COMPARISON OF ANTIBACTERIAL EFFECT OF CRUDE ALKALOID AND SAPONIN
EXTRACT FROM Phyllanthus niruri
By
Ajibade, V. A
Department of Science Technology, Microbiology option, Federal Polytechnic,
P. M. B. 5351, Ado-Ekiti, Ekiti State, Nigeria
E-mail: ajibvijay@yahoo.com
Phone: +2348035781628
ABSTRACT
The antibacterial activity of crude extract of saponin and alkaloid extracted from Phyllanthus niruri against
some test bacteria was investigated and compared. With the activity of saponin Bacillus subtilis,
Salmonella typhi and Klebsiella pneumoniae were resistant at concentrations of 0.01mg/ml and 0.02mg/ml
while they were susceptible at 0.03mg/ml to 0.05mg/ml concentrations. Staphylococcus aureus was
resistant at 0.03mg/ml. With the activity of saponin and alkaloid, Bacillus subtilis, Klebsiella pneumoniae
and Salmonella typhi were resistant at 0.01mg/ml concentration, while Staphylococcus aureus was resistant
to alkaloid at 0.01mg/ml concentration but susceptible to saponin at the same concentration. Escherichia
coli, Pseudomonas aeruginosa and Staph aureus were more sensitive to saponin with Minimum Inhibitory
Concentration (MIC) of 8.00mm, 12.00mm, 12.00mm respectively compared with 5.00mm, 10.00mm,
10.00mm respectively as observed in alkaloid. The entire test organisms were susceptible to saponin at
concentration of 0.03mg/ml. Staph aureus was resistant to alkaloid; B. subtilis, E. coli, kleb pneumoniae, P.
aeruginosa, Sal typhi and Staph aureus were sensitive to both saponin and alkaloid. Combined, saponin
and alkaloid may offer an alternative therapeutic agent against bacterial infections.
Key words: Antibacterial activity, Alkaloid, Phyllanthus niruri, saponin
1
INTRODUCTION
Medical plant are being used in traditional system of medicine from hundreds of years in many
countries of the world (Grewal, 1982) and which is in line with world health organization prescription that
medicinal researchers warrant attention. Phyllanthus niruri originated in India, usually occurring as a
winter weed throughout the hotter parts, it has over 600 species of shrubs, trees, and annual or biannual
herbs distributed throughout the tropical and subtropical areas.
Most of these medicinal plants with hypoglycemic properties are being used immediately as
alternative forms of treatment and management of varieties of disease (Farjou et al., 1987).
Phyllanthus niruri also known as “chanca piedra” belong to the family Eughorbiaceae. It is known as
enyikwonwa in Ibo language of the South Eastern Nigerian and popularly known as Asasa in Yoruba South
West Nigeria. It has been confirmed that it has hypoglycaemic properties (Sivaprakasm et al., 1995), for
treatment of jaundice (Farjou et al., 1987; Kumar et al., 2005) hepatitis (Adedapo et al., 2005), dysentery,
diuretic, typhoid, kidney stone, malaria, influenza, antibacterial and antiviral (Mellinger et al., 2005), and
has protective action on the different organs especially the liver and kidney, hepatoprotective action, lipid
lowering action, anti diabetic action, anti fungal action and that no form of toxicity has been associated
with the usage of this plant extract (Barros, 2003). Popular medicines also consider P. niruri useful to treat
problems associated to women, such as dysmenorrheal (painful menstruation), extracts of P. niruri have
been reported to be effectives against Hepatitis B and other viral infection (Chatterjee, 2006). The alkaloid
extract of P. niruri showed suppressing activities on strain of HIV-1 cells cultured on MT-4 cell line
(Natarraj et al., 2000).
However, chronic consumption of large amounts of traditional remedies must always be taken with
caution as toxicity studies have not be conducted for most of these plants (Ridout et al., 1991).
2
.
Alkaloids are naturally occurring chemical compound containing basic nitrogen atoms. They are
produce by a large variety of organisms, including bacteria, fungal, plants and animals and are part of the
group of natural products (Juvekar 2003). Some alkaloid has a bitter taste (Matsuura, 2007).
