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CHAPTER-2

REVIEW OF LITERATURE

In this chapter an attempt has been made to review the work done on the identification

of traditional medicinal plants of Himachal Pradesh and characterization of plant derived

compounds that exhibit synergism with commercial antibiotics against clinical pathogenic

bacteria. Due to paucity of literature available on Colebrookea oppositifolia, studies on other

plants have been incorporated in order to find synergism. The literature is reviewed under

following aspects:

2.1 Antibiotics and their mode of actions

2.2 Drug resistance and mechanism of resistance

2.3 Importance of traditional medicinal plants

2.4 Historical uses of essential oils and terpenoids

2.5 Synergistic effect of medicinal plants and antibiotics

2.6 Characterization of phytochemicals

2.1 ANTIBIOTICS AND THEIR MODE OF ACTIONS

Understanding the role of microorganisms in disease took many years for scientists to

establish the connection between microorganisms and illness. Robert Koch, for the first time

demonstrated the role of bacteria Bacillus anthracis causing disease anthrax; later Louis

Pasteur and Robert Koch observed that airborne Bacillus led into the inhibition of Bacillus

anthracis to which they gave the name antibiosis in 1877. The term was renamed by Selman

Waksman into antibiotics in 1942 and has been defined as a chemical substance derivable from

microorganisms or produced by chemical synthesis to kill or inhibit the microorganisms and

cure infections. Antibiotics have been classified into three categories derived from various

sources; 1) Natural antibiotics: derived from fungus e.g. benzylepenicillin and gentamycine; 2)

Semi-synthetic antibiotics are chemically-altered natural compounds e.g. ampicillin and

amilkacin; 3) Synthetic antibiotics are chemically designed in laboratories e.g. moxifloxacin

and norfloxacin. The antibiotics work with two phenomenons; 1) Inhibition of microbial

multiplication known as bacteriostatic effect; 2) By killing the microbial population known as

bactericidal effect. The characteristic feature of an antibiotic for prescription is taken into the

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account in such a way that it should be selective target, bactericidal, narrow spectrum so that it

does not kill the normal gut flora, high therapeutic index, few adverse effects, various route of

administration, good absorption and emergence of resistance is low.

Antibiotics work with different mode of action and have been classified according to their

properties;

1. Inhibitors of cell wall synthesis: The various classes of antibiotics which comes under

this category are;

A. β- lactams: it is the part of core structure of several antibiotics families like

penicillins, cephalosporins, carbapenems and monobactams. They generally work as

inhibitor of cell wall biosynthesis.

Figure 2.1: Chemical structures of β-lactam class antibiotics: A) Penicillin: These antibiotics

inhibit cross links in peptidoglycan in cell wall; B) Cephalosporins: They disrupt the synthesis of peptidoglycans; C) Carbapenems: They inhibit L,D-transpeptidases synthesis during cell synthesis in bacteria; D) Monobactams: They are genrally employed against aerobic Gram’s negative bacteria (Neisseria sp., Pseudomonas sp.)

A.1 Penicillin: There are wide range of antibiotics falling under this class for

example; a) Natural penicillin (Penicillin G): they do not produce β-lactamase; b)

Penicillinsase resistant penicillins (Methicillin); c) Extended-spectrum penicillins

(Aminopenicillins e.g. amoxicillin, carboxypenicillins and ureidopenicillins); d) β-

lactamase inhibitors, widely used antibiotics as they produce β-lactamase

(Amoxicillin+clavulanic acid, ampicillin+sulbactum and piperacillin + tazobactum).

9

Figure 2.2: Chemical structures of various classes of penicillins: A) Chemical structure

of Penicillin G; B) Methicillin (Penicillinsase resistant penicillin); C) Amoxycillin (Aminopenicillins, Extended-spectrum penicillin); D) Amoxicillin and Clavulanic acid (β-lactamase inhibitors)

A.2 Cephalosporins: They are the inhibitors of cell wall synthesis in bacteria and

have been characterized as; A) 1st generation cepahlosoprins: A narrow spectrum

fight against Gram’s positive bacteria e.g. cefazolin; B) 2nd generation

cephalosporins: Better Gram’s negative bacteria coverage e.g. cefuroxime; C) 3rd

generation cephalosporins: Much active against Enterobacteriaceae and

Psuedomonas aeruginosa e.g. ceftriaxone; D) 4th generation cephalosporins: Broad

spectrum of cepahlaosporins against against Enterobacteriaceae and Psuedomonas

aeruginosa e.g. cefepime.

.

Figure 2.3: Chemical structures of various classes of cephalosporins: A) 1st generation

cephalosporins e.g. Cefazolin, mainly prescribed for bacterial infection of skin; B) 2nd generation cephalosporin e.g. Cefuroxime, widely used for various bacterial infection like against Haemophilus influenzae, Neisseria gonorrhoeae and Lyme disease; C) 3rd generation cephalosporin e.g. Ceftriaxone, widely used for the treatment of pneumonia, bacterial meningitis, skin infection, urinary tract infection etc.; D) 4th generation cephalosporins e.g. Cefepime used against Enterobacteriaceae and Psuedomonas aeruginosa

A.3 Carbapenems: They are the β

Gram’s positive bacteria, except, MRSA, Gram’s negative (

aeruginosa) e.g. meropenem

A.3 Monobactams:

(Enterobacteriaceae and

Figure 2.4: Chemical structures of carbapenem and monobactumstructure of meropenem (carbapenem), broad spectrum antibiotics used for Gram’s positive and negative bacteria; B) Chemical structure of aztreonam (monobactum), anitratus, Escherichia coli

B. Glycopeptides:They work as inhibitor of cell wall synthesis o

infection e.g. vancomycin.

C. Fosfomycins: They act

than the penicillin and cephalosporins.

Figure 2.5: Chemical structures of glycopeptides and fvancomycin (glycopeptides), widely used foS.aureus (MRSA) Chemical structure of fosfomycin, generally used for urinary tract infections

A

They are the β-lactams with broad spectrum antibiotics against

Gram’s positive bacteria, except, MRSA, Gram’s negative (

g. meropenem.

Monobactams: They generally act on Gram’s negative bacteria

(Enterobacteriaceae and Psuedomonas) e.g. aztreonam.

