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10 - Chapter 3 PDF
10 - Chapter 3 PDF
3.1. INTRODUCTION
(Solanki, 2010). Diseases caused by bacteria, viruses, fungi and other parasites are major
causes of death, disability, and social and economic disruption for millions of people
(Breman et al., 2004; WHO, 2008; UNAIDS, WHO, 2008). The frequency of life-
multi-drug resistant strains of bacteria and the recent appearance of strains with reduced
adds urgency to the search for new infection-fighting strategies (Zy et al., 2005; Rojas et
al., 2006).
In India around 20,000 medicinal plant species have been recorded recently (Dev,
1997), however 500 traditional communities use about 800 plant species for curing
plant-derived medicine for the first line of primary health care for human alleviation
(Veale et al., 1992). According to World Health Organization, medicinal plants would be
the best source to obtain a variety of drugs. Therefore, such plants should be investigated
to better understand their properties, safety and efficacy (Nascimento et al., 2000) and
As mentioned earlier, the state of Tripura is situated in one of the mega diversity
region of the world because of her location in the sub-Himalayan area and the state is
very rich in flora and fauna. Two medicinal plants of the state with known ethno
medicinal properties have been collected and systematically investigated regarding their
medicinal properties. The chapter deals with the antibacterial properties of the studied
plants.
their secondary metabolites (Erdemeier et al., 1996, Hassan and Ahmed, 1996; Darokan
et al., 1999; Cutter, 2000; Babu et al., 2002) and plants have always been a source of
potential antibacterial drugs (Iyengar, 1985; Chopra et al., 1992; Behl and Arora, 1993;
D'Souza, 1993; Youvraj et al., 1995; Ibrahim and Osman, 1995). The effects of plant
extracts on bacteria have been studied by a very large number of researchers in different
parts of the world (Santos Filho et al., 1990; Nascimento et al., 1990; Anesini and Perez,
1993; Akpulu et al., 1994; Saxena et al. 1994; Perez and Anesini, 1994; Alonso-Paz et
al., 1995; Vlietinck et al., 1995; Martinez et al., 1996; Vincenzo et al. 1998; Kudi et al.,
1999; Larshini et al. 1999; Kelmanson et al., 2000; Gislene et al. 2000; Reddy et al.,
2001; Ateb and ErdoUrul, 2003; Ajaiyeoba, 2002; Janovska et al., 2003; Abu-Shanab et
al., 2004; Seher et al., 2006; Rojas et al., 2006; El-Mahmood and Ameh 2007; Mothana
et al. 2008; Sassi et al. 2008; Rahman et al., 2008; Ibrahim et al. 2009; Koshy et al.
2009; Dogan et al., 2010; Joshi et al., 2011; Obeidat et al., 2011; Bisi-Johnson et al.,
2011).
al., (1998) tested eighty two Indian traditional medicinal plants against pathogenic and
opportunistic microorganisms. The results indicated that fifty six out of eighty two
exhibited activity against one or more test pathogens. The great potentiality of spices was
studied by Arora and Kaur in the same year and they suggested that spices might have a
great potential to be used as antimicrobial agents. Samy and Ignacimuthu (2000) tested
30 Indian folklore medicinal plants used by traditional healers for antimicrobial activity.
Among them, the leaf extracts of Cassia occidentalis and Cassia corniculata exhibited
Staphylococcus aureus. Nair et al., (2005) observed potentiality of nine Indian medicinal
plants as antibacterial agents and found that Sapindus emarginatus, out of these nine
plants showed strong activity against the tested bacterial strains. In the year 2006,
Duraipandiyan et al., assessed antimicrobial activity of eighteen Indian plant extracts that
are used in folk medicine in the treatment of skin diseases, venereal diseases, respiratory
problems and nervous disorders against nine bacterial strains (Bacillus subtilis,
coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Ervinia sp, Proteus vulgaris) and
one fungal strain (Candida albicans). They observed that out of 18 plants, 10 plants
exhibited antimicrobial activity against one or more of the tested microorganisms at three
different concentrations of 1.25, 2.5 and 5 mg/disc. Among the plants tested, Acalypha
granatum and Syzygium lineare were most active. The ethnobotanical efficacy of Indian
hysterophorus and Chromolaena odorata were examined by Sukanya et al. in the year,
2009 against clinical bacteria (Escherichia coli and Staphylococcus aureus) and
assessed the activity of five traditional Indian medicinal plants (Delonix elata,
used for treatment of various diseases against two gram positive (Bacillus subtilis,
strains. In vitro antibacterial activity of crude leaf extracts of three plants, Tecoma stans,
Coleus forskohlii and Pogostemon patchouli commonly practiced medicinal plants in the
villages of Salem District, Tamilnadu (India) was investigated by Senthil Kumar et al.
