7

REVIEW OF LITERATURE

2.1 Drug resistance in bacteria

Antimicrobial resistance continues to grow quickly among key microbial

pathogens such as Staphylococcus aureus, Pseudomonas spp, Streptococcus spp, and

Enterobacteriaceae all around the world. Development of new antimicrobial agents is

imperative. The increased prevalence of antibiotic-resistant bacteria due to the extensive

use of antibiotics may render the current antimicrobial agents insufficient to control some

bacterial diseases. Global antibacterial resistance is becoming an increasing public health

problem. Bacterial resistance to almost all available antibacterial agents has been

reported. The pharmaceutical industry and biotechnology companies are intensifying

efforts to discover novel antibacterial agents in attempts to overcome bacterial resistance.

Most of the bacteria and other micro organisms have developed resistance to the existing

line of drugs and due to this most of developing countries are affected by health

problems. To overcome these problems, it is necessary to investigate newer molecules to

treat infections at affordable costs.

2.2 Efflux Pumps in Bacteria

The first incident of resistance due to efflux was observed in Escherchia coli against

tetracycline (Mcmurry et al., 1980; Ball et al., 1980). In bacteria there are five major

families of efflux transporters: 1. MF (major facilitator) 2. MATE (multidrug and toxic

efflux) 3. RND (resistance-nodulation-division) 4. SMR (small multidrug resistance) and

5. ABC (ATP binding cassette). All these families utilize the proton motive force as an

energy source, apart from the ABC family, which utilizes ATP hydrolysis for the export

of substrates (Li and Nikaido, 2004). MFS and RND are the most abundant pumps. MFS

is found in both Gram positive and Gram negative bacteria while RND is found only in

Gram negative bacteria (Han et al., 2007).

8

Fig. 2.1 Schematic illustration of main types of bacterial efflux pumps

In Gram-negative bacteria, most of the efflux pumps that contribute to resistance to most

antibiotics are three component structures that traverse both inner membrane and outer

membrane. This structural organization allows extrusion of substrates directly into the

external medium bypassing the periplasmic space and makes efflux pumps more efficient

(Zgurskaya and Nikaido, 1999).

2.2.1 Development of mdr in E. coli

Multidrug resistance (mdr) is an increasing public health concern worldwide (Nikaido,

2009; Poole, 2005). There is a growing epidemic of mdr Gram-negative pathogens and a

dwindling arsenal of antibiotic options. Multiple-antibiotic- resistance (Mar) mutants of

E. coli express elevated levels of resistance to a wide range of structurally unrelated

antibiotics (Morgan et al., 2009; Nishino and Yamaguchi, 2001) and the resistance to

some agents, such as tetracycline and fluoroquinolones, has been shown to result from

increased levels of active efflux (Morgan et al., 2009). Increased efflux pump expression

9

has been documented in association with resistance to several antibiotic classes, including

the fluoroquinolones. Of more than 40 putative transporters in E. coli, acrAB-tolC, mdfA,

and norE affect fluoroquinolone MICs when expressed with their own promoters under

laboratory growth conditions (Sulavik et al., 2001). Only AcrAB-TolC overproduction,

however, has been shown to contribute to clinical fluoroquinolone resistance.

Additionally, plasmid-borne efflux pump gene qepA was found in a small percentage of

E. coli isolates (Perichon et al., 2007; Yamane et al., 2007) and it confers resistance to

fluoroquinolones and aminoglycosides (Perichon et al., 2008).

2.2.2 Development of mdr in P. aeruginosa

P. aeruginosa has acquired multiple mechanisms of resistance against all available

antipseudomonal agents (Hancock, 1998; Sefton, 2002). Specifically, target-based

mutation in gyrase or topoisomerase and overexpression of efflux pumps (EPs) contribute

to fluoroquinolone (FQ) resistance (Hancock, 1998). IN VITRO data indicate that at FQ

concentrations near the MIC, efflux mutants are preferentially selected before target

mutations (Kohler et al., 1997). Efflux pumps present in P. aeruginosa serve physiologic

functions such as the removal of intracellular toxic substances or metabolites as well as

excretion of signaling molecules into the environment to facilitate cell-to-cell

communication (Gotoh et al., 1998; Kohler et al., 2000; Neyfakh, 1997; Pearson et al.,

1999; Poole, 2000). Six EPs have been identified so far: MexAB-OprM, MexCD-OprJ,

MexEF-OprN, MexXY-OprM, MexJK-OprM, and MexVW-OprM (Chuanchuen et al.,

2002 ; Li et al., 2003 ; Poole, 2002). The first three have been well characterized. Each

pump consists of a tripartite system: cytoplasmic membrane exporter protein in the RND

family, an outer membrane protein, and a membrane fusion protein linking the exporter

protein and the outer membrane protein. All of the pumps can expel a variety of

compounds ranging from detergents to structurally unrelated antimicrobial agents from

the cytoplasm or periplasmic space (Gotoh et al., 1998; Poole, 2002). While each pump

has a preferential set of antimicrobial agent substrates, the fluoroquinolones are universal

substrates for all known Mex pumps (Poole, 2002). Thus, flouroquinolone exposure has

10

the potential to select for mutants with the mdr phenotype via efflux pump

overexpression.

2.2.3 Development of mdr in S. aureus

S. aureus is a major human pathogen that produces many virulence factors which

contribute to its pathogenicity (Gemmel et al., 1997; Bolsuck et al., 2005). This

bacterium is a cause for considerable concern, because of its ability to acquire resistance

towards the currently used antibacterial agents (Stavri et al., 2007). S. aureus

chromosomes encode a range of mdr transporters. Several of these efflux pumps have

been identified and demonstrated to cause resistance to various compounds (Bolsuck et

al., 2005). Fluoroquinolone resistance of several clinical isolates of S. aureus is provided

by the membrane protein NorA encoded in the bacterial chromosome (Markham et al.,

1999; Neyfakh, 1997). Also, over-expression of NorB leads to decrease in the

susceptibility to fluoroquinolones, tetracycline, disinfectants, and dyes (Piddock, 2006;

Bolsuck et al., 2005). Mechanism involved in quinolone resistance in S. aureus is

overexpression of norA. This gene encodes a multidrug efflux protein (NorA) capable of

transporting FQ outside the bacteria (Kaatz et al., 1993; Kaatz and Seo, 1995; Kaatz and

Seo, 1997; Yamada et al., 1997; Yoshida et al., 1990). Overexpression of norA has been

related to mutations 89 bp upstream from the putative ATG start codon (Kaatz et al.,

1993; Ng et al., 1994). NorA-mediated resistance has been described in the apparent

absence of mutations in topoisomerase genes (Kaatz and Seo, 1997). Moreover, NorA-

mediated resistance can appear both in the presence (Kaatz et al., 1993; Ng et al., 1994)

and in the absence of promoter mutations (Ng et al., 1994). S. aureus strains derived from

a single strain (SA-1199) that can overexpress norA in a constitutive or inducible manner

have been reported. Inducible strains lack promoter mutations (Ng et al., 1997). NorA

has been shown to play a role even in quinolone susceptible strains, since norA disruption

leads to MIC eightfold lower than for the parent strain (Yamada et al., 1997).

