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6.1.1 Introduction

Antibiotics provide the main basis for the therapy of microbial (bacterial and

fungal) infections. Since the discovery of these antibiotics and their uses as

chemotherapeutic agents there was a belief in the medical fraternity that this would

lead to the eradication of infectious diseases. However, excessive and over usage of

antibiotics has become the most important factor for the emergence and dissemination

of multi-drug resistant strains of several groups of microorganisms (Harbottle et al.,

2006). The worldwide emergence of Escherichia coli, Klebsiella pneumoniae,

Haemophilus and many other ß-lactamase producers has become a major therapeutic

problem. Multi-drug resistant strains of E. coli and K. pneumoniae are widely

distributed in hospitals and increasingly being isolated from community acquired

infections (Akram et al., 2007; Khan and Musharraf, 2004). Candida albicans, also a

nosocomial pathogen has been reported to account for 50-70% cases of invasive

candidiasis (Paula et al., 2006). Alarmingly, the incidence of nosocomial candidemia

has risen sharply in the last decade (Kao et al., 1999). All this has resulted in severe

consequences including increased medicinal cost and mortality of patients.

Thus, with the evidence of rapid global spread of resistant clinical isolates, the

need to search new antimicrobial agents is of paramount importance. However, the

past record of rapid, widespread and emergence of resistance to newly introduced

antimicrobial agents indicates that even new families of antimicrobial agents will

have a short life expectancy (Coates et al., 2002). For this reason, researchers are

increasingly turning their attention to plants, looking for new leads to develop better

drugs against strains of MDR microbes (Braga et al., 2005).

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For thousands of years, natural products have been used in traditional

medicine all over the world and predate the introduction of antibiotics and other

modern drugs. The antimicrobial efficacy attributed to some plants in treating diseases

has been beyond belief. It is estimated that local communities, to treat various

infections, have used about 10% of all flowering plants on earth throughout the world

but only 1% have gained recognition by modern scientists (Kafaru, 1994). Owing to

their popular use as remedies for many infectious diseases, searches for substances

with antimicrobial plants are frequent (Betoni et al., 2006). Plants are rich in a wide

variety of secondary metabolites such as tannins, alkaloids and flavonoids, which

have been found in vitro to have antimicrobial properties (Lewis and Ausubel, 2006).

A number of phytotherapy manuals have mentioned various medicinal plants for

treating infectious diseases due to their availability, fewer side effects and reduced

toxicity (Lee et al., 2007). There are several reports on antimicrobial activity of

different herbal extracts (Islam et al., 2008; de Boer et al., 2005; Bonjar, 2004). Many

of the plants have been found to cure urinary tract infections, gastrointestinal

disorders, respiratory diseases and cutaneous infections (Somchit et al., 2003;

Brantner and Grein, 1994). Cytotoxic compounds have been isolated from the species

of Vismia (Hussain et al., 2003). Antibacterial activity of the essential oil as well as a

purified eugenol of Ocimum gratissimum to treat pneumonia, diarrhea and

conjunctivitis has been reported (Nakamura et al., 1999). According to WHO,

medicinal plants would be the best source for obtaining variety of drugs (Santos et al.,

1995). These evidences contribute to support and quantify the importance of

screening natural products.

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120

The aim of the present study was to investigate the antibacterial and antifungal

activity against multi-drug resistant strains isolated from nosocomial and community

acquired infections of ethanolic extracts of Acacia nilotica, Terminalia arjuna,

Eucalyptus globulus, Syzygium aromaticum and Cinnamomum zeylanicum.

6.1.2 Experimental procedure

Leaves of A. nilotica, E. globulus and bark of T. arjuna were collected from

the gardens of AMU, Aligarh, India. C. zeylanicum and S. aromaticum were collected

from local market of Aligarh. The taxonomic identity of these plants was confirmed at

Department of Botany, AMU, Aligarh, India. Extracts were prepared as outlined in

section 2.2.3. Strains used in this study have been mentioned in section 2.2.1. The

susceptibilities of the microbial strains to different antibiotics were tested using disc

diffusion method (Section 2.2.21). The extracts were tested for antimicrobial activity

using agar diffusion and determination of minimum inhibitory concentration and

minimum bactericidal/fungicidal concentration was carried out as mentioned in

section 2.2.22.

6.1.3 Results

6.1.3.1 Determination of sensitivity of the strains to antibiotics

In this study, we have tested the ethanolic extracts of five plants for their

antimicrobial activity against multi-drug resistant strains as well as standard ATCC

strains of gram-negative bacteria, gram-positive bacteria and yeast species. All the

plant extracts showed antimicrobial activity in regards to at least four types of

microorganisms tested, as exhibited by agar diffusion assay (Table 6.1.1). Extracts of

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121

A. nilotica, C. zeylanicum and S. aromaticum showed most potent activity against all the

microorganisms studied. E. faecalis, S. aureus, S. bovis and S. mutans were the most

susceptible to all the plant extracts tested. On the contarary, S. typhimurium, K.

pneumoniae, E. coli, P. aeruginosa and C. albicans strains were sensitive only against

extracts of A. nilotica, C. zeylanicum and S. aromaticum.

