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Antibacterial, antifungal and insecticidal activities of Ruellia tuberosa (L.) root

extract

Article in Journal of Bio-Science · January 2012

DOI: 10.3329/jbs.v20i0.17720

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J. bio-sci. 20: 91-97, 2012 ISSN 1023-8654
http://www.banglajol.info/index.php/JBS/index

ANTIBACTERIAL, ANTIFUNGAL AND INSECTICIDAL ACTIVITIES OF

RUELLIA TUBEROSA (L.) ROOT EXTRACT
Md Abdul Kader∗, Shumaia Parvin, Md Aktar uzzaman Chowduri, Md Ekramul Haque
Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh
Abstract
Context: Plants as therapeutics are popularized for thousands of years and people continue to rely on
them for health care until now due to their effectiveness, easy availability, low cost and comparatively
being devoid of serious toxic effects. Microorganisms such as bacteria, fungi and insects are developing
resistance to the current therapies very easily and the currently available antibacterial, antifungal agents
and pesticides are very much costly and toxic. So the current shift to the use of herbal antibacterial,
antifungal agents and pesticides may be more effective, economic and advantageous.
Objectives: The present research was performed to investigate the antibacterial, antifungal and
insecticidal activities of the methanolic extract of the dried root of the plant Ruellia tuberosa (L.).
Materials and Methods: Five Gram (+) ve bacteria namely Staphylococcus aureus, Staphylococcus
agalactiae, Bacillus cereus, Bacillus megaterium, Bacillus subtilis; five Gram (-) ve bacteria namely
Pseudomonus aeruginosa, Escherichia coli, Shigella flexneri, Shigella dysenteriae, Shigella sonnei were
used as test bacteria for testing the antibacterial activity of the plant extract. Antifungal activity was
observed against six fungi namely Candida albicans, Aspergillus niger, Aspergillus ochreus, Aspergillus
ustus, Rizopus oryzae and Trichophyton rubrum. The disc diffusion assay method was used in both the
cases and standard Kanamycin disc (30µg/disc) was used as the reference standard. The test for
insecticidal activity was performed by using surface film activity testing method and Tribolium castaneum
(Herbst) was used as the test insect.
Results: The methanol extract was active against all the bacteria and fungi tested and showed
significant antibacterial and antifungal properties with the zone of inhibition 9 to 23 mm for antibacterial
screening and 8 to 15 mm for antifungal screening. The insecticidal assay by surface film activity test
also revealed strong insecticidal activity with 80% mortality rate of Tribolium castaneum (Herbst) at a
dose of 50 mg/ml in 48 hours.
Conclusion: From our experiment it is informed that Ruellia tuberosa (L.) may be used to treat bacterial
and fungal diseases and also as insect repellant and it is also possible to isolate antibacterial, antifungal
and insecticidal drug from this plant.
Key words: Ruellia tuberosa, methanol extract, antibacterial, antifungal and insecticidal activity.
Introduction
Globalization interferes with infectious disease control at the national level while microbes move freely
around the world, unhindered by borders, human responses to infectious diseases and are conditioned by
jurisdictional boundaries (Stepanovic et al. 2003). According to WHO, important progress has been made in
controlling major infectious diseases. About 43% of total deaths occured in developing countries due to
infectious diseases in recent years (Carballo et al. 2002). Similarly freedom from insect infestation and
contamination has become an important consideration in storage of grain and to maintain high quality food
product by preventing them from attack of the most frequently invading organisms (Coolins 1998).

∗
Corresponding author E-mail: [email protected]
92 Kader et al.