Maximum growth inhibition and effectiveness was remarkably observed in the extracts of
Coleus forskohlii, Pogostemon patchouli and then in Tecoma stans. Abhilash et al., in
the same year evaluated seven Indian plant extracts Moringa oleifera (Murungai) , Musa
coli, Bacillus subtilis, Vibrio cholerae and Klebsiella pneumoniae. The ethanolic extract
of Azardiratica indica showed considerably high activity against Escherichia coli than
other extracts. Leaf extracts of Aloe vera, Datura stromonium, Pongamia pinnata,
Lantana camara , Calotropis procera was studied by Johnson et al. in the year 2011 for
concluded that Aloe vera had the highest and strong activity against S.aureus and E.coli.
Lanata camara does not show antibacterial and antifungal activity. Pongamia pinnata
aqueous extract showed better anti E.coli activity than its alcoholic extract. Datura
stramonium showed better activity against Staphylococcus aureus & showed little anti
Staphyloccus aureus & E.coli and did not show anti-Aspergillus activity.
Ethno medicinal use of Parkia species have been reported by many authors
(Irvine, 1961; Sinha, 1996; Nacoulma, 1999; Bhardwaj and Gakhar, 2005; Majumder et
al., 2009). Antibacterial and antifungal properties of P. biglobosa and P. bicolor were
reported by Ajaiyeoba (2002). The screening was done using standard strains of
Aspergillus niger and Candida utilis and broad spectrum activity of the extracts was
reported.
authors (Chitralekha et al., 1964; Jain, 1991; Manandhar and Manandhar, 2002; Goyal &
Singh, 2005; Saini et al., 2007). Anti helminthic activity of E. nummularius has been
reported by Dash et al., (2003). Pavithra et al., (2009), studied antibacterial properties of
E. nummularius. According to them the methanol extract was found to be active against
Escherichia coli and Bacillus subtilus and MIC values were found to be 12.50 mg/ml and
The plant material, stem bark and leaves of Parkia javanica (P. javanica) and
whole plant of Evolvulus nummularius (E. nummularius) were air-dried in shade and
5 kilo gram (kg) grinded powder of P. javanica was extracted in 15 liter (lit) of
methanol by percolation method. The mixtures were kept for 2-5days in tightly sealed
vessels at room temperature at 220C, protected from sunlight. This mixture was filtered
through muslin cloth and the residue, was adjusted to the required concentration (15 lit of
methanol for the residue of 5 kg of powdered plant material) with the extraction fluid for
further extraction. Further extraction of the residue was repeated 3-5 times until a clear
colorless supernatant extraction liquid was obtained indicating that no more extraction
5.0 lit of methanol by percolation method. The mixtures were kept for 2-5days in tightly
sealed vessels at room temperature. This mixture was filtered and processed as mentioned
above.
methanol. After drying, the residues were weighed in order to know the amount of extract
of each individual plant and percentage of yield. The % yield of crude plant extract was
% yield of crude plant extract = (Wt. of dry extract) × 100/ Wt. of sample powder taken
After rota evaporation 560 g of gummy residue of P. javanica was obtained giving
11.2 % yield and 25 g of gummy residue of E. nummularius was obtained giving 1.67 %
yield.
After solvent removal, the extract was successively partitioned between water
(H2O) and benzene (Bz), H2O and chloroform (CHCl3), and H2O and n-butanol (BuOH).