: Chemical structures of carbapenem and monobactum:structure of meropenem (carbapenem), broad spectrum antibiotics used for Gram’s positive and negative bacteria; B) Chemical structure of aztreonam (monobactum), used to treat bacterial infections caused by

Escherichia coli and Proteus mirabilis

They work as inhibitor of cell wall synthesis of Gram positive

ancomycin.

They act on the inhibition of cell wall synthesis at a stage earlier

than the penicillin and cephalosporins.

tructures of glycopeptides and fosfomycins: A) Chemical structure of vancomycin (glycopeptides), widely used for the treatment of methicillin

(MRSA) and multi-resistant Staphylococcus epidermidisChemical structure of fosfomycin, generally used for urinary tract infections

B

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lactams with broad spectrum antibiotics against

Gram’s positive bacteria, except, MRSA, Gram’s negative (Psuedomonas

negative bacteria

: A) Chemical

structure of meropenem (carbapenem), broad spectrum antibiotics used for Gram’s positive and negative bacteria; B) Chemical structure of aztreonam

used to treat bacterial infections caused by Acinetobacter

Gram positive

at a stage earlier

hemical structure of

r the treatment of methicillin resistant Staphylococcus epidermidis (MRSE); B)

Chemical structure of fosfomycin, generally used for urinary tract infections (UTI)

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2. Inhibitors of protein synthesis: They generally bind to the RNA of 30S ribosomal

sub-unit which affects normal protein synthesis The various classes of antibiotics which

comes under this category are;

A. Aminoglycosides: They are bactericidal and broad spectrum killing both Gram’s

positive and Gram’s negative bacteria by inhibiting the protein synthesis which is

done by aminoglycosides, perturbing the protein elongation at 30S ribosomal

subunit leading into the inaccurate mRNA translation, hence, inaccurate translated

protein product is produced e.g. kanamycin, amikacin and gentamycin etc.

B. MLSK: They are macrolids, lincosamides, streptogramins and ketolides. They are

generally confined to the Gram’s positive bacteria. All four classes are of different

structure but same mode of action e.g. erythromycin, telithromycin, clindamycin

etc. They inhibit the protein synthesis as discussed above and also by

immunomodulation in diffuse panbronchiolitis (DBP).

Figure 2.6: Chemical structures of aminoglycosides (kenamycin A) and MLSK

(macrolide-lincosamide-streptogramin-ketolide) (erythromycin): A) Kenamycin interferes with 30S subunit of prokaryotic ribosomes inhibiting the protein synthesis; B) Erythromycin is the macrolid, which binds with 50S subunit of ribosomes and interfers with aminoacyl translocation inhibiting the protein synthesis

C. Tetracyclines: They are generally bacteriostatic and broad spectrum antibiotics

genrally act by inhibiting the binding of aminoacyl tRNA to mRNA-ribosome

complex.

D. Phenicols: They are also bacteriostatic as well as broad spectrum e.g.

chloramphenicol. They prevent the protein elongation by inhibiting the peptidyl

transferase activity.

A B

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Figure 2.7: Chemical structures of tetracyclines and chloramphenicol: A) Tetracyclines are the braod spectrum antibiotics used in urinary tract infections, respiratory tract infections and intestinal infections, especially prescribed for β-lactama and erythromycin allergic patients; B) Chloramphenicol is a broad spectrum antibiotic and used in treating ocular infections caused by a number of bacteria including Staphylococcus aureus, Streptococcus pneumoniae and Escherichia coli

E. Oxazolidinones: They are broad spectrum and narrow spectrum antibiotics e.g.

linezolid.

F. Ansamycins: Generally works on Gram’s positive bacteria and some Gram’s

negative bacteria e.g. rifamycin.

Figure 2.8: Chemical structures of Oxazolidinones and ansamycin (rifampicin): A) Oxazolidinones

are the inhibitor of protein synthesis and are widely used against gram-positive pathogens (Staphylococcus aureus, Enterococcus sp., and Streptococcus pneumoniae); B) Rifampicin, an antibiotic of ansamycin group, inhibits bacterial DNA-dependent RNA synthesis by inhibiting bacterial DNA-dependent RNA polymerase and widely used in the treatment of tuberculosis, MRSA etc.

3. Inhibitors of membrane function: They target on the membrane phospholipids

(lipopolysachharides and lipoproteins. The various categories are;

A. Polymyxins: They act as narrow spectrum for Gram’s negative bacteria by

disrupting the cell membrane after interacting with phospholipids present on the

wall.

A B

B A

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B. Colistin: Colistin has poly-cationic regions and they interact with the bacterial outer

membrane due which displacement in bacterial counter ions in

the lipopolysaccharide occurs. solubilizing the membrane in an aqueous

environment.

Figure 2.9: Chemical structures of Polymyxins and Colistin: A) Polymyins, it contains a cyclic

peptide with longhydrophobic tail, which disrupt the bacterial cell membrane by interacting with phospholipids, typical use of drug is against multidrug resistant Psuedomonas aeroginosa and Enterobacteriaceae; B) Colistin, is a polymyxins antibiotics, effective against most Gram-negative bacilli (Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter)

4. Inhibitors of nucleic acid synthesis: They work as inhibitor of nucleic acid synthesis.

The various classes fall under this category are;

A. Quinolones: They target on DNA gyrase which is responsible for cutting one of

the chromosomal DNA strands at the beginning of the supercoiling process e.g.

ciprofloxacin, levofloxacin, gatifloxacin and moxifloxacin.

B. Furanes: They are broad spectrum and bactericidal drugs act on damaging

bacterial DNA e.g. nitrofurantoin.

