7

CHAPTER II

REVIEW OF LITERATURE

Plants as a source of medicinal compounds have continued to play a dominant role in

the maintenance of human health since ancient times. Nature has bestowed upon us a very

rich botanical wealth and a large number of diverse types of plants grown wild in different

parts of our country. In India, the use of different parts of several medicinal plants to cure

various disorders has been in vogue from ancient times (Bhattacharjee 1998). India is one of

the 12-major biodiversity centers having about 10% of the world‟s biodiversity wealth, which

is scattered across 16 agro-climatic zones (Shiva, 1996). Around 20,000 medicinal plant

species have been recorded in our country (Dev, 1997).

Bacterial pathogens are a serious problem to health and major cause of morbidity and

mortality worldwide. These microbes cause human diseases involving the skin and mucosal

surfaces which constitute a serious illness particularly in tropical and subtropical developing

countries (Portillo et al., 2001). Antibiotics are generally used against pathogenic bacteria.

These are the chemical substances produced by one group of microorganisms that inhibits the

growth of other pathogenic microbes (Benjar, 2004). Conventional antibiotics are strong

medicines, which if not used in precise way may cause harmful effects. In recent years, drug

resistance to human pathogenic bacteria has been commonly reported from all over the world.

So the major thrust is to establish alternative antimicrobial agent in order to treat microbial

infections.

In view of it, natural compounds extracted from plants, have been suggested as

alternative sources for antibiotics. Recently there has been a shift in universal trend from

synthetic to natural medicine, which can be said „Return to Nature‟. Several plants have

ability to treat the multiple drug resistance strains (Kayser and Ferreira 2001; Kayser and

Kiderlen 2001). Generally all medicinal preparations are derived from plants, whether in the

simple form of raw plant materials or in the refined form of crude extracts, mixtures etc.

(Krishnaraju et al. 2005) because they produce wide array of bioactive molecules, most of

which probably evolved as chemical defense against predation or infection. It is estimated

that only one percent of 2, 65,000 flowering plants on earth have been studied exhaustively

for their chemical composition and medicinal value (Cox and Balick, 1994). Most of the

developing countries have implemented traditional system of medicine as an integral part of

8

their culture. Investigation of the activities of plants during the past two centuries has yielded

compounds for the development and discovery of novel and more effective therapeutic agents

(Nair et al., 2007). A large proportion of such medicinal compounds have been discovered

with the aid of ethno botanical knowledge of their traditional uses. The rich knowledge base

of countries like India and China in medicinal plants has led to the keen interest of

pharmaceutical companies to use this knowledge as a source for research and development

programs in the pursuit of discovering novel drugs (Krishnaraju et al., 2005). The increasing

use of traditional therapies demands more scientifically sound evidence for the principles

behind such therapies and for effectiveness of medicines. Local flora exploited in traditional

medicine for various biological activities is a necessary first step in the isolation and

characterization of the active principle and further leading to drug development.

In view of these following medicinally significant Indian traditional plants were

studied in the present work:

Acacia nilotica L. (Babul)

A. nilotica belongs to the family Leguminosae. The plant is reported to have various

medicinal uses such as appetite enhancer, for sore joints, stomach ache and clear out wounds.

The bark, root, gum, leaves and flowers have found use for skin diseases, dysentery, cough,

diabetes, eczema, burning sensation and as an astringent, anti-asthmatic (Basu et al., 1947). It

has anticancer, antimutagenic (Meena et. al., 2006; Farzana et al., 2014), anti-inflammatory,

antiplasmodial (Kirira et al., 2006), antidiarrhoeal (Agunu et al., 2005), antihypertensive,

molluscicidal, antifungal, antimicrobial, immunomodulatory, inhibitory activity against

Hepatitis C and HIV-I (Hussein et al., 1999; Sharma et al., 2014).

Brassica campestris L. (Mustard)

B. campestris belongs to the family Brassicaceae. Brassino steroids, a group of

steroidal substance, has also been reported in seed of Brassica campestris, which were shown

to have substantial anti-viral activity against pathogenic viruses including herpes simplex

virus type 1, RNA viruses and measles virus (Cao et al., 1997). Earlier literature reported

about the biocidal, bio herbicidal, anti-oxidant and anti-cancer activities of glucosinolates of

Brassicaceae (Rosa et al., 1997; Fahey et al., 2001; Halkier and Gershenzon, 2006).

Cynodon datylon L. (Dhoob, Indian doob)

C. dactylon belongs to the family Poaceae. Cynodon plant is brain and heart tonic,

expectorant, carminative, and aphrodisiac (Agharkar, 1991). According to Ayurveda, the

plant destroys foulness of breath, useful in leucoderma, bronchitis, pains, toothache, piles,

asthma, tumors, and enlargement of the spleen. In Homoeopathic systems of medicine, it is

9

used to treat all types of bleeding and skin problems (Ghosh, 1988; Oudhia et al., 1998). A

preparation of C. dactylon plant mixed with sugar is useful in the problem of urine retention

(Saikia et al., 2006; Rajakumar and Shivanna, 2009). Rhizome extract is said to have anti-

inflammatory action (Sindhu et al., 2009).

Emblica officinalis Gaertn. (Indian Goose berry, Amla)

E. officinalis is a small to medium sized deciduous tree belonging to the family

Euphorbiaceae. The plant parts are used as aphrodisiac, carminative, stomachic, laxative,

rejuvenative, diuretic and antipyretic. They are useful in diabetes, ophthalmopathy,

dyspepsia, colic, cough, asthma, hyperacidity, peptic ulcer, skin diseases, leprosy, bronchitis,

haematogenesis, inflammations, anemia, hepatopathy, jaundice, diarrhoea, dysentery,

hemorrhages, leucorrhoea, cardiac disorders, intermittent fevers and greyness of hair

(Dasaroju and Gottumukkala, 2014; Khosla and Sharma 2012)

Foeniculum vulgare Mill. (Fennel, Saunff)

F. vulgare is a biennial or short lived perennial herb belonging to family Apiaceae.

The seeds are oval, ribbed, 5-10 mm long with strong and sweet smell. Fennel and its

preparations are used to cure various diseases and also useful as carminative, digestive and

diuretic agent. Fennel increases elasticity of connective tissues and act as anti-aging agent

(Arslan et al., 1989). Herbal drugs and essential oils of fennel have hepatoprotective effects

(Ozbek et al., 2003), as well as antispasmodic effects (Reynolds, 1982). They are also known

for their diuretic, anti-inflammatory, analgesic and antioxidant activities (Choi and Hawang,

2004). Anand et al., 2008, reported that fennel seed possesses anticancer activity.