Examination of the S. aureus genome reveals numerous potential mdr efflux-pump-

encoding genes. Some of those that have been studied in detail including QacA and

QacB, highly similar MFS pumps that are encoded on plasmids, NorA and MdeA, both

11

chromosomally encoded MFS pumps and MepA and a MATE family mdr pump that

also is chromosomally encoded (Kaatz et al., 2005; Paulsen et al., 1996b). More recently

described are the NorB, NorC and SdrM MFS pumps, the genes for which are also

chromosomal (Bolsuck et al., 2005; Yamada et al., 2006). SepA gene is of considerable

interest, positioned immediately downstream of sdrM, which encodes a structurally

unique transporter that has some similarity to the SMR family of proteins (Narui et al.,

2002). Unlike all other S. aureus SMR family pumps described to date, SepA is encoded

on the chromosome.

2.2.4 Development of mdr in S. typhimurium

Salmonellae posses multiple drug efflux systems including the AcrAB-TolC system (Li

and Nikaido, 2004; Li, 2008). Laboratory- selected and naturally occurring FQ resistant

S. typhimurium strains showed increased expressions of acrA, acrB, acrE, acrF, emrB,

emrD and mdlB as well as, to a lesser extent, of mdtB, mdtC and emrA. A

complementary result is that ciprofloxacin –resistant S. typhimurium mutants are

difficult to select in the absence of AcrB and TolC (Ricci et al., 2006). In S. typhimurium

DT204 overexpression of acrAB plays a dominant role in floroquinolone resistance and

selection of floroquinolone resistant mutant in an acrB background resulted in the

isolation of strains overexpressing acrEF through insertion of IS1 or IS10 elements. AcrD

and MdtABC pumps are also involved in metal resistance (Nishino et al., 2007).

Table 2.1 Efflux pumps of some important pathogens (Bambeke et al., 2010, Omote et

al., 2006; Putman et al., 2000; Ana, 2012).

S.no. Efflux pump

family

Nature of substrate Antibiotics used Bactria containing efflux

pump

1. SMR Lipophilic,

multicationic

Substrates

Tetracycline,

erythromycin,

sulfadiazine

Staphylococcus aureus

and Acinetobacter

baumannii

2. RND Aphiphilic, charged

substrates

Tetracycline,

fluoroquinolone,

Escherchia coli and

Pseudomonas aeruginosa

12

erythromycin, rifampicin,

β- lactam, fusidic acid,

chloramphenicol,

aminoglycosides

3. MFS Amphiphilic, mono

or dicationic

substrates

Tetracycline,

fluoroquinolone,

erythromycin,

lincosamides, rifampicin,

pristinamycin,

chloramphenicol,

aminoglycosides

Staphylococcus aureus

and Escherchia coli

4. ABC Amphiphilic neutral

or cationic

substrates

Teracycline,

fluoroquinolone,

macrolids, lincosamides,

rifanpicin,

chloramphenicol,

aminoglycosides

Staphylococcus aureus

and Lactococcus lactis

5. MATE Low molecular

weight Cationic

substrates

Norfloxacin,

Fluoroquinolone, ami

oglycosides

Staphylococcus aureus,

Escherichia coli and

Vibrio parahaemolyticus

2.3 Resistance modulating compound from plants

Plants have traditionally provided a source of hope for novel drug compounds, as plant

herbal mixtures have made large contributions to human health and well-being (Iwu et

al., 1999). Owing to their popular use as remedies for many infectious diseases, searches

for substances with antimicrobial activity in plants are frequent (Betoni et al., 2006;

Shibata et al., 2005). The rich chemical diversity in plants promises to be a potential

source of antibiotic resistance modifying compounds.

2.3.1 Active compounds with resistance modifying activities from plant sources

Some isolated pure compounds of plant origin have been reported to have resistance

modifying activities in vitro. Examples of some of the compounds are given in Table 2.2.

13

This has prompted the search for such compounds from a variety of medicinal plants.

Some of the compounds which have been observed to have direct antimicrobial activity

have also been shown to potentiate the activity of antibiotics when used at below MIC

levels.

The antimicrobial properties of tea (Camellia sinensis) have been found to be a result of

the presence of polyphenols (Yam et al., 1998; Stapleton et al., 2004; Si et al., 2006).

Bioassay directed fractionation of the extracts revealed that epicatechin gallate (ECG),

epigallocatechin gallate (EGCG), epicatechin (EC) and caffeine (CN) are the bioactive

components. ECG and CG reduced MIC values for oxacillin from 256 and 512 to 1 and 4

mg/L against MRSA (Shibata et al., 2005). Ethyl gallate, a conginer of alkyl gallates

purified from a dried pod of Tara (Caesalpinia spinosa) native to South America,

intensified beta-lactam susceptibility in MRSA and MSSA strains (Shibata et al., 2005).

The abietane diterpenes, (carnosic acid carnosol) isolated from the aerial parts of

Rosmarinus officinalis by fractionation of the chloroform extract at 10 µg/mL,

potentiated the activity of erythromycin (16 -32 fold) against strains of S. aureus that

express the two efflux proteins MsrA and TetK. Additionally, carnosic acid was shown to

inhibit ethidium bromide efflux in a NorA expressing S. aureus strain (Oluwatuyi et al.,

2004). A penta-substituted pyridine, 2, 6-dimethyl-4-phenylpyridine-3, 5-dicarboxylic

acid diethyl ester and proparcine have been isolated

from an ethanol extract of rhizome of Jatropha elliptica by bioassay guided fractionation.

The pyridine at a concentration of 75 µg/mL was shown to increase by 4 -fold, the

activity of ciprofloxacin and norfloxacin against NorA expressing S. aureus when tested

at sub-inhibitory concentrations (Marquez and Beatrice, 2005; Smith et al., 2007).

Screened active compounds from the cones of Chamaecyparis lawsoniana for resistance

modifying activities and observed that Ferruginol and 5-Epipisiferol were effective in

increasing the efficacy of tetracycline, norfloxacin, erythromycin and oxacillin against

resistant S. aureus. The majority of researches on the combinations between plant

extracts and antibiotics have been focused on the identification and isolation of potential

resistance modifiers from such natural sources which are considered to be positive

results. However, it is likely that such combinations could produce antagonistic

interactions that most studies have considered irrelevant and therefore ignored.