B A

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Figure 2.10: Chemical structures of Quinolones (ciprofloxacin) and Furanes (nitrofurantoin): A)

Ciprofloxacin, it is a second class fluoroquinolones, which inhibits the bacterial growth by inhibiting DNA gyrase, a type II topoisomerase and topoisomerase IV, enzymes, it is used against respiratory tract, abdominal tract, gastrointestinal and urinary tract infection; B) Nitrofurantoin, they react with flavoproteins and reduces to various multiple reactive intermediates that attack the ribosomal protein, DNA, pyruvate metabolism and other macromolecules within the cell, the drug has been used to treat the urinary tract infections

2.2 DRUG RESISTANCE AND MECHANISM OF RESISTANCE

Drug resistance is an ever increasing worldwide health threat that involves all major

microbial pathogens and antimicrobial drugs (Stuart and Bonnie, 2005). Antimicrobial can be

categorized according to their principal mechanism of action, which includes, 1) Interference

with cell wall synthesis (beta-lactams and glycopeptide agents), 2) Inhibition of protein

synthesis (macrolides and tetracyclines), 3) Interference with nucleic acid synthesis

(fluoroquinolones and rifampin), 4) Inhibition of a metabolic pathway (trimethoprim-

sulfamethoxazole), 5) Disruption of bacterial membrane structure (polymyxins and

daptomycin) (Tenover, 2006), as discussed above (2.1). The microbial resistance is the ability

of microbes to grow in the presence of antibiotics. The difference between non- resistant and

resistant bacteria is described in Figure 2.11, which shows drug resistant bacteria are not

controlled or killed my antibiotics, whereas in the case of non- resistant bacteria they die in the

presence of antibiotics. Antibiotic resistance has increased substantially in recent years and

considered as fast growing therapeutic problem (Guillemot 1999; Austin et al., 1999). The

development of antibiotic resistance can be natural or intrinsic and acquired which can be

transmitted within same or different species of bacteria. In inherent (natural) resistance is the

innate ability of bacteria to resist activity of a particular antibiotic through its inherent

structural or functional characteristics, due to which it can tolerate a particular drug or

antimicrobial class. It may be because of lack of transport system, inaccessibility of the drug

into the bacterial cell, extrusion of the drug by chromosomally encoded active exporters or

innate production of enzymes that inactivate the drug. Acquired resistance includes vertical

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gene transfer in which the resistance genes may be transferred directly to all bacteria during

DNA replication. The second mode is horizontal gene transfer in which genetic material

(DNA) can be transferred between individual bacteria of the same species or even between

different species (Todar, 2011) shown in the Figure 2.12 (A). The genetic mutations are also

responsible for the drug resistance in the bacteria shown in the Figure 2.12 (B). As shown in

Figure 2.13 (B), there is no change in the final population of antibiotic-sensitive and antibiotic-

resistant bacteria in the absence of any drug, whereas when the antibiotic (tetracycline) was

given the resistance has increased resulting in the formation of more resistant bacteria.

The different mechanism of antibiotic resistance was observed in bacteria. These

mechanisms can either chemically modify the antibiotic, makes it inactive by removing it from

the cell or modify target site so that it is not recognized by the antibiotic. Mostly, the resistance

mechanism was controlled by extra chromosomal plasmids, which are transmissible to other

bacteria. 1) Efflux pumps are reverse transport systems present in the membrane that transport

the antibiotic out of the cell e.g this is resistance mechanism for tetracycline; 2) The enzyme

modifies the antibiotic in a way that to make it inactive e.g the streptomycin is chemically

modified so that it cannot bind to the ribosome and inhibit protein synthesis; 3) the enzyme

degrades the antibiotic and inactivate it e.g the penicillinases (beta-lactamase enzymes) cleaves

the beta lactam ring of the penicillin molecule Figure 2.13 (Todar, 2011).

Traditional methods that are being used currently to overcome antimicrobial resistance

include: a) identification of new drugs; b) chemical modification of the existing drug (for

example semisynthetic penicillins like methicillin and flucloxacillin); c) Use of a combination

of two or more antibiotics with different mechanisms of action (Beringer, 1999). However,

none of the current clinical therapies utilize natural plant products as antibiotics.

Figure 2.11: Drug resistance in bacteria (resistant and resistant bacteria

Figure 2.12: Acquired bacterial resistance to antibiotics (

phenomenon of gene transfer

A

Drug resistance in bacteria (www.niaid.nih.gov) shows difference between nonresistant and resistant bacteria

Figure 2.12: Acquired bacterial resistance to antibiotics (www.niaid.nih.gov).

phenomenon of gene transfer

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difference between non-

A: Describing the

Figure 2.12: Acquired bacterial due to regular exposure of antibiotics

Figure 2.13: Mechanism of drug resistance in bacteria (Todar, 2011):

different mechanisms of drug resistance; 1) Enzymes which degrades the antibiotics 2)

Efflux pump expelling antibiotic outside the cell 3) Antibiotic altering enzymes which

change the structure of antibiotic, to make it inactive

B

quired bacterial resistance to antibiotics (www.niaid.nih.gov): due to regular exposure of antibiotics

Mechanism of drug resistance in bacteria (Todar, 2011): A) Plasmid which modulates

different mechanisms of drug resistance; 1) Enzymes which degrades the antibiotics 2)

Efflux pump expelling antibiotic outside the cell 3) Antibiotic altering enzymes which

change the structure of antibiotic, to make it inactive

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B: Gene mutation

A) Plasmid which modulates

different mechanisms of drug resistance; 1) Enzymes which degrades the antibiotics 2)

Efflux pump expelling antibiotic outside the cell 3) Antibiotic altering enzymes which

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2.3 IMPORTANCE OF TRADITIONAL MEDICINAL PLANTS

Plant-based therapies offer an excellent alternative with an immense potential to screen

the biodiversity of the plant kingdom. The medicinal plants of Himalayan region have been

used by folklore and are still utilized in modern medicine nowdays. The medicnal plants are

known for their historical uses in medicine. The medicinal plant contains variety of

phytochemicals (phenols, coumarins, tannins, alkaloids, gylcosides, sterols, flavanoids,

terpenoids and saponins) which can be exploited for therapeutic uses. Medicinal plants; Acacia

burkei, Brachylaena discolor, Ozoroa engleri, Parinari capensis, subsp. capensis,

Portulacaria afra, Sida pseudocordifolia, Solanum rigescens, Strychnos

madagascariensis and Drimia delagoensis were used for the treatment for skin disorders (De

Wet et al., 2013). Caesalpinia pulcherrima, Euphorbia hirta and Casuarina equisetifolia were

observed to be antibacterial against K. pneumoniae and B. cereus (Parekh and Chanda 2007).