Lawsonia inermis L. (Henna, Mehandi)

L. inermis belongs to family Lythraceae. It is a perennial plant. Henna leaves, flowers,

seeds, stem bark and roots are used in traditional medicine to treat a variety of ailments as

rheumatoid arthritis, leprosy, fever, headache, ulcers, diarrheoa, diabetes, leucorrhoea,

cardiac disease and hepatoprotective agent (Chopra et al., 1956; Reddy, 1988). Lawsone, the

antimicrobial agent in henna (Malekzadeh, 1968; Sharma et al., 1995) exerted inhibitory

effects upon common nosocomial urinary tract pathogens such as Escherichia coli, P.

mirabilis, K. pneumoniae, P. aeroginosa and S. aureus (Bhuvaneswari et al., 2002).

Mangifera indica L. (Mango)

M. indica is a large evergreen tree which belongs to the family Anacardiaceae. The

leaves have been reported to contain saponins, glycosides, unsaturated sterols, polyphenols,

mangiferine and tannins etc. It is traditionally known to be useful for the treatment of various

diseases like throat infection, burns, and scalds. Mango possesses antidiabetic, anti-oxidant,

10

anti-viral, cardiotonic, hypotensive, anti-inflammatory properties. Various effects like

antibacterial, anti-fungal, anthelmintic, anti-parasitic, anti-tumor, anti HIV, antispasmodic,

antipyretic, antidiarrhoeal, antiallergic, anti-microbial, hepatoprotective, gastroprotective

have also been studied (Shah et al., 2010).

Myristica fragrans Houtt. (Nutmeg, Jaiphal)

M. fragrans belongs to the family Myristicaceae. The fruits are fleshy, drooping,

yellow, and with a longitudinal ridge. Nutmeg has been used as a folklore medicine for

treating diarrhea, mouth sores and insomnia (Somani and Singhai, 2008). In traditional

medicine, it has been widely used as antithrombotic, carminative, astringent, antifungal,

aphrodisiac (Sonavane et al., 2002). The essential oil of nutmeg is used externally for

rheumatism and possesses analgesic and anti-inflammatory properties (Olajide et al., 2000).

Ocimum sanctum L. (Tulsi)

O. sanctum belongs to the family Lamiaceae. In traditional systems of medicine,

different parts (leaves, stem, flower, root, and seeds) of O. sanctum have been suggested for

the treatment of bronchitis, dysentery, skin diseases, arthritis, asthma, malaria, diarrhea,

chronic fever, insect bite etc. It also possesses antifertility, anticancer, hepatoprotective,

cardioprotective, antidiabetic, antifungal, antispasmodic and analgesic actions. In a study it

has been found that the aqueous extract of O. sanctum significantly increases the activity of

anti-oxidant (Gupta et al., 2006) enzymes such as superoxide dismutase and catalase level in

extract-treated group compared to control.

Piper nigrum L. (Black pepper)

P. nigrum is a flowering vine in the family Piperaceae and cultivated for its fruit.

Black pepper is used to treat asthma, obesity, sinus, chronic indigestion, congestion, fever

(Ravindran, 2000), colic, gastric ailments cold extremities, and diarrhea (Ao et al., 1998). It

has been shown to have antimicrobial activity (Dorman and Deans, 2000). The major

constituent of black pepper is Piperine. It is bioactive compound and has been reported to be

the major contributors to the antimicrobial activity (Chaudhry and Tariq, 2006).

Psidium guajava L. (Guava)

P. guajava belongs to the family Myrtaceae. Guava has been used for the treatment of

diarrhea, fever, gastritis and ulcers (Robineau and Soejarto, 1996) and possess numerous

therapeutic uses including analgesic, anti-inflammatory (Garrido et al., 2001), antiamoebic

(Tona et al., 2000) antihelminthic, antiallergic (Garcia et al., 2003) and antibacterial (Bairy et

al., 2002). The boiled water extract of guava plant leaves and bark are used in medicinal

preparations which are utilized as remedies for dysentery, diarrhoea and upper respiratory

tract infections, cholera, external ulcers, leucorrhea and skin diseases (Dutta, 1998).

11

Rosa indica L. (Rose)

R. indica is a perennial flower shrub of family Rosaceae. Rose water is used as an

antiseptic. Rose tea can bring down fever and also works as diuretic. Rose petals possess

antimicrobial activity (Koday et al., 2010). It was reported that leaves, stem and flower of R.

indica have bacteriocidal effects on pathogenic microorganisms (Mishra et al., 2011). Gram-

negative bacteria were found to have more susceptibility to the extracts of different parts of

the rose plants as compared to Gram-positive bacterial species (Kumar et al., 2012).

Sesamum indicum L. (Til, Sesame)

S. indicum belongs to the Pedaliaceae family. Sesame oil is a natural antibacterial,

antifungal, antiviral and anti-inflammatory and also used for treatment of hepatitis, diabetes

and migraines (Kandangath et al., 2010). Analgesic activity of ethanol extract of S. indicum

has been tested in mice (Nahar and Rokonuzzaman, 2009). Oil has been found to inhibit

growth of melanoma in vitro and the proliferation of human colon cancer cells (Smith and

Salerno, 1992).

Terminalia chebula Retz. (Haritkari, Harad)

T. chebula belongs to the family Combretaceae. It has reported to have antioxidant

and free radical scavenging activities (Cheng et al., 2003). It is effective in inhibiting

Helicobactor pylori (Malekzadeh et al., 2001), Xanthomonas campestris (Afzalakhtar et al.,

1997) and Salmonella typhi (Rani and Khullar, 2004). It is reported to be hepatoprotective

(Tasaduq et al., 2003; Tasduq et al., 2006), adaptogenic (Rege et al., 1999), anti-

inflammatory (Pratibha et al., 2004) and immunomodulatory activities (Srikumar et al.,

2005).

Ziziphus mauritiana Lam. (Indian Jujube, Ber)

Z. mauritiana belongs to the family Rhamnaceae. The leaves are eaten with A.

catechu as an astringent. They are regarded as diaphoretic and are prescribed for typhoid in

children. A decoction of the bark is used for the treatment of diarrhea and dysentery. The

bark is also used as an astringent in gingivitis (Pareek 2001; Peto et al., 2006; Awasthi and

More, 2009; Bhandari, 2012). Z. mauritiana also possess anticancer, antidiarrhoeal,

antihyperglycemic, antioxidant, hepatoprotective, immunomodulatory and antimicrobial

activities (Manoj et al., 2012).

In the present investigation with a focus of upcoming problem of antibiotic resistance

the above mentioned plants were evaluated for their antibacterial, antioxidant antiurease and

anticollagenase potential.