6.1.3.2 Agar diffusion method

K. pneumonia, amongst the tested gram-negative bacteria, was found to be most

sensitive while S. typhimurium was the most resistant bacteria. In case of gram-positive

bacteria, E. faecalis was the most sensitive while S. aureus was the most resistant strain.

C. albicans was found to be highly sensitive to the action of A. nilotica (least MIC 4.9

µg/ml) followed by C. zeylanicum and S. aromaticum with the least MIC being 19.5

µg/ml and 156 µg/ml, respectively (Table 6.1.2). On the contrary, C. albicans was

completely resistant against T. arjuna and E. globulus at the concentrations tested.

6.1.3.3 Determination of minimum inhibitory concentration and minimum

bactericidal/fungicidal concentration

Amongst the five plants, extracts of A. nilotica, C. zeylanicum and S. aromaticum

showed good antimicrobial activity against multidrug resistant strains of K. pneumoniae,

E. coli and C. albicans isolated from nosocomial and community acquired infections

(Table 6.1.3). Extracts of A. nilotica was found to be the most active extract against the

nosocomial as well as community acquired isolates. The MIC value of the extract of A.

nilotica against different isolates was found to be in the range of 4.9-313µg/ml. As per

our results, the MIC values for most of the extracts were lower than their MBC/MFC

values, suggesting that these extracts inhibited growth of the test microorganisms while

being bactericidal /fungicidal at higher concentrations.

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Table 6.1.1: Susceptibility pattern of ethanolic herbal extracts against different

microorganisms.

#Susceptibility pattern of crude herbal extract against different microorganisms

Microbial Strains A.nilotica* T.arjuna* E.globulus* S.aromaticum* C.zeylanicum*

S. mutans

ATCC-700610 +++ +++ ++ +++ +++

S.aureus

ATCC-29213 +++ +++ ++ +++ +++

E.faecalis

ATCC-29212 ++ ++ + ++ +++

S.bovis

ATCC 9809 ++ ++ + +++ +++

P.aeruginosa

ATCC-27853 +++ - - +++ +++

S. typhimurium

ATCC-13311 ++++ - - +++ +++

E.coli

ATCC-25922 +++ - - ++ ++

C.albicans

ATCC-10231 +++ - - ++++ ++++

K.pneumoniae

ATCC-700603 ++ - - + ++

E.coli [10]a) ++ (10/10) - (10/10) - (10/10) + (10/10) ++ (10/10)

- (1/16) - (1/16) - (1/16)

E.coli [16]b) + (1/16) - (16/16) - (16/16) + (5/16) + (13/16)

++ (14/16) ++ (10/16) ++ (2/16)

++ (3/18)

C.albicans [18]c) ++ (18/18) - (18/18) - (18/18) +++ (12/18)

++++

(18/18)

++++ (3/18)

K.pneumoniae

[14]d) + (9/14) - (14/14) - (14/14) + (12/14) ++ (14/14)

++ (5/14) ++ (2/14) # = Diameter of inhibition zone: no inhibition (-); 5-15mm (+); 16-25mm (+ +); 26-35mm

(+ + +); >40mm (+ + + +);

*=values in parentheses indicate number of isolates out of total isolates tested;

a) & c) = isolates of nosocomial infection; b) & d) = isolates of community acquired infection.

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Table 6.1.2: MIC and MBC/MFC values for crude extracts of plant parts against multi-drug resistant strains of nosocomial and community

acquired infections and susceptible standard strains MIC(µg/ml) and MBC/MFC(µg/ml) of Crude herbal extracts A. nilotica* T. arjuna E. globulus S. aromaticum C. zeylanicum Microorganism

MIC MBC/MFC MIC MBC/MFC MIC MBC/MFC MIC MBC/MFC MIC MBC/MFC S. mutans ATCC-700610 78 313 1560 3130 3130 6250 390 780 195 390

S.aureus ATCC-29213 39 78 780 1560 6250 12500 780 1560 390 1560

E.faecalis ATCC-29212 9.75 78 1560 3130 3130 12500 195 1560 97.5 1560

S.bovis ATCC-9809 39 78 1560 3130 3130 12500 780 1560 195 390

P.aeruginosa ATCC-27853 39 39 - - - - 390 1560 390 1560

S.typhimurium ATCC-13311 9.75 39 - - - - 1560 1560 1560 3130

E.coli ATCC-25922 19.5 39 - - - - 780 1560 390 1560

C.albicans ATCC-10231 4.9 19.5 - - - - 156 156 19.5 78

K.pneumoniae ATCC-700803 9.75 78 - - - - 390 6250 195 3130

E.coli [10] a)

156 313

313(3/10) 625(7/10)

- -

- -

- -

- -

6250

12500(10/10)

3130 6250

6250(7/10) 12500(3/10)

E.coli [16] b)

19.5 39

39(3/16) 156(13/16)

- -

- -

- -

- -

390 1560

1560(2/16) 3130(14/16)

195 780

1560(11/16) 3130(5/16)

C.albicans [18]c)

9.5 39

39(9/18) 78(9/18)

- -

- -

- -

- -

390 780

780(3/18) 3130(15/18)

780

1560(18/18)

K.pneumoniae [14]d) 156 313 (11/14) - - - - 780 1560(4/14) 390 1560(11/14) 313 1250(3/14) - - - - 1560 3130(10/14) 780 3130(3/14)

MIC= minimum inhibitory concentration, MBC= minimum bactericidal concentration, MFC= minimum fungicidal concentration; a) & c) = isolates of nosocomial infection; b) & d) = isolates

of community acquired infection; =value in parentheses indicates number of isolates out of total isolates tested; - = No activity at the concentration of the extracts tested.

