6

2. Literature Review

A reviewer is one who gives the best jeers of his life to the author.

- Anonymous

________________________________________________________________________

2.1. Bioactive constituents in plant seeds

2.2. Plant Materials

2.3. Test Organisms

2.4. Extraction

2.4.1. Microwave assisted extraction (MAE)

2.5. Antioxidant Activity

2.5.1. Phenolic antioxidants

2.6. Antimicrobial Activity

2.6.1. Antimicrobial susceptibility tests

2.7. Characterization / Fractionation of Active Extracts

2.7.1. Ultraviolet and visible spectroscopy

2.7.2. Thin-layer chromatography (TLC)

2.7.3. High pressure liquid chromatography (HPLC)

2.8. Synergy

7

2. Literature Review

The universal role of plants in the treatment of disease is exemplified by their

employment in all the major systems of medicine. There is a great wealth of knowledge

concerning the medicinal and other properties of plants that is transmitted from

generation to generation by tribal societies. Now monographs on crude plant preparations

have made their way in modern pharmacopoeia. The use of modern isolation techniques

means that new plant drugs usually find their way into medicine as purified substances

rather than in the form of galenical preparations. The use of single pure compounds,

including synthetic drugs, is not without its limitations, and in recent years there has been

an immense revival in interest in the herbal system of medicine, which rely heavily on

plant sources. The editor of Journal of Natural Products, 1999, writes that in response to

the increasing prominence of herbal remedies, additional contributions describing

scientific investigations of a rigorous nature are welcomed. Undoubtedly, the plant

kingdom still holds many species of plants containing substances of medicinal value

which have yet to be discovered; large numbers of plants are constantly being screened

for their possible bioactivity (Evans, 2002).

2.1. Bioactive constituents in plant seeds

Since seeds are the primary stage of plant life cycle, have strong defence mechanism

possibly due to presence of phytoconstituents contributing to antioxidant and/or

antimicrobial activity. Many fruits have inedible seeds and are not part of human diet.

However, such seeds are a part of ayurvedic preparations against many diseases (Kothari

and Seshadri, 2010).

Numerous reports are there in literature concerning presence of bioactive

substances in plant seeds. Seeds from the genus Lupinus are known to accumulate large

quantities of poisonous quinolizidine alkaloids. Lignans in seeds either help defend

against various pathogens or act as antioxidants. Sesamin, from the sesame seed, has in

vitro antioxidant properties that stabilize sesame oil against turning rancid during

commercial storage. Red sorghum produces proanthocyanidin antifeedant compounds-

8

condensed tannins, which deter birds from feeding on the seed. Coumarins in seeds coats

appear to possess antimicrobial, antifeedant, and germination inhibitor properties

(Croteau et al., 2000). Methanol extract of Garcinia kola seeds were reported for their

bactericidal action against B. anthracis and E. coli (Akinpelu, 2008). Prunus armeniaca

kernels were reported to possess antioxidant and antimicrobial activities (Yigit et al.,

2009).

2.2. Plant Materials

A brief description of the plants selected as the subject of this study follows (Khare,

2007).

2.2.1. Annona squamosa Linn.

This member of family Annonaceae is a native to South America and the West Indies,

now cultivated throughout India, commonly known as custard apple, sugar apple, and

sitaaphal. Its seeds are reported to be abortifacient. Seed powder mixed with leaf juice is

used as insecticide, especially against lice. A fraction of total alkaloid from roots exhibits

antihypertensive and bronchodilatory properties. Leaves contain a cardiotonic alkaloid-

quinoline. Another alkaloid- annonaine has been reported in its bark, seeds and leaves.

2.2.2. Carica papaya Linn.

It belongs to family Caricaceae, commonly known as papaya or papitaa. Seeds are

abortifacient, emmengagogue, and vermifuge. Juice of seeds is administered in enlarged

liver and spleen, and in bleeding piles. Papain, extracted from latex of C. papaya has

been included among unapproved herbs by German Commission E. Green parts of the

plant and seed contain an alkaloid carpaine. Seeds also contain carpasemine, which is

responsible for its anthelmintic action against Ascaris lumbricoides. There are reports of

unripe C. papaya pulp, meat and seeds exhibiting bacteriostatic action against few

enteropathogens. Same parts of the fruit were reported to have radical scavenging ability

(Osato et al., 1993).

9

2.2.3. Citrus limon (Linn.) Burm.f.

This member of family Rutaceae is cultivated all over India, with common name lemon.

Its leaves and stems are reported to have antibacterial action. All parts of the plants of

citrus sp. contain coumarins and psoralins. Fruit is reported to have antioxidant and

anticancer properties. It has been used to tone and purify liver, in treatment of ulcers,

arthritis, gout, and acne (http://plants.usda.gov).

2.2.4. Manilkara zapota L

It belongs to family Sapotaceae, and popularly known as chikoo. Fruit usually have about

3-12 hard, black shiny seeds. The seed kernel which forms 50% of the whole seed

contains 1% saponin, and 0.08% sapotinin

(http://www.hort.purdue.edu/newcrop/morton/sugar_apple.html#Other%20Uses). Fruit is

known to have high latex content. Seeds are used for their diuretic action, and to cure

kidney stones.

2.2.5. Phoenix sylvestris Roxb.

It belongs to family Palmae (Arecaceae), commonly referred as Khajur or wild date palm.

Seeds are used in ague. The fruit is sweet, cooling, oleaginious, cardiotonic, aphrodisiac,

good in heart complaints, abdominal complaints, fever, vomiting, wandering of mind,

loss of consciousness (Nadkarni, 2002). Central tender part of the palm is useful in

gonorrhoea and gleet. Root is used in toothache and is good in nervous debility. A paste

of the kernels with the root of Achyranthes aspera Linn. is eaten with betel leaves as a

remedy for ague.