Ruellia tuberosa (L.) is an erect, sub erect or diffuse perennial herb up to 60-70 cm tall and belongs to the
family Acanthaceae, a native of Central America introduced into Indian gardens as ornament and widely
distributed in South East Asia including Thailand and Laos. It is used medicinally in West Indies, Central
America, Guiana and Peru. Ruellia tuberosa (L.) is commonly known as “Cracker plant” (Pandey 2005,
Medicinal plants of the Guiana’s and Chothani et al. 2010). In Siddha system of medicine, leaves are given
with liquid copal as remedy for gonorrhea and ear diseases (Suseela and Prema 2007), used in stomach
cancer (Reddy et al. 1991). Dried and ground roots in dose of two ounces cause abortion and also used in
sore eyes (Kirtikar and Bashu 1935). The herb also exhibits emetic activity and employed as substitute of
ipecac also used in bladder stones and decoction of leaves is used in the treatment of bronchitis (The wealth
of India 1972). In Suriname’s traditional medicine system, it is used as anthelmintic and also in the
management of joint pain and strained muscles. In folk medicine, it has been used as diuretic, antipyretic,
antidiabetic, antidotal, thirst- quenching agent and analgesic and antihypertensive agent (Chiu and Chang
1995, Chen et al. 2006). Ruellia tuberosa (L.) is used as cooling agent in urinary problems and uterine
fibroids (Lans 2001 and 2006). It has recently been incorporated as a component in an herbal drink in
Taiwan (Balick et al. 2000). It is reported that it contains flavonoids, steroids, triterpenoids and alkaloids (Lin
et al. 2006, Subramanian and Nair 1974, Singh et al. 2002, Andhiwal and Varshney 1985). The aim of this
study was to explore the antibacterial and antifungal activity of Ruellia tuberosa (L.) roots to determine the
scientific basis for its use in folk medicine to treat microbial pathogen and other infectious diseases. Besides
this, we made an attempt to investigate the importance of Ruellia tuberosa (L.) roots as strong natural
insecticide.
Materials and Methods
Collection of plant Materials
The fresh roots of Ruellia tuberosa (L.) were collected during the month of July 2009, from the village
Haibotpur, situated near Natore district of Bangladesh and identified by Md. Arshed Alom, Taxonomist,
Department of Botany, University of Rajshahi, Bangladesh.
Extraction of plant Materials
The collected roots were sun dried for 10-12 days and then kept in an electric oven for 72 hrs at 40°C. Then
the dried roots were pulverized into coarse powder with the help of a grinding machine. The ground powder
(900 gm) was extracted with methanol (4.5 Liters) in an air tight clean flat bottomed container for 12 days at
room temperature with occasional stirring and shaking (Trease and Evans 1997).The extract was then
filtered first through a fresh cotton plug and finally with a whatman filter paper. The filtrate was then
evaporated to dryness in vacuum by a rotary evaporator at 40 - 50°C to afford a brownish mass (35 gm) and
kept for further analysis.
Collection of microorganisms
Antibacterial activity was determined against five Gram (+) ve bacteria (Staphylococcus aureus,
Streptococcus agalactiae, Bacillus cereus, Bacillus megaterium and Bacillus subtilis) and five Gram (-) ve
bacteria (Pseudomonus aeruginosa, Escherichia coli, Shigella flexneri, Shigella dysenteriae and Shigella
sonnei). The antifungal screening was carried out against six fungi (Candida albicans, Aspergillus niger,
Aspergillus ochreus, Aspergillus ustus, Rizopus oryzae and Trichophyton rubrum). All these organisms were
collected from the Microbiology Research Laboratory of Pharmacy Department, University of Rajshahi,
Rajshahi, Bangladesh.
Activities of Ruellia tuberosa root extract 93

