discussion -...
TRANSCRIPT
Chapter V
Discussion
DISCUSSION
Out of the sixty one selected medicinal plants screened for
antibacterial activities, all of them were found to have inhibitory
action on bacteria in one or other extract. Certain plants showed
strong antibacterial activities, while others were found to have
moderate activity and only a few showed negligible effects.
The plants which showed least antibacterial effect in
aqueous and alcoholic extracts were Derris indica, Mentha piperita,
Tribulus terrestris, Achyranthes aspera and Emilia sonchifolia,
which showed inhibitory effect to only less than three among the
fourteen bacterial species tested. When compared with the other
plants tested, these plants, though they possess some inhibitory
effects, in one or other extracts, can be considered as plants with
negligible antibacterial activity. Among these plants the
antibacterial activity of Achyranthes aspera against Gram positive
bacteria was reported by Valsaraj et al., (1997).
Aqueous and alcoholic extracts of Thottea siliquosa, Aloe
vera, Mimosa pudica, Vetiveria zizanoides, Emblica officinalis,
Crocus sativus, Santalum album, Pterocarpus santalinus and
Phyllanthus niruri were also found to have lesser antibacterial
128 Chapter V
effects, when compared with others. Phyllanthus niruri had been
reported for antibacterial effect by E Thomas et al., (1999). The
antimicrobial activity against Gram positive and Gram negative
bacterial species by Thottea siliquosa was reported by Valsaraj et al.,
(1997). Agarry et al., (2005) reported the antimicrobial activity of
Aloe vera.
Highest broad spectrum activity was exhibited by Punica
granatum, among the tested plants. Plants like Ocimum sanctum,
Camelia sinensis, Psidium guajava, Cinnamomum verum,
Tabernemontanum divericata, Calotropes procera, Alstonia
scholaris, Pedilanthus thythamoides, Allium sativum, Holarrhena
antidysenterica, Eugenia caryophyllata and Myristica fragrans
showed higher antibacterial activities in either aqueous or alcoholic
preparations.
Other plants showed moderate activity against the fourteen
bacterial species tested.
Among the ten plants whose ethyl acetate and ether extracts
showed more activity than aqueous and alcoholic extracts, against
the bacterial species, Zingiber officinale showed least activity.
Curcuma aromatica and Glycosmis cochinsinensis also produced
Discussion 129
negligible effect. Highest activity was exhibited by Kaempferia
galanga and Curcuma decipiens. Ether and ethyl acetate extracts of
other five plants showed moderate broad spectrum activity in either
of the extracts.
Effects against Gram positive bacteria
It is interesting to note that all plants except Derris indica
inhibited any of the three Staphylococcus species tested. Alcoholic
extract of Alstonia scholaris, Leucas aspera, Eupatorium odoratum,
Aloe vera, Coleus aromaticus, Murraya koenigii, Cymbopogon
citratus, Myristica fragrans, Curcuma longa, Trichosanthus
cucumerina, Cinnamomum verum and Calotropes procera and
aqueous extract of Aristolochia indica, Allium sativum, Pedilanthus
thythamoides and Saraca indica showed considerably large zone of
inhibition of growth of Staphylococcus aureus. The highest activity
against S.aureus was shown by the aqueous and alcoholic extract of
Punica granatum. Psidium guajava, Camelia sinensis, Eugenia
caryophyllata, Curcuma longa and Holarrhena antidysenterica
extracts were found to have high inhibitory activity. The ether extract
of Cyperus rotundus, Ricinus communis and ethyl acetate extract of
Tinospora cordiofolia and Ocimum tenniflorum also exhibited
moderate inhibition towards S.aureus.
130 Chapter V
Maximum activity against Staphylococcus epidermidis was
exhibited by alcoholic extract of Punica granatum and Psidium guajava.
Antibacterial activity of alcoholic extract of Lawsonia alba, Alstonia
scholaris, Leucas aspera, Coleus aromaticus, Murraya koenigii,
Cymbopogon citratus, Holarrhena antidysenterica, Saraca indica,
Myristica fragrans, Cyclea peltata, Eugenia caryophyllata and
Trichosanthus cucumerina were significant as they produced an
inhibition zone above 10 mm. Aqueous extract of Trichosanthus
cucumerina, Curcuma longa, Saraca indica and Pedilanthus
thythamoides also produced a considerable inhibition to
Staphylococcus epidermidis. Ether extract of Kaempferia galanga,
Cyperus rotundus, Ricinus communis and ethyl acetate extract of
Tinospora cordifolia showed moderate inhibitory effects to the
organism, as in the case of aqueous extract of Camelia sinensis and
alcoholic extract of Calotropes procera.
Staphylococcus saprophyticus, which is an opportunistic
pathogen was inhibited by many plant extracts. Alcoholic extract of
Psidium guajava, Calotropes procera, Alstonia scholaris, Leucas
aspera, Murraya koenigii, Eugenia caryophyllata, Trichosanthus
cucumerina; aqueous extract of Pedilanthus thythamoides, Allium
sativum and Camelia sinensis showed inhibitory zone more than
Discussion 131
10mm. Here also aqueous and alcoholic extract of Punica granatum
showed maximum activity, that is more than 20mm. Tamarindus indica,
Mentha piperita, Pimenta dioica, Coleus aromaticus, Moringa oleifera,
Emilia sonchifolia, Derris indica, Saraca indica, Sida retusa, Mimosa
pudica, Vetiveria zizanoides, Citrus aurentium, C.aurantifolia, Crocus
sativus, Santalum album, Pterocarpus santalinus, Achyranthes aspera
and Tribulus terrestris showed no inhibition either in aqueous or in
alcoholic extract. The ethyl acetate or ether extract of Glycosmis
cochinsinensis, Ricinus communis, Zingiber officinale, Ocimum
sanctum, Camelia sinensis, Psidium guajava also had no activity
against Staphylococcus saprophyticus.
Growth of Streptococcus faecalis which is a commensal of
human intestine was inhibited by aqueous and alcoholic extracts of
Alstonia scholaris, Coleus aromaticus, Moringa oleifera, Holarrhena
antidysenterica, Ocimum sanctum, Cinnamomum verum, Punica
granatum; aqueous extract of Allium sativum and Curcuma longa and
alcoholic extracts of Citrus aurentium, C.aurantifolia and Eugenia
caryophyllata. S.faecalis was found to be susceptible to the ethyl
acetate extract of Carica papaya and Ocimum tenniflorum, while
Streptococcus viridans, a human throat commensal was found to be
susceptible to aqueous and alcoholic extracts of Leucas aspera, Emilia
132 Chapter V
sonchifolia, Psidium guajava, Camelia sinensis and Cyclea peltata;
aqueous extract of Aristolochia indica and Ocimum sanctum, ethyl
acetate and ether extract of Ocimum tenniflorum and Cyperus
rotundus. Ethyl acetate extract of Camelia sinensis and ether extract of
Ocimum sanctum also inhibited it.
Bacillus cereus, an organism which can cause food poisoning
was showing susceptibility to aqueous and alcoholic extracts of
Tabernemontanum divericata, Alstonia scholaris, Leucas aspera,
Eupatorium odoratum, Allium sativum, Allium cepa, Pimenta dioica,
Moringa oleifera, Myristica fragrans, Psidium guajava and Punica
granatum; alcoholic extract of Cymbopogon citratus, Holarrhena
antidysenterica, Citrus aurentium, Citrus aurantifolia, Syzygium cumini,
Curcuma longa, Cyclea peltata, Eugenia caryophyllata and Santalum
album and ethyl acetate extract of Ricinus communis, Ocimum
sanctum, Camelia sinensis and Cinnamomum verum. The ether
extract of Ocimum sanctum and Psidium guajava inhibited the growth
of Bacillus cereus. Maximum activity was shown by Allium sativum.