10

A. squamosa M. zapota

C. papaya C. limon T. dioica

S. cumini P. sylvestris

T. indica

Plate 1. Plant materials investigated during this work

11

2.2.6. Syzygium cuminii (Linn.) skeels

This plant also bears other names- S. jambolanum (Lam.) DC., and Eugenia jambolana

Lam. It is a member of family Myrtaceae, popularly called Jaamun or black plum. Leaves

are reported for antibacterial and antidysenteric action. Seed is believed to possess

hypoglycemic action (Sagrawat et al., 2006), they are also reported to be useful as

astringents in diarrhea as well as dysentery. Seeds contain tannin (about 19%), ellagic

acid, gallic acid (1-2%), beta-sitosterol, and essential oil (0.05%). Myricyl alcohol is

present in the unsaponifiable matter. Phenols, including methylxanthoxylin and 2,6-

dihydroxy-4-methoxyacetophene have been isolated from the seed. Chloroform fraction

of seed extract exhibited anti-inflammatory, anti-arthritic, antipyretic, and analgesic

activities. Water extract of the seed exhibited antibacterial property against S. boydi and

S. dysentrae. Methanol and ethyl-acetate extracts of the seeds were reported to exert

antibacterial action against gram-positive as well as gram-negative bacteria (Sagrawat et

al., 2006). Vibrioicidal activity of the S. cumini bark has been reported to be due to

tannins present in them (Sharma et al., 2009). Aqueous extract of the seed have been

shown to contain α-amylase inhibitors (Karthic et al., 2008). Flavonoids isolated from

Myrtaceae species, including S. cumini, are known to exert a potent inhibitory effect on a

variety of enzymes related to cell activation and to the production of inflammatory

mediators (Brito et al., 2007). Aqueous extract of S. cumini were reported for their

antifungal activity (Satish et al., 2007).

2.2.7. Tamarindus indica Linn.

This member of family Caesalpiniaceae bears synonyms- T. occidentalis Gaertn., and T.

officinalis HK. Common name is Amli or Tamarind. Ethanolic extract of the seed coat

has antioxidant activity. A bitter principle (tamarindienal) isolated from the fruit pulp

showed bactericidal and fungicidal activity. The ethanolic extract of dry fruit pulp was

found to be active against E. coli, Kleibsiella pneumoniae and S. paratyphi A. The

aqueous extract was found to be highly active against P. aeruginosa and moderately

against E. coli and K. pneumoniae (Daniyan and Muhammad, 2008). The extracts of

stem, bark, and leaves of tamarind in water and organic solvents were found to be active

against both gram-positive and gram-negative bacteria (Doughari, 2006). The ethanolic

12

extracts of pulp and leaves of tamarind exhibits inhibitory activity against Fusarium

oxysporum, while Aspergillus flavus was inhibited by the ethanolic extract of leaves only

(Tyagi and Bohra, 2003).

2.2.8. Trichosanthes dioica Roxb.

It is included in family Cucurbitaceae, and popularly called patola or parwal. Fatty acids

from the seeds comprise elaeostearic, linoleic, oleic and saturated acids. Extracts of seeds

exhibit antimicrobial, hypoglycemic, and haemagglunating activity. The plant cures

bronchitis; it is an alternative tonic; useful in heart troubles, obstinate fevers, boils.

Whole plant is antipyretic, antihelmentic, aphrodisiac, stomachic, appetiser, and cathartic.

The root is cathartic, it is drastic purgative and useful in jaundice anasarea and ascites.

Leaves are cholagogue, aperients, tonic febrifuge, expectorant and used in case of

enlarged liver and spleen, haemorrhoids, fever, leprosy, intrinsic haemorrhage erysipelas,

alopecia, diseases of mouth, inflammation and wounds. Fruit is cooling, laxative,

febrifuge, antibilious, cholagogue, aperients and used in diarrhoea, oedema, fever, and as

an adjunct to alternative medicine. In Gujarat it is used as a remedy for spermatorrhoea

(Nadkarni, 2002).

2.3. Test Organisms

Brief notes on the species of bacteria which served as test organisms during this work are

presented below.

2.3.1. Aeromonas hydrophila

A. hydrophila is an ubiquitous, heterotrophic foodborne pathogen which is widely

distributed in aquatic environments like fresh, marine, estuarine, salt, etc. A. hydrophila

is a gram-negative, facultatively anaerobic gammaproteobacteria. It can also grow even at

low temperature upto -0.1°C (Peter, 2000). Due to its capacity to grow at low temperature

it has become a problem for food industry (Adams and Moss, 2003). Since they are found

in water and sewage, they are potential contaminants of foodstuffs like vegetables and

other foods of animal origin like milk and dairy products. Their presence can be detected

in raw, refrigerated or frozen food. A. hydrophila is also found to be associated with

variety of extra-intestinal infections like cellulitis, myonecrosis, bacteraemia, septicemia,

13

and eczema. When A. hydrophila crosses the blood–occular barrier to reach the eye via

blood stream, it causes a sight-threatening condition known as endogenous

endophthalmitis (Palu et al., 2006). A. hydrophila shows resistance to antibiotics such as

cabenicillin, vancomycin, ampicillin, cephalothin, rifampicin, penicillin, cefoxitin,

sulbactam, erythomycincefoxitin, bacitracin, and trimethoprim. In 1982, this organism

was found involved in an outbreak of gastroenteritis at Louisiana, of which 472 cases

were associated with oysters. This suggests that it is not out of place to suspect this

organism when oysters are involved in gastroenteritis (Sohn et al., 2007).

2.3.2. Bacillus subtilis

This gram-positive bacterium is a common mesophilic, endospore-forming saprophyte.

This bacterium being thermoduric, may cause concerns in food and dairy industries.

Bacillus species may survive milk pasteurization or inadequate heat treatment during

canning of foods. This bacterium has been listed among foodborne pathogens involved in

outbreaks from contaminated food and water (Bhunia, 2008). It produces rope in the open

texture of bread, and is capable of spoilage in underprocessed foods (Adams and Moss,

2003). It is known to produce an antibiotic- bacitracin, which is used for topical treatment

of infections caused by gram-positive bacteria (Pelczar et al., 1993).