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Table 6.1.3: Resistance profile of multi-drug resistant isolates of nosocomial and

community acquired infections

Microorganisma)

Source of Infection

Resistance Pattern of Antibacterial/Antifungal Agent Isolatesb)

E. coli (10) Nosocomial Ch,Ci,Cpm,Ac,Ao,Pc,G,Tb,Na,Cf,T Ch,Ca,Ci,Cpm,Ac,Ao,Pc,G,Na,Cf,T Ch,Ca,Ci,Cpm,Ac,Ao,Pc,G,Tb,Na,Cf,T Ch,Ca,Ci,Cpm,Ac,Ao,Pc,G,Na,Cf,T,C Ch,Ca,Ci,Cpm,Ac,Ao,Pc,G,Tb,Na,Cf,T,C

8E,9E,10E 2E,7E 3E,6E 1E 4E,5E

E. coli (16) Community Acquired

Ch,Ci,Cpm,Ac,Pc,Na,Nf,C Ch,Ca,Cpm,Ac,Ao,Pc,Ak,Tb,Cf Ch,Cpm,Ac,Pc,Tb,Na,Nf,T,C Ch,Ca,Ci,Cpm,Ao,Pc,Na,Cf,T Ch,Ci,Cpm,Ac,Pc,Ak,Tb,Na,Cf,Nf,T Ch,Ca,Cpm,Ac,Ao,Pc,Ak,Na,Cf,Nf,T,C Ch,Ca,Ac,Pc,Ak,G,Tb,Na,Cf,Nf,T,C Ch,Ca,Cpm,Ac,Ao,Pc,G,Tb,Na,Cf,Nf,T,C Ch,Ca,Ci,Cpm,Ac,Ao,Pc,Ak,G,Tb,Na,Cf,T Ch,Ca,Ci,Cpm,Ac,Ao,Pc,Ak,G,Tb,Na,Cf,Nf,T Ch,Ca,Ci,Cpm,Ac,Ao,Pc,Ak,G,Tb,Na,Cf,Nf,T,C

128E 68E 92E, 112E 137E 186E 61E 93E 67E, 144E 158E 103E, 133E 59E,90E,152E

K. Pneumoniae (14)

Community Acquired

Ch,Cpm,Ac,Pc,Ak,Tb,Cf,T Ch,Ci,Cpm,Ac,Pc,Ak,G,Cf,T Ch,Cpm,Ac,Ak,G,Tb,Cf,Nf,T Ch,Ca,Ci,Cpm,Ac,Ao,Pc,G,Tb,T Ch,Cpm,Ac,Pc,Ak,G,Tb,Na,Cf,Nf,T,C Ch,Ca,Ci,Cpm,Ao,Pc,G,Tb,Na,Cf,Nf,T Ch,Ca,Ci,Cpm,Ac,Ao,Pc,Ak,G,Tb,Na,Cf,Nf Ch,Ca,Ci,Cpm,Ac,Ao,Pc,G,Tb,Na,Cf,Nf,T Ch,Ca,Ci,Cpm,Ac,Ao,Pc,Ak,G,Tb,Na,T,C Ch,Ca,Ci,Cpm,Ac,Ao,Pc,Ak,G,Tb,Na,Cf,Nf,T,C

173K 63K 66K, 155K 111K, 141K 153K 159K 150K 164K,174K, 192K 165K, 194K

C. albicans (18)

Nosocomial It,Ns,Ap It,Fu,Ap It,Ns,Fu,Ap It,Ns,Cc,Ap It,Kt,Cc,Ap It,Kt,Ns,Cc,Ap It,Kt,Ns,Fu,Ap It,Ns,Cc,Fu,Ap It,Kt,Ns,Cc,Fu,Ap

2C,10C,15C 17C 14C 11C 5C,13C 12C 9C,16C 3C,4C 1C,6C,7C,8C,18C

a) = No. of isolates tested in parentheses; b) = Name of the strains studied in our lab. Antibacterial Agent:

Cephalosporins:Ch=Cephalothin(30µg), Ca=Ceftazideme(30µg), Ci=Ceftriaxone(30µg), Cpm=Cefepime(30µg). Other

ß-lactam:Ac=Amoxyclav(30µg), Ao=Aztreonam(30µg), Pc=Piperacillin(100µg).