Bacillus subtilis, a saprophytic Gram positive sporulating
bacillius was found to be susceptible to Aegle marmelos,
Tabernemontanum divericata, Alstonia scholaris, Pedilanthus
thythamoides, Leucas aspera, Eupatorium odoratum, Allium cepa,
Discussion 133
Pimenta dioica, Moringa oleifera, Holarrhena antidysenterica, Sida
retusa, Mimosa pudica, Myristica fragrans, Hemidesmus indicus,
Ocimum sanctum, Cinnamomum verum, Trichosanthus cucumerina,
Calotropes procera and Punica granatum in aqueous and alcoholic
extract. The growth was inhibited by ether extract of Calotropes
procera, Cinnamomum verum, Psidium guajava and Camelia sinensis;
ethyl acetate extract of Ocimum sanctum; aqueous extract of
Azadirachta indica, Derris indica and Vetiveria zizanoides; alcoholic
extract of Citrus aurentium, Citrus aurantifolia, Emblica officinalis,
Crocus sativus, Eugenia caryophyllata, Santalum album, Achyranthes
aspera and Plumbago zeylanica; ether extract of Carica papaya,
Camelia sinensis, Psidium guajava, Cinnamomum verum and
Calotropes procera and ethyl acetate extract of plants like Ricinus
communis ,Curcuma decipiens and Ocimum sanctum.
Many workers reported the antimicrobial activity of plant extracts
to Gram positive bacteria. In a study of antimicrobial properties of
seventy eight plants, Valsaraj et al., (1997) observed that alcoholic
extracts of Alstonia scholaris, Calotropes gigantea, Leucas aspera,
Tinospora cordifolia, Cypers rotundus, Cymbopogon citratus, Murraya
koenigii and Cyclea peltata were inhibitory to Staphylococcus aureus.
The antistaphylococcal effect of aqueous, alcoholic and ethyl acetate
134 Chapter V
extract of Tinospora cordifolia was reported by Rajurkar and
Vadlamudi, (2002). According to E Thomas et al., (1999) alcoholic
extracts of Leucas aspera and Murraya koenigii were inhibitory to
Staphylococcus aureus. Activity against Staphylococcus aureus by
Curcuma longa was reported by Sasidharan et al., (1998) and
Banerjee and Nigam (1978). Myristica fragrans was found to possess
antimicrobial properties against Gram positive bacteria by Dorman and
Deans, (2000). Cinnamomum verum was reported to have anti
Staphylococcus aureus activity by Sasidharan et al., (1998). Psidium
guajava was observed for very high activity against S.aureus by Gnan
and Demello, (1999). Holarrhena antidysenterica was reported to
possess anti S.aureus activity by Asima and Branter, (1999). According
to Toda et al., (1999), Camelia sinensis could inhibit even MRSA. Ether
extract of Kaempferia galanga also was observed for
antistaphylococcal activity by E Thomas et al., (1996).
Staphylococcus epidermidis was found to be inhibited by
Lawsonia alba, Azadirachta indica, Leucas aspera, Phyllanthus niruri,
Murraya koenigii and Ocimum sanctum as in the case of
Staphylococcus aureus according to E Thomas et al., (1999). Deans
and Ritchie, (1987) reported that Cinnamomum verum, Citrus
aurantifolia and Eugenia caryophyllata oils inhibited Streptococcus
Discussion 135
faecalis, while Zingiber officinale, Myristica fragrans and Citrus
aurentium could not inhibit it. Streptococcus mutans was found to be
inhibited by Terminalia chebula (Jagtap and Karkera, 1999). Gnan and
Demello, (1999) reported that Psidium guajava extracts had inhibitory
activity on Streptococcus pyogenes.
Bacillus cereus was reported to be inhibited by Allium sativum
(Saleem and Al-Delaimy, 1982) and Luffa tuberosa (Kulkarni et al.,
1992). Reports are available for the inhibition of Bacillus subtilis by
plant extracts of Curcuma caesia (Garg and Jain, 1998), Cinnamomum
verum, Eugenia caryophyllata [Deans and Ritchie, (1987); Agnihotri
and Vaidya, (1996)] and Feronia elephantum (Garg, 2001).
Valsaraj et al., (1997) reported that alcoholic extracts of Achyranthes
aspera, Alstonia scholaris, Thottea siliquosa, Calotropes gigantea,
Hemidesmus indicus, Cassia fistula, Carica papaya, Cyperus rotundus,
Leucas aspera, Sida rhombifolia, Cyclea peltata, Tinospora cordifolia,
Moringa oleifera, Cymbopogon citratus, Aegle marmelos and Murraya
koenigii inhibited the growth of Bacillus subtilis.
It is interesting to note that the plant extracts were found more
effective against Gram positive bacteria rather than Gram negative
bacteria.
136 Chapter V
Effects against Gram negative bacteria
Seven Gram negative bacterial species were tested for
susceptibility towards various extracts of sixty one plants.(Table IX
and X).
Escherichia coli was found to be inhibited by aqueous and
alcoholic extracts of plants like Aegle marmelos, Tabernemontanum
divericata, Eupatorium odoratum, Tamarindus indica, Allium
sativum, Allium cepa, Murraya koenigii, Azadirachta indica, Moringa
oleifera, Cymbopogon citratus, Holarrhena antidysenterica, Myristica
fragrans, Curcuma longa, Hemidesmus indicus, Eugenia
caryophyllata, Trichosanthus cucumerina, Achyranthes aspera,
Phyllanthus niruri, Tribulus terrestris, Ocimum sanctum, Camelia
sinensis, Psidium guajava, Cinnamomum verum, Calotropes procera
and Punica granatum. Among these, highest activity (more than 20
mm) was shown by Punica granatum.
Ocimum sanctum was the next plant with considerable effect
against Escherichia coli. Aqueous extract of Mentha piperita;
alcoholic extract of Lawsonia alba, Alstonia scholaris, Thottea
siliquosa, Pimenta dioica, Cassia fistula and Syzigium cumini; ether
and ethyl acetate extract of Tinospora cordifolia, Curcuma
aromatica, Cyperus rotundus and Zingiber officinale; ether extract of
Discussion 137
Kaempferia galanga; ethyl acetate extract of Carica papaya,
Glycosmis cochinsinensis, Punica granatum and Curcuma decipiens
etc., also showed inhibitory activity. An inhibition zone ranging from
10-20 mm was shown by Eupatorium odoratum, Tamarindus indica,
Allium sativum, Murraya koenigii, Moringa oleifera, Cymbopogon
citratus, Holarrhena antidysenterica, Hemidesmus indicus, Eugenia
caryophyllata, Phyllanthus niruri, Tinospora cordifolia, Curcuma
decipiens, Cinnamomum verum and Calotropes procera. Inhibitory
action of various plant extracts to E.coli were reported for Ocimum
sanctum (Geetha et al., 2001), Allium sativum [Al-Delaimy and Ali,
(1970), Elizabeth,(2001)], Feronia elephantum (Garg 2001), Eugenia
caryophyllata [J Briozzo et al., (1988), Agnihotri and Vaidya, (1996),
Deans and Ritchie, (1987)], Cinnamomum verum [Agnihotri and
Vaidya, (1996), Deans and Ritchie (1987)], Myristica fragrans
(Deans and Ritchie,1987), Delonix elata and D. regia (Seetharam et
al., 2002), Evolvulus alsinoids (Purohit et al., 1994), Curcuma caesia
(Garg, 1998), Euphorbia hirta, Lawsonia alba and Phyllanthus niruri
(E Thomas et al.,1999), Alstonia scholaris, Thottea siliquosa, Cassia
fistula, Cyperus rotundus, Tinospora cordifolia, Cymbopogon
citratus, Aegle marmelos, Murraya koenigii (Valsaraj et al., 1997),
Tamarindus indica, Moringa oleifera, Eupatorium odoratum,
138 Chapter V
Cinnamomum verum, Cymbopogon citratus (Sasidharan et al.,
1998) etc.