2.3.3. Escherichia coli

E. coli is a member of the large bacterial family, Enterobacteriaceae, the enteric bacteria,

which are facultatively anaerobic, gram-negative rods that live in the intestinal tracts of

animals in health and disease. Pathogenic strains of E. coli are responsible for three types

of infections in humans: urinary tract infections (UTI), neonatal meningitis, and intestinal

diseases (gastroenteritis).

When ingested, the following strains can cause diarrhoea:

Enterotoxigenic Enteroinvasive Enteropathogenic Enterohaemorrhagic Enteroaggregative

14

Some strains are emerging as potentially important causes of persistent diarrhoea in

patients with AIDS and in children in tropical areas (Burke and Cunha, 2009). Over 700

antigenic types (serotypes) of E. coli are recognized based on O, H, and K antigens. In

northern India, 302 E. coli isolates from human and animal populations were checked for

their antibiotic susceptibility and the results obtained showed the prevalence of multidrug

resistant strains which accounted 41%. Other isolates were reported to be resistant to

ampicillin (43.5%), oxytctracycline (36·4%) and trimethoprim-sulphamethoxazole (9.3%)

Similarly, in a recent study in London, E. coli isolates from urine samples of different

people were tested for their ability to resist various antibiotics, commonly used as

empirical oral treatments for urinary tract infections. It was found that out of 11,865

isolates of E. coli, only 55% and 40% of isolates showed resistance to ampicillin and

trimethoprim respectively. While 94% isolates were susceptible to nitrofurantoin

followed by gentamicin (93.7%), and cefpodoxime (92%) (Bean et al., 2008). According

to the US Centre for Disease Control and Prevention (CDC), E. coli is one of the leading

causes of food borne illness in the US. Yearly, an estimated 76 million Americans fall ill

from some form of food-borne illness, 325,000 land in the hospital, and 5,000 die

(http://www.foodpoisoningblog.org/).

2.3.4. Psuedomonas oleovorans

P. oleovorans is a gram-negative, methylotrophic bacterium that is a source of rubredoxin

(part of the hydroxylation-epoxidation system). It was first isolated in water-oil

emulsions used as lubricants and cooling agents for cutting metals. Based on 16S rRNA

analysis, P.oleovorans has been placed in the P. aeruginosa group (Anzai et al., 2000).

They are straight or slightly curved rod shaped cells that occur singly or in pairs or in

short chains. They do not possess pili and normally grow at 28-30°C. (Moore et al., 2006)

When grown on agar, the cells are almost coccoid (0.5×0.8 µm), but the length increases

to about 1.5 µm during the exponential phase in broth. Gelatine is not liquefied and starch

is hydrolysed by this organism (Garrity et al., 2005).

15

2.3.5. Salmonella typhi

S. typhi is a gram-negative rod shaped facultatively anaerobic motile bacterium with

peritrichous flagella. It is a human specific pathogen and a major cause of classic

typhoid. Its infection results from ingestion of water or food contaminated with human

faeces or close contact with an individual who has typhoid fever (Ellermeier and Slauch,

2006). Salmonellas are responsible for a number of clinical syndromes grouped as

enteritis (Adams and Moss, 2003).

2.3.6. Salmonella paratyphi A

S. paratyphi A is a member of the Enterobacteriaceae family. It is a gram-negative

motile aerobic rod which is facultatively anaerobic and there is serological identification

of somatic and flagellar antigens. It causes a milder form of enteric fever. There has

been a tremendous increase in the cases of enteric fever caused by S. paratyphi A in

various parts of India since 1996. In New Delhi, there has been an increase in S.

paratyphi A infection from 6.5% in 1994 to 44.9% in 1998 (Sood et al., 1999). Similarly,

there has been a rise from 11.1% in 2001 to 59% in 2003 in Calicut (Lalith et al., 2004).

It was 46.15% in Nagpur (Tankhiwale et al., 2003) and 53.33% in Sevagram (Mendiratta

et al., 2004). Recently a prospective study was carried out in the microbiology

department of Government Medical College and Hospital, Chandigarh from January

2006 to April 2007 on Salmonella species and an increase in number of S. paratyphi A

isolates was found from 34.18% to 40.63% (Gupta et al., 2009). The reasons for the

increase in the number of isolates may be changing host susceptibility, change in

virulence of the organism or increase in resistance to various antibiotics. It is resistant to

nalidixic acid and ampicillin but is susceptible to ciprofloxacin and chloramphenicol.

However, nalidixic acid resistant strain could not be treated with ciprofloxacin too. This

is due to mutation in DNA gyrase which is responsible for resistance to ciprofloxacin

(Ruiz et al., 1999).

2.3.7. Staphylococcus aureus

S. aureus is a gram-positive, spherical bacteria occurring in short chains or grape like

clusters. It is commonly found on the skin and in the nose of healthy people. It produces

16

numerous toxins that cause toxic-shock syndrome and staphylococcal scarlet fever. It is

responsible for a number of diseases in humans like food poisoning, wound infections,

skin infections, pneumonia, and toxic shock syndrome. It has been reported by the

World Health Organization (WHO) that all over the world S. aureus has developed

resistance to most potent antibiotics which were used against S. aureus infections. 95%

strains of S. aureus are resistant to penicillin and 60% are resistant to methicillin. Apart

from these antibiotics, it has also developed resistance to quinolone and vancomycin

(Sakoulas and Moellering, 2008). The factors behind this resistant potential are

numerous like resistance to penicillin is mediated by blaZ, the gene that encodes β-

lactamase (Kernodle, 2000), in case of methicillin resistance it is due to the presence of

mecA gene in the genome of S. aureus (Hiramatsu, 2001; Katayama, 2000),

chromosomal mutations in topoisomerase IV or DNA gyrase or by the induction of

multidrug efflux pump for resistance to fluoroquinols (Hooper, 2002) and for resistance

to vancomycin it is chromosomally mediated and the conjugal transfer of vanA gene

from Enterococcus faecalis. Recently methicillin resistant S. aureus (MRSA) is also

shown to be resistant to pristinamycin (Keshari et al., 2009).