Saponins one of the phytochemicals in P. niruri are naturally occurring surface – active glycosides
that derived their name from their ability to form stable, soap-like foams with aqueous solution. They are
known for their hypocholesterolic, anticarcinogenic, immune boostering and antibacterial activities
(Ovesna et al., 2004). Their physiological, immunological and pharmacological properties have provoked
considerable clinical interest in these substances. Saponins are divided into three major groups,
triterpenoid, basic steroidal and steroid saponins. Saponins and alkaloid extracted from Phyllanthus niruri
have been discovered to be very effective in vivo against infections caused by Salmonella typhi and
Escherichia coli without deleterious effect on organs of the body of test rabbits (Ajibade and Egbebi,
2011). This work tends to compare the potency of saponin and alkaloid extract from whole plant of P.
niruri on clinical pathogenic microorganisms.
MATERIALS AND METHODS
Collection and Processing of Plant Material
The plant was collected from farmlands in the Federal Polytechnic Ado–Ekiti during the raining
season between the months of September and December 2010. The plant was dried at room temperature
(28oC ± 1oC). The plant was milled using centrifugal milling machine (GallenKamp) and stored in air-tight
plastic container. Identification and authentication of plant was done in the Department of Science
Technology, Federal polytechnic Ado–Ekiti, Nigeria where a voucher specimen (No. MED. PLT 0-200810) was kept.
3
Extraction of Crude Alkaloid
The method of Naik and Javekars (2003) was employed for the extraction. A 15g portion of the
method plant material was soaked in 100ml of 95% ethanol and allowed to stand for three days. The
ethanol solution was filtered and evaporated under reduced pressure with Bibby rotary evaporator (EVF530-010k) and the residue suspended in 30ml distilled waters. 20ml chloroform was added and passed
through Whatman - No 4 filter paper. The extract was dried at room temperature (28oC ± 1oC) for six days
and 100% sodium sulphate added. It was flitted and evaporated under reduced pressure to obtain a light
brown powder.
Extraction of Saponin
The method described by Marston et al. (2000) was employed. The dried and powder plant
materials (500g) was defatted in a Soxhlet with Petroleum spirit at (40-60) oC for 16 hours. The residue
was added to 100ml of absolute methanol and left overnight under reflux at 70 oC. It was filtered and
dissolved in 100ml of distilled water, extracted in a separating funnel with 1-butanol three times and dried
by evaporation. Finally, the extract was dissolved in 25ml of absolute methanol and the saponin compound
was precipitated by adding 75ml of diethyl ether.
Collection of Bacteria
Five strain each of Escherichia coli, Staphylococcus aureus, Salmonella spp, Bacillus subtilis,
klebsiella pneumoniae and Pseudomonas aeruginosa from different source were collected at the Medical
laboratory section of the University Teaching Hospital, Ado-Ekiti, sub cultured on a nutrient agar slant
medium and stored in culture collection box at the Microbiology Laboratory of the Department of Science
Technology, Federal Polytechnic, Ado-Ekiti
4
Bacteriological Assay
Inoculums was removed from stock culture into 5ml of nutrient broth and incubated at 370C for
24hrs to reactivate the bacterium. The bioassay monitoring of crude extracts was done with the agar disc
diffusion technique (Denni and Hussain, 1991). 20l aliquots of the extracts were placed on susceptibility
test discs. These were then applied to the surface of over dried nutrient agar plates, which had been seeded
with an overnight culture of test organism. Plates were incubated overnight at 370C and zones of inhibition
estimated semi-quantitatively. Gentamicin sulphate (Nicholas Laboratories, U.K.) and ampicillin were used
as positive control.
RESULT AND DISCUSSION
The results of this work are shown in the tables below. Diameter of zone of inhibition (mm) of
saponin at different concentration to Bacillus subtilis, Escherichia coli, klebsiella pneumonia,
Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus are shown in table1. Bacillus
subtilis and Salmonella typhi were resistant at concentration of 0.01mg/ml and 0.02mg/ml, Klebsiella
pneumoniae was resistant at concentration 0.01mg/ml while others are susceptible at 0.03mg/ml,
0.04mg/ml and 0.05mg/ml concentration.