Terminalia arjuna which constitutes arjunolic acid, gallic acid, terminic acid,

pyrocatechols, β-sitosterol, calcium, magnesium, zinc, copper etc. and showed anticancer,

antidiabetic, antiacne, antihelmintic, antiinflammatory, anticholinesterase, antioxidant,

antiasthmatic, wound healing, cardioprotective and insecticidal activities. It is used to cure

cancer, coronary artery disease, hypertension and ischemic cardiomyopathy (Khan et al., 2013)

and has been reported as antibacterial against Escherichia coli, Klebsiella aerogenes, Proteus

vulgaris, Pseudomonas aerogenes and MDR Salmonella typhi (Perumal samy et al., 1998;

Rani and Khullar, 2004). Eugenia Jambolana was known for its anti- diabetic (Grover et al.,

2000), antibacterial (Bhuiyan et. al., 1996) and ulcer protective (Chaturvedi et al., 2009). Rhus

sp. has been reported as antibacterial against Bacillus megaterium, Bacillus brevis, Bacillus

subtilis, Bacillus cereus, Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa,

Staphylococcus aureus, Listeria monocytogenes and Micrococcus luteus, Candida tropicalis

and Candida albicans (Digrak et al., 2001). The extracts of Cotinus coggygria was observed

antifungal against Candida albicans (Matic et al., 2011) and major constituents of this plant

were limonene (48.5%), (Z)-β-ocimene (27.9%) and (E)-β-ocimene (9.7%) (Demirci et al.,

2003). Allium cepa and Allium sativum have been reported as antifungal and antibacterial

plants (Hughes and Lawson 1991; Benkeblia, 2004). Allium sativum was observed to be

effective against Gram’s positive, Gram’s negative and yeast species but Allium cepa showed

no effect on Gram’s negative bacteria (Dankert et al., 1979; Hughes and Lawson 1991).

9

Berberis aristata has antibacterial, antiamoebic, antifungal, antihelminthic, leishmanicidal and

tuberculostatic activity (Soffar et al., 2001; Singh et al., 2007).

Juglans regia showed antioxidant properties and strong antibacterial activity against

S.aureus (Pereira et al., 2007; Wojdyło et al., 2007). Pinus roxburghii was reported for anti-

inflammatory property (Kaushik et al., 2012), oil extracted from Pinus was observed to be

antibacterial for various Gram’s negative and Gram’s positive strains (Chopra et al., 1960;

Iqbal et al., 2011). All the parts of Rhododendron arboretum were found to be powerful drugs

exhibiting the antimicrobial activity against Escherichia coli and Staphylococcus aureus

(Chhetri et al., 2008). The phytochemical screening of the flowers of R. arboreum showed the

presence phenols, saponins, tannins and coumarins (Nisar et al., 2011). Antibacterial activity of

Piper nigrum was reported against Salmonella typhimurium, Staphyloccus aureus, Bacillus

cereus and Escherichia coli (Pradhan, 1999; Dorman, 2000). The compounds isolated from P.

nigrum were pellitorine, trachyone, pergumidiene and isopiperolein. These compounds were

antibacterial against Bacillus subtilis, Bacillus sphaericus and Staphylococcus aureus,

Klebsiella aerogenes, Chromobacterium violaceum (Reddy, 2004). Insecticidal (Siddiqui,

2005) and antitumor activities (Sunila and Kuttan, 2004) of Piper nigrum were already

reported. Withanolides are a group of naturally occurring triterpenoids of Withania somnifera

which showed the tumor inhibition and antiangiogenic properties (Mirjalili et al.,

2009). Leaves of Datura metel consists of a new pyrrole derivative which was characterised as

2β-(3, 4-dimethyl-2, 5-dihydro-1H-pyrrol-2-yl)-1′-methylethyl pentanoate which observed to

be antifungal against Aspergillus fumigates (Dabur et al., 2004), whereas in the presence of

steroids, triterpinoids, reducing sugars, alkaloids, phenolic compounds, flavonoids, tannins in

Datura metel showed growth inhibitory effects on Xanthomonas campestris (De Britto and

Gracelin, 2011).

Phytochemical screening has shown the presence of phenolics, vitamin C and

carotenoids in Capsicum annum (Marin et al., 2004). The presence of antioxidant (Deepa et al.,

2007) and ascorbic acid content was higher in “Chile”, “yellow wax” and “ancho” peppers than

“jalapeno” peppers (Lee et al., 1995). Punica granatum was known for its anti-cancerous, anti-

inflammatory (Lansky and Newman, 2007) and antibacterial activity (Prashanth et al., 2001).

Lawsonia alba exhibits broad-spectrum antimicrobial activity against various multidrug

resistant microbes (Ahmad and Beg, 2001). Zingiber officinale showed immuno-modulatory,

9

anti-tumorigenic, anti-inflammatory, anti -apoptotic, anti-hyperglycemic, anti-lipidemic, anti-

emetic and strong anti-oxidant. It is considered safe herbal medicine and used by folklore from

the back history (Ali et al., 2008).

Curcumin extracted from Curcuma longa, exhibits anti-inflammatory, anti-human

immunodeficiency virus, antibacterial against methicillin resistant S. aureus (Kim et al., 2005),

antioxidant effects and nematocidal activities (Araujo and Leon, 2001). Emblica officinalis was

observed anti-pyretic and analgesic due to alkaloids, tannins, phenolic compounds,

carbohydrates and amino acids (Perianayagam et al., 2004). The seed oil of Nigella sativa

provides protection against nephrotoxicity, hepatotoxicity and exhibit anti-inflammatory,

analgesic, antipyretic, antimicrobial and antineoplastic activity (Ali and Blunden 2003). The

ethanolic extract of bark of Zanthoxyllum armatum exhibit anti- inflammatory and antioxidant

activities (Sati et al., 2011).