2.3.8. Staphylococcus epidermidis

S. epidermidis is a gram-positive, nonspore forming, cocci, nonmotile and facultatively

anaerobic microorganism. It is chemoorganotrophic, can cause endocarditis and urinary

tract infections. It is associated with intravascular diseases and may produce life-

threatening bloodstream infections. It is a common cause of infections associated

with vascular catheters, cerebrospinal shunts, prosthetic joints, peritoneal catheters,

vascular grafts and prosthetic cardiac valve (Raad et al., 1992; Inman et al, 1984;

Karchmer et al., 1983). Multiple antibiotic-resistant S. epidermidis was reported to be

involved in nosocomial septicemia (Christensen et al., 1982). It is also the most common

cause of nosocomial bacteremia in neutropenic patients with cancer (Koll et al., 1993)

and in bone marrow transplant patients with leukemia (Wade et al., 1982). The isolates

of S. epidermidis from patients suffering from above disease are found to be plasmid

mediated methicillin resistant (Moller, 1988; Lowy et al., 1982). Kotilainen et al., (1990)

17

has reported that S. epidermidis exhibits resistance to ciprofloxacin and vancomycin due

to the production of slime over clinical devices. It is also a common agent of bacterial

keratitis. The virulence factor responsible for keratitis is slime production (Nayak et al.,

2007; Nayak and Satpathy, 2000).

2.3.9. Streptococcus pyogenes

S. pyogenes is a gram-positive, nonmotile, nonspore forming coccus. It has a capsule

composed of hyaluronic acid. It is a respiratory pathogen causing acute diseases in

respiratory tract and blood stream like acute rheumatic fever and acute

glomerulonephritis. It is the leading cause of pharyngitis and tonsillitis. Apart from

these, it is also responsible for causing pneumonia, obits and sinusitis. In India, it is

sensitive to penicillin but resistant to tetracyclines and macrolides (Raghunath, 2008).

On the basis of a retrospective study carried out at two academic institutions in New

York, it was reported that S. pyogenes is resistant to erythromycin and clindamycin but

penicillin resistance has not been seen yet (Karen et al., 2009). A reason for resistance to

erythromycin and clindamycin can be attributed to erythromycin resistant methylase

gene erm TR (Seppala et al., 1998). 2.3.10. Shigella flexneri

S. flexneri is a gram-negative, nonspore forming, nonmotile facultative anaerobe. It is a

human intestinal pathogen causing a number of health problems from mild diarrhoea to

dysentery. This pathogen is relatively resistant to stomach acid. Therefore the bacteria

can pass into the small intestine, multiply and finally invade colonic mucosal cells where

it induces an intense inflammatory response leading to formation of colonic mucosal

ulcerations. The annual number of Shigella episodes throughout the world is estimated

to be 164.7 million, with 69% of all deaths attributed to shigellosis involving children

less than 5 years old (Kotloff, 1999). In a five year study from 1995 to 2000 carried out

in Kolkata, it was observed that in India, S. flexneri showed more than 90% resistance to

trimethoprim-sulfamethoxazole and 69% for ampicillin (Dutta et al., 2002), while no

resistance was reported against nalidixic acid, ciprofloxacin and ceftriaxone (Jesudason,

2002). But recently it is confirmed that S. flexneri shows high resistance to nalidixic acid

18

and resistance to ciprofloxacin is emerging (Taneja, 2007).

2.3.11. Vibrio cholerae

V. cholerae is a gram-negative, small, slightly curved, facultative anaerobic bacterium

(Peter, 2000). It causes cholera, which is one of the world’s most communicable acute

intestinal infection diseases (Madigan and Martinko, 2006). V. cholerae produces cholera

toxin, the model for enterotoxins, whose action on the mucosal epithelium is responsible

for the characteristic diarrhoea of the disease cholera. In its extreme manifestation,

cholera is one of the most rapidly fatal illnesses known. In April 1997, a cholera outbreak

occurred among 90,000 Rwandan refugees residing in temporary camps in the

Democratic Republic of Congo. During the first 22 days of the outbreak, 1521 deaths

were recorded, most of which occurred outside of health-care facilities (Todar, 2009). It

has led to an epidemic in Viet Nam between 5th March and 22nd April 2008. There were

2,490 reported cases of severe acute watery diarrhoea including 377 cases that were

positive for V. Cholerae (http://www.irinnews.org/report.aspx?ReportId=77970). There

has been a sharp increase in number of cholera cases reported to WHO during 2005 with

a total of 1,31,943 cases including 2,272 deaths notified from 52 countries (Sharma et al.,

2009). The WHO estimates that during any cholera epidemic, approximately 0.2-1% of

the local population will contract the disease. Death rates associated with untreated or

poorly treated cholera are often 20% - 50%, can be even over 50% during severe

epidemic. However, with prompt treatment, death rate may be as low as 1-2%

(Choudhury, 2009).

2.4. Extraction

Extraction as a pharmaceutically used term can be defined as the technique used for the

separation of therapeutically desired active constituent(s) and elimination of unwanted

insoluble material by treatment with selective solvents. Extraction mainly involves the

release of complex plant constituents and solubilization of secondary metabolites from

the matrix, followed by separation of soluble target compounds from the crude extract

through selective use of solvents (Yrjonen, 2004).

19

The basic parameters influencing the quality of an extract are (Evans, 2002)- the

plant part used as starting material, the solvent used for extraction, the extraction

technology used with the type of equipment employed, and crude drug : extract ratio.

Other important parameters affecting the yield of the extraction procedure are the

moisture content of the plant material and temperature.