14

There are several modes of action of efflux pumps: a) The EPI may bind directly to the

pump in a competitive or non competitive manner with the substrate, causing the

blocking of the efflux pump; b) EPI may also cause a depletion of energy, through the

inhibition of the binding of ATP or the disturbance of the proton gradient across the

membrane; c) EPI may have affinity for substrates, and bind them, forming a complex

that facilitates the entry of the drug in the cell and prevents its efflux (Zloh et al., 2004;

Marquez and Beatrice, 2005).

2.4 Efflux Pump Inhibitors

The use of efflux pump inhibitors can facilitate the re-introduction of therapeutically

ineffective antibiotics back into clinical use and might even suppress the emergence of

mdr strains (Stavri et al., 2007).

2.4.1 Synthetic EPIs against different bacteria

Synthetic compounds remain to be the major approach in finding bacterial efflux

pump inhibitors, because little is known about substrate-pump binding interaction.

1. L-phenylalanyl-L-arginyl-b-naphthylamide (PAβN): It is a dipeptide amide

and had potentiated the activity of levofloxacin by 8 fold at 10 µg/mL against P.

aeruginosa (Barrett, 2001). It also increases the susceptibility of erythromycin 8-

32 fold and rifampicin 8-64 folds against Campylobacter jejuni and

Campylobacter coli (Hannula and Hanninen, 2008). It also inhibited the efflux

pump in E. coli and reduced the susceptibility of rifaximin (Gomes et al., 2013).

PAβN convertes ciprofloxacin resistant strains of Pseudomonas aeruginosa,

Acinetobacter baumannii and Escherchia coli to susceptible ones (Cetinkaya et

al., 2008). In combination with fluoroquinolones, it seems to have inhibitory

activity against the MexCD-OprJ and MexEF-OprN pumps of P. aeruginosa, and

against the AcrAB-TolC efflux pump of Gram-negative bacteria, including K.

pneumoniae, E. coli, S. typhimurium and E. aerogenes (Hasdemir et al., 2004;

15

Renau et al., 1999; Mazzariol et al., 2000; Baucheron et al., 2002; Mallea et al.,

2002).

2. Arylpiperidines and arypiperazines: Some of the members of Arylpiperazines

family are Capable of reversing multidrug resistance in E. coli overexpressing

RND Efflux Pumps, eg: 1-(1- Naphthylmethyl) - piperazine enhanced the

susceptibility of E. coli to fluoroquinolones and levofloxacin (Bohnert and

Winfried, 2005).

3. Quinoline derivatives: They have been proved as promising inhibitors of

antibiotic efflux pump in multidrug resistant Enterobacter aerogenes isolates.

Various quinoline derivatives significantly increased the intracellular

concentration of chloramphenicol and thereby inhibit the transport of drug by

AcrAB-TolC pump (Mahamoud et al., 2006).

4. Alkoxyquinolone derivatives: Alkoxyquinolone derivatives such as 2,8-

dimethyl-4-(2’-pyrrolidinoethyl)-oxyquinoline, inhibit efflux pumps in E.

aerogenes and K. pneumoniae. This EPI increased the efficacy of

chloramphenicol, norfloxacin, tetracycline and cefepime by up to 8-fold

(Chevalier et al., 2004).

5. Carbonyl cyanide m-chlorophenylhydrazone (CCCP): CCCP is a

protonophore. It considerably affects the energy level of the membrane and cell

viability by causing a dissipation of the proton motive force of the membrane,

affecting the transporters that depend on this mechanism. Besides its high toxicity

for the cell, it is described as a substrate of bacterial efflux pumps (Mahamoud et

al., 2007; Alvarado and Vasseur, 1998). It has shown inhibitory activity in

Mycobacterium smegatis (Choudhuri et al., 1999) and in Mycobacterium

fortuitum by inhibition of the MFS efflux pump (Garcia et al., 2006).

6. Phenothiazines: Thioridazine an EPI belongs to neuroleptic drugs,

phenothiazines. Thioridazine is known due to its inhibitory effect on multidrug

efflux pumps (Thorsing et al., 2013). It inhibits efflux pumps in M. tuberculosis

(Amaral and Viveiros, 2012). Phenothiazines also act as EPIs against S. aureus,

B. pseudomallei, E. coli, P. aeruginosa and S. typhimurium (Chan and Chua,

2005).

16

7. Sodium Orthovanadate: Sodium orthhovanadate (Na3VO4) is an inhibitor of

ABC efflux pumps of S. pneumoniae. It also abolishes both the efflux and

resistance to ciprofloxacin and ethidium bromide (Garvey and Piddock, 2008).

8. Amide derivatives: Two compounds of this family named 5,9-dimethyl-deca-

2,4,8-trienoic acid amides and 9-Formyl-5-methyl-deca- 2,4,8-trienoic acid

enhance the activity of ciprofloxacin against S. aureus (Sumithra et al., 2012).

9. Substituted Polyamines: N-benzylated polyazaalkanes and N-benzylated

polyaminoalkanes have ability to behave as EPIs against Haemophilus influenzae

(Sumithra et al., 2012).

10. Nocardamines: They are iron chelators and acts as EPIs against TetB and TetK

efflux pump of S. aureus (Rothstein et al., 1993).

11. Arylated benzothiophenes and tiophenes: These compounds act as EPIs against

NorA efflux pump of S. aureus by restoring the activity of ciprofloxacin against a

resistant S. aureus strain in which this efflux pump is overexpressed (Chabert et

al., 2007).

12. Indole derivatives: Indole derivatives like INF-55 and INF-271 act as EPIs

against NorA efflux pump of S. aureus (Ambrus et al., 2008). Indole derivatives

like 3-amino-6-carboxyl-indole and 3-nitro-6-amino-indole had potentiated

antibacterial effects of chloramphenicol, tetracycline, erythromycin and

ciprofloxacin against E. coli YD2 and FJ307 overexpressing AcrAB-TolC efflux

pump and also decreased MIC at 2-64 folds (Zeng et al., 2010).

13. GG918, biricodar (VX-710) and timcodar (VX-853): These are the compounds

which show synergism with fluoroquinolones against S. aureus, S. pneumoniae

and E. faecalis and also reduced the MIC of ethium bromide upto 2 to 31 fold

against these three pathogens.

14. Verapamil: It is a drug used in the treatment of hypertension, cardiac arrhythmia

and cluster headaches. It acts as an EPI against Mycobacterium tuberculosis and

also enhances the activity of isoniazid, rifampin and pyrazinamide (Gupta et al.,

2013). It also acts as an EPI of LmrA efflux pump of Lactococcus lactis

(VanVeen et al., 1996).

17

15. Phenylpiperidine selective serotonin reuptake inhibitors: P-SSRIs are

inhibitors of MFS and RND efflux pumps of different Gram positive and Gram

negative bacteria (Wei et al., 2004). P-SSRIs particularly inhibited the NorA

efflux pump of S. aureus (Kaatz et al., 2003).