12

A. nilotica B. campestris C. dactylon E. officinalis

F. vulgare L. inermis M. indica M. fragrans

O. sanctum P. nigrum P. guajava R. indica

S. indicum T. chebula Z. mauritiana

Figure 2.1 Images of plants used in the study

13

Antibiotic resistance

The successful use of any therapeutic agent is compromised by the potential

development of tolerance or resistance to that compound from the time it is first

employed. This is true for agents used in the treatment of bacterial, fungal, parasitic, and viral

infections and for treatment of chronic diseases such as cancer and diabetes; it applies to

ailments caused or suffered by any living organisms, including humans, animals, fish, plants,

insects, etc. The most striking examples, and probably the most costly in terms of morbidity

and mortality, concern bacteria (Davies and Davis, 2010). Antibiotics have revolutionized

medicine in many respects, and countless lives have been saved; their discovery was a

turning point in human history. Regrettably, the use of these wonder drugs has been

accompanied by the rapid appearance of resistant strains. Antibiotic resistance is considered a

global health concern and has been called one of the world‟s most pressing public health

problems. The rates of some communicable diseases have started to increase again as a result

of the rise in antibiotic resistance (Levy, 1998).

The evolution of microbs towards antibiotic resistant patterns has been clearly

indicated by 440000 new cases of Multi-Drug-Resistant resistant (MDR) Tuberculosis along

with widespread of Extensively Drug Resistant (XDR) Tuberculosis in 64 countries; 41%

cases of Hospital- Acquired MRSA and increased upsurge of Vancomycin Resistant

Enterococci (VRE) and Human Immunodeficiency Virus (HIV) infections and/or Gonorrhoea

etc (Giovanni et al., 2011).

The uncontrolled and inappropriate use of antibiotics today may reduce future

effectiveness of the antibiotics. The emergence of antimicrobial resistance has its roots in the

use of antimicrobials in animals and the subsequent transfer of resistance genes and bacteria

among animals, animal products and the environment (McEwen and Fedorka-Cray, 2002).

Extra-chromosomal genes were found responsible for these antimicrobial resistant

phenotypes that may communicate resistance to an entire antimicrobial class. The DNA

mobile elements transfer genetic determinants for antimicrobial resistance mechanisms and

may cause dissemination of resistance genes among different bacteria (McDermott et al.,

2002).

The increasing misuse of antibiotics has led to an international public health

nightmare, with increasing bacterial resistance to many antibiotics that once readily cured

bacterial diseases (Levy, 1998). With each passing time bacteria that defy not only single but

also multiple antibiotics have become increasingly common and extremely difficult to

control. During the past decade there has been an emergenence of carbapenem-resistant

14

Enterobacteriaceae that produce carbapenemases enzymes that efficiently hydrolyze

carbapenems, as well as most β-lactam drugs (Queenan and Bush, 2009). Among the most

recent carbapenemases to appear in the United States is the newly described New Delhi

metallo β-lactamase (MDM). First reported in 2009, NDM-1 was initially identified in K.

pneumoniae and E. coli clinical solates obtained from a Swedish patient who was

hospitalized in India (Rasheed et al., 2013). Drug-resistant gram-negative bacteria that

produce NDM have been found in community and health care settings in India in a wide

range of gram-negative genera. Resistant bacteria have various mechanisms to disable the

harmful actions of certain antibiotics, ensuring bacterial survival (Berkowitz, 1995; Lewis,

1995), such as:

production of enzymes that destroy the active antibiotics

changing cell wall permeability to antibiotics

rapid effluence/discharge of antibiotics form the interior of the bacteria

developing structural alteration in the attachment site for antibiotics

The search for new effective antimicrobial agents may alleviate the difficulties

associated with treatment of antibiotic resistant infections. The investigation and discovery of

novel effective antimicrobial agents should be accompanied with an appreciation and rational

use of current antibiotics. Scientific investigation of traditionally used medicinal plants for

antimicrobial properties may serve as effective approach in the treatment of antibiotic

resistant infections.

2.1 Plants as antibacterial agents

Plants are used medicinally in different countries and are the source of potential and

powerful drugs. In India, extracts of medicinal plants are used both directly as folk medicines

in different indigenous systems of medicine like Siddha, Ayurveda and Unani and indirectly

in the pharmaceutical preparations (Srinivasan et al., 2001). The use of plant extracts with

known antimicrobial properties, are of great significance to therapeutic treatments (Nagesh

and Shanthamma, 2009). However, this area is not much developed when compared to

modern system of medicine, mainly because of the lack of scientific documentation in this

field (Kalimuthu et al., 2010). Scientists realized that the effective life span of any antibiotic

is limited, so new sources especially plant sources are being investigated. Plants are rich in a

wide variety of secondary metabolites such as tannins terpenoids, alkaloids, flavonoids, etc,

which have been found in vitro to have antimicrobial properties (Cowan, 1999; Dahanukar et

15

al., 2000). There are several reports on antimicrobial activity of different herbal extracts

(Adelakun et al., 2001; Camporese et al., 2003; Bonjar, 2004; Boer et al., 2005; Nair et al.,

2005).

Kumar and Reetha, (2009) reported that methanol extract of Cassia auriculata and

Aegle marmelos extract showed good antibacterial activity to a group of bacterial pathogens.

Similarly, Ruta graveolens and Zingiber officinale plant extracts traditionally revealed an

inhibitory potential against Bacillus cereus strains (Alzoreky and Nakahara, 2003). It was

also reported that the aqueous extract of Cynara scolymus and the ethanol extracts from

Achyrocline satureioides inhibited the growth of Bacillus cereus, P. aeruginosa, Bacillus

subtilis and S. aureus (Asolini et al., 2006). Antimicrobial assays performed on methanol

extracts from leaves of Mikania glomerata, Psidium guajava, Baccharis trimera, Mentha

piperita and Cymbopogon citratus, revealed their antibacterial activity against S. aureus

(Betoni et al., 2006).

Voravuthikunchai et al., 2011 investigated the aqueous and ethanolic extract of ten

traditional Thai medicinal plants for their ability to inhibit 35 hospital isolates of Methicillin

resistant Staphylococcus aureus (MRSA). Nine medicinal plants displayed activity against all

isolates tested. Ethanolic extracts of Garcinia mangostana, Pucinia granatum and Quercus

infectoria against MRSA isolates.

Murugan and Saranraj, 2011 tested the various extracts of herbal plant Acalypha

indica for its antibacterial activity against nosocomial infection causing bacteria and

concluded that the solvent methanol was able to leach out antimicrobial principle very

effectively from the plant than the other solvents.