Achyranthes aspera, leaf of Hemidesmus indicus and Leucas
aspera, seed of Carica papaya, root of Cyclea peltata, stem bark of
Moringa oleifera etc., in the study of Valsaraj et al.,(1997) were
found to be ineffective against E.coli at the tested concentration.
Aqueous extract of Aegle marmelos leaf was observed ineffective
against E.coli at 5% concentration, by Sasidharan et al., (1998).
Klebsiella pneumoniae, a high risk pathogen was inhibited
only by a lesser number (fourteen out of sixty one) of plants. Punica
granatum showed the maximum activity against this notorious
pathogen, among the plants screened. Considerable activity was
exhibited by alcoholic extract of Murraya koenigii and ether extract of
Ricinus communis with an inhibition zone diameter of above 10 mm.
Other plants which showed activity were Aegle marmelos,
Tabernemontanum divericata, Moringa oleifera, Saraca indica,
Cassia fistula, Hemidesmus indicus, Trichosanthus cucumerina and
Ocimum sanctum in aqueous or alcoholic extract, Curcuma
aromatica, Cyperus rotundus and Kaempferia galanga in ether or
ethyl acetate extract.
Discussion 139
Many of the plant extracts were observed ineffective against
Klebsiella pneumoniae. Rabe and Van Staden, (1997) in a study of
twenty one plants for antibacterial properties, found out that none of
the plants were inhibiting K. pneumoniae in methanolic extract, and
only one was showing activity against Escherichia coli. Klebsiella
pneumoniae was reported to be inhibited by the aqueous extract of
Ocimum sanctum better than the alcoholic extract by Geeta et al.,
(2001). J Briozzo et al., (1989) reported the anti-klebsiella effect of
Eugenia caryophyllata oil. Curcuma longa was observed inhibitory
to Klebsiella pneumoniae by Anup Banerjee and Nigam, (1978).
E Thomas et al., (1999) reported that Murraya koenigii was the only
plant which inhibited Klebsiella pneumoniae out of the twenty one
plants tested.
Serratia marcescens, a pigmented Gram negative bacillus
can cause infections in human beings, mainly in urinary tract. It was
found that aqueous and alcoholic extract of plants like Aegle
marmelos, Lawsonia alba, Tabernemontanum divericata, Alstonia
scholaris, Pedilanthus thythamoides, Eupatorium odoratum,
Tamarindus indica, Pimenta dioica, Cymbopogon citratus,
Holarrhena antidysenterica, Saraca indica, Sida retusa,
Hemidesmus indicus, Eugenia caryophyllata, Trichosanthus
140 Chapter V
cucumerina, Plumbago zeylanica, Ocimum sanctum, Camelia
sinensis, Cinnamomum verum, Calotropes procera and Punica
granatum inhibited this bacterium. Ether extract of Camelia sinensis,
Carica papaya, Tinospora cordifolia, Ricinus communis and
Myristica fragrans; ethyl acetate extract of Kaempferia galanga,
Ocimum sanctum and Punica granatum; alcoholic extract of
Eupatorium odoratum, Myristica fragrans, Citrus aurentium, Citrus
aurantifolia, Emblica officinalis, Cyclea peltata and aqueous extract
of Curcuma longa were capable of preventing the growth of Serratia
marcescens up to certain extent. Punica granatum showed high
activity both in aqueous and in alcoholic preparations. Serratia
marcescens was reported to be inhibited by oils from Eugenia
caryophyllata, Cinnamomum verum, Myristica fragrans, Mentha
peperita and Zingiber officinale (Deans and Ritchie, 1987).
According to Kulkarni et al., (1992) Luffa tuberosa inhibited Serratia
marcescens. Gnan and Demello, (1990) reported that aqueous
extract of Allium sativum, Comnelina beghlensis and Psidium
guajava had no inhibitory effect towards Serratia marcescens.
Salmonella typhi is a human pathogen causing enteric fever
which is a multisystem illness affecting many vital organs. Typhoid is
endemic in India and many epidemics occur in different parts of the
Discussion 141
country including Kerala. This bacterium was very effectively
inhibited by Punica granatum extracts and some other plant extracts
like aqueous and alcoholic extracts of Allium sativum, Pimenta
dioica, Cyclea peltata, Eugenia caryophyllata, Trichosanthus
cucumerina, Ocimum sanctum, Camelia sinensis, Cinnamomum
verum and Calotropes procera. Maximum activity was observed for
Camelia sinensis against this pathogen. Other than this, no other
plants showed inhibitory action against Salmonella in ethyl acetate
extract. Ether extracts showed negative results for all the plants
screened. Alcoholic extract of Myristica fragrans, Sida retusa and
Curcuma aromatica showed moderate inhibitory activity. Among the
sixty one plants screened, only thirteen plants showed anti
salmonella activity.
Extracts from plants were studied by researchers for inhibitory
effect on Salmonella typhi. Al – Delaimy and Ali, (1970) reported
that 4% fresh garlic extract inhibited growth of Salmonella typhi.
Elizabeth, (2001) also reported that Allium sativum extracts were
inhibitory to S.typhi. Agnihotri and Vaidya, (1996) reported the
inhibitory effect of Cinnamomum verum and Myristica fragrans on
S.typhi. Ciraj et al., (2001) in a study found out that twenty seven
142 Chapter V
strains of S.typhi out of sixty four were sensitive to 2% alcoholic
extract of Camelia sinensis.
Rajarajan et al., (2002) reported that there were no inhibitory
effect by Plectranthus ambonicus towards Salmonella typhi,
Salmonella paratyphi A and Salmonella paratyphi B. Salmonella
typhimurium was observed to be inhibited by Curcuma longa (Singh
et al., 2002), Ocimum sanctum (Geeta et al., 2001) and Allium
sativum (Elizabeth, 2001). It has been reported that Salmonella
gallinarum was inhibited by Tinospora cordifolia (Rajurkar and
Vadlamudi, 2002), Salmonella pullorum by Cinnamomum verum,
Eugenia caryophyllata, Myristica fragrans, Mentha piperita,
Pimenta dioica and Citrus aurantifolia (Deans and Ritchie, 1987)
and Salmonella paratyphi by Curcuma longa (A Banerjee and
Nigam, 1978) and Camelia sinensis (Ciraj et al., 2001).
Two species of the genus Proteus, namely Proteus vulgaris
and P. mirabilis were included in the preliminary screening. It was
observed that P.vulgaris was more resistant to plant extracts than
P.mirabilis as some plant extracts which inhibited P. mirabilis could
not inhibit P.vulgaris as in the case of Pimenta dioica, Saraca indica,
Cassia fistula, Curcuma longa, Cyclea peltata, Pterocarpus
santalinus, Trichosanthus cucumerina, Glycosmis cochinsinensis and
Discussion 143
Curcuma decipiens. Against this bacterium also maximum,
antibacterial effect was observed for Punica granatum.