Ethyl acetate extract of Ruta graveolens roots yielded rutacridone epoxide which

showed strong activity in comparison to commercial fungicides captan and benomyl against

pathogenic fungi such as Colletotrichum fragariae, C. gloeosporioides, C. acutatum,

and Botrytis cineara and Fusarium oxysporium (Meepagala et al., 2005). The alkaloids,

saponin, flavonoids present in roots of Vitex negundo L and coumarin in leaves of Aegle

marmelos have shown antifilarial effect against Brugia malayi (Sahare et al., 2008). Cannabis

sativa inhibited the microbial growth and could be used to control spoilage and food-borne

pathogens and plant pathogens (Nissen et al., 2001). Myristica fragrans has shown

antimicrobial activity against Gram’s positive and Gram’s negative bacteria (El Malti et al.,

2008; Cherian et al., 2013). The flavonoids, isolated from Populus sp. namely 5-hydroxy-7-

methoxy-flavone, 5, 7-dihydoxy-flavone and 5, 7-dihydroxy-flavonol, have shown

antimicrobial activity against Pseudomonas lachrymans, Ralstonia solanacearum,

Xanthomonas vesicatoria and Magnaporthe oryzae (Marcucci et al., 2001). Cinnamonum

tamala (leaf) contains fats, carbohydrates, proteins, flavonoids, saponin, tannins, alkaloids,

polyphenols (Dandapat et al., 2013) and oil extracted showed potent antifungal activity against

Aspergillus niger, A. fumigatus, Candida albicans, Rhizopus stolonifer and Penicillium spp.

(Pandey et al., 2012). The antimicrobial activity has been reported in Callistemon citrinus

against S. aureus, P. aureoginosa, E. coli, B. cereus and B. subtilis (Chitemerere and

Mukanganyama, 2011). Syzygium aromaticum was observed antimicrobial against bacteria

9

(Bacillus subtilis, B. megaterium, B. polymyxa, B. sphaericus, Staphylococcus aureus and

Escherichia coli) and molds (Penicillium oxalicum, Aspergillus flavus, A. luchuensis, Rhizopus

stolonifer, Scopulariopsis sp. and Mucor sp.) (Pundir et al., 2010). The antimicrobial activity

and antihemorrhagic activity against Vibrio cholerae was observed in Azardica indica

(Thakurta et al., 2007) and also against the typhoidal pathogen Salmonella enterica serovar

typhi (Mandal et al., 2007). The sesquiterpene coumarins isolated from Ferula assafoetida has

been reported as antifungal, anti-diabetic, anti-inflammatory, anti-mutagenic and antiviral

activities (Iranshahy and Iranshahi, 2011). Ajuga reptans has been used to cure fever, asthma

and gout (Brimani, 1987) and as vasoconstrictor (Zargari, 1992). Antimicrobial activity was

well documented in Thymus vulgaris against clinical isolate of Pseudomonas aeruginosa and

Bacillus subtilis (Adwan et al., 2006), Euphorbia hirta against Staphylococcus aureus,

Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa (Ogbulie, et. al, 2007),

Hypericum perforatum (Dulger and Gonuz, 2005) and Taraxacum officinale (Sengul, et. al.,

2009).

Camellia sinensisis (Chinese green tea) showed antibacterial activity against Listeria

monocytogenes (Mbata et al., 2008) and contains high amount of antioxidants (Farrukh et al.,

2006). The red form of Abrus precatorius observed to be anti-cancerous (Sivakumar and

Alagesaboopathi, 2008) and antimicrobial (Mistry et al., 2010). The antimicrobial activity was

observed in Cymbopogon citrates (Souza et al., 2005), Cuscuta reflexa (Pal et al., 2006) and

Musa paradisiaca (Imam and Akter 2011). Anti-inflammatory activity was reported in Bombax

ceiba (Anandarajagopal et al., 2013) and antibacterial and antifungal activity was shown by

Tinospora cordifolia (Mahesh and Satish, 2008).

Chrysanthemum indicum showed antibacterial activity against B. cereus (Pitinidhipat

and Yasurin 2012) and arresting the cell cycle of human HCCMHCC97H cells (Li et al.,

2009). Mentha spp. was reported for its antioxidants activity (Couladis et al., 2003), whereas

Berginia ciliate was documented for its anti-inflammatory activity (Kumar et al., 2002). The

water extract of Urtica dioica exhibited antioxidant, analgesic, antiulcer activities (Gulcin et

al., 2004) and cardiovascular activity (Testai et al., 2002). Trigonellia foenum-graecum

exhibited anti-inflammatory and antipyretic properties (Lim, 2012). The strong antioxidant

activity was observed in Prunus amygdalus (Sang et al., 2002), Cocos nucifera (Chakraborty

and Mitra 2008) and Colocasia esculenta (Lako et al., 2007). Thymol extracted from

9

Trachyspermum ammi was reported insecticidal and repellent against Anopheles stephensi

(Pandey et al., 2009) Bauhinia variegata bark contained tannins, alkaloids, saponins and

showed antibacterial activity (Parekh et al., 2006). Mimosa pudica is reported for its wound

healing activity (Kokane et al., 2009). Brassica nigra was reported for its antioxidant and anti-

inflammatory activities (Alam et al., 2011), Rumex hastatus as antidiarrhoeal (Shakuntala et

al., 2011) and Asclepias curassavica as antibacterial against Pseudomonas solanacearum and

Escherichia coli (Hemavani and Thippeswamy, 2012). Podophyllum hexandrum was reported

anti-cancerous (Giri and Narasu, 2000). Ageratum conyzoides exhibited the wound healing

(Sachin et al., 2009) and insecticidal activity (Moreira et al., 2007). Cichorium intybus have

inulin, esculin, volatile compounds (monoterpenes and sesquiterpenes), coumarins, flavonoids,

vitamins and the root extracts observed to be antibacterial against Bacillus subtilis,

Staphylococcus aureus, Salmonella typhi, Micrococcus luteus and Escherichia coli

(Nandagopal and Kumari, 2007).

The plant Colebrookea oppositifolia of Lamiaceae family exhibited antioxidant

(Barman et al., 2012) and cardioprotective activity (Pallab et al., 2011). Plant juice has been

used to treat fever and headache, leaves used to treat dysentery, roots used to treat peptic ulcers

and haemostatic. Leaves of this plant have been used in the treatment of wounds, bruises and

fracture, roots have been used in the treatment of epilepsy and oil exhibited fungitoxic property

(Yoganarasimhan 2000; Gupta et al., 2001; Singh et al., 1983). Antiulcer activity has been in

Colebrookea oppositifolia root extract (Ghaisas et al., 2010). Synergistic interaction has been

documented between amphotericin B (AmB) and acteoside, isolated from the aerial parts of the

shrub Colebrookea oppositifolia. Acteoside alone exhibited no activity but in combination with

amphotericin B, a potent synergism has documented against Candida albicans, Cryptococcus

neoformans and Aspergillus fumigates (Ali et al., 2011).