Collection of test insect

For insecticidal screening the insect Tribolium castaneum (Herbst) used in the experiment was provided from
the stock cultures of the Crop Protection and Toxicology Laboratory, University of Rajshahi, Bangladesh.
Growth media and conditions
Nutrient agar media (Difco Laboratories) pH 7.2 and Sabouraud dextrose agar media (Biolife Vole Monza)
pH 5.6 were used for antibacterial and antifungal screening respectively.
Antibacterial screening
The invitro antibacterial activity of the extract was determined by disc diffusion method (Bauer et al. 1996).
The test sample was prepared by dissolving 50 mg of the methanol extract in 2 ml of respective solvent to
give a concentration of 25 µg/µl. Sample discs were prepared by allowing each sterile disc (6 mm in
diameter) of filter paper to absorb 20 µl of a test solution in aseptic condition. The discs were allowed to dry
until complete evaporation of solvent. Dried and sterilized filter paper discs, each containing a test sample of
500 µg of the test agent was placed on nutrient agar medium uniformly seeded with the test microorganisms.
Kanamycin disc (30 µg/disc) and blank disc were used as the positive and negative control respectively. The
plates were incubated at 37°C for 24 hours for optimum growth of the organisms. The antibacterial activity of
the extract was determined by measuring the diameter of the zone of inhibition expressed in millimeter.
Antifungal screening
The extract was screened for its antifungal activity by disc diffusion method at the concentration of
500µg/disc. Sabouraud dextrose (20 ml) plates were prepared and incubated by spread plate method under
aseptic conditions. The sterile impregnated discs with plant extracts were placed on the agar surface with
framed forceps and gently pressed down to ensure complete contact of the disc with agar surface. Control
discs of Kanamycin were prepared and placed on the agar surface. All the plates were incubated at 37°C for
72 hours and the size of the inhibition zones were measured. The mean zone of inhibition of the three
replicated tests (triplicate analysis) of the plant extract is expressed in millimeter.

Insecticidal screening
For the conduction of surface film activity test of the plant extract, 60 mm petridishes were taken. The plant
extract (50 mg) was dissolved into 1 ml methanol. This was poured into the lower part of the petridish. A
control experiment applying only the solvent into the petridish was also set at the same time under the same
conditions (Bousquet 1990). After completing all the arrangements, treated petridishes were placed in a
secured place at room temperature. The whole experiment was observed from time to time and mortality was
observed first after 30 minutes and then after 12 hrs, 24 hrs, 36 hrs and finally after 48 hrs of exposure and
data were recorded. A simple microscope was used to observe each and every beetle by tracing natural
movements of each organism. In some cases hot needle was taken closer to the bodies (without movement)
to confirm death. Attention was also paid to recover the insects if occurred.
Results and Discussion
The result representing antibacterial activity of methanol extract of the roots of Ruellia tuberosa (L.) is
presented in Table 1. The highest activity of the plant extract was 23 mm diameter of zone of inhibition found
against Gram (-) ve bacteria, Shigella dysenteriae followed by Shigella sonnei (22 mm), Shigella flexneri (21
mm), Escherichia coli (18 mm) and Pseudomonus aeruginosa (17 mm) at a concentration of 500 µg/disc.
The plant extract was less effective against Gram (+) ve than Gram (-) ve bacteria. The lowest antibacterial
94 Kader et al.

activity was observed against Bacillus megaterium with the least zone of inhibition, 9 mm diameter and then
10 mm against Bacillus cereus, 11 mm against Bacillus subtilis and 13 mm against Staphylococcus aureus
and Streptococcus agalactiae. The reason for different sensitivity could be due to morphological difference
between microorganisms.
Table 1. Antibacterial activities of the methanolic extract of Ruellia tuberosa (L.) roots
Diameter of Zone of Inhibition(mm)
Test bacterial strains Methanol extract 500 µg/disc Std. Kanamycin 30 µg/disc
Gram (+ve) bacteria
Bacillus subtilis 11 21
Bacillus megaterium 09 22
Bacillus cereus 10 23
Staphylococcus aureus 13 22
Streptococcus agalactiae 13 20

Gram (- ve bacteria

Escerichia coli 18 28
Shigella dysenteriae 23 29
Shigella sonnei 22 28
Shigella flexneri 21 26
Pseudomonus 17 27
aeruginosa