Proteus mirabilis was found to be susceptible to the aqueous
and alcoholic extract of Ocimum sanctum by Geeta et al., (2001). Oil
of Feronia elephantum was reported to have inhibitory action on
Proteus vulgaris (Gary 2001). Plants like Cinnamomum verum,
Eugenia caryophyllata, Myristica fragrans, Pimenta dioica, Mentha
piperita(Deans and Ritchie, 1987) Delonix elata, D.regia (Seetharam
et al., 2002) and Evolvulus alsinoids (Purohit et al.,1995) also posses
antibacterial activity against P.vulgaris. Aqueous extract of Psidium
guajava was reported negative for inhibition of this bacillus, by Gnan
and Demello, (1999).
Pseudomonas aeruginosa is highly resistant to various
antibiotics and disinfectants and is notorious in hospital infections, as
in the case of Staphylococcus aureus and Klebsiella pneumoniae. It
was observed that plants like Allium sativum, Pimenta dioica, Eugenia
caryophyllata, Trichosanthus cucumerina, Glycosmis cochinsinensis,
Ocimum tenniflorum, Ricinus communis, Curcuma aromatica,
Curcuma decipiens, Zingiber officinale, Kaempferia galanga, Ocimum
sanctum, Camelia sinensis, Psidium guajava, Cinnamomum verum,
144 Chapter V
Calotropes procera and Punica granatum in one or other extract could
inhibit the growth of Pseudomonas in vitro. Commendable activity
was produced by Punica granatum against Pseudomonas. Reports
are available regarding different effects on Pseudomonas aeruginosa
by many plant extracts. Those having reported inhibitory activity
include Ocimum sanctum aqueous and alcoholic extract, (Geeta
et al., 2001) Feronia elephantum (Garg, 2001), Allium sativum
(Elizabeth, 2001), Eugenia caryophyllata [J Briazzo et al., (1989);
Agnihotri and Vaidya, (1996); Deans and Ritchie, (1987)],
Cinnamomum verum and Mentha piperita (Deans and Ritchie, 1987),
Delonix elata and D.regia (Seetharam et al.,2002), Evolvulus
alsinoids (Purohit et al.,1995), Alpinia galanga, Curcuma longa,
Curcuma caesia , Curcuma zedoana, Curcuma aromatica, Curcuma
decipiens, Kaempferia galanga, Kaempferia rotunda, Zingiber
casumunar, Zingiber officinale and Elettaria cardamomum (E Thomas
et al., 1996), Mutisia acuminata (Catalano et al., 1998) and Psidium
guajava (Gnan and Demello, 1999). Valsaraj et al., (1997) observed
antipseudomonas effects by alcoholic extracts of Alstonia scholaris,
Thottea siliquosa, Cassia fistula, Cyperus rotundus, Cyclea peltata,
Tinospora cordifolia, Moringa oleifera, Cymbopogon citratus, Aegle
marmelos and Murraya koenigii and no effect for Achyranthes aspera,
Discussion 145
Hemidesmus indicus, Carica papaya, Leucas aspera and Sida
rhombifolia. Ali –Shtayeh et al., (1997) reported that among the
organisms tested against extracts of Micromeria nervosa, the least
susceptible was Pseudomonas aeruginosa. Rana et al., (1998)
reported that Eupatorium adinophorum had negligible effect on
Pseudomonas. In these circumstances, the present results of
antipseudomonas activity of many plant extracts are promising.
Among the sixty one plants screened, results of six plants,
which were found to have inhibitory action against more than ten
bacterial species out of the fourteen species used in the study are
separately given (Table XI). The inhibitory effect of ethyl acetate or
ether extract of the forty five plants shown in Table IX was negligible
and so these are not included in the table. Like wise, the aqueous and
alcoholic extract results of ten plants showed in Table X are also not
included as these were negligible.
Antibacterial effects of Punica granatum
Among the plants tested in the present study, very promising
results were exhibited by Punica granatum. Its alcoholic or aqueous
extract inhibited all the tested bacterial species except Streptococcus
viridans. More over, the inhibition zone was larger for many bacteria
146 Chapter V
when compared with other plants with broad spectrum activity like
Ocimum sanctum, Camelia sinensis, Cinnamomum verum,
Calotropes procera etc. Different extracts of fruit peel of Punica
granatum in water, alcohol, ethyl acetate, acetone, ether and
chloroform were examined for antibacterial activity and as alcoholic
extract showed maximum activity, further tests were performed using
alcoholic extract against many strains of different bacterial species
including pathogens and commensals.
Pathogens isolated from various clinical specimens like urine,
pus, pus swab, throat swab, sputum, ear and nasal swabs, aspirates,
faeces, blood, bone marrow, cerebrospinal fluid, semen etc., were
included in the study. Organisms isolated from these samples include
Gram positive pathogens like Staphylococcus aureus, S.epidermidis,
S.saprophyticus and Streptococcus pyogenes; and Gram negative
pathogens like Escherichia coli, Klebsiella pneumoniae, Serratia
marcescens, Salmonella typhi, Shigella sonnei, Proteus mirabilis,
Pr.vulgaris and Pseudomonas aeruginosa. The number of strains
under each species from different specimen are separately shown in
Table XII to XVI. The results are analysed in detail in the following
pages.
Discussion 147
Thirty two strains of Staphylococcus aureus were highly
susceptible to the pomegranate extract among the hundred and
twenty seven strains (25.2%) exhibiting an inhibition zone above 20
mm. Ninety one strains showed considerable inhibition with a zone
diameter ranging from 11 to 20 mm (71.6%). Only four strains showed
negligible effect (3.1%). Among the pathogenic bacterial species
tested, S.aureus showed maximum susceptibility to the extract. Out
of the thirty two strains nine showed inhibition of 23 mm. More over,
all strains were inhibited by the extract. This is giving a new hope for
the development of a natural antibacterial against S. aureus.
Reports regarding the antibacterial effect of natural products
against large number of clinical strains of Staphylococcus aureus are
limited in number, though reports are available against standard
strains of S.aureus. Mathabe et al., (2006) reported that the methanol,
ethanol, acetone and aqueous extract of pomegranate were equally
effective against S.aureus. Braga et al., (2005) reported that
pomegranate extract inhibited S.aureus growth and subsequent
enterotoxin production. Elizabeth, (2001) reported that S.aureus was
not inhibited by Punica granatum pericarp, but in the present study all
the one hundred and twenty seven strains showed susceptibility,
among which 25% had very high susceptibility.
148 Chapter V
Staphylococcus epidermidis and Staphylococcus saprophyticus
strains (sixteen each) also exhibited large zone of inhibition of growth.
None showed inhibition below 10 mm. Fourteen and twelve strains
respectively showed higher inhibition (87.5% and 75%). Two strains
of S.epidermidis and four strains of S.saprophyticus had moderate
susceptibility. In case of Streptococcus pyogenes only two strains out
of six showed moderate inhibition while four showed negligible zone.
It was observed that the extract had higher activity towards
Staphylococcus species rather than Streptococcus species.
Reports of anti-streptococcal activities of pomegranate are
limited in number. Vasconcelos et al., (2006) suggest pomegranate
gel to control adherence of different microbes in the oral cavity
including Streptococcus mitis. Menezes et al., (2006) suggest
pomegranate extract as an alternative antibacterial for the treatment
of dental plaque caused by bacteria. No reports are available
regarding the activity to Streptococcus pyogenes.
Most of the natural products are acting against Gram positive
bacteria more effectively rather than Gram negative bacteria.
Products having activity against Gram negative bacteria and human
pathogens are limited in number (Banerjee and Nigam, 1978).
Punica granatum is a plant having antibacterial effects against Gram
Discussion 149
negative bacteria also along with the Gram positive bacteria. This is
evident from the results shown in Table XXIV.
