Inhibitory action of solanine from Solanum melongena

(eggplant) against Listeria monocytogenes and

Streptococcus pyogenes

Ainimei Juliet A. Castillo1, Jenica O. Albuñan1, Perlie Flor Caguete1, Michaela E.

Holgado1, Liza Louise P. Perez1, Ira Angela L. Tanyag1, and
Dianne Kristine F. Fajarillo2
College of Allied Medical Professions, Lyceum of the Philippines University,
Capitol Site, Batangas City

¹Student Researcher
²Faculty Researcher
*Correspondence: julietcastillo@yahoo.com
________________________________________________________________________

Abstract: Action of solanine from Solanum melongena interferes against Listeria

monocytogenes and Streptococcus pyogenes. Solanine is a bitter-tasting steroidal alkaloid
saponin that has been isolated from all nightshades, including tomatoes, capsicum,
tobacco, and eggplant. It is very toxic even in small quantities. The organisms that will be
used in this study are the common species of gram-positive motile bacilli that cause
listeriosis and a non-invasive food-borne diarrheal disease. Thin layer chromatography with
chloroform will be used in testing solanine against Listeria monocytogenes and
Streptococcus pyogenes. The aim of the study is to evaluate the inhibitory effect of
solanine against Listeria monocytogenes and Streptococcus pyogenes.