Traditional extraction processes involve extraction with water or organic solvents.

Water is almost universally the solvent used to extract activity. Initial screenings of plants

for possible antimicrobial activities typically begin by using crude aqueous or alcohol

extractions. Starches, polypeptides, and lectins are better extracted in water. Coumarins

and fatty acids are better extracted in ether, whereas methanol is reported to be good for

extracting lactones and phenones (Cowan, 1999).

The choice of extraction procedure depends on the nature of the plant material

and the components to be isolated. Various procedures that can be used for the extraction

of medicinal plants include- maceration, infusion, percolation, decoction, Soxhlet

extraction, counter current extraction, sonication, supercritical fluid extraction, steam

distillation, etc. The head space trapping technique, microwave assisted extraction

(MAE), solid phase microextraction, and molecular distillation are some of the newer

methods of extraction.Ultrasound may enhance the extraction process for some plant

materials, e.g., in the preparation of the ethanolic solution of opium for the assay of

alkaloids (Evans, 2002; Armstrong, 1999).

2.4.1. Microwave assisted extraction (MAE)

In recent years, the use of microwave for extraction of constituents from plant material

has shown tremendous research interest and potential. Microwaves are non-ionizing

electromagnetic waves of frequency between 300 MHz to 300 GHz. Microwave heating

is governed by two phenomenons- ionic conduction and dipole rotation. The efficiency

with which different solvents heat up under microwave depends on the dissipation factor

(tanδ), which is indeed the measure of the ability of the solvent to absorb the microwave

energy (∞ Appendix C). The moisture when heated inside of the plant cell due to

20

microwave effect, evaporates and generates tremendous pressure on the cell wall due to

swelling of the plant cell. The pressure pushes the cell wall from inside, stretching and

then ultimately rupturing it, which facilitates leaching out of the active constituents from

the ruptured cells to the surrounding solvent, thus improving the yield of

phytoconstituents. In some cases only selective heating of sample matrix is brought about

by immersing the sample in a microwave transparent solvent (hexane, chloroform). This

approach is particularly useful for thermolabile components to prevent their degradation

(Mandal et al., 2007).

In contrast with conventional liquid-solid extraction methods (e.g. Soxhlet

extraction) in which a relatively long extraction time (typically 3-48 h) is required, the

use of microwave energy for solution heating results in significant reduction of the

extraction time (usually less than 30 min). MAE also enables a significant reduction in

the consumption of organic solvent. The use of pure, microwave-transparent solvents

such as hexane could result in the rapid extraction of essential oil components (Huie,

2002). MAE offers the advantage of providing agitation during extraction, which

improves the mass transfer phenomenon. Solvent recovery however is not possible in this

method. Instrumental set up like Soxwave combines both the features of Soxhlet and

advantages of microwave, thus making extraction even more effective (Ahuja and

Jespersen, 2006; Sarkar et al., 2006; Mitra, 2003).

There are two types of commercially available MAE systems: (a) closed

extraction vessels and (b) focused microwave ovens. Even a modified multimode

domestic microwave oven operates as an open vessel extraction system. Latter has been

applied by different workers for extraction of essential oils, pectin, artemisnin,

diterpenes, etc (Mandal et al., 2007). Further, MAE has been applied for extraction of

flavonoids from Herba epimedii (Chen et al., 2007), for fast extraction of plant phenolic

compounds (Prestos and Comaitis, 2007), and for the leaching of lupin alkaloid

(sparteine) from seeds (Huie, 2002).

21

2.5. Antioxidant Activity

Free radicals contribute to more than one hundred disorders in humans including

atherosclerosis, arthritis, and reperfusion injury of many tissues. Oxidation process is one

of the most important routes for producing free radicals in food, drugs and even living

systems. Strong restrictions have been placed on the application of synthetic antioxidants,

and there is a trend to substitute them with naturally occurring antioxidants. Moreover,

synthetic antioxidants such as butylated hydroxyl anisole (BHA) and gallic acid esters

also show low solubility and moderate antioxidant activity. Recently there has been an

upsurge of interest in the therapeutic potentials of medicinal plants as antioxidants in

reducing such free radical induced tissue injury.

Antioxidant activity of plants might be due to their phenolic compounds.

Flavonoids are a group of polyphenolic compounds with known properties which include

free radical scavenging, inhibition of oxidative enzymes and anti-inflammatory action.

There have been reports of relationship of total flavonoid and phenol contents with

antioxidant activity. In the longer term, plant species (or their active constituents)

identified as having high levels of antioxidant activity in vitro may be of value in the

design of further studies to unravel novel treatment strategies for disorders associated

with free radicals induced tissue damage (Pourmorad et al., 2006). It may be that such

simple and cost-effective measures as improving our diet through supplementation with a

number of key antioxidants (antioxidant therapy) can dramatically improve the health of

the general population and the individual person (Frei, 1994). Antioxidants, however,

when administered in higher amounts exert detrimental effects on health. The dose and

the route of administration of antioxidants are important factors to be considered before

taking in any exogenous supplements of antioxidants.

Many research groups are focusing on the appraisal of antioxidant properties of

various parts of plants. Metabolic engineering of plants can improve the yield of

antioxidants. In order to identify the compatible targets for metabolic engineering,

screening of a large number of plant species for their antioxidant activity needs to be

22

done. More research on natural antioxidants can certainly help in increasing our average

life expectancy in the coming decades.

Capacity of polyphenolic antioxidants to scavenge free radicals has been

evidenced by a large number of tests measuring the antioxidant activity in vitro (Silva et

al., 2007). Antioxidant tests can be classified into two groups: those assays used to

evaluate lipid peroxidation, and those assays used to measure free radical scavenging

ability. In view of the diversity of the methods, there is a great need to standardize them

for measurement of antioxidant activity (Moreno, 2002).