Aminoglycosides:Ak=Amikacin(30µg), G= gentamycin(10µg), Tb=Tobramycin(10µg) Fluoroquinones:Na=Nalidixic

acid(30), Cf=Ciprofloxacin(5µg) Others:Nf=Nitrofurantoin(300µg), T=Tetracycline(30µg), C=Chloramphenicol(30µg)

Antifungal Agents: It=Itraconazole(10 µg), Kt=Ketoconazole(10µg), Ns=Nystatin(100 units), Cc=Clotrimazole(10µg),

Fu=Fluconazole(10µg), Ap=Amphotericin(100 units)

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6.1.4 Discussion

Our data revealed that standard ATCC strains of gram-positive bacteria were more

sensitive than gram-negative strains, against the plant extracts studied. This data is also

supported by previous workers (Nair and Chanda, 2006). It has been proposed that the

mechanism responsible for the antimicrobial effects involves the inhibition of microbial

respiration followed by increase in plasma membrane permeability and finally ion leakage

from the cells (Walsh et al., 2003).

In contrast to the findings that gram-negative bacteria are hardly susceptible to the

plant extracts in doses less than 2 x 105 µg/ml (Suffredini et al., 2006) , our results showed

inhibition at concentrations as low as 9.75 µg/ml (A. nilotica). The variation of

susceptibility of the tested microorganisms could be attributed to their intrinsic properties

that are related to the permeability of their cell surface to the extracts. Due to the

emergence of antibiotic resistant pathogens in hospitals and homes, plants are being

looked upon as an excellent alternate to combat the further spread of multidrug resistant

microorganisms.

Our data showed that strains isolated from nosocomial infection were more

resistant to the extracts than the strains of community acquired infections. It was also

reported earlier that the resistance to antibiotics as well as mortality is almost two times

higher in case of nosocomial infections than in community-acquired infections (Kang et

al., 2006).

Acacia nilotica was found to be the most potent antimicrobial extract (Table 6.1.2).

It is reported to have antimicrobial, antihyperglycemic and antiplasmodial properties

(Meena et al., 2006; El-Tahir et al., 1999; Sotohy et al., 1995). Cinnamum zeylanicum

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showed next highest activity followed by Syzygium aromaticum. These two plants are

known to possess antipyretic activity (Lopez et al., 2005; Kurokawa et al., 1998).

Essential oils from these two species have been shown to possess antibacterial activities

(Prabhuseenivasan and Jayakumar, 2006). Eugenol, a compound found in S. aromaticum

is reported to have strong antifungal (Chamin et al.,2004), anti-inflammatory activities

(Dip et al., 2004), and has been investigated for its potential anticarcinogenic effect (Dorai

and Aggarwal, 2004). The essential oil from C. zeylanicum shows antioxidant (Dhuley,

1999), antibacterial and antifungal activities (Wang et al., 2005).

Terminalia arjuna, a well known herbal cardiac tonic and also known to possess

antimicrobial activity (Rani and Khullar, 2004; Miller, 1998) and Eucalyptus globulus

traditionally used to treat diabetes (Duke, 1985) showed antimicrobial effects only on

gram-positive bacteria (Table 6.1.2). T. arjuna contains ellagic acid, ethyl gallate, gallic

acid and luteolin that exhibits antimutagenic property (Kandil et al., 1998; Kaur et al.,

1997). It also possesses significant antioxidant effect that is comparable with vitamin E

(Gupta et al., 2001). Plants of the genus Eucalyptus have been shown to produce a number

of phloroglucinol-sesquiterpene- or -monoterpene-coupled compounds, namely, the

macrocarpals and euglobals. Reports have revealed their biological activities such as HIV-

RTase inhibition, granulation inhibition and antiviral effects (Nishizawa et al., 2001;

Yamakoshi et al., 1992).Globulol isolated from the fruit of this plant has been shown to be

the major constituent for its antimicrobial activity (Tan et al., 2008).

The antimicrobial potency of plants is believed to be due to tannins, saponins,

phenolic compounds, essential oils and flavonoids (Aboaba and Efuwape, 2001). It is

interesting to note that even crude extracts of these plants showed good activity against

multidrug resistant strains where modern antibiotic therapy has failed. The ethanolic

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extracts of A. nilotica, C. zeylanicum and S. aromaticum could be possible source to obtain

new and effective herbal medicines to treat infections caused by multi-drug resistant

strains of microorganisms from community as well as hospital settings. However, it is

necessary to determine the toxicity of the active constituents, their side effects and

pharmaco-kinetic properties.

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6.2.1 Introduction

Many efforts have been made to discover new antimicrobial compounds from

various kinds of sources such as plants, animals and microorganisms. A large number

of plant products have long been utilized as a source of therapeutic agents worldwide

(Gottlieb et al., 2002; Gibbons, 2005; Khan et al., 2009). Recently, herbal medicines

have increasingly been used to treat many diseases including several infections. Plants

produce certain chemicals which are naturally toxic to bacteria (Singh and Bhat,

2003) and many plants have been investigated for the development of novel drugs

with therapeutic properties (Tomoko et al., 2002). As opposed to synthetic drugs,

antimicrobials of plant origin are not associated with many adverse effects and have

an enormous therapeutic potential to heal many infectious diseases.

Drug resistance to pathogenic microorganisms has been commonly reported

worldwide. Antibiotic resistance refers to the ability of a microorganism to withstand

the effects of an antibiotic. The increasing frequency of microorganisms that are

resistant to common and generally accepted antibiotics is on the increase.