Keywords: Listeria monocytogenes, Streptococcus pyogenes, Solanum melongena,

solanine

INTRODUCTION
Human listeriosis is a common
health problem of low incidence but high
mortality and must have prompt diagnosis
and adequate antibiotic therapy. (Rodas-
Suárez et al., 2006). It is a disease caused
by Listeria monocytogenes, a facultative
intracellular pathogen. The disease affects
primarily pregnant women, newborns and
patients with degenerative diseases. In
immunocompromised individuals, it is
clinically shown as meningitis and
septicemia. The disease has a high
mortality rate, between 20 and 30% of
cases, and causes neurological sequelae
in some cases. The widespread
distribution of epidemiologically important
serotypes of L. monocytogenes and their
resistance to frequently used antibiotics
indicate a potential public health risk.
(Reis et al., 2011). Because
ampicillin/penicillin and gentamicin are the
primary antibiotics for listeriosis therapy,
resistance to these antibiotics has been
the focus of many previous studies. (Wu S
et al., 2015).
Similarly, Streptococcus pyogenes
(group A streptococcus [GAS]) is an
etiological agent for diverse human
diseases. GAS is one of the major causes
of acute respiratory tract infections such
as acute sinusitis, acute otitis media,
pharyngitis and acute bronchitis. GAS is
wide spread and represent a major health
problem particularly in low-resource
settings. In developing countries, they
contribute significantly to morbidity and
mortality in the pediatric setting, with an
estimated lethality rate of 15% in young
children. In addition, the pathogen is
associated with potentially life-threatening
sequelae such as poststreptococcal
glomerulonephritis and acute rheumatic
fever (Dieng & Boye, 2013).
Solanum melongena from the
family of Solanaceae is commonly known
as eggplant (English) or aubergine
(Europe). It is a coarse and half-woody
plant with oval leaves that are irregularly-
lobed. Its flowers are purplish, and its
fruits have varied sizes and shapes. The
plants are cultivated in countries with
temperate climates including the
Philippines. The roots, leaves, and fruits of
this plant contains an array of nutritional
values due to its vitamins, minerals,
proteins and lipid contents. Proteins and
lipids are present at very low levels.
(Rodriguez et al., 1999). Eggplants are
also a good source of fat-soluble vitamin K
and it is important in blood clotting.
Phytochemical studies showed that it
contains alkaloids, saponins, steroids,
tannins, and flavonoids. Some of the
alkaloids present are tropane, pyrrolidine,
steroid alkaloids and glycoalkaloids
(Evans, 2002). A bioflavonoid glycoside
named solanoflavone is present in the
leaves and fruits of Solanum melongena
(Shen et al., 2005). Two steroidal
saponins - melongoside L and
melongoside M and three new saponins
melongoside N, O and P, have been
isolated from seeds (Kintia et al., 1985).
The folkloric uses of this plant include
decoction of roots taken internally for
asthma and as a general stimulant.
Leaves are used for piles. The boiled root
of the wild plant, mixed with sour milk and
grain porridge, has been used for the
treatment of syphilis. The ashes of the
peduncle are used in the treatment of
intestinal hemorrhage, piles and
toothache. The extract of roots, dried stalk
and leaves has been used as astringent
for hemorrhage from the bladder. It is
bruised with vinegar and used as a
poultice for cracked nipples, burns,
abscesses, cold sores. In India, the juice
of the pulp of eggplant has been used for
various ailments such as diabetes, otitis,
toothaches, cholera, bronchitis, asthma,
dysuria, among many others. In Taiwan
folk medicine, roots are used for
rheumatism, inflammation and foot pain. In
medicine, Solanum melongena is used as
anti-asthmatic, hypo-cholesterolemic, anti-
cancer, anti-herpetic, anti-inflammatory.
(Das & Barua, 2015). Eggplants have
been also known to have lowering effect in
blood cholesterol levels (Guimarães et al.,
2000). The fruit has antihaemorrhoidal and
hypotensive effect. It is also used as an
antidote to poisonous mushrooms. The
plant's antioxidant property is due to the
flavonoids. The terpenes (steroids) make it
useful for bronchitis. Analgesic property is
because of the alkaloids (Shrivastava et
al., 2012). The roots considered anti-
asthmatic and stimulant. Leaves
considered anodyne. Its fruit considered
cooling, digestive and phlegmatic.
There are numerous studies
conducted on this plant but there is no
document so far that proves its inhibitory
actions against Listeria monocytogenes
and Streptococcus pyogenes. This study
aims to extract the solanine compound
and test the inhibitory effects of the
different solanine concentrations against
the test organisms.
RELATED LITERATURE
Alkaloids
Alkaloids are a chemically related
group of substances found in a variety of
foods, including cocoa, coffee, tea, black
pepper, honey, potatoes, tomatoes and
eggplants. Alkaloids in foods can have
health benefits, sometimes including
antioxidant related benefits and cancer
preventive properties. These same
potential health benefits have been linked
in research studies to some of the
alkaloids found in nightshades. Among the
nightshade plants most commonly enjoyed
as foods are, tomatoes, potatoes and
eggplant (Manrique-Moreno et al., 2014).
Solanine
It is a very potent glycoalkaloid
found in various solanaceous plants,
including potatoes which have gone green
through exposure to light. Glycoalkaloids
are natural pesticides produced by
nightshade plants and found throughout
the plant, but their concentrations are
especially high in leaves, flowers, and
unripe fruits. They are there to defend
plants against bacteria, fungi, viruses, and