16. Valinomycin and Dinitrophenol (DNP): They are used to abolish completely

the efflux of different molecules. DNP dissipate the proton-motive force of the

membrane by modifying the trans-membrane potential. Whereas valinomycin

dissipates the electrochemical gradients generated by K+

(Mahamoud et al., 2007;

Pages et al., 2005; Mallea et al., 1998 ).

Out of the EPIs discussed above, only few like PAβN and CCCP are found to be of some

use and are the most common synthetic EPIs. PAβN is routinely used in the laboratories

to indicate efflux-mediated antibiotic resistance in Gram-negative bacteria. However, it is

not in clinical use due to toxicity and bioavailability issues (Ricci et al., 2006). It can be

considered as a broad spectrum efflux pump inhibitor because it can restore the activity

of unrelated antibiotics such as chloramphenicol and macrolides (Lomovskaya et al.,

2001; Lomovskaya and Bostain, 2006). CCCP is another important EPI which is an

inhibitor of proton motive force in bacteria. Conventionally, 0.1 mM or 1mM CCCP with

incubation time less than 10min is used to detect the bacterial efflux system (Cho et al.,

2001).

2.4.2 Plant derived EPIs against different bacteria

Plants produce many cytotoxic compounds which protect them from pathogenic microbes

that is the reason why very less infective diseases are seen in wild plants (Stavri et al.,

2007).

2.4.2.1 Gram-positive bacteria

Multidrug resistant Gram positive bacteria represent a major public health problem

(Woodford and Livermore, 2009). Gram positive cocci are a major cause of nosocomial

and community acquired infections. They frequently show a high natural, intrinsic

resistance to antimicrobials (Jeljaszewicz et al., 2000).

18

1. Bacillus: Bacillus cereus causes minority of foodborne illnesses like severe nausea,

vomiting and diarrhoea (Kotiranta et al., 2000). B. subtilis causes disease in severely

immune compromised patients, and can be used as a probiotic in healthy individuals

(Oggioni et al., 1998). Drug resistance due to efflux is a common problem in Bacillus.

Reserpine a compound belonging to a class of rauwolfia alkaloids, first isolated from the

roots of Rauwolfia vomitoria is used in the treatment of high blood pressure. It is also

used to treat the patients in case of severe agitation in mental disorders and works by

slowing down the activity of the nervous system, causing the heartbeat to slow and the

blood vessels to relax (Neyfakh et al., 1991). Bmr efflux pump found in Bacillus is

inhibited by reserpine. The antihypertensive reserpine was first shown to block Bmr-

mediated multidrug resistance in Bacillus subtilis (Li and Nikaido et al., 2004;

Mahamoud et al., 2007). Chalcones belonging to flavonoids class of chemicals found

mainly in plants like Bridelia ferruginea Benth and Dalea versicolor as natural defence

mechanisms is an inhibitor of NorA efflux pump (Chambers, 1997). These compounds,

along with the stilbene, also increased the activity of tetracycline against Bacillus cereus

(Hiramatsu et al., 2001).

2. Staphylococcus: Staphylococcus aureus is one of the most important community and

major hospital-acquired pathogen (Perl, 1999; Rotun et al., 1999). S. aureus is of major

concern due to its ability to acquire resistance towards the newest antibacterial drugs

currently in the market (Blumberg et al., 1991). Reserpine enhances the activity of

fluoroquinolones on mdr Gram-positive bacteria and also decreases the emergence of

resistant mutant strains of S. aureus and S. pneumoniae (Mallea et al., 1998; Brenwald et

al., 1997; Aeschlimann et al., 1999). Although reserpine has been used to treat

hypertension from a long time, it cannot be used in combination with antibiotics for the

treatment of staphylococcal infections, as the concentrations required to inhibit NorA

efflux pump are neurotoxic (Schmitz et al., 1998). Caffeoylquinic Acids from Artemisia

absinthium showed efflux pump inhibitory activity against Gram-positive pathogenic

bacteria like S. aureus and E. faecalis (Markham and Neyfakh, 1996). NorA efflux pump

of S. aureus is inhibited by several natural products, such as the porphyrin pheophorbide

and the flavoligan 5 �- methoxy-hydnocarpin (5 �-MHC), isolated from Berberis plant

19

(Fiamegos et al., 2011; Stermitz et al., 2000). A study on Geranium has led to the

isolation of acylated neohesperidosides an inhibitor of S. aureus NorA, from Geranium

caespitosum (Michalet et al., 2007; Cetinkaya et al., 2000). The carnosic acid and

carnosol are isolated from herb Rosemary (Rosmarinus officinalis) and potentiate

tetracycline and erythromycin against S. aureus strains possessing the Tet(K) and Msr(A)

efflux pumps, respectively (Roberts, 2005; Piddock, 2006). Dalea versicolor ‘mountain

delight’ contains phenolic metabolites that enhanced the activity of berberine,

erythromycin and tetracycline against S. aureus (Belofsky et al., 2004). The catechin

gallates are a group of phenolic metabolites that was reported by Hamilton-Miller's group

to reverse methicillin resistance in MRSA (Hamilton and Shah, 2000; Gibbons et al.,

2004; Roccaro et al., 2004). An extract of Lycopus europaeus (Lamiaceae) was

investigated by Gibbons et al., in 2003. Lipophilic extract of Lycopus europaeus caused a

potentiation of tetracycline and erythromycin against strains IS-58 and RN4220 of S.

aureus possessing multidrug efflux pumps Tet(K) and Msr(A), respectively (Gibbons et

al., 2003). Baicalein a trihydroxy flavone isolated from the leaves of the thyme (Thymus

vulgaris) was identified as possessing a strong synergistic activity with tetracycline or the

β-lactam antibiotics oxacillin, cefmetazole and ampicillin against MRSA (Fujita et al.,

2005). In 2005, Marquez and Beatrice, studied extract of Jatropha elliptica

(Euphorbiaceae) and led to the isolation of the penta-substituted pyridine, 2, 6-dimethyl-

4-phenyl-pyridine-3, 5-dicarboxylic acid diethyl ester, which is not antibacterial but does

augment ciprofloxacin and norfloxacin activity against S. aureus SA-1199B. Study of

Ipomoea violacea species by Pereda et al., in 2006, led to the isolation of three

oligosaccharides exerting a potentiation effect of norfloxacin against the NorA

overexpressing S. aureus strain SA-1199B. Piperine, a major plant alkaloid isolated from

the family Piperaceae including black pepper (Piper nigrum) and long pepper (Piper

longum), has recently been reported to increase the accumulation of ciprofloxacin by S.

aureus (Khan et al., 2006). Salicylic acid, a phenolic compound present in many plants

like Salix alba, has been proved to induce a reduction of both the antibiotic ciprofloxacin

and mdr substrate ethidium bromide for S. aureus (Price et al., 2002). Several Berberis

spp such as Berberis repens, B. aquifolia and B. Fremontii produce an inhibitor of the S.