The fractions were concentrated following the same procedure using rotavapor (Natrori et
al., 1981). These fractions along with the crude methanol part were sterilized by passing
The calculated amount of each dried plant extract obtained from different solvents
were dissolved in a calculated volume of 1:1 ethanol/ DMSO solvent (used as control) in
the ratio of 500mg/ml to make stock solution and were used for determination of Zone of
Culture medium used for growth of bacteria was Nutrient broth, Nutrient Agar,
Bacterial strains used during the investigation were kind gift of Dr. Prasanta Bag,
Micrococcus luteus, and three standard strains of gram negative bacteria, Pseudomonas
The stock culture of each organism was prepared by taking two nutrient agar
slants and sub culturing each confirmed test organism aseptically. One set slant was kept
as stock culture and another as working set. The cultures of bacteria in their appropriate
agar slants were stored at 4°C and used as stock cultures. One counter glycerol stock was
Antibacterial activity of plant extracts was determined by using the agar cup-plate
diffusion and dilution method (Cruickshank et al., 1975; Sousa et al., 1991). On each
sterile Nutrient Agar plate 100 l of bacterial suspension was added by a glass spreader.
On each dry bacteria seeded plate wells of 5 mm diameter was made by a sterilized micro
tip and solution (25 μl) of the extract was added on each well and on one well only
solvent was used as a control. The plates were kept in this condition for 30 minutes so
that solutions in the wells can spread through the agar medium. Then they were incubated
at 37oC for 24 hr. After this period, the positive antibacterial activity was ascertained by
Two fold serial Dilution Technique was used to determine the Minimum
Inhibitory Concentration (MIC). This technique was recommended by WHO (1991) and
used successively by other groups (Devkota et al., 2000; Pokharel 2000 and Devkota and
Dutta 2001).
3.4. RESULTS
nummularius, was carried out on three gram positive bacteria namely Bacillus subtilis,
Staphylococcus aureus, Micrococcus luteus and three gram negative bacteria namely
The extraction was first done with methanol and further fractionated with water
(H2O) and organic solvents- benzene (Bz), chloroform (CHCl3), and n-butanol (BuOH)
according to increasing polarity. The activity of each fraction was separately determined.
The result of antibacterial activity of the crude methanol extracts of P. javanica and E.
nummularius are summarized in Table 3.1 and Table 3.2. Crude extract of P. javanica
showed activity against all the three strains of gram positive and one gram negative
bacteria (P. aeruginosa) (Table 3.1). E. nummularius crude extract showed activity
against two gram positive (Bacillus subtilis, Micrococcus luteus) and two gram negative
bacteria (Pseudomonas aeruginosa, Escherichia coli) (Table 3.2). Therefore both the
plant extracts were active against both gram positive and gram negative bacteria. The
plant extracts showed maximum activity against P. aeruginosa (more than 15 mm)
(Table 3.1, Table 3.2). Klebsiella aerogens did not show any sensitivity towards any
extract (Table 3.1, Table 3.2). In addition, E. nummularius was also inactive against
Activity of all the three fractions of P. javanica crude extract i.e., benzene,
chloroform and n- butanol soluble fractions, are presented in Table 3.3, 3.4, and 3.5. P.
javanica benzene extract showed activity against B. subtilis (16 mm), S. aureus (13 mm),
M. luteus (18 mm) and P. aeruginosa (21 mm) at 250 mg/ ml. Chloroform fraction
exhibited activity against B. subtilis (18 mm), S. aureus (11 mm), M. luteus (18 mm) and
P. aeruginosa (20 mm) at 250 mg/ ml. Similarly, butanol fraction showed activity against
B. subtilis (17 mm), S. aureus (15 mm), M. luteus (18 mm) and P. aeruginosa (21 mm) at
250 mg/ ml. No activity was observed against bacteria K. aerogens and E. coli. Bacteria
P. aeruginosa showed maximum sensitivity towards all the three fractions of P. javanica.
Activity of the fractions are similar to the activity of the crude extract.