insects. Glycoalkaloids kill bacteria and
viruses. It should also not be surprising
that glycoalkaloids have been shown in
laboratory studies to possess antibiotic
and antiviral properties (Milner, 2011).
Leaves, roots, flowers stems, and fruits
can all have solanine in varying levels.
(Izawa & Kuroda, 2010). Solanine intake
can cause nausea, vomiting, dry mouth,
abdominal pain, mydriasis, and seizures
due to anticholinergic properties. (Bock &
Norris, 2016)
Solanum melongena
Eggplants are vegetables long
prized for its beauty as well as its unique
taste and texture. Eggplants belong to the
plant family of Solanaceae, also
commonly known as nightshades, and are
kin to the tomato, bell pepper and potato.
Eggplant is a very good source of dietary
fiber, potassium, manganese, copper, and
thiamin (Knapp et al., 2010).
The nutrient analysis of 100 grams
raw eggplant includes 92.30 g of water, 25
kcal of energy, 0.98 g of protein, 0.18 g of
total lipid, 5.88 g of carbohydrate by
difference, 3.0 g of total dietary fiber, 3.53
g of total sugar, 9 mg of calcium, 0.23 mg
of iron, 14 mg of magnesium, 24 mg of
phosphorus, 229 mg of potassium, 2 mg
of sodium, 0.16mg of zinc, 2.2 mg of
Vitamin C, 0.039 mg of thiamine, 0.037
mg riboflavin, 0.649 mg of niacin, 0.084
mg of vitamin B6, 22 µg of folate, 23 IU of
vitamin A, 0.30 mg of vitamin E, 3.5 µg of
vitamin K. (USDA National Nutrient
Database for Standard Reference, 2018)
In a country like India, Solanum
melongena is a common fruit consumed
by majority of the people. It is also used as
disinfectants in case of minor injuries like
cuts and bruises to the farm laborers
working with field of eggplants (Knapp et
al., 2010).
Solanum has immense natural
diversity and natural host plant resistance.
It includes many species of plant resistant
to attack by pathogenic organisms such as
insects. This resistance is attributed to the
presence of leptine glycoalkaloids which is
solanine and chaconine (AL-Janabi and Al
Rubeey, 2010).
Investigation of the ethanolic
extract of leaves of the green variety, have
been found to have antimicrobial action
against common wound infecting
organism like Micrococcus aureus. It is
due to the presence of a wide variety of
siloxanes which are good enough to retard
the growth of such wound infecting
organisms. This finding is important as this
extract can be used as an alternative
disinfectant for first-aid dressing of minor
wounds and bruises (Knapp et al., 2010)
Inhibitory effects of plant extracts have
varying effect to microorganisms as
presented in the study of AL-Janabi, AL-
Rubeey (2010) the concentration depends
on the inhibition activity of the egg plant.
The higher the concentration of the egg
plant, the greater the inhibitory effect of
the plant extracts associated with the test
organism
Streptococcus pyogenes
It belongs to the group A
Streptococcus, an exclusive human
bacterial pathogen with tremendous
virulence potential. Interactions with
humans range from asymptomatic
carriage over mild and superficial
infections of skin mucosal membranes up
to systemic purulent toxic-invasive disease
manifestations. Particularly the latter are a
severe threat for predisposed patients and
lead to significant death tolls worldwide.
This places GAS among the most
important Gram-positive bacterial
pathogens. Many recent reviews have
highlighted the GAS reported of virulence
factors, regulators and regulatory
circuits/networks that enable GAS to
colonize the host and to deal with all levels
of the host immune defence (Low, 2010).
It is one of the most aggressive
pathogens encountered in clinical
microbiology. Streptolysin S and O are
factors contributing beta-hemolytic pattern
on blood agar plates. Streptolysin S is an
oxygen stable, non-immunogenic
hemolysin capable of lysing erythrocytes,
leukocytes and platelets. While,
Streptolysin O is immunogenic, capable of
lysing the same cells and cultured cells, is
broken down by oxygen and will produce
hemolysis only in the absence of room air
(Patterson, 2010). Acute Streptococcus
pyogenes infections may take the form of
pharyngitis, scarlet fever (rash), impetigo,
cellulitis, or erysipelas. Invasive infections
can result in necrotizing fasciitis, myositis
and streptococcal toxic shock syndrome.
Patients may also develop immune-
mediated sequelae such as acute
rheumatic fever and acute
glomerulonephritis. S agalactiae may
cause meningitis, neonatal sepsis, and
pneumonia in neonates; adults may
experience vaginitis, puerperal fever,
urinary tract infection, skin infection, and
endocarditis (Patterson, 2010).
Streptococci are Gram-positive,
nonmotile, non-spore forming, catalase-
negative cocci that occur in pairs or
chains. Older cultures may lose their
Gram-positive character. Most
streptococci are facultative anaerobes,
and some are obligate anaerobes
(Patterson, 2010).
Listeria monocytogenes
It is a gram positive ubiquitous,
intracellular pathogen which has been
implicated within the past decade as the
causative organism in several outbreaks
of foodborne disease. Organism occurs
widely in food with the highest incidences
being found in meat, poultry, and sea food
products.
Listeria can be found in soil, water
and animal feces. Humans typically are
infected by consuming raw vegetables that
have been contaminated from the soil or
from contaminated manure used as
fertilizer, contaminated animal meat,
unpasteurized milk or foods made with
unpasteurized milk and certain processed
foods such as soft cheeses, hot dogs and
deli meats that have been contaminated
after processing (CDC, 2015).
Listeriosis with a mortality rate of
about 24%, is found mainly among
pregnant women, their foetuses, and
immunocompromised persons with
symptoms of abortion, neonatal and