Reports are available on the use of several plant by-products, which possess

antimicrobial properties against several pathogenic bacteria and fungi (Bylka et al., 2004;

Shimpi and Bendre, 2005). Flavonoids may act through inhibiting cytoplasmic membrane

function as well as by inhibition of DNA gyrase and β-hydroxyacyl-acyl carrier protein

dehydratase activities (Cushnie and Lamb, 2005). It has been suggested that terpenes promote

membrane disruption, tannins act on the membranes of microorganism as well as bind to

polysaccharides and coumarins cause reduction in cell respiration (Chung et al., 1998;

Cowan, 1999). In vitro anti-bacterial activity of a glycoside, phenyl ethyl β-D-

glucopyranoside from the plant Sida rhombifolia was studied by Ekramul et al., 2002 and

16

reported that it had significant antibacterial activity against most of the tested bacteria. The

functions of triterpene, saponin in plants for their antimicrobial, analgesic, anti-inflammatory,

fungicidal, antibacterial, antiviral, immunostimulant, antihelmintic, antitumor, cytotoxic, and

antitussive activities, have been known for many years (Hostettmann and Marston, 1995).

Saranraj et al., 2012 reported the bioactivity of M. indica ethanol extract against

human pathogenic bacteria and fungi.

Furthermore, the results of a recent study described a potent inhibitory activity of

Vernonia polyanthes extract against Leishmania strains (Braga et al., 2007). Antibacterial

study was done on extracts from Allium sativum, Caryophyllus aromaticus, Madia glomerata,

C. citratus, Z. officinale, and P. guajava against E. coli, Enterococcus sp., S. aureus and

Salmonella. The extracts from A. sativum and Z. officinale showed the most significant

activity against gram-negative bacteria. Gram-positive strains were more susceptible to P.

guajava extracts and C. aromaticus extracts (Ushimaru et al., 2007).

Saranraj and Sivasakthivehan, 2012 tested the antibacterial activity of Phyllanthus

amarus was tested against the urinary tract infection (UTI) causing bacterial isolates viz., S.

aureus, Serratia marcescens, E. coli, Enterobacter sp., Streptococcus faecalis, K.

pneumoniae, P. mirabilis and P. aeruginosa. It was found that methanol extract of P. amarus

showed more inhibitory activity against UTI causing bacterial pathogens when compared to

other solvent extracts.

Rosmarinus officinalis hydroalcoholic extract was assayed against Streptococcus

mitis, Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and

Lactobacillus casei standard strains, and its antimicrobial activity was proven in all tests,

except against S. mitis (Silva et al., 2008). In another study, essential oils from R. officinalis,

Z. officinalis, C. citratus, Caryophyllus aromaticus, Mentha piperita and Cinnamomum

zeilanicum were tested against S. aureus and E. coli strains. Ginger essential oil was the most

efficient against S. aureus while cinnamon and clove were the most effective against E. coli

(Silva et al., 2009).

Ali et al., 2013 assessed three medicinal plants viz., Phyllanthus amarus, Tribulus

terrestris and Cassia auriculata were investigated for their antibacterial activity against

urinary tract infection causing pathogens.

17

Table 2.1 List of few plants showing Antimicrobial activity

S.No. Plant name Activity against microbe Reference

1. Acalypha wilkesiana S. aureus (Antimethicillin resistant) Akinyemi et al.,

2005

2. Adiantum capillus veneris E. coli, K. pneumoniae, Salmonella

typhimurium, P. vulgaris, P. aeruginosa,

Staph. Aureus, Vibrio cholerae

Ishaq et al., 2014

3. Alangium salviifolium B. subtilis, Micrococcus luteus,

Staphylococcus epidermis, E. coli, P.

aeruginosa,

Pandian et al., 2006

4. Amomum subulatum and

Elettaria cardamomum

Dental caries causing microbes Aneja and Joshi,

2009

5. Artemisia annua S. aureus, Salmonella enterica, K.

pneumoniae, Shigella dysenteriae, E.

coli

Tajehmiri et al.,

2014

6. Azadirachta indica S. aureus, E. coli, P.

aeruginosa

Orhue et al., 2014

7. Bixa orellana S. aureus, B. cereus, E. coli and Candida

albicans

Rojas et al., 2006

8. Caesalpinia pulcherrima S. epidermis, B. subtilis, Pseudomonas

pseudoalcaligenes, P. vulgaris,

Salmonella typhimurium

Parekh et al., 2005

9. Calotropis procera S. aureus, B. cereus Meena et al., 2010

10. Capsicum annum P. vulgaris, P. aeruginosa, S.

typhimurium,

Keskin et al., 2011

11. Casuarina equisetifolia S. epidermis, B. subtilis, P.

pseudoalcaligenes, P. vulgaris, S.

typhimurium

Parekh et al., 2005

12. Cinnamomum verum Streptococcus faecalis, V. cholerae, B.

subtilis

Mishra et al., 2008

13. Cisampelos pareira S.

aureus, S. typhimurium, K. pneumoniae,

E. coli, P. vulgaris and Streptococcus

pneumoniae

Ngoci et al., 2014

14. Coriandrum sativum P. pseudoalcaligenes, P. vulgaris, S.

typhimurium

Cao et al., 2012

15. Delonix regia S. epidermis, B. subtilis Parekh et al., 2005

16 Eucalyptus camaldulensis S. aureus, B. subtilis Babayi et al.,2004

17. Euphorbia hirta S. epidermis, B. subtilis, P.

pseudoalcaligenes, P. vulgaris, S.

typhimurium

Parekh et al., 2005

18 Euphorbia tirucalli S. epidermis, B. subtilis, P.

pseudoalcaligenes, P. vulgaris, S.

typhimurium

Parekh et al., 2005

19. Ficus benghalensis S. epidermis and B. subtilis Parekh et al., 2005

18

20. Gmelina asiatica B. subtilis and P. pseudoalcaligenes Parekh et al., 2005

21. Hypericum perforatum S. aureus, S. mutans, Staphylococcus

oxford, E. coli, P. vulgaris,

Streptococcus sanguis, P.