and n- butanol soluble fractions, are presented in Table 3.6, 3.7, and 3.8. Benzene soluble
fraction of E. nummularius crude extract showed activity against B. subtilis (10 mm) and
P. aeruginosa (14 mm) at 250 mg/ ml. Chloroform soluble extract showed against B.
subtilis (11 mm) at 250 mg/ ml. The butanol soluble fraction showed little activity against
M. luteus (8 mm), P. aeruginosa (10 mm) and E. coli (8 mm) at 250 mg/ ml.
The minimal inhibitory concentration (MIC) values of crude extracts and its
WHO. The values are presented in Table 3.9. The lowest the MIC value means highest
the activity. The lowest MIC value in case of P. javanica extracts was found against
bacterium P. aeruginosa (5 mg/ ml), in case of E. nummularius lowest MIC value was
Table 3.1. Antibacterial activity of P. javanica methanol (MeOH) extract against different
bacterial strains.
1 0 0 0 0 0 0
5 0 0 0 13 ± 0.158 0 0
10 0 0 0 14 ± 0.158 0 0
20 0 10 ± 0.158 0 16 ± 0.158 0 0
bacterial strains.
1 0 0 0 0 0 0
5 0 0 0 0 0 0
10 0 0 0 0 0 0
20 0 0 0 0 0 0
40 11 ± 0.158 0 0 12 ± 0.418 0 0
60 11 ± 0.158 0 0 12 ± 0.224 0 0
Table 3.3. Antibacterial activity of P. javanica benzene (Bz) extract against different
bacterial strains.
5 0 0 0 12 ± 0.224 0 0
10 0 0 0 13 ± 0.158 0 0
20 11 ± 0.418 10 ± 0.158 0 15 ± 0 0 0
(mg/ml)
B. subtilis S. aureus M. luteus P. K. E.
aeruginosa aerogens coli
1 0 0 0 0 0 0
5 0 0 0 13 ± 0 0 0
10 0 0 0 14 ± 0.158 0 0
20 0 0 0 15 ± 0.158 0 0
Table 3.5. Antibacterial activity of P. javanica n-butanol (BuOH) extract against different
bacterial strains.
(mg/ml)
B. subtilis S. aureus M. luteus P. K. E.
aeruginosa aerogens coli
1 0 0 0 0 0 0
5 0 0 0 14.5 ± 0 0 0
10 0 0 0 15 ± 0.224 0 0
20 0 0 0 17.5 ± 0.158 0 0
40 10 ± 0.418 11 ± 0.224 0 19 ± 0 0 0
bacterial strains.
aerogens
1 0 0 0 0 0 0
5 0 0 0 0 0 0
10 0 0 0 0 0 0
20 0 0 0 0 0 0
40 0 0 0 9.5 ± 0.224 0 0
60 0 0 0 10 ± 0.418 0 0
80 9 ± 0.158 0 0 11 ± 0.5 0 0
bacterial strains.
1 0 0 0 0 0 0
5 0 0 0 0 0 0
10 0 0 0 0 0 0
20 0 0 0 0 0 0
40 0 0 0 0 0 0
60 0 0 0 0 0 0
80 10 ± 0.158 0 0 0 0 0
100 10 ± 0.158 0 0 0 0 0
250 11 ± 0.224 0 0 0 0 0
bacterial strains.
aeruginosa aerogens
1 0 0 0 0 0 0
5 0 0 0 0 0 0
10 0 0 0 0 0 0
20 0 0 0 0 0 0
40 0 0 0 0 0 0
60 0 0 0 0 0 0
P. javanica 40 20 80 5 -
methanol extract
P. javanica 20 20 100 5 -
benzene extract
P. javanica 40 40 100 5 -
chloroform
extract
P. javanica 40 40 100 5 -
butanol extract
E. nummularius 40 - 80 40 80
methanol extract
E. nummularius 80 - - 40 -
benzene extract
E. nummularius 80 - - - -
chloroform
extract
E. nummularius - - 80 80 80
butanol extract
3.5. DISCUSSION
The presence of anti bacterial substances in the higher plants is well established
(Srinivasan, 2001). Being situated in one of the megadiversity region of the world, the
state of Tripura is bestowed with the rich resource of medicinal plants. Two medicinal
plants Parkia javanica and Evolvulus nummularius with known ethnomedicinal uses
(sections 2.1.4 and 2.2.4), had been undertaken to investigate their medicinal properties
To study the antibacterial properties, stem bark and leaves of Parkia javanica (P.