Furthermore, the rate of resistance to these drugs is higher in developing countries as

compared to developed countries because of extensive and indiscriminate use of

antibiotics over the last few decades (Akram et al., 2007) and people’s ability to self-

medicate without a prescription from a physician. Among the wide array of

antibiotics, beta (β)-lactams are the most varied and widely used (Bronson and Barret,

2001). The most common cause of bacterial resistance to β-lactam antibiotics is the

production of β-lactamases. Bacterial resistance to β-lactam antibiotics has been

attributed to the spread of plasmid-mediated extended spectrum β-lactamases

(ESBLs) (Sanders and Sanders, 1987). Medicinal plants are natural resources for

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129

valuable products that can be used in the treatment of various ailments. Plant

materials remain an important resource for combating illnesses, including infectious

diseases, and many plants have been investigated for novel drugs for the development

of new therapeutic agents. Thus the emergence of multiple drug resistance of

pathogenic organisms has necessitated a search for new antimicrobial substances from

other sources including plants (Lin et al., 2005).

In the present study, extracts of Prosopis spicigera (P. spicigera), Zingiber

officinale (Z. Officinale) and Trachyspermum ammi (T. ammi), which were prepared

using solvents with different polarities, were tested to screen their antimicrobial

activity (MIC and minimum bactericidal/fungicidal concentration [MBC/MFC])

against multi-drug resistant Escherichia coli (E. coli, ESBL positive), Candida

albicans (C. albicans), Candida krusei (C. krusei), Candida tropicalis (C. tropicalis),

Candida glabrata (C. glabrata), Streptococcus mutans (S. mutans) and Streptococcus

bovis (S. bovis).

6.2.2 Experimental procedure

Seeds of T. ammi and dried roots of Z. officinale were collected from the local

market of Aligarh while the leaves of P. spicigera were collected from the campus of

Aligarh Muslim University (AMU), Aligarh, India. The taxonomic identities were

confirmed by Prof. Wazahat Husain, Taxonomist, ex-chairman of the Department of

Botany, AMU. The plant materials were washed under running tap water, air dried

and then homogenized to fine powder and stored in airtight bottles. The method of

preparation of the extract is detailed section 2.2.3, while the phytochemical analysis

mentioned in section 2.2.23. The determination of minimum inhibitory concentration

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130

and minimum bactericidal/fungicidal concentration was performed as explained in

section 2.2.22.

6.2.3 Results

6.2.3.1 Preliminary phytochemical analysis

Results obtained in the present study revealed that the three plant extracts

tested possess potential antibacterial activity against multidrug resistant (MDR) E.

coli, S. mutans and S. bovis as well as antifungal activity against MDR C. albicans, C.

tropicalis, C. glabrata and C. krusie (Table 6.2.1). The active fractions found in P.

spicigera, T.and Z. officinale were ethanol, petroleum ether and ethyl acetate,

respectively. The crude extracts of the three plants as well as their most active fraction

showed positive tests for alkaloids, amino acids, and proteins while none of them

exhibited the presence of phenols and flavonoids (Table 6.2.2). Tests for resins and

reducing sugars were positive only in the case of Z. officinale, while T. ammi and Z.

officinale tested positive for sterols and terpenes. Glycosides were found in crude

extracts of T. ammi, Z. officinale and in the PE fraction of T. ammi.

6.2.3.2 Determination of minimum inhibitory concentration

When tested for MIC (Table 6.2.3), the ethanolic fraction of P. spicigera

showed significant activity against all the microorganisms with the least MIC being

4.88 µg/ml. The highest antibacterial activity exhibited by this fraction of 4.88 µg/ml

was against S. bovis and the least activity of 312.5 µg/ml was recorded in two strains

of C. albicans. The petroleum ether fraction of T. ammi showed highest activity

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131

against C. albicans (78.125 µg/ml) and the lowest in E. coli. Z. officinale ethyl acetate

fraction possessed maximum activity against C. albicans and S. mutans (625 µg/ml).

6.2.3.3 Deternination of minimum bactericidal/fungicidal concentration

In the majority of the tests, the MBC or MFC (Table 6.2.4) was found to be

two-fold higher than the MIC. T. ammi exhibited lowest activity to C. krusei, E. coli 4

and S. bovis whereas Z. officinale did not show any inhibitory effect on C. albicans 1,

E. coli 3, or S. bovis with MIC ≥ 5000µg/ml. C. tropicalis 2, E. coli 3 and S. bovis

were found resistant to P. spicigera.

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Table 6.2.1: Strains used in the study.

Strain Description of resistant markers

Candida albicans It, Kt, Fu

Candida albican It, Fu, Cc

Candida albicans It, Kt, Ns,Fu

Candida glabrata It, Kt, Fu

Candida krusei It, Kt, Fu, Cc

Candida tropicalis It, Kt, Amp, Fu, Cc

Candida tropicalis It, Fu, Cc

Escherichia coli ESBL+ve (TEM-1)

Escherichia coli ESBL+ve (TEM-1)

Escherichia coli ESBL+ve (TEM-1, CTXM)

Escherichia coli ESBL+ve (CTXM)

Escherichia coli ESBL+ve (CTXM)

Streptococcus mutans ATCC-700610

Streptococcus bovis ATCC-9809

Antifungal Agents: It=Itraconazole (10 µg), Kt=Ketoconazole (10 µg), Ns=Nystatin (100 units),

Cc=Clotrimazole (10 µg), Fu=Fluconazole (10 µg), Ap=Amphotericin (100 units); ESBL= Extended Spectrum

β Lactamase producing strains.TEM-1 and CTXM are ESBL types confirmed by PCR amplification of their

genes in the strains used.