2.5.1. Phenolic antioxidants

Whereas phenol itself is a rather ineffective antioxidant, when other radical-stabilizing

features are accentuated, extremely potent antioxidants can result. Electron-donating

groups at the ortho positions of a phenol characterize a number of efficient antioxidants.

A further feature of the structure of phenols is their acidity, or ability to ionize; the

phenolate anion is even more readily oxidized than the protonated form. Naturally

occurring poplyphenols have repeatedly been shown to scavenge peroxyl radicals. Many

phenolic compounds, in addition to being potent quenchers of free radical reactions, also

react quite rapidly with singlet oxygen. Some flavonoids, too, which are also well-known

radical scavengers, appear to be reactive with singlet oxygen. The most chemically

reactive quencher types are flavonols such as quercetin and fisetin. The flavonoid

quenches singlet oxygen without undergoing much change in concentration.

Flavonoids represent a large and diverse group of phenolic compounds derived from

higher plants. Derived from the type structure, flavone, these heterocyclic compounds

display a wide range of substitution patterns and oxidation states including flavonols,

flavanols, flavanones, and flavans or catechins. These compounds appear to have the

capacity of radical scavenging and metal ion complexation. Quercetin is known to form

stable complexes with Cu(II). Flavonoids having greater numbers of hydroxyl groups are

more effective antioxidants. Isoflavonoids are to be noted for their distribution being

restricted to a single plant family, the Leguminosae. Among free phenolic acids,

23

compounds derived from the C6-C3 phnylpropanoid unit are especially abundant in seeds

and bark.

In addition, gallic acid and related phenols in red wines have been suggested to be

responsible for the “French paradox”, that is the fact that residents of France have lower

rates of cardiovascular disease than those of other countries, despite consuming a diet

high in fats. Gallic acid derivatives are often powerful antioxidants (Larson, 1997).

2.6. Antimicrobial Activity

Clinical microbiologists become interested in the antimicrobial plant extracts because it is

very likely that these phytochemicals will find their way into the arsenal of prescribed

antimicrobial drugs. It is also recognized that the effective lifespan of any antibiotic is

limited, accordingly there is an increase in worldwide spending on finding new anti-

infective agents (Cowan, 1999). Characteristics of plants that inhibit microorganisms

have been investigated in laboratories since 1926. In modern times a large number of

research groups are enganged in this area of biology, some of them particularly targeting

drug-resistant bacterial strains (Mohanty and Cock, 2010; Srividya et al., 2010; Sharma et

al., 2009; Rahman et al., 2008; Ates and Erdogrul, 2003).

Exploiting plants for their natural products as templates for new antibacterial

substances is a much needed exercise, given the continuing and developing problems of

bacterial resistance, and in particular multidrug-resistance. Certain plant natural products

are reported to modulate or modify bacterial resistance. Specific examples include plant-

derived efflux pump inhibitors (EPIs) which inhibit bacterial antibiotic efflux

mechanisms that are problematic due to their broadness in substrate specificity (Gibbons,

2008). A schematic representation of targeted screening of plant extracts for

antimicrobial activity is presented in Figure 2 (Ahmed et al., 2006).

Why should time and effort be invested into characterizing new antimicrobial

substances from plants? Plants produce antimicrobial metabolites as part of their

chemical defence strategy. There are countless examples of plants which are used

24

topically and systemically to treat bacterial infections in the ethnobotanical setting. It is

extensive functional group chemistry, chirality and ultimately chemical diversity of

phytochemicals, and natural products in general, which mark them out as a valuable pool

of bioactive molecules. Phytochemicals are structurally distinct from microbially derived

antibiotic natural products, it is likely that this chemical uniqueness will give rise to

classes of antibacterials which have modes of action distinct from existing compounds.

Perhaps phytochemicals display new mechanisms of action, or activity toward some of

the newer targets. However despite all this, the fact that markets for antibacterial drugs

are small does have an impact on investment in this area of research. Plant-derived

antibacterials also have potential as topical materials rather than systemic drugs. There

are considerable benefits to the topical route over the conventional systemic drug, in

particular the speed to market and smaller amount of data needed to achieve this. Plant

antimicrobials also offer potentially new classes of agents to deal with the threat of

biowarfare (Gibbons, 2008).

Plants have an almost limitless ability to synthesize aromatic substances, most of

which are phenols or their oxygen-substituted derivatives. Most are secondary

metabolites, of which at least 12,000 have been isolated, a number estimated to be less

than 10% of the total. Useful antimicrobial phytochemicals can be divided into following

major categories (Cowan, 1999):

(i) Phenolics and polyphenols

(ii) Terpenoids and essential oils

(iii) Alkaloids

(iv) Lectins and polypeptides

Initial screenings of plants for possible antimicrobial activities typically begin by

using crude aqueous or alcohol extractions. Nearly all of the identified components from

plants active against microorganinsms are aromatic or saturated organic compounds.

Water-soluble compounds are commonly more effective as inhibition of pathogen

adsorption and would not be identified in the screening techniques commonly used.

25

Figure 2. Schematic representation of targeted screening of plant extracts for antimicrobial activity (Ahmed et al., 2006).

Plant samples (Roots, shoots, leaves, fruits, seeds etc.)

Extraction in organic solvents

Aqueous and organic extracts

Antimicrobial screening

Active extracts Antimicrobially inactive extracts

Activity guided fractionantion & MIC determination

Targeted screening approaches (1-6)

High potency fractions

Low potency

Targetted screening approaches (1-6)

Activity in one or more assay

No activity

Characterization & identification of active compounds

Molecular mechanism of action

Toxicity, efficacy, stability, safety, pharmacodynamics and availability in vitro & in vivo

Targeted screening approaches: 1. Quorum sensing inhibition 2. Beta-lactamase inhibition 3. Antibiotic plants interation 4. Multidrug efflux pump inhibition 5. Decreasing virulence & Pathogenecity 6. R-plasmid elimination & inhibition of R-plasmid transfer

26

2.6.1. Antimicrobial susceptibility tests

The two most commonly used screens to determine antimicrobial susceptibility are the

broth dilution assay and the disc or agar well diffusion assay. Clinical microbiologists are

very familiar with these assays.