Barbagallo and

Chisari, 1987

22. Justicia secunda E. coli and C. albicans Rojas et al., 2006

23 Laurus nobilis E. coli, P. aeruginosa, S. aureus, B.

cereus, Sarcina lutea, , B. subtilis,

Perez and Anesini,

1994

24. Matricaria chamomilla E. coli, K. pneumoniae, P. aeruginosa, S.

aureus, S. epidermidis, Sterptococcus

salivarius, Moraxella gleucidolytica

Kedzia, 1991

25 Musa paradis iaca E. coli, K. aerogenes, P. aeruginosa, S.

aureus, Staphylococcus albus,

Streptococcus hemolyticus, B. cerus,

Bacillus coagulans, Bacillus

stearothermophilus,Clostridium

sporogenes

Sharma et al., 1989

26. Ocimum gratissimum S. aureus (Antimethicillin resistant) Akinyemi et al.,

2005

27. Phylantus discoideus S. aureus (Antimethicillin resistant) Akinyemi et al.,

2005

28. Piper pulchrum B. cereus and E. coli Rojas et al., 2006

29. Psidium guajava

E. coli, S. enteritidis, S. aureus, B.

cereus

Biswas, et al., 2013

30. Santalum album B. subtilis Parekh et al., 2005

31. Tecomella undulata S. epidermidis, B. subtilis Parekh et al., 2005

32. Terminalia avicennioibes S. aureus (Antimethicillin resistant) Akinyemi et al.,

2005

33. Terminalia catappa S. aureus, B. subtilis Babayi et al.,2004

34. Withania somnifera K. pneumoniae Bokaeian et al.,

2014

Plants have traditionally provided a source of hope for novel drug compounds against

infectious bacterial diseases (Iwu et al., 1999). Owing to their popular use as remedies for

many infectious diseases, searches for substances with antimicrobial activity in plants are

frequent (Shibata et al., 2005; Betoni et al., 2006). The literature on antimicrobial activity of

plant products is very broad, including an increasing number of publications per year.

Therefore, it is difficult to integrate all those numerous studies on the antimicrobial action of

plant products in the present review.

2.2 Plants as antioxidants

The oxidative stress, defined as „„the imbalance between oxidants and antioxidants in

favor of the oxidants potentially leading to damage‟‟ has been suggested to be the cause of

various disease in humans. The production of oxidants is associated with aerobic metabolism.

19

When oxygen is supplied in excess or its reduction is insufficient, free radicals such as

superoxide anions, hydroxyl radicals and hydrogen peroxide are generated (Kris-Etherton et

al., 2004). Accumulation of these free radicals in body organs or tissues can cause oxidative

damage to bimolecules and membranes of cell, eventually leading to many pathological

conditions, such as cancer, inflammation, aging, cardiac disfunction, diabetes, and other

degenerative diseases (Wang et al., 2004). So the balance between antioxidants and oxidants

is believed to be a critical concept maintaining a healthy biological system (Davies, 2000).

In the past few years, there is an increased preference for antioxidants from natural

sources rather than from synthetic sources because of the health risks and toxicity of synthetic

antioxidants (Buxiang et al., 1997). Natural products from medicinal plants serve as a

potential source of antioxidant agents possibly with novel mechanism of action. Studies have

proven the reverse correlation between the intake of fruits, vegetables and the morbidity and

mortality from degenerative diseases (Rimm et al., 1996). Many plants have been identified

as having potential antioxidant activities and their consumption recommended (Velioglu et

al., 1998; Kitts et al., 2000; Lee and Shibamoto, 2000; Liu and Ng, 2000; Wang and Jiao,

2000; Tiwari, 2001; Lee et al., 2003).

In India several plants are used in the form of crude extracts, infusions or plaster to

treat common infections without scientific evidence of efficacy (Ahmad et al., 1998). Hence,

it is of interest to determine the scientific basis for the traditional use of these medicinal

plants. The antioxidant properties of plant extracts lie in their metabolites including alkaloids,

tannins, flavonoids, glycosides, triterpenoids and other compounds of phenolic nature (Rajos

et al., 1992). In last two decades the number of publications on the potential health benefits

of polyphenols, has increased enormously (Friedman and Kimball, 1986; Dreosti, 1991;

Ahmad, 1995; Pietta, 2000; Tiwari, 2001; Lee et al., 2003; Modun et al., 2003). Aqueous

extract from the bark of M. indica was reported to contain anti-inflammatory,

immunomodulatory and antioxidant activities (Garrido et al., 2004). The extract is composed

of a variety of phenolic acids, phenolic esters, flavanols and the xanthone mangiferin (Janet et

al., 2006). Fruit such as strawberry, raspberry and red plum, which are rich in anthocyanins

had the highest antioxidant activities, followed by those rich in flavanones, such as orange

and grapefruit, and flavonols (e.g. onion, leek, spinach and green cabbage), while the

hydroxycinnamate containing fruit (e.g. apple, tomato, pear and peach) consistently elicited

lower antioxidant activities (Proteggente et al. 2002).

Sharma et al., 2013 reported a list of plants exhibiting antioxidant potential which is

mentioned below:

20

Achillea millefolium, Acorus calamus, Alliaria petiolata, Allium sativum, Allium ursinum,

Angelica sylvestris, Anthriscus cerefolium, Anthriscus sylvestris, Anthyllis vulneraria,

Arctium lappa, Artemisia absinthium, Artemisia vulgaris, Bellis perennis, Betula pendula,

Bidens tripartita, Calluna vulgaris, Capsella bursa-pastoris, Carum carvi, Carlina acaulis,

Carthamus tinctorius, Catharanthus roseus, Cichorium intybus, Cirsium arvense, Citrus

aurantifolia, Coleus ferscoli, Commiphora myrrha, Cornus mas, Corylus avellana, Cotinus

coggygria Scop., Curcuma domestica, Cymbopogon citratus, Daucus carrota, Elaeagnus

angustifolia, Equisetum arvense, Eryngium campestre, Eugenia caryophylla, Evonymus

europaeus, Genista tinctoria, Ginkgo biloba, Hieracium pilosella, Hippophae rhamnoides,

Humulus lupulus, Juniperus communis, Lotus corniculatus, Matricaria recutita, Melilotus

officinalis, Mentha piperata, Myristica fragrance, Nasturtium officinale, Ocimum sanctum,

Olea europaea, Onopordum acanthium, Piper nigrum, Rosemarionus officinalis, Salvia

sclarea, Sambucus ebulus, Sambucus nigra, Sanicula europaea, Santalum album, Scutellaria

barbata, Solidago virgaurea, Taraxacum officinale, Trifolium arvense, Tussilago farfara,

Vaccinium myrtillus, Viburnum lantana, Viburnum opulus, Vitis vinifera, Withania

somnifera, Zingiber officinalis. Trouillas et al., 2003 investigated the antioxidant, anti-

inflammatory and antiproliferative properties of sixteen French herbal teas and found some

herbs exhibiting high tested activities.

Kaur and Mondal, 2014 assessed the anti-oxidant activity and total phenolic content

of alcoholic extracts from seven medicinal plants (Asparagus racemosus, Ocimum

sanctum, Cassia fistula, Piper betel, Citrus aurantifolia, Catharanthus roseus, and Polyalthia

longifolia) by using a model system consisting of β-carotene, DPPH free radical and Folin-

Ciocalteu method.