meningitis. Listeriolysin O being identified
as playing a crucial role in the organism’s
ability to multiply within host phagocytic
cells and to spread from cell to cell (CDC,
2015). The condition caused by Listeria is
a serious infection usually caused by
eating food contaminated with the
bacterium.
An estimated 1,600 people get
listeriosis each year, and about 260 died.
The infection is most likely to sicken
pregnant women and their newborn,
adults aged 65 or older, and people with
weakened immune systems are also at
higher risk of life-threatening
complications (CDC, 2015).
Hypocholesterolemic
Solanum melongena is widely
consumed in Brazil and in other countries
and its infusion has been used in popular
medicine as a hypocholesterolemic agent.
However, there are few controlled studies
of its effect on cholesterol metabolism,
and its possible hypocholesterolemic
effect in humans has not been proved by
rigorous studies (Schaefer et al., 1997).
Mitschek (1970) showed that a
preparation of S. melongena prevented
both hypercholesterolemia and the
formation of atheromas in the aorta of
rabbits fed high cholesterol diets.
However, Kritchevsky et al. (1975) did not
confirm these findings in rats fed diets
containing 1% S. melongena fruit or leaf
powders. Likewise, Jorge et al. (1998)
studied the effects of S. melongena on
plasma lipid levels, lipid peroxidation and
endothelial dysfunction in experimental
hypercholesterolemia.
Antiherpetic/Antioxidant/ S. melongena
Genotype
Study evaluated the antioxidant
and antiherpetic properties of DR2
Solanum melongena genotype. Samples
exhibited antioxidant properties as
evidenced by scavenging of different
radical species and blocking of ROS
generation by chelating mechanisms. The
DR2-C extract exhibited antiherpetic
properties, likely due to an impairment of
specific steps of the virus life-cycle. The
antioxidant properties could be involved in
the antiviral effects. Results suggest
potential application as dietary supplement
and as topical formulations for herpetic
skin lesions (Di Giacomo et al., 2015).
Anticancer / Fruit Peels
Study evaluated methanol extracts
of peels of Solanum melongena against
five human cancer cell lines. Study yielded
five steroidal compounds; three steroidal
alkaloids: solasodine (S1), solamargine
(S4) and solasonine (S5) together with two
steroidal glycosides: β-sitosterol-3-O- β-D-
glucoside (S2) and poriferasterol-3-O- β-
D-glucoside (S3). Results showed
moderate to potent activities against the
tested cancer cell lines (Shabana et al.,
2013).
Anti-Inflammatory
Study evaluated an aqueous
extract of leaves of S. melongena for anti-
inflammatory activity using carrageenan-
induced paw edema in Wistar albino rats.
Aspirin was used as standard drug.
Results showed significant anti-
inflammatory activity. In doses of 200
mg/kg and 400 mg/kg, the aqueous
extract showed percentage inhibition of
42.62% which is than standard aspirin at
64.5% inhibition (Umamageswari &
Maniyar, 2015).
MATERIALS AND METHODS
Plant material
The variety of Solanum
melongema (eggplant) to be used in this
study is the Italian eggplant and will be
harvested directly from the plantation and
will be authenticated at the National
Museum. Fresh flesh with seeds will be
used in this study.
Figure 1: Italian eggplant (www.google.com)
Test Strains
The organisms to be used in this
study are Listeria monocytogenes
(BIOTECH 1958) and Streptococcus
pyogenes (UST CMS 1101) which will be
procured from Philippine National
Collection of Microorganisms National
Institute of Molecular Biology and
Biotechnology (BIOTECH) UP Los Baños,
College, Laguna 4031 and UST
(University of Sto. Tomas) Manila,
Philippines.
Crude Extract Preparation
The preparation of the crude
extract of Solanum melongema is based
on previous study of Sakhare (2014).
Maceration will be done by soaking 100
grams of eggplant’s flesh and seed with
5% acetic acid (300ml) in a beaker for 24
hours. It will be subjected to a boiling
temperature and will be cooled after. The
pH of the cooled solution will be adjusted
up to 9.5 with ammonia and will be cooled
again. It will be centrifuged, and the
precipitate will be washed with 1%
ammonia solution and centrifuged after.
Supernatant will be discarded, and its
residue will be dried and weighed. This
isolated solanine will be resuspended to
be able to get 100ug/ml and will be tested
for its antimicrobial activity.
Inhibitory test
The following procedures will be
used to test the inhibitory effect of S.
Melongena. (Sakhare, 2014)
Preparation of base layer medium
(nutrient agar)
1.68gm of nutrient agar will be
dissolved in 30ml of distilled water and will
be subjected to sterilization by autoclaving
at 15 p.s.i. for 20 minutes.
Sterilization of equipment.
Petri dishes, pipettes, cork borer,
test tubes will be sterilized by dry heat
sterilization at 1600c for 1 hr in hot air
oven.
Preparation of subcultures
24 hours prior to testing,
inoculations of bacterial cultures will be
made on nutrient agar.
Preparation of test solution
100mg of isolated solanine will be
dissolved in 1ml of acetone to give a 100
g/ml of these stock solution. It will be
pipetted out with the help of micropipette
and will be used for testing.
Method of testing microorganisms by using thin layer
chromatography method. One-way
A 25 ml of sterilized nutrient agar analysis of variance (ANOVA) with
on a petri dish will be added with about Tukey’s multiple comparison post hoc test
0.1ml of bacterial inoculum (Listeria will be used to assess the data obtained to
monocytogenes and Streptococcus compare the level of significance between
pyogenes). The cups will be prepared with control and experimental groups.
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