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Table 6.2.2: Qualitative test for various phytochemical constituents in different

extracts and fractions.

Compound

P.s.

ET extract (Crude)

T.a.

ET extract

(Crude)

Z.o.

ET extract

(Crude)

P.s.

ET fraction

T.a.

PE fraction

Z.o

EA fraction

Alkaloid +ve +ve +ve +ve +ve +ve

Amino

acids +ve +ve +ve +ve +ve +ve

Flavonoids -ve -ve -ve -ve -ve -ve

Phenols -ve -ve -ve -ve -ve -ve

Proteins +ve +ve +ve +ve +ve +ve

Resins -ve -ve +ve -ve -ve +ve

Sterols

terpenes -ve +ve +ve -ve +ve +ve

Reducing

sugar -ve -ve +ve -ve -ve +ve

Tannins -ve -ve -ve -ve -ve -ve

Glycosides -ve +ve +ve -ve +ve -ve

P.s.= Prosopis spicigera; T.a.= Trachyspermum ammi; Z.o.= Zingiber officinale ET= Ethanol; PE= Petroleum

ether; EA=Ethyl acetate

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Table 6.2.3: MIC (µg/ml) values for different fractions of plant extracts studied

against multi-drug resistant strains of fungus and bacteria

Fractions Strain No. Plant PE DE CH EA AC ET MT

T.a. 78.125 2500 2500 2500 2500 1250 1250 Z.o. 5000 5000 2500 2500 2500 5000 5000 C.a.1 P.s. >5000 5000 1250 2500 1250 156.25 625 T.a. 625 625 1250 1250 1250 1250 1250 Z.o. 1250 2500 1250 625 1250 1250 1250 C.a.2 P.s. 2500 1250 1250 2500 1250 312.5 1250 T.a. 312.5 625 1250 1250 2500 2500 5000 Z.o. 2500 625 1250 1250 1250 2500 5000 C.a.3 P.s. >5000 2500 2500 5000 1250 312.5 1250 T.a. 625 1250 1250 2500 2500 2500 5000 Z.o. 1250 2500 2500 2500 2500 2500 2500 C.g. P.s. 2500 5000 1250 1250 1250 156.25 625 T.a. 312.5 >5000 >5000 1250 2500 2500 >5000 Z.o. 2500 5000 5000 2500 2500 2500 5000 C.k. P.s. 1250 2500 625 1250 1250 78.125 312.5 T.a. 625 >5000 5000 1250 1250 1250 2500 Z.o. 1250 2500 1250 1250 1250 2500 2500 C.t.1 P.s. 2500 1250 2500 1250 625 78.125 156.25 T.a. 625 5000 5000 1250 1250 1250 2500 Z.o. 1250 2500 1250 1250 1250 1250 1250 C.t.2 P.s. 2500 2500 625 2500 1250 78.125 78.125 T.a. 1250 2500 2500 2500 2500 2500 5000 Z.o. 2500 5000 2500 1250 625 2500 5000 E.c.1 P.s. 2500 1250 2500 2500 2500 156.25 156.25 T.a. 2500 2500 2500 2500 2500 2500 2500 Z.o. 1250 2500 2500 2500 1250 5000 5000 E.c.2 P.s. 1250 2500 1250 2500 2500 78.125 312.5 T.a. 1250 2500 1250 2500 2500 2500 5000 Z.o. 2500 5000 1250 1250 5000 5000 5000 E.c.3 P.s. 2500 2500 5000 5000 1250 1250 2500 T.a. 1250 5000 5000 2500 2500 5000 2500 Z.o. 5000 5000 1250 1250 1250 1250 2500 E.c.4 P.s. 1250 1250 1250 2500 2500 78.125 156.25 T.a. 1250 2500 2500 2500 1250 2500 2500 Z.o. 5000 2500 2500 1250 2500 2500 5000 E.c.5 P.s. 1250 1250 1250 1250 2500 78.125 156.25 T.a. 1250 1250 1250 1250 1250 1250 2500 Z.o. 1250 1250 625 625 1250 1250 2500 S.m. P.s. 1250 1250 1250 1250 1250 9.76 625 T.a. 625 2500 5000 1250 2500 2500 2500 Z.o. 1250 2500 1250 1250 1250 2500 >5000 S.b. P.s. 2500 1250 1250 2500 1250 4.88 1250

MIC= Minimum Inhibitory Concentration; Strains C.a.=Candida albicans,C.g.=Candida glabrata, C.t.