Table XXIV: Effect of Punica granatum extract on different pathogenic bacterial species isolated from various samples.
No. of strains showing inhibition zone Sl.
No. Bacterial species No. of strains tested 0-10
mm 11-20 mm
21-30 mm
1. Staphylococcus aureus 127 4 91 32
2. Staphylococcus epidermidis 16 0 2 14
3. Staphylococcus saprophyticus 16 0 4 12
4. Streptococcus pyogenes 6 4 2 0
5. Escherichia coli 115 14 86 15
6. Klebsiella pneumoniae 67 8 55 4
7. Serratia marcescens 18 0 6 12
8. Salmonella typhi 12 0 10 2
9. Shigella sonnei 2 0 2 0
10. Proteus vulgaris 29 4 17 8
11. Proteus mirabilis 35 6 18 11
12. Pseudomonas aeruginosa 38 5 24 9
Total strains 481 45 317 119
150 Chapter V
In the case of Escherichia coli, 74.7 % of the total strains
showed moderate susceptibility while higher inhibition was observed
in 13% of the strains tested. Around same percentage showed
negligible susceptibility.
The effects of alcoholic extract of Punica granatum on
enterohaemorrhagic Escherichia coli has been already reported by
Voravuthikunchai et al., (2005). In the present study E.coli from
different specimens like urine, faeces, pus etc., were included so
that those which cause urinary tract infection (UTI), diarrhoea and
pyogenic infections are exposed to the extract. It is clear that E.coli
can be effectively controlled by P.granatum extract. In Indian and
other Asian folk practices, pomegranate is advised for diarrhoeal
diseases and UTI. Among the seven pathogenic E.coli strains isolated
from faecal sample tested, six were found to have inhibition zone
ranging from 11-20mm. Only one was resistant to the extract. This
justifies the old folk practice of using pomegranate to control
diarrhoea because different types of E.coli, like enterohaemorrhagic,
enteroinvasive or enteropathogenic can cause diarrhoea. In the case of
E.coli isolated from patients with UTI, 78% of the strains were showing
considerable inhibition as antibiotics like gentamicin, norfloxacin etc do.
Seventeen percent showed inhibition above 20 mm, like ciprofloxacin
Discussion 151
and cefotaxime against Gram negative bacteria. Only four strains
showed negligible zone out of which only two was completely resistant
to the extract. This indicates that a drug derived from the fruit peel
extract can be developed in the future for curing UTI caused by E.coli.
It is promising that other urinary pathogens also were effectively killed
by the extract. Out of the twenty one strains of E.coli isolated from pus,
twelve showed moderate zone, two were resistant and seven showed
negligible zone.
Klebsiella pneumoniae is a notorious bacterial pathogen in
drug resistance and nosocomial infection. (The recent neonatal
death incidences reported in a hospital at Thiruvananthapuram, was
attributed to this culprit!). In the present study, though only four
strains of this bacterial species were found to be inhibited with a
larger zone, fifty five strains out of sixty seven were moderately
inhibited by the extract. Six strains were totally resistant and two
showed negligible zone. Larger number of strains were isolated
from urine samples that is twenty nine, among which twenty three
had inhibition upto 20 mm and four with more than that. Most of the
natural products remain ineffective or possess slight inhibitory
effects on K.pneumoniae. But P.granatum showed a considerable
effect on this pathogen. The results of pus samples reveal that
152 Chapter V
K.pneumoniae causing pyogenic infections were also susceptible to
the pomegranate extract. Here three out of eighteen strains were
totally resistant, while the remaining fifteen exhibited (83%) growth
inhibition zone below 21 mm. No strain showed high susceptibility.
However 62.5% strains from respiratory specimens showed
moderate zone of inhibition and 37.5% strains showed complete
resistance. Here also as in the case of urinary pathogens, no strains
showed very large zone of growth inhibition. All the twelve strains
isolated from other samples showed moderate inhibition, none
showed resistance, none showed very high susceptibility.
Klebsiella pneumoniae was found resistant to methanolic
extracts of all the twenty one South African plants traditionally used
for therapy, by Rabe and Van Staden (1997). E Thomas et al.,
(1999) reported that among the plants tested for antibacterial activity
only Murraya koeinigii exhibited activity against Klebsiella
pneumoniae.
Serratia marcescens, which is a pigmented Gram negative
bacilli and can cause urinary tract infections showed high
susceptibility to the punica extract that is 67% of the total strains had
a growth inhibition zone above 20 mm and 33% showed moderate
inhibition. No strains were found to be resistant or with negligible
Discussion 153
effect. It is interesting to note that many of these strains were multi-
drug resistant. Results of the present study point to the development
of a new drug from pomegranate extract, as urinary pathogens
isolated in laboratories recently are multidrug resistant, including
S. marcescens.
Plate 12: Effect of different concentrations of the pomegranate extract on S. marcescens 1: control 2,3,4,12,13: lower concentrations 5,6: higher concentrations 7,8,9,10,11: medium concentrations
Salmonella typhi is a potential pathogen causing typhoid
which affects many vital organs. Twelve strains of S.typhi were
included in the study in which none showed resistance towards
Punica granatum extract and two strains were highly susceptible
with growth inhibition zone diameter above 20 mm. Ten strains
154 Chapter V
showed medium zone of inhibition. The finding is very relevant as
multi-drug resistance is very common in S.typhi. Rani and Khullar,
(2004) reported that P.granatum was effective in inhibiting multidrug
resistant S.typhi along with other plants like Myristica fragrans,
Ocimum sanctum etc.
Only two strains of Shigella sonnei, the bacilli causing
dysentery, were tested and these two showed medium zone of
inhibition. Alanis et al., (2005) reported the inhibitory effect of
P.granatum against Sh.sonnei and Sh.flexneri.
Two species of Proteus were checked in the study, that is
thirty five strains of Proteus mirabilis and twenty nine strains of
Proteus vulgaris. Out of these, eleven and eight strains remained
highly susceptible while eighteen and seventeen showed medium
zone of growth inhibition respectively. Three strains of P.vulgaris
and two strains of P.mirabilis were resistant to the extract. Two
strains of P.mirabilis exhibited narrow zone as in case of one strain
of P.vulgaris. Proteus mirabilis strains were found more susceptible
rather than P.vulgaris strains. No reports are available for the
inhibitory effect of Punica granatum to Proteus.
Discussion 155
Pseudomonas aeruginosa is notorious in hospital infections
as it is highly resistant to many of the routine antibiotics and
disinfectants used in hospitals. The analysis of results of present
study reveals that pomegranate extract is a very good remedy for
Pseudomonas infections, as 63% of strains tested showed medium
inhibition zone, 24% showed high susceptibility and only 13%
showed negligible zone of growth inhibition. In addition, no strain
showed complete resistance to the alcoholic extract of pomegranate.
These findings are very encouraging as in many researches natural
products having activity against Pseudomonas are very few in
number. Many workers observed that among the bacteria tested,
Pseudomonas was the least susceptible organism [R Rana et al.,
(1998); Ali-Shtayeh et al., (1997)].
When analyzing the sensitivity results of the bacterial strains
towards the commercially available antibiotics by disc diffusion
method, it was seen that many pathogenic strains were drug
resistant, and even showed multiple drug resistance. (The results
are shown in Table XII to XVI). So further experiments were
concentrated on multi drug resistant strains of five bacterial species.