The rich medicinal diversity of Himachal Pradesh was explored for the comparative

analysis of antimicrobial activity against E. coli and C. albicans by Sharma et al. (2014). In

this study, extracts of H. perforatum, C. citrate and T. arjuna were observed to be strong

antimicrobial against E. coli and C. albicans.

9

2.4 TRADITIONAL USES OF ESSENTIAL OILS AND TERPENOIDS

Although perfume extracted from spices contains flavor and preservative properties

since antiquity (Bauer et al., 2001), only oil of turpentine was mentioned by Greek and Roman

historians (Guenther, 1948). The extraction of volatile oil by distillation was first used in the

East (Egypt, India and Persia) (Guenther, 1948) more than 2000 years ago and improved in the

9th century by the Arabs (Bauer et al., 2001).The first authentic written proof of distillation of

essential oil was given by Villanova, a Catalan physician (Guenther, 1948). By the 13th

century essential oils were being made by pharmacies and their pharmacological effects were

described in pharmacopoeias (Bauer et al., 2001).

The greatest use of essential oil is in food, perfumes and pharmaceuticals (Bauer and

Garbe, 1985; Van Welie, 1997; Van de Braak and Leijten, 1999). The well-known use of

essential oil in aromatherapy constitutes approximately 2% of the total market (Van de Braak

and Leijten, 1999). Individual components of oils are also used as food flavourings; either

extracted from plant material or synthetically manufactured (Oosterhaven et al., 1995). The

documented proof of use of tea tree oil for medical purposes was of the 18th century, although

it was used by the native people of Australia prehistorically (Carson and Riley, 1993). The

antimicrobial properties of some essential oils were reported lately (Guenther, 1948; Boyle,

1955; Nychas, 1995) with the antimicrobial properties of spices cloves, cinnamon, sage and

oregano contains eugenol, carvacrol and thymol as the major antimicrobial compounds (Shelef,

1983). The essential oils extracted from various plants were reported antibacterial e.g.,

Pelargonium hortorum (Koheil et al., 2012), Cymbopogon citratus (Onawunmi et al., 1984),

Syzygium aromaticum, Piper nigrum, Myristica fragrans (Dorman and Deans, 2008) etc. The

antibacterial properties of essential oils and their components are exploited in such diverse

commercial products as dental root canal sealers (Manabe et al., 1987), antiseptics (Bauer and

Garbe, 1985; Cox et al., 2000) and feed supplements (Van Krimpen and Binnendijk, 2001;

Ilsley et al., 2002). Essential oils of different plants of Lamiaceae family have shown

antimicrobial activity against mutidrugresistant bacteria (Niculae et al., 2009). The antifungal

activity of Agastache rugosa was reported in combination with ketoconazole against

Blastoschizomyces capitatus (Shin and Kang, 2003).

Besides antibacterial properties (Deans and Ritchie, 1987; Carson et al., 1995; Mourey

and Canillac, 2002), essential oils or their components have been shown to exhibit antiviral

9

(Bishop, 1995), antimycotic (Azzouz and Bullerman, 1982; Akgul and Kivanc, 1988;

Jayashree and Subramanyam,1999; Mari et al., 2003), antitoxigenic (Akgul et al., 1991; Ultee

and Smid, 2001; Juglal et al., 2002), antiparasitic (Pandey et al., 2000; Pessoa et al., 2002) and

insecticidal (Konstantopoulou et al.,1992; Karpouhtsis et al., 1998) properties. These

characteristics are possibly related to the secondary metabolites in plants (Guenther, 1948;

Mahmoud and Croteau, 2002). The phenolic components are chiefly responsible for the

antibacterial properties of essential oils (Cosentino et al., 1999). Essential oils of Nutmeg

(Myristica fragrans) showed a potent hepatoprotective activity against liver damage caused by

lipopolysaccharide (LPS) plus D- galactosamine (D- GaIN) (Morita et al., 2003). The

protective activity was correlated with a major constituent myristicin (Ahmed et al., 1997).

The terpenoids are found in almost all plant species. Terpenoids are mixtures of

isomeric hydrocarbon along with their oxygenated derivatives like alcohols, aldehydes, ketones

etc. The isoprene unit i.e. C5H8 (2-methyl-1, 3-butadiene) is the building block of all

terpenoids, thus all terpenoids have multiple of (C5) in their structure. Approximately 25,000

terpene structures are reported. Many terpenoids are essential for plant growth, development

and general metabolism. As the largest class of natural products, terpenes/terpenoids have a

variety of roles in mediating antagonistic and beneficial interactions among organisms

(Chiasson et al., 2001) like reproduction, defence or symbiosis (Bohlmann and Christopher,

2008).

2.5 SYNERGISTIC EFFECT OF MEDICINAL PLANTS AND ANTIBIOTICS

Plant-derived chemicals (phytochemicals) can be used in combination with traditional

antibiotics for enhancing the activity of antibiotics (synergism) and making them more potent

at low dosage. These synergistic combinations represent a largely untapped source of

pharmaceutical products with novel and multiple mechanisms of action that can overcome

microbial resistance. In the recent years, increasing attention has been focused on plant

extracts/phytocompounds exhibiting strong antibacterial activity which are expected to interact

synergistically with antibiotics.

In one study, combinational effect of protoanemonum isolated from Ranunculus

bulbosus with 22 antibiotics was evaluated. In one combination, protoanemonum showed

strong synergism with cefamendole against the growth of Staphylococcus aureus (Didry et al.,

9

1993). Antibacterial activity of flavones isolated from Sophora exigua against MRSA

(Methicillin Resistant Staphylococcus aureus) and its interaction with antibiotics has also been

reported (Sato et al., 1995). Iinuma et al. (1996) demonstrated the synergistic activity of two

xanthones, mangostin and rubraxanthon, isolated from Garcinia mangostana with antibiotics

against MRSA bacterial strains. Nascimento et al. (2000) demonstrated the antibacterial

activity of 11 medicinal plants against several gram-negative and few gram-positive bacteria,

with the highest activity from extracts of Caryophyllus aromaticus and Syzygium aromaticum.