aureus NorA MEP identified as 5’-methoxyhydno-carpin (5’-MHC) (Fiamegos et al.,

20

2011). Momordica balsamina were evaluated for their ability to inhibit the activity of

bacterial efflux pumps of meticillin-resistant S. aureus (MRSA). Some compounds

isolated from Momordica balsamina significantly inhibited efflux of ethidium bromide

by MRSA (Ramalhete et al., 2011). Isopimaric acid isolated from Pinus nigra blocks the

Nor (A) efflux pump of MRSA and shows synergism with reserpine (Simonetti et al.,

2004). According to Schmitz et al., 1998, Isoflavones isolated from Lupinus argenteus

inhibits mdr pump in S. aureus (Aeschlimann et al., 1999). Two flavonols from Artemisia

annua potentiate the activity of berberine and norfloxacin against a resistant strain of S.

aureus, possessing the mdr pump (Stermitz et al., 2002). Murucoidins from Ipomoea

murucoides inhibits NorA efflux pump in S. aureus (Cherigo et al., 2008). According to

Silva et al., 2009, Kaempferol glycoside from Herissantia tiubae inhibits norA efflux

pump in S. aureus (Falco et al., 2009). A plant named Persea lingue also contains a

compound Kaempferol-3-0-L-(2,4-bis-E-p-coumaroyl) rhamnoside which inhibits NorA

efflux pump in S. aureus (Holler et al., 2012). Nor A efflux pump of S. aureus is also

inhibited by an active polyphenolic amide: N-trans-feruloyl 4'-O-methyldopamine

present in Mirabilis jalapa. Reserpine shows synergism with norfloxacin against S.

aureus (Neyfakh et al., 1993).

Some oils also have shown the EPI activity like grapefruit oil contains some of the

components that act as potential modulators of efflux pumps in MRSA strains

(Kristiansen et al., 2003). Except these EPIs discovered so far some plant extracts also

have shown EPI like activity eg: Some Kuwaiti plants are known to produce piperidine

alkaloids such as julifloridine, juliflorine and juliprosine, their methanol extract was

identified to possess resistance-modifying activity by causing a reduction in MIC of

norfloxacin against S. aureus 1199B (Ahmed et al., 1986). According to the study done

by Dickson et al., in 2006 extracts of Mezoneuron benthamianum and Securinega virosa

exerted a potentiation activity against fluoroquinolone, tetracycline and erythromycin-

resistant strains of S. aureus (Dickson et al., 2006). The methanolic extract of Punica

granatum caused an increase in ethidium bromide uptake in S. aureus RN-7044, having

an ethidium bromide efflux mechanism (Braga et al., 2005). Ethanolic extracts of

Mangifera indica, Callistemon citrinus and Vernonia adoensis are a potential source of

21

EPIs against Staphylococcus aureus, Bacillus cereus and Bacillus subtilis (Chitemerere

and Mukanganyama, 2011). From the review of previous literature it can be concluded

that a large number of EPIs has been discovered against S. aureus. Epigallocatechin

gallate is the most abundant catechin in tea and is a potent antioxidant and used in the

treatment of cancer also acts as an EPI. It is obtained from Camellia sinensis and

increases the activity of tetracycline upto four-fold against Staphylococcus epidermidis

(Stavri et al., 2007).

3. Lactococcus: Lactococcus lactis is generally considered to be non-pathogenic, but it

appears that pathogenicity may be emerged (Ramirez et al., 2013). Two types of efflux

pumps are responsible for multidrug resistance in Lactococcus lactis, these are LmrA and

LmrP. LmrP confers resistance to lincosamides, macrolids, streptoGramins and

tetracyclines. Verapamil and quinine inhibit the LmrP efflux pump competitively while

nicardipin and vinblastin inhibit it non-competitively (Putman et al., 1999). Reserpine is

also able to inhibit LmrA efflux of Lactococcus lactis (Poelarends et al., 2002; Gibbons

and Udo, 2000).

4. Mycobacterium: Mycobacterium tuberculosis is one of the oldest and most common

causes of infection and death in the World, Mycobacterium avium often causes blood

infection in AIDS patients, and Mycobacterium smegatis is also an opportunistic

pathogen. The active multidrug efflux pump (EP) has been described as one of the

mechanisms involved in the natural drug resistance in Mycobacteria (Han et al., 2007).

Piperine an alkaloid responsible for the pungency of black pepper and long pepper was

reported as an inhibitor of Rv1258c efflux pump of Mycobacterium tuberculosis (Sharma

et al., 2010). Farnesol a natural 15-carbon organic compound is a colourless liquid

extracted from oils of many plants has been reported as inhibitor of mycobacterial efflux

pumps (Jing et al., 2010).

5. Enterococcus: Enterococci are Gram-positive commensals that inhabit the

gastrointestinal tracts of almost all animals. It can cause diseases like endocarditis, UTI

22

(urinary tract infection) and surgical wound infections. EfrAB, an ABC multidrug efflux

pump in Enterococcus faecalis is inhibited by reserpine (Lee et al., 2003). Karavilagenin

C a triterpenoid isolated from Momordica balsamina significantly inhibited efflux of

Enterococcus faecalis ATCC 29212 (Ramalhete et al., 2011). 4’, 5’-O-dicaffeoylquinic

acid (4’, 5’-ODCQA), a caffeoylquinic acid from Artemisia absinthium is a pump

inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive

human pathogenic bacteria including Enterococcus faecalis (Markham and Neyfakh et

al., 1996).

2.4.2.2 Gram-negative bacteria

Multidrug-resistance phenotype is a very common problem in Gram negative bacteria. As

per the literature studied not so much of EPIs have been discovered against Gram

negative bacteria as they contain, efflux pump complexes consisting of an inner-

membrane pump, a periplasmic adaptor protein and outer-membrane channel, providing

them an efficient means for the export of structurally unrelated drugs (Pages et al., 2005).

With a decrease in the number of new agents and in antibiotic development, there is a

need to search the compounds that will restore the activity of previous antibiotics against

Gram negative bacteria (Garvey et al., 2010).

Very few compounds given below had so far proved to be working for the given Gram-

negative bacteria.

Baicalein, a flavone is an efflux pump inhibitor isolated against efflux pumps of E. coli

from Thymus vulgaris (Fujita et al., 2005). Isopimarane derivatives obtained from

Lycopus europaeus act as efflux pump inhibitors against efflux pumps of Enterobacter

aerogenes (Gibbons et al., 2003). The obromine a bitter alkaloid isolated from

Theobroma cacao plant had shown synergism with ciprofloxacin against RND efflux

pump family of different Gram- negative bacteria like Klebsiella pneumoniae,

Salmonella Typhimurium, Enterobacter cloacae and Pseudomonas aeruginosa.

23

Cathinone, a monoamine alkaloid isolated from Catha edulis shows synergism with

ciprofloxacin against Salmonella Typhimurium (Piddock et al., 2010).