156 Chapter V
Antibacterial effects against multi-drug resistant bacterial strains
For studying the effect of alcoholic extract of Punica
granatum, multidrug resistant strains of Staphylococcus aureus,
Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and
Pseudomonas aeruginosa were used as these are the common
clinical isolates which show multi-drug resistance. The diameter of
zone of inhibition exhibited by different strains of the above five
organisms are shown in Table: XVII. All Staphylococcus aureus
strains were sensitive to the extract with a minimum of 10 mm and a
maximum of 22 mm zone of inhibition of growth. Here two strains
showed 10 mm zone, fifteen strains showed 11-20 mm and three
strains showed inhibition diameter more than 20 mm. Out of these
twenty strains, five strains were resistant to cloxacillin also.
The potency of many natural products against S. aureus
including MRSA were already reported by different researchers
[Gnan and Demello,(1999); Toda et al., (1991); Geetha et al.,(2001);
Elizabeth,(2001); Valsaraj et al., (1997); Rajurkar and Vadlamudi,
(2002); Asima and Branter, (1999); Singh et al.,(2002); Arora and
Kaur, (1999)]. But reports regarding the inhibitory effect of
pomegranate on clinical strains of S.aureus which are multidrug
Discussion 157
resistant are very limited in number and researches are going on
very actively only during the recent years. Voravuthikunchai and
Kitpit, (2005) reported the inhibitory activity of pomegranate against
MRSA and found out that the MIC ranges from 0.2-0.4 mg per ml. In
a study, Aquil et al., (2005) observed that Punica granatum and
other plants like Camelia sinensis, Delonix regia and Lawsonia
inermis were effective against clinical isolates of beta lactamase
producing MRSA. Machado et al., (2003) reported that MRSA
strains were susceptible to Punica granatum extracts.
Twenty multi-drug resistant strains of Escherichia coli were
tested against the alcoholic extract of pomegranate and it was
observed that two strains were resistant to it at the concentration
tested, while nine strains showed medium zone of 11-20 mm, seven
showed an inhibition zone of 10 mm and two strains showed
inhibition zone above 20 mm. The lethal effect of alcoholic extract of
pomegranate on enterohaemorrhagic E.coli was reported recently by
Voravuthikunchai et al., (2004 and 2005). Escherichia coli was found
inhibited by pomegranate in the studies of Alanis et al., (2005) and
Mathabe et al., (2006) also.
158 Chapter V
Klebsiella pneumoniae strains were found to be the least
susceptible ones among the tested multidrug resistant organisms to
punica fruit peel extract. In the case of this bacterium, out of the
sixteen strains tested, none showed larger zone of inhibition of
growth, that is above 20 mm. The highest zone was 18 mm
produced by one strain only. Two strains were completely resistant
and thirteen stains showed an inhibition zone diameter ranging from
8-15 mm.
The comparatively lesser effect towards Klebsiella
pneumoniae by pomegranate extract is not surprising as the
organism is found insusceptible to many antibiotics and natural
products. However, strains which did not show multi-drug resistance
were more susceptible to the extract than to the tested antibiotics.
Twelve strains of Serratia marcescens were included in the
study. One strain was resistant to the extract, one showed above 20
mm inhibition zone diameter, four strains showed 10 mm and six
strains had a zone of inhibition ranging from 12-15 mm. Serratia
marcescens was found as the least sensitive organism to plant
extracts tested by Gnan and Demello, (1999). But in the present
study it is observed that strains of this bacillus also are susceptible
to the pomegranate extract.
Discussion 159
Among the fifteen multi-drug resistant strains of
Pseudomonas aeruginosa tested, all were sensitive to the extract
eventhough the inhibition zone did not exceed 20 mm for any of the
strains. This is very encouraging as Pseudomonas aeruginosa is
highly resistant to synthetic drugs (Panizzi et al., 1993) and extracts
from plants like Cinnamomum verum (Agnihotri and Vaidya, 1996).
Eight strains of Pseudomonas aeruginosa showed a negligible zone
of 8-10 mm, seven strains showed a range of 12-15 mm and one
showed 20 mm diameter. Six of these strains showed resistance to
all the drugs tested. The results support for the search for a new
drug from pomegranate, as natural products having activity against
Gram negative bacteria are limited in number as reported by Lin et
al., (1999). In a study using nine traditional medicinal plants they
observed that none of the extracts were capable of inhibiting
Escherichia coli and most of the extracts showed poor inhibitory
activity towards Salmonella and Shigella species. Vlietinck et al.,
(1995), Martin, (1995), Paz et al., (1995) and Rabe and Van Staden,
(1997) also had similar results with lesser activity towards Gram
negative bacteria by plant extracts.
Among the four hundred and eighty one bacterial strains of
different species tested, only forty five strains showed negligible
160 Chapter V
effect, that is an inhibition zone of 10 mm or less. This is
approximately 9.3% only. Majority of the strains showed a
susceptibility ranging from 11 to 20 mm, that is three hundred and
seventeen strains out of four hundred and eighty one (65.9%). This
correlates with the standard zone of inhibition of growth produced by
many antibiotics like ampicillin (for Gram negative enteric organisms
and enterococci), ceftazidime, cephalexin, chloramphenicol,
cloxacillin, co-trimoxazole, gentamicin and other aminoglycosides
like amikacin, kanamycin etc., norfloxacin, pefloxacin ofloxacin,
sulphadiazine and other sulfa drugs, tetracycline and doxycycline,
azithromicin etc. Out of the four hundred and eighty one strains, one
hundred and nineteen strains showed inhibition zone above 20mm.
(24.7%). This is similar to the inhibition zone required for broad
spectrum antibiotics to be considered as effective, like ciprofloxacin,
cefotaxime, ceftriaxone etc.
When taking into account, the above facts and multiple drug
resistance of many bacteria, the findings of the present study is
useful in search for a new clinically effective antimicrobial agent.
Effect on commensal bacteria
The effect of the alcoholic extract of Punica granatum fruit
peel on different strains of five commensal bacterial species is
Discussion 161
shown in Table XVIII. Staphylococcus epidermidis, Staphylococcus
aureus, Streptococcus viridans, Escherichia coli and Klebsiella
pneumoniae strains were isolated from human skin, nostrils, throat
and faeces. The details are shown in Table XXV.
Table XXV: Effect of Punica granatum extract on commensal bacterial strains from human beings.
No. of strains showing inhibition zone Sl.
No. Bacterial species Source No. of strains tested 0-10
mm 11-20
m 21-30 mm
1. Staphylococcus epidermidis Skin 46 16 25 5
2. Staphylococcus aureus Skin and Nostrils 44 13 27 4
3. Streptococcus viridans Throat 34 29 5 0
4. Escherichia coli Faeces 40 12 25 3
5. Klebsiella pneumoniae Faeces 38 23 15 0
Total strains 202 93 97 12
Here also it was observed that the extract is inhibitory to the
commensals as in the case of pathogens, in a rather similar way.
The least susceptible organisms were Klebsiella pneumoniae and
Streptococcus viridans, but no strains were completely resistant.
Eighty five percent of S.viridans strains showed growth inhibition
upto 10mm, and the rest 15% had inhibition zone diameter ranging
162 Chapter V
from 11-20 mm. None produced larger zone. Similar results were
produced by K.pneumoniae strains, that is 61% with inhibition zone
upto 10 mm, 39% with zone diameter 11-20 mm, and none with
larger zone.
In the case of Staphylococcus epidermidis, isolated from skin
of human beings, 11% showed larger inhibition zone that is above
20 mm, 54% showed medium zone and 35% had zone diameter
below 11 mm and no strains showed complete resistance. Similar
results were observed in case of Staphylococcus aureus strains, that
is 9% showed larger zone, 61% medium and 30% smaller zone of
inhibition of growth.