Darwish et al. (2002) reported that sub-inhibitory levels of methanolic extracts of Jordanian

plants, specifically Punica granatum showed synergistic interactions in combination with

chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin against resistant and

sensitive strains of S. aureus. Polyphenols (epicatechin gallate and catechin gallate) have been

reported to reverse beta-lactam resistance in MRSA strains (Stapleton et al., 2004). Synergistic

interactions against MRSA have been reported for the extracts from Acorus calamus and

Holarrhena antidysenterica with tetracycline and ciprofloxacin, while other plant extracts such

as Hemidesmus indicus, Plumbago zeylanica, Camellia sinensis, and Cichorium intybus

showed synergy with tetracycline only (Aqil et al., 2005). The ethanol extracts of two Chinese

plants, Isatis tinctoria and Scutellaria baicalensis in combination with ciprofloxacin had

synergistic activities against antibiotic resistant Staphylococcus aureus (Yang et al., 2005),

while the combinations of pencillin with ethanolic extracts of Paederia scandens and

Taraxacun monilicum showed a strong bactericidal activity on two strains of S. aureus (Yang

et al., 2005). Marquez et al., (2005) found that addition of ciprofloxacin at sub-inhibitory

concentrations to the crude chloroform extracts of Jatropha elliptica enhanced the activity of

the extract when assayed against NorA expressing S. aureus. These findings suggest the

presence of an inhibitor of the pump in the extracts of Jatropha, which could restore the

activity of ciprofloxacin. Diterpenes, triterpenes, alkyl gallates, flavones and pyridines have

also been reported to have resistance modulating abilities on various antibiotics against

resistant strains of Staphylococcus aureus (Marquez et al., 2005; Smith et al., 2007). Betoni et

al. (2006) observed synergistic interactions between extracts of Mikania glomerata (guaco),

Psidium guajava (guava), Syzygium aromaticum (clove), Allium sativum (garlic), Cymbopogon

citratus (lemongrass), Zingiber officinale (ginger), Baccharis trimera (carqueja) and Mentha

piperita (mint) from Brazil and antibiotics against Staphylococcus aureus. The use of Catha

9

edulis extracts at sub-inhibitory levels has been reported to reduce the minimum inhibitory

concentration (MIC) values of tetracycline and penicillin G against resistant oral pathogens

like Streptococcus oralis, Streptococcus sanguis and Fusobacterium nucleatum (Al- Hebshi et

al., 2006).

Three antibiotics including ampicillin, tetracycline and chloramphenicol in combination

with extracts of Thai medicinal plants namely Dracaena loureiri, Mansonia gagei and

Myristica fragrans exhibited inhibitory effect on the growth of A. baumannii and P.

aeruginosa, whereas an antagonistic effect was observed for Dracaena loureiri in combination

with ampicillin against MRSA strains (Stapleton et al., 2004). Adwan et al. (2008) studied the

synergistic effects of different plant extracts (Rhus coriaria, Psidium guajava, Lawsonia

inermis and Sacropoterium spinosum) and oxytetracycline HCl, enrofloxacin, gentamicin

sulphate and sulfadimethoxine against four clinical isolates of methicillin-resistant

Staphylococcus aureus (MRSA). It was shown that ethanolic extracts increase the inhibition

zones of oxytetracycline HCl, gentamicin sulphate and sulfadimethoxine, while combinations

between these plant extracts and enrofloxacin decrease inhibition zone. Adwan and Mhanna

(2008) demonstrated synergistic interaction between the water extracts of Psidium guajava,

Rosmarinus officinalis, Salvia fruticosa, Majorana syriaca, Ocimum basilicum, Syzygium

aromaticum, Laurus nobilis and Rosa damascena alone and known antimicrobial agents of

different mechanisms against five S. aureus isolates including one methicillin-resistant S.

aureus (MRSA) and four methicillin-sensitive S. aureus (MSSA). Anthonia and Olumide

(2010) have characterized the antibacterial potency and synergistic effect of aqueous and

methanol extracts of Carica papaya (leaves, fruit), Citrus aurantifoliia, Anana sativus, Citrus

paradisi, Cymbopogon citratus, Cocos nucifera chaffs, leaves of Euphorbia heterophylla and

Gossypium spp. of South-Western Nigerian plants against multi-drug resistant Salmonella typhi

strains. All the tested extracts were found to be antibacterial against S. typhi. Ahmad and Aqil

(2006) observed that crude extracts of some Indian medicinal plants, Acorus calamus,

Hemidesmus indicus, Holarrhena antidysenterica and Plumbago zeylanica exhibit synergistic

interactions with tetracycline and ciprofloxacin against multidrug-resistant enteric bacteria that

produce extended spectrum β-lactamase (ESβL). Synergism between natural products and

antibiotics against infectious diseases has been reviewed by Hemaiswarya et al. (2008), in

which the various antibiotics and natural product synergism was explained. Natural products

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like carnosic acid, carnosol, totatrol, berberine etc were observed to increase the antibacterial

activity of tetracycline, erythromycin, methicillin and methoxyhynocarpin, with the different

mode of mechanisms like MDR inhibitor pump (Oluwatuyi et al., 2004), β- lactamase inhibitor

(Hu et al., 2001), PBP 2a production (Pao et al., 1998) and Nor (A) MDR pump (Smith et al.,

2005) respectively. In an extensive study, Chatterjee et al., (2009) provided compelling

evidence for the synergistic effect between ethanolic leaf extracts of Vangueria spinosa and

doxycycline and ofloxacin against four pathogenic bacteria (Staphylococcus aureus,

Escherichia coli, Pseudomonas aeruginosa and Klebseilla pneumoniae).

Similarly, Ahmed et al. (2009) showed that extracts of Salvadora persica could

enhance the inhibitory effect of tetracycline and penicillin against Staphylococcus aureus. It

was found that the synergistic effect was much more effective and caused an inhibition more

than the antibiotics alone. In a significant development, Indian Institute of Integrative Medicine

(IIIM), Jammu, in public private partnership with Cadila Pharmaceutical Ltd, Ahmedabad, has

released a new drug formulation against tuberculosis called ‘risorine’ that contains reduced

dosages of the drugs rifampicin (200 mg) and isoniazid (300 mg) along with piperine (10 mg),

a compound derived from the common black pepper. Risorine was found to be bioequivalent to

standard rifampicin regimen (Chawla, 2010).