Some of the plant extracts has also shown EPI like activity against Gram negative

bacteria eg: extracts of Helichrysum italicum, Thymus maroccanus, Thymus broussonetii

and Callistemon citrinus showed synergistic activity when combined with different

antibiotics and they contain some EPI-like compounds that inhibit the efflux pumps of

Pseudomonas aeruginosa (Lorenzi et al., 2009; Fadli et al., 2011). Extracts of

Commiphora molmol, Centella asiatica, Daucus carota,Citrus aurantium and

Glycyrrhiza glabra showed good activity against three strains of Salmonella enteric

serovar Typhimurium that overexpress the AcrAB-TolC efflux protein (Piddock, 2006).

The chloroform extract of Berberis aetnensis had shown EPI activity against E. coli in

combination with ciprofloxacin. Extracts of Mellisa officinalis and Levisticum officinale

had shown activity against strains of Salmonella that overproduced AcrAB efflux pump.

These extracts had also shown synergistic activity with ciprofloxacin (Garvey et al.,

2010). Ethanolic extracts of Mangifera indica, Callistemon citrinus and Vernonia

adoensis are a potential source of EPIs against P. aeruginosa and E. coli (Chitemerere

and Mukanganyama, 2011). According to the study done by Stavri et al., 2007,

chloroform extract of the leaves of Berberis aetnensis had shown synergistic activity with

the ciprofloxacin against E. coli and P. aeruginosa. Methanol plant extracts of some

Cameroonian spices like Aframomum citratum, Dorsentia psilurus and cinnamomum

zeylanicum have shown synergistic activity with aminoglycosides against mdr

phenotypes of Enterobacter aerogenes and Klebsiella pneumoniae (Piddock, 2006).

24

Table 2.2 Efflux pumps, antibiotics involved and EPIs derived from plants for

various different bacteria

S.

No

Bacteria EPI Antibiotic Efflux

Pump

Plant source References

1. B. subtilis Reserpine Tetracycline Bmr Rauwolfia

vomitoria

Mahamoud et al.,

2007; Li and

Nikaido, 2004

2. B. cereus Chalcone berberine,

erythromycin

and tetracycline

NorA Nicotiana

tobacum,

Dalea versicolor

Hiramatsu et al.,

2001

3. S. pneumonia Reserpine Ciprofloxacin NorA Rauwolfia

vomitoria

Mallea et al., 1998,

Brenwald et al.,

1997; Aeschlimann

et al., 1999; 67

4. S. aureus Reserpine Norfloxacin,

Tetracycline

TetK,

NorA

Rauwolfia

vomitoria

Mallea et al., 1998,

Brenwald et al.,

1997; Aeschlimann

et al., 1999;

Neyfakh et al.,

1993

Porphyrin,

Pheophorbide

Ciprofloxacin,

Norfloxacin

NorA Berberis

aetnensis

Fiamegos et al.,

2011; Stermitz et

al., 2000

Polyacylated

neohesperidoside

s

ciprofloxacin,

norfloxacin,

rhein, berberine

NorA Geranium

caespitosum

Michalet et al.,

2007; Cetinkaya et

al., 2000

Carnosic acid

and Carnosol

tetracycline and

erythromycin

Tet (K)

and

Msr (A)

Rosmarius

officinalis

Roberts, 2005;

Piddock, 2006.

Chalcone berberine,

erythromycin

and tetracycline

NorA Dalea versicolor Belofsky et al.,

2004.

Epicatechin

gallate and

Norfloxacin NorA Camellia sinensis Hamilton and Shah

2000., Gibbons et

25

Epigallocatechin

gallate

al., 2004; Roccaro,

et al., 2004

Baicalein Tetracycline tetK Thymus vulgaris Fujita et al., 2005

Citropten and

Furocoumarins

Norfloxacin NorA,

ermA,

ermB

Citrus paradise Kristiansen et al.,

2003

Orizabin Norfloxacin Nor A Ipomoea

violacea

Pereda et al., 2006.

Piperine Ciprofloxacin MdeA

and Nor

A

Piper nigrum,

Piper longum

Khan et al., 2006.