Strains of Escherichia coli, the intestinal commensal of
human beings, were susceptible to the extract, as in the case of
pathogenic E.coli strains. Growth inhibition zone ranging from 11-20
mm was shown by 62.5%, 21-30 mm by 7.5% and 30% strains had
inhibition zone upto 10 mm.
A total of two hundred and two commensal strains were
included in the study. All showed susceptibility to the alcoholic
extract of pomegranate in which 46% produced negligible effect,
Discussion 163
48% moderate effect and 6% were showing large zones, that is
more than 20 mm.
These results are significant because a commensal can turn
into a pathogen when the host’s immunity is diminished or when the
commensal invade to a different site in the host tissue (opportunistic
pathogen).
Antifungal properties of plants
The aqueous, alcoholic, ethyl acetate and ether extracts of
ten selected plants were tested against six fungal species. The
selection of plants were done based on reported medicinal effects.
The plants selected were Lawsonia alba, Glycosmis
cochinsinensis, Allium sativum, Ocimum sanctum, Cinnamomum
verum, Cymbopogon citratus, Myristica fragrans, Eugenia
caryophyllata, Kaempferia galanga and Punica granatum. The
fungal species included in the study were Aspergillus niger,
Asp.flavus, Penicillium species, Rhizopus stolonifer, Trichophyton
rubrum and Candida albicans.
The results are summarized in Table XIX.
Aspergillus niger, a common food contaminant fungus was
inhibited by aqueous extracts of Allium sativum and Cymbopogon
164 Chapter V
citratus; alcoholic extracts of Allium sativum, Ocimum sanctum,
Cinnamomum verum and Cymbopogon citratus; ethyl acetate
extracts of Glycosmis cochinsinensis, Cinnamomum verum,
Myristica fragrans and Kaempferia galanga and ether extracts of
Glycosmis cochinsinensis, Cymbopogon citratus, Myristica fragrans,
Eugenia caryophyllata and Kaempferia galanga. Among the tested
plants, Lawsonia alba and Punica granatum did not inhibit
Aspergillus niger in any of the four extracts. Aspergillus niger was
reported to be inhibited by plant extracts like 5% alcoholic extract of
Aegle marmelos, Murraya koenigii, Tamarindus indica,
Cinnamomum verum, Cymbopogon citratus, Eupatorium odoratum
and Moringa oleifera (Sasidharan et al., 1998); Feronia elephantum
(Garg, 2001); Curcuma longa (Anup Banerjee and Nigam, 1978);
Delonix elata and D.regia (Seetharam et al., 2002); Evolvulus
alsinoids (Purohit et al., 1995); Curcuma caesia (Garg and Jain,
1998); Cassia fistula (Valsaraj et al., 1997) and Eugenia
caryophyllata (Meena and Sethi, 1994).
Aspergillus flavus, a contaminant fungus which can produce
aflatoxin, was inhibited by alcoholic and ethyl acetate extract of
Lawsonia alba and Glycosmis cochinsinensis. Allium sativum,
Ocimum sanctum and Kaempferia galanga could not inhibit it.
Discussion 165
Alcoholic extract of Cinnamomum verum, Cymbopogon citratus,
ethyl acetate and ether extract of Myristica fragrans and Eugenia
caryophyllata also inhibited its growth in SDA tubes. It was observed
that alcoholic and ethyl acetate extract of Punica granatum inhibited
Aspergillus flavus.
The oil of oregano and thyme (Paster et al., 1994), Eugenia
caryophyllata [Hintokoto et al., (1980);M A Azzuouz and Bullerman,
(1982)], Curcuma longa (Anup Banerjee and Nigam, 1978) etc.,
were reported for activity against aflatoxin producing Aspergillus
flavus.
Penicillium growth was prevented by the alcoholic extract of
Lawsonia alba, Ocimum sanctum, Cinnamomum verum, Myristica
fragrans and Eugenia caryophyllata. The ethyl acetate and ether
extract of Cymbopogon citratus, Kaempferia galanga and Allium
sativum; ethyl acetate extract of Ocimum sanctum, Cinnamomum
verum and Eugenia caryophyllata also showed inhibitory effect on
Penicillium. Glycosmis showed no inhibitory action in any of the four
extracts. Different species of Penicillium was reported to be inhibited
by Curcuma longa (Anup Banerjee and Nigam, 1978); Eugenia
caryophyllata, Cinnamomum verum, Allium sativum, Pimenta dioica
166 Chapter V
(M A Azzouz and Bullerman, 1982); Ocimum sanctum (Grover and
Rao, 1977) etc.
Rhizopus stolonifer, the bread mold, was susceptible to the
aqueous and alcoholic extract of Ocimum sanctum; alcoholic
extract of Allium sativum, Cinnamomum verum, Myristica fragrans
and Punica granatum; ethyl acetate extract of Kaempferia galanga,
Eugenia caryophyllata, Cymbopogon citratus; ether extract of
Cinnamomum verum and Eugenia caryophyllata. Lawsonia alba
and Glycosmis cochinsinensis could not inhibit it. Feronia
elephantum was reported to have inhibitory effect on Rhizopus
nodosus (Garg, 2001), and Curcuma caesia on Rhizopus oryzae
(Garg and Jain, 1998).
Dermatophytes are pathogenic fungi which cause ring worm
infection or tinea in humans and animals. Trichophyton rubrum, a
common but highly resistant dermatophyte was included in the
study. It was observed that the alcoholic, aqueous and ethyl acetate
extract of Lawsonia alba was very effective against Trichophyton
rubrum. Ether extract of Cymbopogon citratus and alcoholic extract
of Punica granatum also prevented its growth. Other plant extracts
tested were ineffective against it. Dermatophytes are inhibited by
plant extracts very effectively and were reported by various
Discussion 167
researchers. The bark tinctures of Punica granatum, Psidium
guajava, Syzigium cumini and Emblica officinalis were reported for
antidermatophytic activity including activity against Trichophyton
rubrum by Dutta et al., (2000). Fruits of Aglaia roxburghiana possess
activity against dermatophytes (Janaki and Vijayasekaran, 1998).
Lawsonia alba also was reported to have anti-ringworm action by
Natarajan et al., (2000). Trichophyton rubrum was found inhibited by
Feronia elephantum (Garg, 2001).
Candida albicans is an yeast like fungus causing a wide variety
of infections which include cutaneous, mucocutaneous and systemic
infections. It is an opportunistic pathogen which can cause disease
when the host’s resistance is lowered by other factors. Lawsonia alba,
Allium sativum, Cinnamomum verum, Myristica fragrans,
Cymbopogon citratus, Eugenia caryophyllata and Punica granatum in
one or other extract showed inhibitory activity against it. Glycosmis
cochinsinensis and Ocimum sanctum were found ineffective against
Candida. Among the plants tested, Punica granatum showed activity
against Candida albicans in three extracts – aqueous, alcoholic and
ethyl acetate.
168 Chapter V
The anticandial effect of many plants were reported
previously. Aqueous and alcoholic extract of Ocimum sanctum was
reported to possess anticandidal activity. (Geeta et al., 2001).
Elizabeth, (2001) reported that Candida albicans was inhibited by
Allium sativum. According to Suresh et al., (1997), Santolina
chamaecyparissus oil was effective in controlling experimental
candidiasis in vivo and in vitro and had a synergistic effect on
clotrimazole in controlling Candida in vitro. Vaginal, systemic and
superficial cutaneous candidiasis were reported to be controlled by
it. Ali-Shtayeh et al., (1997) reported that thymol and carvacrol
present in Micromeria nervosa inhibited Candida albicans. Lin et al.,
(1999) observed that all the nine plant extracts tested produced high
inhibitory activity to Candida albicans. Cassia fistula was reported
negative for anticandidal effect by Valsaraj et al., (1997). Alglaia
roxburghiana also was found ineffective against Candida albicans
(Janaki and Vijayasekaran, 1998). According to Dutta et al., (2000),
different extracts from four fruit plants produced only fungistatic
activity against Candida albicans, but these showed fungicidal
activity against dermatophytes.