2.6 CHARACTERIZATION OF PHYTOCHEMICALS

The commonly used techniques for the purification and structural elucidation of

bioactive compound from crude extracts are TLC (Thin layer chromatography), GC-MS (Gas

chromatography mass spectrometry), FTIR (Fourier transform Infrared spectra), HPLC (High

performance liquid chromatography) and NMR (Nuclear magnetic resonance).

TLC has been used for detection of 18 mycotoxins produced by various fungal

pathogens (Scott et al., 1970). The bands of various compounds of 13 Brazilian medicinal plant

extracts were observed on TLC plate, developed by CHCl3/MeOH/H2O (65:35:5) solvent and

the spray used was vanillin/sulphuric acid (Holetz et al., 2002). GC- MS technique has been

used for identification of compounds in extract fractions and essential/ volatile oils

(Prabuseenivasan et al., 2006; Aligiannis et al., 2001; Milos et al., 2000; Wang et al., 1994).

GC-MS technique has been used for identification of phenolic compounds (Proestos et al.,

2006) and metabolic profiling (Talaty et al., 2005; Lisec et al., 2006) in plants.

9

FTIR spectroscopy is an established tool for the structural characterization of proteins,

chemical compounds and phytocompounds. FTIR spectra have been used to analyze 21

globular proteins obtained at 2 cm−1 resolution from 1600 to 1700 cm−1 in deuterium oxide

solution (Byler and Susi, 1986). FTIR and UV- visible spectroscopy have been used to identify

the functional groups of bioactive compound of Heterostemma tanjorense (Asclepiadaceae)

(Thevasundari and Rajendran, 2011). The UV- visible spectroscopy has been used to detect the

functional group presence in plant extracts and different solvents fractions of plant extracts

(Raj and Peter, 2012; Peter and Venkatesan, 2012). FTIR technique is useful to analyze the

appearance or disappearance of peaks of functional groups (drugs and terpenoids) and their

combinations. The defined range of functional group is given in Table 2.1.

Table 2.1: Characteristic IR absorption frequencies of functional groups

Functional Group

Type of Vibration

Characteristic Absorptions (cm-1)

Intensity

Alcohol

O-H (stretch, H-bonded)

3200-3600 Strong, broad

O-H (stretch, free) 3500-3700 Strong, sharp C-O (stretch) 1050-1150 Strong

Alkane C-H Stretch 2850-3000 Strong -C-H Bending 1350-1480 Variable

Alkene =C-H Stretch 3010-3100 Medium =C-H Bending 675-1000 Strong C=C Stretch 1620-1680 Variable

Alkyl Halide C-F Stretch 1000-1400 Strong C-Cl Stretch 600-800 Strong C-Br Stretch 500-600 Strong C-I Stretch 500 Strong

Alkynes C-H Stretch 3300 strong, sharp

C≡C Stretch 2100-2260 variable, not present in symmetrical alkynes

Amine

N-H Stretch 3300-3500 medium (primary amines have two bands; secondary have one band, often very weak)

9

C-N Stretch 1080-1360 medium-weak N-H Bending 1600 Medium

Aromatic C-H Stretch 3000-3100 Medium

-c≡c- Stretch 1400-1600 medium-weak, multiple bands

Analysis of C-H out-of-plane bending can often distinguish substitution patterns C=O Stretch 1670-1820 Strong

(conjugation moves absorptions to lower wave numbers) Ether

C-O Stretch 1000-1300 (1070-1150) Strong Nitrile

CN Stretch 2210-2260 Medium Nitro

N-O Stretch 1515-1560 & 1345-1385 strong, two bands

Table 2.2: IR absorption frequencies of functional groups containing a carbonyl (C=O)

Functional Group

Type of Vibration

Characteristic Absorptions (cm-1)

Intensity

Carbonyl C=O Stretch 1670-1820 Strong

(conjugation moves absorptions to lower wave numbers) Acid

C=O Stretch 1700-1725 Strong O-H Stretch 2500-3300 strong, very broad C-O Stretch 1210-1320 Strong

Aldehyde C=O Stretch 1740-1720 Strong =C-H Stretch 2820-2850 & 2720-2750 medium, two peaks

Amide C=O Stretch 1640-1690 Strong N-H 3100-3500 Un-substituted have two bands N-H Bending 1550-1640

Anhydride C=O Stretch 1800-1830 & 1740-1775 two bands

Ester C=O Stretch 1735-1750 Strong C-O Stretch 1000-1300 two bands or more

Ketone Acyclic Stretch 1705-1725 Strong

Cyclic Stretch 3-membered - 1850 4-membered - 1780

Strong

9

5-membered - 1745 6-membered - 1715 7-membered – 1705

,-unsaturated

Stretch 1665-1685 Strong

aryl ketone Stretch 1680-1700 Strong

HPLC has been used to separate, identify and quantify the components in a mixture. It

has been used in quantification of thymoquinone, dithymoquinone, thymohydroquinone, and

thymol in the oil of Nigella sativa (seed) (Ghosheh et al., 1999). The extractions of phenolic

compounds from white wine and apple extract were performed by reverse phase HPLC

(Fernandez de Simon et al., 1992). It has been used to quantify flavonols, flavones and

flavanones in fruits, vegetables and beverages (Justesen et al., 1998)

NMR analysis has been used to confirm the identity of chemical (Fraissard and Ito,

1988; Aime et al., 1992; Badia et al., 1997) and biological (Tsai et al., 1996; Wuthrich 2003;

Saito and Ando, 1989) compounds. It is a powerful diagnostic tools used to elucidate structures

in the lignin polymer, provides knowledge about the chemical and biochemical changes (Ralph

et al., 2006). In the present study NMR was used to see the structural changes of compounds

alone and in combinations with other compounds.

As it is evident from the above literature update, only few scientific groups in India are

working on the synergistic effects of medicinal plants. To the best of our knowledge, the

medicinal plants of Himachal Pradesh have not yet been ventured for their synergistic

properties by any research group in India. Therefore, the major objective of the present project

was to screen for anti-microbial properties of the medicinal plants of Himalayan region and

discover new combinations of plant extracts with traditional antibiotics to control clinical

bacteria that are prevalent in Indian hospitals. As the medicinal value of plants increases from

lower to higher altitudes, attempts would be made to discover rare medicinal potential of plants

of upper Himalayas.