=Candida Tropicalis, C.k.=Candida krusei, E.c.= Escherichia coli, S.m.=Streptococcus mutans,

S.b.=Streptococcus bovis; Plants T.a.= Trachyspermum ammi, Z.o.= Zingiber Officinale, P.s.=Prosopis

spicigera; Fractions PE=Petroleum Ether, DE=Diethyl Ether, CH= Chloroform, EA= Ethyl Acetate, AC; =

Acetone, ET= Ethanol, MT= Methanol

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Table 6.2.4: MBC/MFC (µg/ml) values for different fractions of plant extracts

studied against multi-drug resistant strains of fungus and bacteria

Fractions Strain No. Plant PE DE CH EA AC ET MT T.a. 78.125 2500 5000 2500 2500 2500 1250 Z.o. >5000 >5000 2500 5000 5000 >5000 >5000 C.a.1 P.s. >5000 5000 2500 5000 5000 625 1250 T.a. 625 1250 2500 1250 1250 5000 2500 Z.o. 1250 2500 1250 1250 2500 1250 2500 C.a.2 P.s. 2500 2500 2500 2500 2500 312.5 1250 T.a. 312.5 625 1250 2500 2500 5000 >5000 Z.o. 5000 2500 1250 1250 2500 2500 >5000 C.a.3 P.s. >5000 5000 2500 >5000 2500 312.5 1250 T.a. 625 2500 1250 2500 5000 5000 >5000 Z.o. 2500 2500 5000 5000 5000 2500 2500 C.g. P.s. 5000 >5000 2500 2500 2500 156.25 1250 T.a. 625 >5000 >5000 1250 >5000 5000 >5000 Z.o. 5000 >5000 >5000 5000 2500 5000 >5000 C.k. P.s. 2500 2500 2500 2500 2500 312.5 312.5 T.a. 625 >5000 >5000 1250 2500 2500 5000 Z.o. 1250 2500 1250 2500 1250 5000 5000 C.t.1 P.s. 2500 2500 2500 1250 1250 312.5 625 T.a. 1250 >5000 >5000 1250 5000 2500 5000 Z.o. 2500 2500 2500 2500 2500 1250 1250 C.t.2 P.s. 2500 2500 2500 2500 2500 78.125 156.25 T.a. 2500 5000 5000 2500 5000 5000 >5000 Z.o. 5000 5000 5000 2500 2500 5000 >5000 E.c.1 P.s. >5000 5000 5000 2500 2500 312.5 312.5 T.a. 2500 2500 5000 2500 >5000 2500 5000 Z.o. 2500 5000 2500 2500 2500 >5000 >5000 E.c.2 P.s. 2500 5000 2500 2500 5000 156.25 625 T.a. 2500 5000 2500 2500 5000 5000 >5000 Z.o. 2500 >5000 1250 2500 >5000 >5000 >5000 E.c.3 P.s. 2500 5000 >5000 >5000 2500 2500 2500 T.a. 2500 >5000 >5000 5000 2500 >5000 5000 Z.o. >5000 >5000 2500 2500 1250 2500 2500 E.c.4 P.s. 2500 1250 1250 2500 5000 156.25 156.25 T.a. 2500 2500 >5000 5000 2500 5000 2500 Z.o. >5000 2500 2500 2500 5000 5000 >5000 E.c.5 P.s. 2500 1250 1250 2500 2500 78.125 156.25 T.a. 312.5 2500 1250 2500 1250 2500 2500 Z.o. 1250 1250 1250 1250 2500 2500 5000 S.m. P.s. 2500 1250 2500 2500 1250 9.766 625 T.a. 1250 2500 5000 1250 2500 5000 2500 Z.o. 1250 2500 1250 1250 2500 5000 >5000 S.b. P.s. 5000 2500 2500 2500 1250 9.766 1250

MB/FC= Minimum Bactericidal/Fungicidal Concentration; Strains C.a.=Candida albicans,C.g.=Candida

glabrata, C.t. =Candida Tropicalis, C.k.=Candida krusei, E.c.=Escherichia coli, S.m.=Streptococcus mutans,

S.b.=Streptococcus bovis ; Plants T.a.= Trachyspermum ammi, Z.o.= Zingiber Officinale, P.s.=Prosopis

spicigera; Fractions PE=Petroleum Ether, DE=Diethyl Ether, CH= Chloroform, EA= Ethyl Acetate, AC=

Acetone, ET= Ethanol, MT= Methanol

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6.2.4 Discussion

Plant herbal mixtures have made a large contribution to human health and

well-being by providing a source of novel compounds and for the development and

synthesis of new chemotherapeutic agents. There exists vast literature on the antiviral,

anticariogenic, anthemintic, antibacterial, antifungal, anti-inflammatory and

antimolluscal properties of different plants parts (Islam et al., 2009; Islam et al., 2008;

Govindarajan et al., 2006; Betoni et al., 2006; Stepanovic et al., 2003). Their uses as

remedies for many infectious diseases as well as searches for additional substances in

plants with antimicrobial activity are frequent (Lewis and Ausubel, 2006). Plants are

rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids,

and flavonoids, which have been found in vitro to have antimicrobial properties

(Norman, 1990).

T. ammi belongs to the family Umbelliferae and is known as a popular

aromatic herb and spice. Its fruit has been used in cooking and as medicine, primarily

to control indigestion and flatulence. It is prescribed for colic, diarrhoea, antibacterial

and other bowel disorders, and in the treatment of asthma (Kaur and Arora, 2009). Z.

officinale (family Zingiberaceae) is widely used as a spice, food, and herbal medicine.

