
The findings of the antibacterial tests
indicate that its action is fairly weak against E.
coli and B. subtilis germs. The level of sensitivity
of these bacteria can influence the size of the gap between the inhibition
zones they produce (Pan et al. 2009). B. Subtilis displays more
potent antibacterial action than E. coli. This is because its
fundamental components all have slightly different cell layouts. Gram-positive
bacteria, such as B. subtilis, are distinguished from gram-negative
bacteria, such as E. coli, by their surface morphology. Gram-positive
bacteria have a structure that is made up of peptidoglycan for the most part
(90 percent) and they also contain a thin layer of negatively charged teichoic
and teicuronic acids on their surface. It has been discovered that the
outermost layer of the cell wall in gram-negative bacteria contains between 5
and 20% peptidoglycan as part of its makeup. The centre of the structure
contains this layer, which is the second lipid layer and is also known as the
lipopolysaccharide layer. This layer is made up of phospholipids,
polysaccharides, and proteins, which are the three components that make it up
(Madigan et al. 2000).
Conclusion
In this investigation, the cytotoxic activity of the ethanolic extract
of RX leaves was shown to be extremely high, with an LC50 value of
10.93 ppm. In addition, the leaf extract possesses extremely significant
anti-inflammatory activity with an IC50 value of 7.55 ± 0.2 mg/L, although its
antibacterial activity against E. coli and B. subtilis is quite poor. According to these findings, the leaves of the R. nasutus have
the potential to act as agents that both reduce inflammation and prevent
cancer.
Acknowledgement
Thanks to the Faculty of Pharmacy, Universitas
Indonesia, and Politeknik AKA Bogor for the laboratory facilities that have
been used during this research.
Author Contributions
CI designed the experiments,
conducted the experiments, compiled the findings, interpreted the results, and
statistically analyzed the results; BE designed the experiments, compiled the
findings, and interpreted the results and MH and FCS compiled the findings,
statistically analyzed and interpreted the results.
Conflict of Interest
The authors have stated that they have no competing
interests related to this study.
Data Availability
On reasonable request, the corresponding author will
provide access to the data given in this work.
Ethics Approval
Outside the scope of this paper.
Funding
Source
The research presented here
was funded by a Doctoral Dissertation Research Grant 2022 from the Indonesian
Ministry of Research and Technology and the Indonesian National Research and
Innovation Agency (Nomor: 021/E5/PG.02.00.PT/2022; 142./PKS/WRIII-DRP/UI/2022;
NKB-782/UN2.RST/HKP.05.00/2022.
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