The aqueous extracts of roots of Tinospora cordifolia showed antioxidant action in

alloxan diabetic rats. The administration of the extract of T. cordifolia roots for 6 weeks

resulted in a significant reduction of serum and tissue cholesterol, phospholipids and free

fatty acids in alloxan diabetic rats (Stanley et al., 1999). Similarly, Capparis decidua powder

showed antioxidant action in alloxan-induced diabetic rats (Yadav et al., 1997). Mc Cune et

al., 2002 tested traditionally used thirty-five plant species from boreal forest by 1, 1-

diphenyl-2-picrylhydrazyl (DPPH) assay. Out of these, 14 % were statistically equal to

ascorbic acid and 23 % had activities similar to green tea and a Trolox positive control.

Ahmad et al., 2006 evaluated antioxidant activity of the extracts from Azadirachta

indica, Carissa carandus, Debregeasia salicifolia, Pistacia integrrima, Vitex negundo and

Zizyphus jujuba using DPPH radical scavenging assay.

21

Ligustrum vulgare (Agati et al., 2009), Stevia rebaudiana (Tadhani et al., 2007),

Casuarina equisetifolia (Zhang et al., 2010), Acacia confusa (Chang et al., 2001), Populus

tremuloides (Diouf et al., 2009), Medicago sativa (Dalton et al., 1998) and Carissa spinarum

(Hegde and Joshi, 2010) were also reported to contain antioxidants.

Dinesh et al., 2014 assessed the preliminary screening of herbal edible plants;

Capsicum annum, Murrya kaneigi, Zingiber officinals, Ocimum sanctum, Foeniculum

vulgare and Curcuma longa for their antioxidant activity. The antioxidant activity was

confirmed by diene-triene-tetrane conjugation for different plant extracts as a sensitive index

of lipid peroxidation. The different plant extracts were observed to inhibit dienetriene- tetrane

conjugation. Curcuma longa extract was shown to inhibit conjugation at all stages effectively

than other extracts and the inhibition was comparable to standards Butylated hydroxyanisole

(BHA) and curcumin.

Antioxidant activity of Rubia cordifolia was investigated by Baskar et al., 2008. The

aqueous extract of R. cordifolia roots was found to inhibit lipid peroxidation (LPO) and

scavenge superoxide (O2•

), nitric oxide (NO•), 1, 1-diphenyl-2-picrylhydrazyl (DPPH

•)

radicals and reducing power in vitro. Ayoola et al., 2008 examined the ethanolic extracts of

the leaves of Carica papaya, stem bark of Mangifera indica, leaves of Psidium guajava and

the leaves of Vernonia amygdalina for DPPH free radical scavenging activity. Among these

P. guajava was found to be most potent radical scavenger.

Agarwal et al., 2010 investigated the antioxidant activity of methanol extract of

Acacia nilotica and Berberis chitria using different in vitro methods. Among the two, Acacia

nioltica had higher antioxidant property. Also the antioxidant property was directly related to

the total phenolic content of the extracts. Another study revealed the antioxidant potentials of

aqueous and alcoholic extracts of Aegle marmelos fruit pulp (Rajan et al., 2011).

In another investigation methanolic extracts of Plumbago zeylanica, Acorus calamus,

Hemidesmus indicus and Holarrhena antidysenterica, were evaluated for their antioxidant

activity by ferric thiocyanate assay. The order of antioxidant potential was found to be

highest in Plumbago zeylanica followed by Holarrhena antidysenterica, Acorus calamus and

Hemidesmus indicus (Zahin et al., 2009).

Hasnah et al., 2009 evaluated the antioxidant activity of fresh and dried plant extracts

of Paederia foetida and Syzygium aqueum using β-carotene bleaching and the 2,2‟-azinobis

(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation assay.

The total phenolic content of the methanolic extract of leaves of Barleria montana

was assessed by using Folin-Ciocalteau method and the antioxidant potential was measured

22

by using Hydrogen peroxide scavenging and DPPH method (Shanaz et al., 2011). B. montana

leaf extract showed strong reducing power and significant antioxidant activity.

Many pharmacological studies have shown that extracts of some antioxidant plant

possess anti-inflammatory, anti-allergic, anti-tumor, anti-bacterial, anti-mutagenic and anti-

viral activities to a greater or lesser extent. Researchers reported that intake of fruits,

vegetables and other foods having high antioxidant activity has been associated with reduced

risks of cancer, cardiovascular disease, diabetes and other diseases (Kris-Etherton et al.,

2004).

Table 2.2 List of common antioxidant plants

S.No. Plant name Family Part used Reference

1. Azadirachta indica Meliaceae Leaves Kapoor, 2014

2. Bixa orellana Bixaceae Seed Abayomi et al., 2014

3. Cinnamomum

zeylanicum

Apiaceae Oil Mancinj et al., 1998

4. Coscinium fenestratum Menispermaceae Stem and leaf Goveas and Abraham,

2013

5. Curcuma longa Zingiberaceae Leaf Sawasha et al., 2003

6. Cuscuta reflexa Convolvulaceae Stem Yadav et al., 2001

7. Cynara scolymus Asteraceae Leaf Gebhardt 1997

8. Datura metel Solanaceae Leaves Ranja et al., 2013

9. Emblica officinalis Euphorbiaceae Fruit Khopde et al., 2001

10 Emilia sonchifolia Asteraceae Leaf Shylesh and Padikkala,

1999

11. Eucommia ulmoides Eucommiaceae Leaf Hesieh and Yen, 2000

12. Foeniculum Vulgare Apiaceae Fruit oil Ruberto et al., 2000

13. Garcinia kola Clusiaceae Fruit Adegoke et al., 1998

14. Glycyrrhiza glabra Fabaceae Root Sam et al., 2001

15. Hibiscus esculentus Malvaceae Seeds Hu et al., 2014

16. Lavandula angustifolia Lamiaceae Arial parts Hohmann et al., 1999

17. Lycium barbarum Solanaceae Fruit Ren et al., 1995

18. Manifera indica Anacardiaceae Root, fruit,

Leaf

Martinez et al., 2000

23

19. Melissa officinalis Lamiaceae Arial parts Hohmann et al., 1999

20. Morinda citrifolia Rubiaceae Fruit Kumar et al., 2014

21. Murraya koenigii Rutaceae Leaf Patel 1979

22. Myrica gale Myricaceae Fruit Mathiesen et al., 1995

23. Ocimum sanctum Lamiaceae Leaf Devi et al., 1999

24. Parkinsonia aculeate Fabaceae Leaves Sharma and Vig, 2013

25. Picrorhiza kurroa Plantaginaceae Leaves Kant et al., 2013

26. Piper nigrum Piperaceae Fruit Manosroi et al., 1999

27. Plantago asiatica Plantaginaceae Seed Toda et al., 1995

28. Prunus domestica Rosaceae Fruit Donovan et al., 1998

29. Psoralea Corylifolia Fabaceae Seed Haraguchi et al., 2000

30. Rhazya stricta Apocyanaceae Leaf Ali et al., 2001

31. Salvia officinalis Lamiaceae Arial parts Hohmann et al., 1999

32. Salvia triloba Lamiaceae Leaf Protogeras et al., 1998

33. Solanum melongena Solanaceae Fruit Sudheesh et al., 1999

34. Solanum nigrum Solanaceae Leaf Sultana et al., 1995

35. Sonchus Oleraceus Asteraceae Whole plant Jain and Singh, 2014

36. Tinospora cordifolia Menispermaceae Root Prince and Menon,

1999

37. Thymus zygis Lamiaceae Arial parts Soares et al., 1997

2.3 Plants as inhibitors of Urease enzyme

Enzyme inhibition is now an important part of the modern drug discovery research.