To perform the disc diffusion test, filter paper discs impregnated with a specified

single concentration of an antimicrobial agent are applied to the surface of an agar

medium that has been inoculated with the test organism. The drug in the disc diffuses

through the agar. As the distance from the disc increases, the concentration of the test

substance decreases logarithmically. In areas where the concentration of drug is

inhibitory, no growth occurs, forming a zone of inhibition around the disc. Diameter of

the zone of inhibition is influenced by the rate of diffusion of the antimicrobial agent

through the agar, which may vary among different drugs depending upon the size of the

drug molecule and its hydrophilicity. The zone size is inversely proportional to the

minimum inhibitory concentration (MIC). The recommended medium for disc diffusion

testing is Mueller-Hinton agar. This medium demonstrates good batch-to-batch

reproducibility, and supports the growth of most nonfastidious bacterial pathogens

(Jorgensen and Turnidge, 2003). MH medium is low in thymine or thymidine content.

Media containing excessive amounts these substances may yield smaller and less distinct

zones.

Minimum inhibitory concentration (MIC) is defined as the lowest concentration

that will inhibit the growth of a test organism over a defined interval related to the

organism’s growth rate, most commonly 18-24 h (Turnidge et al., 2003). It is the lowest

concentration that inhibits the visible growth of an organism. The conventional technique

for measuring the MIC involves exposing the test organism to a series of twofold

dilutions of the antimicrobial agent in a suitable culture system, e.g., broth or agar for

bacteria. MIC measurements are influenced in vitro by a number of factors including the

composition of the medium, the size of the inoculum, the duration of incubation, and the

presence of resistant subpopulations of the organism. Routine dilution testing methods for

MIC determination include- broth microdilution, agar dilution, or gradient methods.

27

Cation-adjusted Mueler-Hinton broth (CAMHB) is the medium recommended for the

routine testing of the commonly encountered nonfastidious organisms. Microdilution

method has gained wide acceptance due to lower volumes (near 0.1 mL) involved. It also

allows multiple assays to be carried out in a single plate. Medium pH is a factor common

to both agar-based and broth-based systems. It should be between 7.2-7.4. Once

determined MICs can be effectively compared to those of a wider range of commercially

used antibiotics. Though the literature abounds with the reports of plant products with

MICs ranging from few µg/mL to few mg/mL, an MIC of less than 10 µg/mL and ideally

less than 2 µg/mL is considered as being of interest to pharma (Gibbons, 2008).

MIC determination requires preparing various dilutions of the compound under

test in a suitable solvent. Dimethyl sulfoxide (DMSO) and ethanol are frequently used as

solvent for natural as well as synthetic antibacterial compounds. Tests to determine the

concentration of solvent above which toxicity occurs should always be carried out before

the experiment proper, and controls with potential solvent toxicity in mind should be

incorporated into the experiment (Wadhwani et al., 2009; Houghton and Raman, 1998). 2.7. Characterization / Fractionation of Active Extracts

Once a particular activity has been confirmed to be possessed by an extract (crude

preparation), investigators next try to analyze it further in terms of its composition.

Efforts are made to isolate the active component(s) responsible for the activity in

question. A variety of separation techniques are available for fractionation of the crude

extracts. After separation is achieved, fractions (simpler mixtures) obtained are subjected

to a number of further analytical investigations in order to obtain more information on the

properties of their constituent substances. Broadly, further investigations involve

(Houghton and Raman, 1998):

(a) Qualitative chemical analyses- determination of the nature of the constituents of a

mixture or the structure of an isolated compound.

(b) Quantitative chemical analyses- determination of the purity of an isolated substances

or the concentration of a single substance or group of substances in a mixture.

28

(c) Bioassay- determination of the biological activity of substances and the dose range

over which they exert their effects.

The amount of fraction available is possibly the most important factor in making

decisions about its future treatment and analysis. Investigative methods can be either non-

destructive or destructive. Usually chemical tests for different chemical classes i.e.

phytochemical screening, is followed by suitable chromatographic and spectroscopic

methods.

In identifying a plant constituent, it is necessary first to determine the class of

compound and then to find out which particular substance it is within that class. The class

of compound is usually clear from its response to colour tests, its solubility and Rf

properties and its UV spectral characteristics. Complete identification within the class

depends on measuring other properties and then comparing these data with those in the

literature. These properties include melting point/boiling point, optical rotation and Rf or

Rt (under standard conditions). However equally informative data on a plant substance

are its spectral characteristics: these include ultraviolet (UV), infrared (IR), nuclear

magnetic resonance (NMR) and mass spectral (MS) measurements. A known plant

compound can usually be identified on the above basis. Direct comparison with authentic

material should be carried out as final confirmation. Identification of new plant

compounds by X-ray crystallography is now routine, and can be applied whenever the

substance is obtained in sufficient amount in crystalline form. It is particularly valuable in

the case of complex terpenoids, since it provides both structure and stereochemistry in the

same operation (Harborne, 1998).

Brief comments on the techniques exploited during the course of present

investigation are provided below (Harborne, 1998; Houghton and Raman, 1998).

2.7.1. Ultraviolet and visible spectroscopy

The absorption spectra (190-750 nm) of plant constituents can be measured in very dilute

solution against a solvent blank. Ethanol, methanol and water are commonly used

solvents for UV-vis spectroscopy. Chloroform is generally to be avoided since it absorbs

29

strongly in the 200-260 nm region; it is, however, quite suitable for making

measurements in the visible region of the spectrum (Harborne, 1998). Cut-off value of

the solvent being used should be considered while making spectral measurements. Such

spectral measurements are important in the identification of many plant constituents, for

monitoring the eluates of chromatographic columns during purification of plant products

and for screening crude plant extracts for the presence of particular classes of compound.