It is traditionally used for the treatment of rheumatism, nervous diseases, gingivitis,

toothache, asthma, constipation, diabetes, and arthritis (Chang et al., 1995). It has

phytoconstituents that have anti-inflammatory, anti-oxidant and anti-cancer effects

(Isa et al., 2008; Ippoushi et al., 2003). P. spicigera, from the family Leguminosae, is

not an extensively studied plant as not much literature is available. It is known to

possess anti-inflammatory properties (Madan et al., 1972). There aredifferences in the

antimicrobial activity of the plants in different solvents as each fraction might possess

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different compounds. This is in agreement with other reports (Parekh et al., 2006;

Parekh et al., 2005). The ability of the extracts to inhibit bacteria as well as fungus

suggests the presence of broad spectrum antibiotic compounds. As reported earlier,

our study also shows that Gram-positive bacteria are more sensitive than Gram-

negative bacteria (Buwa and Staden, 2006; Khan et al., 2009).

With the rise in the emergence of various multidrug resistant microorganisms

and the scenario worsening through the indiscriminate use of antibiotics, new and/or

alternative antimicrobial compounds must be developed to treat common infections.

With the changing patterns of susceptibility and the availability of new antimicrobial

agents, continuous updating of knowledge concerning treatment of disease caused by

such pathogens is required. Extended-spectrum-β-lactamases (ESBLs) have emerged

among Gram-negative bacteria, including Klebsiella pneumoniae (K. Pneumonia) and

E. coli, which has greatly contributed to the enhanced resistance toward a wide range

of antibiotics that are presently in use. ESBLs include TEM, SHV and CTXM

enzymes that are on the rise in enterobacterial isolates (Shakil et al., 2009; Mushtaq et

al., 2003).The search for alternative strategies for the management of disease-resistant

microbes is one of the possible strategies towards this objective and involves the

rational localization of bioactive phytochemicals which have antibacterial activity.

This could be one of the important approaches for the containment of antibiotic

resistance (Bonnet, 2003). The ability of the plants tested in this study against ESBL-

positive bacteria exhibits their efficacy in the treatment of infections caused by such

strains.

An alkaloid sceptrin, isolated from Agelas sceptrum, has been shown to

possess antimicrobial activity against S. aureus, Bacillus subtilis, C. albicans,

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Pseudomonas aeruginosa (P. Aeruginosa), Alternaria (fungus), and Cladosporium

cucumerinum (Soltis et al., 1999). Bromotyrosine alkaloids have demonstrated high

antimicrobial activity against a number of Gram-positive organisms, including

Mycobacteria and Staphylococci, including MRSA, VRSA and VRSH (Pick et

al.,2006). A number of peptides have also been reported to possess antimicrobial

activities. Fallaxin, a 25-mer antibacterial peptide amide, has been shown to inhibit

the growth of several Gram-negative bacteria including E. cloacae, E. coli, K.

pneumoniae, and P. aeruginosa (Nielson et al., 2007). Similarly, antimicrobial

activities of low molecular mass lysine dendrimers against S. aureus, E. coli and C.

albicans have been reported earlier (Janiszewska and Urbańczyk-Lipkowska, 2006).

Proteins such as thanatins, upon chemical modification at the side-chain of cysteine

residues, exhibited eight-fold higher antimicrobial activity against Micrococcus luteus

than wild type thanatin. It was found that there were pathogens by inducing

membrane disruption followed by the efflux of bacterial cytosolic contents

(Malmström et al., 2009). Antibacterial and antifungal terpenes have been isolated

from Pilgerodendron uviferum (D. Don) Florin (Solís et al., 2004). A sterol, 7-

aminocholesterol displayed antibiotic activity against Saccharomyces cerevisiae, S.

aureus, Enterococcus hirae and Bacillus cereus (Dherbomez et al., 1995).

Iyengaroside-A (2), a glycoside isolated from the ethyl acetate soluble part of the

methanolic extract of the marine green alga Codium iyengarii has been reported to

show bactericidal activity (Ali et al., 2002).

The extracts showed significant activity against most of the investigated

microbial strains, which is a promising. It is interesting to note that the extracts are

not pure compounds and in spite of it, antimicrobial results were obtained, which only

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suggests the potency of these extracts. The potential for developing antimicrobials

from plants is rewarding as it will lead to the development of a phytomedicine to act

against microbes. Plant based antimicrobials have enormous therapeutic potential as

they can serve the purpose without any adverse effects that are often associated with

synthetic compounds; hence isolation and purification of phytoconstituents from these

plants may yield significant novel antimicrobials.

All the extracts showed varying degrees of antimicrobial activity against the

resistant strains of microorganisms. The possibility of obtaining phytochemicals was

more apparent in the petroleum ether fraction of T. ammi, ethyl acetate fraction of Z.

officinale and ethanol fraction of P. spicigera. The presence of phytochemicals may

be responsible for their therapeutic effects. These plants could be a source of new

antibiotic compounds which could be more effective against multidrug resistant

strains of bacteria and fungus. To test and identify the specific antimicrobial

compounds, further work is needed.