As Punica granatum inhibited Candida albicans, eight strains
isolated from patients were tested against different extracts. The
Discussion 169
results are shown in Table XX. It is clear that aqueous and alcoholic
extract were equally effective in preventing the growth and were
more effective than ethyl acetate or ether extracts, as the first two
extracts prevented 75% of total strains while ethyl acetate 38% and
ether only 25%. These eight strains were tested by disc diffusion
method also using alcoholic extract which showed an inhibition zone
ranging from 10-15mm diameter.
Eleven commensal strains of Candida albicans isolated from
healthy human beings also were tested by disc diffusion method.
Among these strains, 36% of strains were showing inhibition zone
below 10 mm, 46% of strains 10-15 mm and 18% strains were
resistant to the alcoholic extract of Punica granatum.
Antifungal activity of Punica granatum had been reported
against Trichophyton tonsurans, T.rubrum, Trichosporon beigelli,
Microsporum fulvum, M.gypseum and Candida albicans by Dutta
et al., (2000). According to M A Azzouz et al., (1982), powdered
pomegranate peel is a good inhibitor to four Penicillium species
while it had no effect on Aspergillus flavus and A.parasiticus. The
inhibitory effect may be due to the high content of tannins in
170 Chapter V
pomegranate peel. Dold and Knapp, (1948) reported that spices
which contain tannin or alkaloids are among the most germicidal.
Anti-protozoan effects of plants
For testing the antiprotozoan effects of plants, a fresh water
protozoan, Paramecium caudatum was used. The protozoan was
exposed to the extract for two minutes and then the motility of the
organism was microscopically examined.
It was observed that the plant extracts immobilized different
number of protozoa tested in the alcoholic and aqueous extracts.
Punica granatum in both extract showed the highest activity, that is all
the protozoa were immobilized. Alcoholic extract of Calotropes procera,
Holarrhena antidysenterica and Ocimum sanctum also did the same.
Alcoholic and aqueous extract of Myristica fragrans neutralized the
motility of 86% protozoa. Aqueous extract of Ocimum sanctum and
Calotropes procera immobilized 87.5% protozoa. Eighty three percent
protozoa became non motile on exposure to the aqueous extract of
Holarrhena antidysenterica.
Calotropes procera was reported to have schizonticidal activity.
Sharma and Sharma, (1999) reported that crude fractions of the
flowers, bud and root of it was effective against chloroquin sensitive
and resistant strains of Plasmodium falciparum. Holarrhena
Discussion 171
antidysenterica has been reported to be used against malaria and
vaginitis (Hager Handbuch, 1976). Antiplasmodial activity was reported
for plants like Naucbea latifolia (F B Vical et al., 1998), Phyllanthus
rediculantis, Suregada zanzibariensis, Terminalia spinosa, Dissotis
brazzae (Omulokoli et al., 1997), Hernandia voyronii (Ratsimamanga-
Urverg et al., 1994) etc. Plants with activity against Giardia lamblia
include Butea monosperma, Piper longum (Agarwal et al., 1997)
Strychnos species (Wright et al., 1994) etc.
Trichomonas vaginalis has been reported to be inhibited by
extract from Sophora flavescense (Wang and Cheng, 1994).
Entamoeba histolytica has been reported to be inhibited by alkaloids
from Strychnos species (Wright et al., 1994). While studying twenty
kinds of extracts of traditional Chinese medicine, Yang et al., (1996)
observed that crude extracts of Coptis chinensis and Brucea javanica
were most active against Blastocystis hominis. Punica granatum was
found to be inhibiting protozoa very effectively. Calzada et al., (2006)
observed that methanolic extract of Punica granatum was most
effective against Entamoeba histolytica.
Factors which seem to affect the antimicrobial effects of plants
were the location and season of collection. This may be the reason
172 Chapter V
why different researchers are getting variations in the results.
Moreover, different methods for extract preparation and sensitivity
testing are performed by the scientists using different strains.
Thermal stability and shelf life of the alcoholic extract of pomegranate peel
The thermal stability and shelf life of the pomegranate extract
were studied (Table XXII and XXIII) and it was found that the extract
remain effective even after autoclaving, storage at room temperature
upto three months and refrigeration up to six months without much
variation. This is an added advantage for the extract.
MIC of the partially purified extract of pomegranate peel
MIC of the alcoholic extract was tested and found that it was
ranging from a concentration equivalent to 0.5 to 1 mg per ml for
Staphylococcus aureus and 0.8 to 1.2 mg per ml for Escherichia coli.
Voravuthikunchai and Kitpit, (2005) reported that MIC of pomegranate
to MRSA ranges from 0.2 to 0.4 mg per ml.
From the results of the present study it can be observed that
many plants like Ocimum sanctum, Myristica fragrans, Eugenia
caryophyllata, Camelia sinensis, Cinnamomum verum etc. posses
broad spectrum antimicrobial activity next to Punica granatum.
Discussion 173
Punica granatum leaf, stem, root, seed, fruit and pericarp were
tested for antimicrobial activity in various extracts like aqueous,
alcoholic, acetone, ethyl acetate, ether and chloroform extracts. All
these possessed some antibacterial activity, but aqueous and alcoholic
extract of the pericarp was found most effective. The alcoholic extract
was found stable in antimicrobial activity even after autoclaving, and
was found stable during refrigeration or storage at room temperature. It
was observed that the activity did not diminish considerably on
refrigeration up to six months (Table XXIII). The MIC results show that
minute amounts of the extract are effective against Gram positive and
Gram negative bacteria.
The antimicrobial activity of Punica granatum has been
attributed to the tannins and polyphenols present in it. Tannins from
many plant foods inhibits growth of fungi and bacteria. (Chung et al.,
1998). Tannins are polyphenol group of compounds that damage the
bacterial cytoplasmic membrane. Chelating properties of tannin
coupled with their ability for denaturing bacterial proteins contribute
towards the antibacterial activity (Shimamura et al., 1990). Dold and
Knapp, (1948) reported that spices which contain tannins or alkaloids
were among the most germicidal. Pomegranate peels are high in
tannins, and so it is highly germicidal. Kau, (1980) reported that tannic
174 Chapter V
acid is bacteriostatic or bactericidal to some Gram positive and Gram
negative pathogens.
According to Jagtap and Karkera, (1999), the large number of
phenolic groups presents in tannins provide unique binding properties
so that it may be adsorbed on to bacterial surface. The antidiarrhoeal
activity is also attributed to tannin as reported by Das et al., (1999),
who observed that the seed extract contain tannin which produces anti
secretary activity. Tannins are responsible for protein denaturation,
producing protein-tannate, which reduces secretion from intestinal
mucosa. [Tripathi, (1994); Mukherjee et al., (1995)]. Rani et al., (1999)
also reported that plants containing tannins are antidiarrhoeal.
Punica granatum contains tannins like punicalagin, ellagitannin,
gallotannins etc. which were identified by many workers previously
(Table: II).
The broad spectrum antimicrobial activity may be due to the
presence of tannins in high amount in Punica granatum which adsorb
on to microbial surface and denature the proteins. In vivo experiments
using animals, studies regarding the exact mechanism of action,
toxicity and retention in tissues etc., are to be conducted in detail
before the application of pomegranate peel extract as a safe
therapeutic agent.