Table 2 shows the result of the potency of alkaloid extract of P. niruri on test bacteria at various
concentrations. It was observed that Staphylococcus aureus and Klebsiella pneumoniae were resistant at
0.03mg/ml; Bacillus subtilis and Salmonella typhi were resistant at 0.01mg/ml while E. coli and
Pseudomonas aeruginosa were susceptible at the same concentration.
Table 3 shows the comparison of the potency of saponin and alkaloid to the test bacteria at different
concentrations. At 0.01mg/ml, It was observed that Bacillus subtilis, klebsiella pneumoniae and Salmonella
typhi were resistant to both saponin and alkaloid, while Staphylococcus aureus was resistant to alkaloid but
susceptible to saponin at the same concentration. Escherichia coli, Pseudomonas aeruginosa and
Staphylococcus aureus are more sensitive to saponin with MIC of 8.00mm, 12.00mm and 12.00mm
5
respectively as compared with 5.00mm, 10.00mm and 10.00mm respectively observed in alkaloid. At
0.03mg/ml, it was observed that Kleb pneumoniae and Staph. aureus are resistant to alkaloid while B.
subtilis, E. coli, P. aeruginosa, Salmonella typhi were susceptible with MIC 10.00mm, 10.00mm,
14.00mm, 14.00mm respectively. The entire test organisms were susceptible to saponin at this
concentration. At 0.05mg/ml, it was observed that only Staph aureus was resistant to alkaloid, B. subtilis,
E. coli, kleb pneumoniae, P. aeruginosa, Sal typhi and Staph aureus were sensitive to both saponin and
alkaloid while one Staph aureus was resistant to alkaloid.
The efficacy of phytochemicals acting as antibacterial agent as long been studied and cannot be
under estimated (Adedapo et al., 2005). These non-nutritional components of plants have been investigated
to exhibit high potency when acting in combination because their synergistic nature potentiates each other.
The use of medicinal plants constitutes an important part in traditional or folkloric medicine in African.
Modern orthodox medicine has improved the health of many people all over the world. It is noteworthy
that in many cultures, modern medicine complements traditional practices by what is obtainable in China
(Natarraj, 2000). Saponins are very potent bio-agent in plants and have been known to be very active
against Salmonella typhi and Escherichia coli without adverse effect on cells. Alkaloids have also been
known to be potent against enteropathogenic E. coli (Juvekar, 2003). However, the potency of saponin
against the test bacteria in this work seems to be higher than that of the alkaloid. Because of the potency of
each of these components when acting singly, their combination in this work proved to be highly
therapeutic. It was therefore concluded that the combination of this phytochemicals would serve the useful
purpose of treating diseases caused by or from which Salmonella typhi, Bacillus subtilis, Pseudomonas
aeruginosa and Klebsiella pneumoniae have been isolated.