javanica) and whole plant of Evolvulus nummularius (E. nummularius) were used. Plant
materials was first extracted in methanol and subsequently fractioned in different solvents
parts of both the plants were tested against six bacterial strains, out of which three were
Gram positive bacteria viz. Micrococcus luteus, Staphylococcus aureus and Bacillus
subtilis and three were Gram negative bacteria namely Pseudomonas aeruginosa,
Klebsiella aerogens and Escherichia coli. Staphylococci are primarily responsible for
suppurative lesions in the human body, such as boils, pimples, carbuncle, fruncle,
as well as other infections. One aerobic spore former, Bacillus subtilis, Gram positive,
included in the study are widely distributed in soil, dust, milk and decomposing organic
matter. Among Gram negative enteric bacteria used in the study Escherichia coli are
normal inhabitants of human intestinal tract. They are occasionally pathogenic to man.
Pseudomonas aeruginosa, the other gram negative strain included are commonly
involved in urinary tract infections. Klebsiella aerogens, a non pathogenic Gram negative
bacteria, used for study is non pathogenic by itself but, is closely related to pathogenic
strain Klebsiella pneumoniae which causes infections that include urinary tract, lower
The activity of plant extracts was determined by using agar cup- plate diffusion
method (Kavanagh, 1972) measuring the inhibition zone in mm. For the study extraction
was first done with methanol and then fractionated with different solvents such as
The crude as well as fractionated extracts of both the plants were found more
effective against Gram positive strains compared to Gram negative ones. All the Gram
positive strains, Micrococcus luteus, Staphylococcus aureus and Bacillus subtilis, were
sensitive towards the extracts. Out of three Gram negative strains, only Pseudomonas
aeruginosa, was found to be sensitive. Various workers have already shown that Gram
positive bacteria are more susceptible towards plant extracts as compared to Gram
negative bacteria (Lin et al., 1999; Parekh and Chanda, 2006). These differences may be
attributed to the differences in the composition of cell walls (Yao et al., 1995).
Interestingly, in both the cases maximum activity was observed against P. aeruginosa,
All the solvent fractions of Parkia javanica plants showed almost similar
activities to that of the crude methanol extract. However, methanolic extract showed the
maximum activity. Phytochemical analysis (Dinda et al., 2009) of the P. javanica extract
glucosides along with other mixture of compounds. Regarding fraction distribution of the
compounds, β-sitosterol was found in the lowest polar benzene fraction and iridoid
glucosides, ursolic acid in the highly polar n-butanol fraction. All these compounds are
reported to have antimicrobial activities (Gupta et al., 1980; Davini et al., 1986; Ramesh
et al., 2001; Shokeen et al., 2005). As all the fractions were found to possess anti
bacterial principles, the antibacterial activity of all the fractions may be contributed to
principles they possessed. However, the higher activity of the crude methanolic extract
could be due to the synergistic effect of all the active principles (Bai, 1990).
Regarding E. nummularius, the results of the present study corroborate with the
findings on antibacterial activity reported by Pavithra et al., (2009). The strains, they
used were S. aureus, B. subtilis, P. aeruginosa, K. pneumoniae and E. coli. Out of five
strains, E. coli and B. subtilis were reported to be sensitive with MIC values 12.5mg/ ml
and 3.125 mg/ml respectively. In the present study also, E. coli and B. subtilis were found
sensitive however with higher MIC values, 80 mg/ml and 40 mg/ml respectively. P.
aeruginosa was found to be resistant to the extract. However, in the present study, P.
aeruginosa was found sensitive with MIC value, 40mg/ml. The differences observed
between the studies may be attributed to the differences existing in plants growing in
ecologically different areas. Indeed, Lucas and Lewis, (1994) had indicated that some
Therefore, the differences can logically be explained for the simple reason that plants had
not been collected from the same area and at the same time of the plant growth.
the presence of β-sitosterol and its glucoside, stigmasterol, d-mannitol, ursolic acid and
its isomer oleanolic acid (pentacyclic triterpene acid). Therefore the antimicrobial activity