Salicylic acid Ciprofloxacin,

Ethidium

bromide

SarA Salix alba Price et al., 2002

Balsaminol,

Balsaminagenin,

Karavilagenin

AcrAB-TolC NorA Momordica

balsamnia

Ramalhete et al.,

2011

Isopimaric acid - Nor (A) Pinus nigra Simmonetti et al.,

2004

Crysoplenol and

Crysoplenetin

Berberine,

Fluoroquinolone

s, Norfloxacin

NorA Artemisia annua Stermitz et al.,

2002

Murucoidins Norfloxacin Nor A Ipomoea

murucoides

Cherigo et al., 2008

Kaempferol

Glycoside,

Tiliroside

Ciprofloxacin Nor A Herissantia

tiubae

Falcao et al., 2009

Genistein,

orobol,

Biochanin

Norfloxacin,

Berberine

- Lupinus

argenteus

Morel et al., 2003

Galbanic acid Ciprofloxacin,

Ethidium

bromide

- Ferula

szowitsiana

Fazly et al., 2010

Chrysosplenol-D Berberine - Artemisia annua Stermitz et al.,

2002

26

Orobol - NorA Lupinus

argenteus

Kourtesi et al.,

2013

Biochanin - NorA Lupinus

argenteus

Kourtesi et al.,

2013

Bonducillin Berberine - Caesolpinia

digyna

Kourtesi et al.,

2013

Acetoxycavicola

cetate

Ehidium

bromide

- Alpinia galangal Kourtesi et al.,

2013

Totarol Ehidium

bromide

- Chamaecyparis

nootkatensis

Smith et al., 2007a

Ferruginol Norfloxacin,

oxacillin

NorA Chamaecyparis

lawsoniana

Smith et al., 2007b

Olaanolic acid,

ulvaol

- - Carpobrotus

edulis

Martins et al., 2011

Orizabin Norfloxacin - Ipomoea

violacea

Pereda et al., 2006

Harmaline Ethidium

Bromide

- Peganum

harmala

Mohtar et al., 2009

Ergotamine Norfloxacin - Claviceps

purpurea

Gibbons, 2008

Julifloridine,

Juliflorine and

Juliprosine

Norfloxacin - Prosopis juliflora Stavri et al., 2007

Indoles,

Indirubicin

Ciprofloxacin - Wrightia

tinctoria

Ponnusamy et al.,

2010

Chalcone Ethidium

bromide

Nor A Nicotiana

tobacum

Holler et al., 2012

Pterocarpan

Berberine Nor A Dalea spinosa Kourtesi et al.,

2013

Reserpine - LmrA Rauwolfia

vomitoria

Poelarends et al.,

2002; Gibbons et

al., 2002

Caffeoylquinic

acids

- NorA Artemisia

absinthium

Markham and

Neyfakh 1996

5. S. epidermidis Epigallocatechin

Gallate

Tetracycline Tet(K) Camellia sinensis Marquez and

Beatrice, 2005

6. Mycobacterium Farnesol Ethidium - Cymbopogon Jing et al., 2010

27

spp. bromide Citratus,

Cyclamen

Myricetin, Isoniazid - Allium cepa Lechner et al., 2008

Quercetin Isoniazid - Allium cepa Lechner et al., 2008

Rutin Isoniazid - Dimorphandra

mollis

Lechner et al., 2008

Taxifolin Isoniazid - Sophora

japonica

Lechner et al., 2008

Isorhamnetin Isoniazid - Tagetes lucida Lechner et al., 2008

Kaempferol Isoniazid - Camellia sinensis Lechner et al., 2008

Baicalein,

Biochanin A

Ethidium

bromide

- Oroxylum

indicum

Mossa et al., 2004

Epicatechin Isoniazid - Camellia sinensis Lechner et al., 2008

Genistein Ethidium

bromide

- Glycine max Mossa et al., 2004

Resveratrol Ethidium

bromide

- Fallopia

japonica

Lechner et al., 2008

Plumbagin Isoniazid - Plumbago

zeylanica

Mossa et al., 2004

Sandaracopimeri

c acid

Isoniazid - Juniperus

procera

Mossa et al., 2004

Totarol Isoniazid - Juiperus procera Mossa et al., 2004

Ferruginol Isoniazid - Juiperus procera Mossa et al., 2004

Curcumin,

Demethoxycurcu

min

Isoniazid - Curcuma longa Kourtesi et al.,

2013; Lechner et

al., 2008

Piperine Ethidium

bromide

Rv1258

c

Piper nigrum,

Piper longum

Sharma et al., 2010

7.

E. faecalis Karavilagenin C - - Momordica

balsamina

Ramalhete et al.,

2011

Caeffeoylquinic

acid

Berberine NorA Artemisia

absinthium

Markham et al.,

1999

8. E. cloacae Theobromine Ciprofloxacin AcrAB-

TolC

Theobroma

cacao

Piddock et al., 2010

9. E. coli Baicalein Tetracycline Tet K Thymus vulgaris Fujita et al., 2005

28

2.4.2.3 Structures of some important EPIs

Pheophorbide a

Ciprofloxacin

-

Berberis

aetnensis

Musumeci et al.,

2003

10. S. typhimurium Theobromine Ciprofloxacin AcrAB-

TolC

Theobroma

cacao

Piddock et al., 2010

Cathinone Ciprofloxacin AcrAB-

TolC

Catha edulis Piddock et al., 2010

11.

P. aeruginosa Pheophorbide a Ciprofloxacin MexAB

-OprM

Berberis

aetnensis

Musumeci et al.,

2003

Theobromine Ciprofloxacin MexAB

-OprM

Theobroma

cacao

Piddock et al., 2010

12. K.pneumoniae Theobromine Ciprofloxacin AcrAB-

TolC

Theobroma

cacao

Piddock et al., 2010

29

Fig. 2.2 Structures of important EPIs

30

2.5 Rosmarinus officinalis

Fig. 2.3 Flowers and leaves of R.officinalis

Classification

Kingdom: Plantae

Division: Magnoliophyta

Class: Equisetopsida

Subclass: Magnoliidae

Order: Lamiales

Family: Lamiaceae

Genus: Rosmarinus

Species: officinalis

Rosemary (Rosmarinus officinalis) come from the genus Rosmarinus being derived from

the Latin ros, meaning dew (also similar to rhus meaning small shrub) and marinus

meaning sea. This refers to the plant’s habit of growing near the coast. Rosemary is an

evergreen perennial shrub growing to 2m in height, with thick, dark green, aromatic and

linear leaves. Rosemary thrives in sunlight, needing hot, dry summers and calcareous,

clayey (impoverished) soils not saturated by water to achieve optimal growth. Rosemary

is native to the Western Mediterranean region, growing wild on the islands and in the

countries surrounding the Mediterranean ocean - France, Spain, Tunisia, Morocco,

31

Algeria, Italy, Corsica, Sardinia and the Atlantic coast of Portugal. It is also grown

commercially as a culinary herb and for essential oil production in the USA, Australia

and other temperate climates.

Rosemary is commonly used as a spice and flavoring agent in food processing (Saito et

al., 2004). Also, rosemary is used as an antispasmodic in renal colic and dysmenorrheal,

in relieving respiratory disorders and to stimulate hair growth. Extract of Rosemary

relaxes smooth muscles of trachea and intestine, and has choleretic, hepatoprotective and

antitumergenic activity. Moreover, Rosemary constituents have a therapeutic potential in

the treatment or prevention of bronchial asthma, spamogenic disorders, diabetes mellitus,

peptic ulcer, inflammatory diseases, hepatotoxicity, atherosclerosis, ischemic heart

diseases, cataract, cancer and poor sperm motility (Al-Sereiti et al., 1999; Masuda et al.,

2002; Osakabe et al., 2004).

2.6 Biological effects of R. officinalis

R.officinalis exhibit several biological proporties which are discussed below:

2.6.1 Antioxidant properties

The antioxidant activity of polar extracts of Rosemary is related to the content of

phenolic compounds (i.e. carnosol, carnosic acid). Constituents in Rosemary have shown

a variety of pharmacological activities for cancer chemoprevention and therapy in vitro

and, in vivo models (Shabtey et al., 2008).

Cheung and Tai in (2007) studied anti–proliferative properties of crude extracts of

Rosemary (Rosmarinus officinalis L.) in several human cancer cell lines and their anti-

oxidative properties in vitro in a mouse RAW 264.7 macrophage/monocyte cell line. The

study showed that the crude ethanolic Rosemary extract had anti-proliferative effect on

human leukemia and breast carcinoma cells. The body possesses various antioxidative

systems (free radical scavenging activity, FRSA) that prevent oxidative stress, for

example Saliva exhibits such an activity.

2.6.2 Antidiabetic properties

32

Rosemary is used in traditional Turkish folk medicine for the treatment of

hyperglycaemia. In 2008, BakireI and his coworkers investigated potential effect of

ethanolic extract of leaves on glucose homeostasis in rabbits. Results of this study

showed that ethanolic extracts of leaves of Rosmary reduced blood glucose level in

normoglycaemic and glucose-hyperglycaemicra rabbits. Repeated administration of

Rosemary extract to alloxan-diabetic rabbits led to a decrease in blood glucose level and

significant increase in serum insulin level.

2.6.3 Effect on immunity

The results of Babu et al., (1999) indicated that Rosemary dietary extract might not

result in general enhancing of the immune system in young rats and will probably be

effective under some stress condition, such as protein or antioxidant deficiency.