Urease (urea amidohydrolase) is an enzyme that catalyzes the hydrolysis of urea to ammonia

and carbamate, which is the final step of nitrogen metabolism in living organisms (Okwu,

1999). The reaction catalyzed by Urease is as follows:

(NH2)2CO + H2O → CO2 + 2NH3.

Carbamate rapidly and spontaneously decomposes, yielding a second molecule of

ammonia. These reactions may cause significant increase in pH and are responsible for

negative effects of urease activity in human health and agriculture (Spiro, 1977; Rama et al.,

24

2010). A variety of ureases are found in bacteria, fungi, higher plants, and in soil (Mobley

and Hausinger, 1989). In the mammalian body, no urease activity has been detected, but urea-

splitting bacteria colonizing the human body have urease activity (Odake et al., 1992).

Medically, bacterial ureases are important virulence factors implicated in the pathogenesis of

many clinical conditions, such as pyelonephritis, hepatic coma and the formation of infection-

induced urinary stones (Krajewska and Zaborska, 2007). Due to diverse functions of this

enzyme, its inhibition by potent and specific compounds could provide an invaluable addition

for treatment of infections, and secondary complexes such as gastritis and gastric ulcer

caused by Helicobacter pylori (Menezes et al., 2012; Ramsay et al., 2012). The success of

commercially available drugs in the treatment of diseases caused by bacterial urease is

overshadowed by the various side effects associated with these drugs. There is also the

resistance problem coming up by inappropriate and extensive use of such drugs, then, it is

interesting to control the activity of urease through the use of its inhibitors in order to

counteract these negative effects in medicine, environmental and agronomic. Many urease

inhibitors have been described in the past decades, such as phosphorodiamidates (Pedrazzini

et al., 1987), polyhalogenated benzo- and naphthoquinones (Ashiralieva and Kleiner, 2003)

and imidazoles (Tanaka et al., 2003) and α-hydroxyketones (Tanaka et al., 2004). But until

now, only acetohydroxamic acid has been clinically used for the treatment of infections

caused by bacterial urease (Juszkiewicz et al., 2004). In the urine, it acts as an antagonist of

the bacterial enzyme urease. Unfortunately, it exhibits severe side effects and also it does not

have any direct antimicrobial action. Moreover, its relatively moderate inhibitory activity

requires rather large doses (about 1000 mg/day for adults) (Kosikowska and Berlicki, 2011).

Therefore, compounds with high inhibitory activity and appropriate hydrolytic stability

urgently need to be discovered for the possible development of a therapy for urease mediated

bacterial infections.

It is well known that structural diversity and complexity within natural products are

unique and the functional complexity found in natural products will never be invented de

novo in a chemistry laboratory (Von et al., 2006). Over 75% of new chemical entities

submitted (1981-2004) were based on natural product lead structures, indicating that the

reliance on natural products is so far the most-successful route for new drug discovery

(Newman et al., 2003; Koehn and Carter, 2005). In the past decades, exploration of urease

inhibitors from natural products has attracted much attention (Brigitte et al., 2013, Firdous et

al., 2012; Ramsay et al., 2012).

25

The previous literature revealed the isolation of urease inhibitors from some plants

and herbs (Ayaz et al., 2006; Shafiq et al., 2011). Typically, Allium sativum extract is a

natural inhibitor of urease (Juszkiewicz et al., 2004). Catechins in green tea extract were

reported to inhibit H. pylori urease strongly (Matsubara et al., 2003). In addition, a H. pylori

urease inhibitor has been isolated from Rubus coreanus, and characterized as a proteinaceous

substance (Yang et al., 2004). Hypericum oblongifolium has been assayed for anti-urease

activity (Irfan et al., 2010). Natural urease inhibitors from Euphorbia decipiens (Ahmad et

al., 2003) and sulfated polysaccharide found mainly in various species of brown seaweed

(fucoidan compounds) had been reported previously (Limuro et al., 2003). Urease inhibition

activity of some Pakistani traditional medicinal plants have also been reported recently (Amin

et al., 2013).

The methanolic extract of Melilotus indicus and its subfractions in chloroform, ethyl

acetate, n-butanol and water showed remarkable enzyme inhibitory activities against urease

enzyme (Ahmed et al., 2014). Antiurease activity of eleven methanol extract from different

medicinal plant was investigated against stomach infection associated with pathogenic strains

of H. pylori. Extracts of Taraxacum officinale, Achillea millefolium, Aristolochia bracteata,

Eucalyptus globulus, Adhatoda zeylanica, Cuscuta reflexa and Mentha longifolia are reported

stronger inhibitors of H.pylori (Ghous et al., 2010)

Potent Urease inhibitors were isolated from Hypericum oblongifolium (Arfan and Ali,

2010). Urease inhibitor was also isolated from Cucumis melo seeds which showed inhibition

activity in vitro (Makkar et al., 1980). Tight-binding inhibitors of urease were isolated from

jack bean (Stephen et al., 1995). Methanol extracts of edible plants and seaweeds were tested

for their inhibitory activity against Jack bean urease (Shabana et al., 2010).

Qatouseh et al., 2013 tested methanol extracts of Algerian plants, Mentha rotundi-

folia, Eucalyptus globulus, Malva sylvestris, Inula viscosa, Achille aodorata and Utrica

dioica for their urease inhibition potential. All the tested extracts showed inhibitory effect at

concentration of 250 mg/ml. However, the range of the urease inhibitory concentrations

varied significantly among the extracts with highest activity and widest range found for E.

globulus. In another study significant anti-urease activity i.e. 72 % was observed in the ethyl

acetate fraction of Glycyrrhiza glabra with respect to standard inhibitor Thiourea (Lateef et

al., 2012).

Perveen et al., 2011 investigated two new C-glycosylflavonoids celtisides from Celtis

africana, along with five known C-glycosylflavonoids vitexin, orientin, isoswertiajaponin,

26

isoswertisin, and 200-Orhamnosyl vitexin for biological activities and they showed

significant antioxidant and urease inhibitory activities.