6
Table 1: Degree of potency of different concentration of saponin extract of P. niruri on some test
bacterial
Concentration (mg / ml)
Test organisms
Diameter of zone of inhibition (mm)
0.01
0.02
0.03
0.04
0.05
Bacillus subtilis
0.00
0.00
14.00
14.00
16.00
Escherichia coli
8.00
8.00
16.00
16.00
16.00
Kleb. pneumoniae
0.00
12.00
15.00
16.00
18.00
Pseudo. aeruginosa
12.00
12.00
16.00
16.00
20.00
Salmonella spp
0.00
0.00
14.00
16.00
20.00
Staph aureus
12.00
14.00
16.00
16.00
16.00
Table 2: Degree of potency of different concentration of alkaloid extract of P. niruri on some test
bacterial
Concentration (mg/ml)
Test organisms
Diameter of zone of inhibition (mm)
0.01
0.02
0.03
0.04
0.05
Bacillus subtilis
0.00
10.00
10.00
12.00
14.00
Escherichia coli
5.00
8.00
10.00
10.00
16.00
Kleb. pneumoniae
0.00
0.00
0.00
10.00
14.00
Pseudo. aeruginosa
10.00
12.00
14.00
14.00
14.00
Salmonella spp
0.00
10.00
14.00
20.00
24.00
Staph aureus
0.00
0.00
0.00
0.00
0.00
7
Table 3: Comparison of the degree of potency of saponin and alkaloid at different concentrations
Test organisms
Saponin
Alkaloid
0.01mg/ml
Saponin
Alkaloid
0.03mg/ml
Saponin
Alkaloid
0.05mg/ml
Bacillus subtilis
0.00
0.00
14.00
10.00
16.00
14.00
Escherichia coli
8.00
5.00
16.00
10.00
16.00
16.00
Kleb. pneumoniae
0.00
0.00
15.00
0.00
18.00
14.00
Pseudo. aeruginosa
12.00
10.00
16.00
14.00
20.00
14.00
Salmonella spp
0.00
0.00
14.00
14.00
20.00
24.00
Staph aureus
12.00
0.00
16.00
0.00
16.00
0.00
8
REFERENCES
Adedapo A. A, Asegbayibi A. Y. and Emikpe B. O (2005) Some clinic-pathological changes associated
with the aqueous extract of the leaves of Phyllanthus niruri in rats; Interscience Journal 40 (1):
119-122.
Ajibade, V.A and Egbebi A.O (2012) Effect of Alkaloid Extract of Phyllanthus niruri on Rabbits Infected
with Enteropathogenic Escherichia coli. Int. Journal of Tropical Medicine and Public Health 1 (1):
33-39
Agharkar S. P. (1991). Medicinal plants of Bombay. Presidency scientific Publications Jodhpur, India.
Barros M. E. and Zahad G (1979). Plants with Oral hypoglycaemic action; J. Crude Res. 17: 139-196.
Chatterjes, M. (2006) Herbal (Phyllanthus niruri) protein isolate protects liver from nimesulide induced
oxidative stress. Pathophysiology 13(2):95-102.
Farjou I. B, Al-Ani M and Guiges S. Y (1987) Lowering of blood glucose in diabetic rabbits by Artemisia
extract. J. Faculty Med. Baghdad 92(5): 137-141
Fenwick G. R, Price K..R, Isukamboto C and Okubo K (1991) Saponins as toxic substances in crop plants
(ETPA Mello Duffus and J. H Dufus Editors) Cambridge.
Grewal, R. C (1999) Medicinal plants; Campus Book International: New Delhi, 1st Edition, 298-304.
Juvekar, A. R. (2003): Effects of alkaloid extract of Phyllanthus niruri on HIV replication, Indian J. Med
Sci 57: 387-93.
Mellinger, C. G, Carbonero E. R, Cipriani, T. T; Gorin, P.A and lacommi M (2005) Xylans from the
medicinal herb Phyllanthus niruri. J Nat Prod 68(1): 129-32.
Natarraj, C. G. (2000): Role of herbal extracts in HIV infected patients, proceedings of International
Congress on Ayurveda. 207.
Otsuka, E. A; (2005) Analysis and isolation of saponins from plants materials ; Saponins in food, feed
stuffs and medicinal plants Annual proceeding of the Phytochemical society pg 1-12 (W. Oleszed
and A) (Marston, Editors) Oxford and London, Clarendon press.
9
Prajapati N. D; Purohit S. S; Sharma A. K; Kumar T, A (2005) Hand book of medicinal plants A complete
source Book. (Jodhpur Press); 1st Edi; 392
Ross Ivan, (1999). Medicinal plants of the world Chemical Constituents; Traditional and Modern
Medicinal uses, Humana Press: New Jersey (Totowa); 249-252
Santos, A. R (1994); Analysis effects of callus culture extracts from selected species of Phyllanthus in
mice, J. pharm. Pharmacol 46(9):755-759.
Sivaprakasam, K; Yasodha, R; Sirandam, G. and veluchamy, G. (1995) Clinical evaluation of Phyllanthus
amarus schum and Thonn in Diabetes mellitus; Seminar on research in Ayurveda and Siddha
medicine. CCRAS, New Delhi Pg. 7. (5).
10