The absorption maxima for particular functional groups vary according to features of the

molecule (such as double bonds, etc.) and thus can provide useful clues to the rest of the

molecule. As long as the sample is a pure compound, this kind of non-destructive

spectroscopic investigations should be carried out before final biological tests are

performed. For comprehensive accounts on spectral data one may refer works compiled

by Lang (1966), Scott (1964), etc.

Scope of UV spectroscopy has been recognized in dereplication process in

modern analytical natural product chemistry. Many of the alkaloid and polyketide natural

products have characteristic spectra that can often be associated to a certain subclass

within these major types of natural products. Though a lot of structural information can

be extracted from an UV spectrum, UV approaches should not stand-alone but naturally

be part of a hyphenated approach ultimately using a combination of UV, MS, and NMR

detection (Larsen and Hansen, 2008).

2.7.2. Thin-layer chromatography (TLC)

TLC is widely used for analysis of plant products as it is a rapid method, and provides

qualitative as well as semi-quantitative information on the sample constituents. It

provides a chromatographic fingerprint, hence suitable for monitoring the identity and

purity of phytochemical preparations. When comparing Rf values of a component with

those quoted in the literature, or those recorded in one’s own previous experiments, it is

important to remember that variations may occur due to slight changes in temperature,

stationary phase or mobile phase composition and saturation status of the TLC tank.

Reproducible TLC preparations can be guaranteed only if standardized adsorption layers

are used. Silica gel is an efficient adsorbent for the separation of most plant extracts

30

(Wagner and Bladt, 1996). The system selection entails choosing the mobile phase, the

stationary phase and the detection method. While recovering the separated components

from silica plates, it should be considered that polar eluants, e.g., alcohol, can dissolve

not inappreciable amounts of colloidal silica in it (Stahl, 1969). Ethanol, methanol, and

water can dissolve appreciable amounts of silica gel which appear as a residue when the

eluant is concentrated (Houghton and Raman, 1998).

Analytical TLC plates can deal only with a small amount of sample, for applying

larger sample amounts preparative plates (thickness 0.5-1 mm) are available. However,

resolution in some cases may not be satisfactory on preparative plates. Even on

preparative plates only up to 50 mg of a sample mixture can be separated (Stahl, 1969;

Houghton and Raman, 1998).

For detection of separated substances use of UV lamps offer a non-destructive

approach. Chromatograms are viewed in short (254 nm) and long (365 nm) wave UV

light. If a fluorescent layer such as silica gel GF254 is exposed to short wave UV light, all

substances which absorb in this region stand out distinctly as dark zones on the green

fluorescing layer as background. Details of TLC have been dealt in the most

comprehensive fashion by Stahl (1969).

Use of TLC for investigation of bioactive plant products, including those with

antioxidant and/or antimicrobial properties, has been reported by numerous workers

(Keterere and Eloff, 2005; Horwath et al., 2002).

2.7.3. High pressure (performance) liquid chromatography (HPLC)

HPLC has the ability to provide both quantitative and qualitative data in the single

operation. HPLC is mainly used for those classes of compound which are non-volatile,

e.g. higher terpenoids, phenolics of all types, alkaloids, lipids and sugars. It works best

for compounds which can be detected in the ultraviolet or visible regions of the spectrum.

It is possible to carry out most separations using either a silica microporous particle

column (for non-polar compounds) or a reverse-phase C18 bonded phase column (for

31

polar compounds). The mobile phases used in HPLC range from organic solvents to

mixtures of water or buffer with water-miscible organic modifiers such as methanol,

acetonitrile and tetrahydrofuran. Due to the small volumes needed (5-100 µL), it is

important that the sample solution is sufficiently concentrated for the amounts applied to

be detectable. The mobile phase is to be used as the sample solvent wherever possible,

and in all cases the sample solvent and the mobile phase must be miscible. HPLC is

advantageously used for the quantitative estimation of various flavonoids in plant

extracts. Identification of vibrioicidal compounds in plant extracts through HPLC has

been reported by Sharma et al (2009). This technique has been used for the study of

bioactive tannins, flavonoids and their derivatives in different plant preparations (Brito et

al., 2007; Sharma et al., 2008).

The efficiency of HPLC and slica gel thin layer chromatography was compared

by applying them for the separation and quantitative determination of the compounds in

the leaves of Stevia rebaudiana Bertoni. It was found that all the basic features such as

resolution and reproducibility of both the techniques were nearly the same. A

combination of TLC and HPLC may be the best approach for separating a particular class

of plant compounds (Kothari et al., 2010, Damyanova et al., 2007; Harborne, 1998).

Chromatographic separation of plant saponins has extensively been reviewed by Oleszek

(Oleszek and Bialy 2006; Oleszek, 2002).

2.8. Synergy

Synergy occurs when the effect of two or more components applied together to a

biological system is more than the sum of the effects when identical amounts of each

constituent are used separately. This may be due to the substances acting in different

ways, so that, for example, some rate-limiting step in the process is eased or overcome.

Other mechanisms may exist, such as enhanced penetration to the site of action through

effects on gut or membrane permeability. Synergy may explain why the activity of an

extract or other mixture is greater than might be expected from the amount of the

compound, which is thought to be the major ‘active’ constituent, present. Synergy may

32

also be one of the explanations for the activities of any of the fractions obtained from a

mixture being considerably less than that of the total mixture.

If the total extract shows activity in the test but little is shown by any of the

derived fractions, the active compounds may have decomposed during the fractionation

process. Many natural products are sensitive to light, oxygen, water and changes in pH.

Observed activity in the total extract is due to synergism between components which are

separated as a result of the fractionation process. It is necessary to test combinations of

the fractions to see if this is the case.

The active components may remain in the system used for fractionation. This

possibility should be considered particularly if chromatography with a solid stationary

phase has been applied (Houghton and Raman, 1998).