2.6.4 Cognition-enhancing properties

Several non-toxic European herbal species have pan-cultural traditions as treatment for

cognitive deficit, including that associated with ageing. Particularly promising candidate

species include Sage, Lemon and Rosemary.The essential action of Rosemary essential

oil is in stimulation of the nervous system under sympathetic control resulting in

improved memorizing and concentrating abilities (Sanders et al., 2002).

Rosemary essential oil was found to cause moderate inhibition of acetylcholinesterase

(Orhan et al., 2008). The anti-acetylcholinesterasd activity of Rosemary essential oil is

explained by synergic interaction between 1,8 cineole and 2-pinene in Rosemary essential

oil. Inhibition of acetylcholinesterase is considered one of the treatment strategies against

several neurologic disorders, including Alzeimer's disease, senile dementia and

myasthenia gravis

2.6.5 Effect on bone

In 2003, Muhlbauer et al., tested the effects of some common herbs (Rosemary, Thyme

and Sage) and their constituent essential oils and monoterpenes on bone resorption in

ovariectomized rats. Bone resorption was inhibited by the addition of I g of powdered

33

leaves of each herb and the essential oils extracted from Sage and Rosemary had similar

inhibitory effects (Faixova et al., 2008).

2.6.6 Antimicrobial properties

Moreno et al., in 2006 reported that Rosemary plants are rich sources of phenolic

compounds with high antimicrobial activity against both Gram-positive and Gram-

negative bacteria. High percent of the antimicrobial activity they attributed to carnosic

acid and carnosol. It is clear that Rosemary extracts have bioactive properties, but their

antimicrobial activities have not been deeply characterized. Antimicrobial activities of

plant essential oils have been known for centuries, but their strong flavor limited their use

in food (Campo et al., 2000).

2.7 Carnosol

Fig. 2.4 Structure of carnosol

Carnosol was first isolated from sage (Salvia carnosa) in 1942 and the chemical structure

was first established by Brieskorn et al., in 1964. Rosemary and sage have been known to

contain a variety of polyphenols such as carnosol, carnosic acid, rosmanol and rosmarinic

acid as well as others (Chang et al., 2008). It has been estimated that approximately 5%

of the dry weight of rosemary leaves contains carnosol and carnosic acid, however, this

fraction is estimated to account for > 90% of the antioxidant activity (Aruoma et al.,

1992).

Carnosol is an ortho-diphenolic diterpene with an abietane carbon skeleton (Figure 2.4)

with hydroxyl groups at positions C-11 and C-12 and a lactone moiety across the B ring

34

(Gajhede et al., 1990). Carnosol is the product of oxidative degradation of carnosic acid.

The most popular and expensive way to procure a highly purified form of carnosol is

through extraction and purification from natural sources such as Rosemary. Carnosol was

prepared from carnosic acid in the presence of silver oxide in CH2CL2 with a purified

yield of 67%. Alternatively, carnosic acid in the presence of methanol for 1 week at room

temperature will oxidize carnosic acid to carnosol (Huang et al., 1994; Schwarz et al.,

1992).

2.7.1 Anti -oxidant activity of carnosol

Carnosol has been shown to inhibit Cu2+

induced LDL oxidation and lipid free radicals in

mouse liver microsomes (Zeng et al., 2001) and are good scavengers of peroxyl radicals

(Aruoma et al., 1992). The antioxidant response element is believed to be activated

through the catechol-hydroxyl groups of carnosol and is converted to a carnosol quinine

(Satoh et al.,2008). This quinine derivative is the main anti-oxidation product of carnosol

essentially voiding it of any antioxidant activity (Masuda et al., 2004)

The glutathione-S-transferase (GST) family of phase II detoxification enzymes catalyze

the reaction of glutathione with electrophiles and have been a target of interest for cancer

(Hayes and Pulford, 1995). Carnosol by intra peritoneal administration has been shown to

enhance the in vivo activity of GST and quinine reductase in the liver of the female rat

(Singletary et al., 1996). Carnosol (100-400 mg/kg) increased GST activity by 1.6 to 1.9

fold increase.

2.7.2 Anti-inflammatory activity of carnosol

Deregulated inflammatory signaling including excess nitric oxide (NO) produced by NO

synthase (iNOS) occurs during inflammation and the multi-step process of carcinogenesis

which has led to the search for agents that decrease inflammatory signaling pathways.

Raw 264.7 cells treated with carnosol reduced LPS stimulated NO production with an

IC50 of 9.4 uM (Lo et al., 2002). This led to an inhibition of the NF-kB, p38 and p44/42

mitogen activated protein kinase (MAPK). In another study, carnosol was shown to

35

activate the peroxisome proliferator-activated receptor gamma. Carnosol has also been

shown to reduce the pro-inflammatory leukotrienes in intact polymorphonuclear

leukocytes (PMNL), inhibit 5-lipoxygenase, antagonize the intracellular ca2+

mobilization, and inhibit the secretion of leukocyte elastase (Poeckel et al., 2008). In

addition, carnosol blocks protein kinase C signaling and inhibits the binding of AP-1 to

the COX-2 promoter which should be noted is fundamentally different than the synthetic

COX-2 inhibitors (e.g. celecoxib) that function as direct inhibitors of Cox-2

(Subbaramaiah et al., 2002).

2.7.3 Anti-Cancer properties of carnosol

The anti-cancer properties of carnosol were associated with a potential to modulate

multiple signaling pathways such as the cell cycle related proteins, PI3K/AKT, and

apoptotic related proteins (Khan et al., 2007).

Both carnosol and carnosic acid were shown to have cytotoxic activity against MCF-7

cells with an IC50 of 82 and 96 µM. Further studies are needed to determine if carnosol

preferentially targets ER+ breast cancer cells versus ER

- breast cancer cells.

Carnosol was shown to induce apoptosis by disrupting the mitochondrial membrane

potential in three acute leukemia cell lines which included SEM, RS4:11, and

MV4:11(Dorrie et al., 2001). At 18 µM carnosol did not induce cell death of peripheral

blood mononuclear cells (PBMCs) isolated from healthy volunteers while the same dose

in cell lines resulted in apoptosis. Apoptosis and alterations in mitochondrial membrane

potential resulted from carnosol treatment. Carnosol targets the anti-apoptotic members

of the Bcl-2 family of proteins. A reduction in Bcl-2 protein expression ranged from 33 to

53% in the three cell lines. Interestingly, co-treatments of carnosol and AraC, or

methotrexate, or vincristine resulted in a delay in chemotherapy induced DNA

fragmentation (Zunino et al., 2009). Further studies are needed to understand how co-

treatment of carnosol with chemotherapeutic agents delays DNA fragmentation.

36

The anti-cancer properties of carnosol were associated with a potential to modulate

multiple signaling pathways such as the cell cycle related proteins, PI3K/AKT, and

apoptotic related proteins (Khan et al., 2007). .