The methanolic extract of roots of Ruellia tuberosa (L.) was found to be effective against various fungi as
indicated by the zone of inhibition (Table 2). Maximum inhibition was obtained against Candida albicans (18
mm) followed by Aspergillus niger (16 mm), Aspergillus ochreus (15 mm), Aspergillus ustus (14 mm),
Rizopus oryzae (10 mm) at a concentration of 500 µg/disc in comparison to reference standard kanamycin
30µg/disc. The least zone of inhibition was noted against Trichophyton rubrum (8 mm).
Table 2. Antifungal activities of the methanolic extract of Ruellia tuberosa (L.) roots
Diameter of Zone of Inhibition(mm)
Test fungal strains Methanol extract 500 µg/disc Standard Kanamycin
30 µg/disc
Aspergillus niger 16 23
Aspergillus ochreus 15 21
Aspergillus ustus 14 21
Candida albicans 18 26

Rizopus oryzae 10 19

Tricophyton rubrum 08 17

The insecticidal activity of methanol extract of Ruellia tuberosa (L.) roots has been studied by testing it
against the insect, Tribolium castaneum (Herbst) and the results are represented in Table 3. The maximum
mortality rate of Tribolium castaneum (Herbst) was 80% at a dose of 50 mg/ml in 48 hrs. The results have
shown that the methanolic extract of Ruellia tuberosa (L.) root was highly toxic to insects.
Activities of Ruellia tuberosa root extract 95

Table 3. Insecticidal activities of the methanolic extract of Ruellia tuberosa (L.) roots.
Extract Amount of Number of Number of insect killed Mortality %
extract (mg/ml) insect used
30 min 12 hrs 24 hrs 36 hrs 48 hrs
Methanolic extract
of Ruellia tuberosea 50 15 - 1 5 10 12 80%
Linn. Roots.

As an evolutionary process, plants on which insects, microorganisms and mammals are feeding, usually
acquire self defending capabilities by producing a variety of secondary metabolites such as alkaloids,
terpenoids, steroids and aromatic compounds which are presumably unpleasant or even toxic to the enemy.
Inside the tissue of nearly all the healthy plants, there are a lot of microorganisms which are called
endophytes. Endophytes are mutualistic to their host; at least some of them are thought to be making returns
for the nutrition from the plant by producing special substances such as secondary metabolites to prevent the
host from successful attack of fungi, pest and mammals. As a matter of fact, metabolites of endophytes were
reported to inhibit a number of microorganisms (Fisher et al. 1984, Gurney and Mantle 1993).
Yang and Tang (1998) reviewed the plant used for insect control and found that there is strong connection
between medicinal and pesticidal plants. Not only the world wide annual losses of food grains storage
caused by insects have been estimated to be about 10% of the world’s production, but loses of 25% or more
may also occur in tropical countries through insect attack after harvest (Howe 1965).
Previous phytochemical invstigations on this plant have revealed the presence of flavonoids, steroids,
triterpenoids and alkaloids. So, the insecticidal activity showed by the extract of Ruellia tuberosa (L.) roots
may be due to the presence of such type of phyto-constituents.
Human pathogenic microorganisms, phyto-pathogens are prone to developing drug resistance to decrease
substantially the effectiveness of those pesticides (Rosenberger and Meyer 1981). Therefore, there is an
urgent need to work towards the development of safer antimicrobial agents and bio-pesticides that are
expected to be renewable, non-petrochemical, naturally eco-friendly and easily obtainable.
Conclusion
From the results obtained, it is evident that Ruellia tuberosa (L.) root possesses potential inhibitory activity
against human pathogens (bacteria and fungi) and insects. Hence, there is a need to isolate possibly by
purification of the various phytochemical groups in the extracts. The further isolation of such bioactive
components could perhaps clarify the pharmacological properties of Ruellia tuberosa (L.) root and be further
exploited for pharmaceutical use.

Acknowledgement
The authors would like to thank the Department of Microbiology, University of Dhaka, Bangladesh and the
Microbiological Laboratory of the Institute of Nutrition and Food Science (INFS) for supply of test organisms.
We are also indebted to Md. Arshed Alom, Senior Technical Officer, Herbarium Museum, Department of
Botany, University of Rajshahi, Bangladesh for the identification of the plant botanically.
96 Kader et al.

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