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.