Twenty one randomly selected herbal methanolic extracts were evaluated for their

effect on inhibition of Jack-bean urease (Biglar et al., 2012). Among them, five extracts

whose inhibitory activity was found to be the most potent were: Camelia sinensis, Citrus

aurantifolia, Nasturtium officinale, Punica granatum and Nasturtium officinale.

Laghari et al., 2010 isolated a new flavanenol from ethyl acetate fraction of roots of

Alhagi maurorum. Experiments were carried out to evaluate its urease-inhibition activity.

From the observations it has been noticed that flavanenol possesses remarkable urease-

inhibitory effect.

Ghous et al., 2010 investigated antiurease activity of eleven ethanol and five methanol

extracts of medicinal plants collected from the State of Kashmir. Ethanol of Taraxacum

officinale, Achillea millefolium, Aristolachia bracteata, Eucalyptus globules, Adhatoda

zeylanica, Cuscuta reflexa and Mentha longifolia showed stronger action against urease

activity. Among methanol extracts, A. millefolium and A. bracteata demonstrated stronger

antiurease activity. Results of this study illustrate that most of the studied extracts exhibited

reasonable antiurease activity, however, ethanolic extracts of T. officinale, M. longifolia and

methanolic extracts of A. millefolium and A. bracteata showed significant inhibition potential.

Kaleem et al., 2012 investigated that Zizyphus oxyphylla, n-hexane, ethyl acetate and

butanol fractions showed good to excellent antiurease activity while chloroform fraction

showed non-significant activity. Amongst isolated compounds of the tested plant

Oxyphylline D was the most active of the three isolated cyclopeptide alkaloids followed by

Nummularin R and Nummularin C.

Recently, Xiao et al., 2012 revealed quercetin and its analogues as H. pylori urease

inhibitors. They selected a library of twenty flavonoids for urease inhibition screening which

showed excellent potency. Out of these flavonoids, quercetin 41 was the most active against

H. pylori urease, which is slightly more potent than the positive control, acetohydroxamic

acid. Zhu and co-workers (Xiao et al., 2007) demonstrated the synthesis of a series of twenty

polyphenols and evaluated their effect on H. pylori urease. Among the tested compounds, 4-

(p-hydroxyphenethyl) pyrogallol and 7, 8, 4-trihydroxyisoflavone showed potent inhibitory

activities, and inhibited H. pylori urease. Still the full potential of urease inhibitors from

natural sources has not yet been fully explored. So, seeking novel and efficacious urease

inhibitors with good bioavailability and low toxicity is very substantial.

27

2.4 Plants as inhibitors of Collagenase enzyme

The most abundant protein found in mammalian tissues is type I Collagen. It is the

main structural protein of skin, bone and tendon (Nimni and Harkness, 1988). Collagen

provides structural integrity acting as a scaffold, a matrix, upon which other cells can

proliferate. Collagen is also known to have a role in the control of cell shape and

differentiation, migration, and the synthesis of a number of proteins. Collagen as a protein

has a distinguishing feature, each molecule has a coiled coil structure with three polypeptide

chains, wound together to form a triple helix (Ramachandran and Kartha, 1955; Bella et al.,

1994).

Matrixmetalloproteinases (MMPs) are part of a group of transmembrane zinc

containing endopeptidases which include collagenases and gelatinases. Collagenase cleaves

the X-gly bond of collagen and also synthetic peptides that contain the sequence -Pro-X-Gly-

Pro where X is almost any amino acid provided that the amino terminus is blocked (Van Wart

and Steinbrink, 1981). Collagenase from the bacteria Clostridium histolyticum (ChC) also

degrades extracellular matrix. This bacterial collagenase hydrolyses triple helical collagen in

both physiological conditions and in vitro conditions using synthetic peptides as substrates

(Kim et al., 2004). Native collagen is susceptible to attack only by collagenase at

physiological pH, temperature and ionic strength (Grant and Alburn, 1959; Brodsky and

Persikov, 2005). Collagen molecule is susceptible to attack by other proteases only after

initiation of cleavage of the triple helix by collagenase (Harrington, 1996).

MMPs play an important role not only in physiologic degradation of extracellular

matrix (ECM) mediating tissue morphogenesis, tissue repair, and angiogenesis but also in

pathologic conditions characterized by excessive degradation of ECM such as chronic

inflammation, wrinkle formation, arthritis, osteoporosis, periodontal disease, tumor invasion

and metastasis. Recently, it was reported that ultraviolet B-induced enhancement of

gelatinase activity in the skin contributes to wrinkle formation through the destruction of

basement membrane structure and dermal collagen, and thus, topical application of inhibitors

of MMPs may be an effective way to overcome this problem (Inomata et al., 2003). MMP

inhibitors investigated are mainly synthetic peptides, chemically modified tetracyclines,

bisphosphonates or compounds isolated from natural sources. However, most of these drugs

are reported to exert side effects such as musculoskeletal pain in tendons and joints.

Secondary metabolites and whole extracts from plants have been widely investigated

and found to have anti-collagenase activities. Plants contain a wide variety of compounds

including polyphenols such as flavonoids, tocopherols, phenolic acids and tannins which

28

have been found to provide collagenase inhibitory compounds or a platform on which to

synthesize active molecules. Isolated Camellia sinensis (green tea) polyphenols such as

catechin and epigallocatechin gallate (EGCG) have been found to be inhibitors of collagenase

and elastase (Kim et al., 2004). Aloe gel constituents (aloins) have also been isolated from A.

barbadensis and have been found to show inhibition of collagenase in vitro (Barrantes and

Guinea, 2003). Polyphenols isolated from Diospyros kaki (persimmon) leaf showed anti-

collagenolytic and anti-elastase activity (An et al., 2005). This activity was thought to be due

to the flavonoids present in the polyphenol extract. Twenty three plant extracts were assessed

for anti-elastase and anti-collagenase activities. Anti-elastase activities were observed for

nine of the extracts with inhibitory activity in the following order: Camellia sinensis (white

tea) (89%), Galium aparine (cleavers) (58%), Arctium lappa (burdock) (51%), Fucus

vesiculosus (bladderwrack) (50%), Illicium verum (anise) and Angelica archangelica

(angelica) (32%). Anti-collagenase activities were exhibited by sixteen plants of which the

highest activity was seen in C. sinensis (white tea) (87%), C. sinensis (green tea) (47%), Rosa

centifolia (rose) (41%), and Lavandula angustifolia (lavender) (31%). Nine plant extracts had

activities against both elastase (E) and collagenase (C) and were ranked in the order of C.

sinensis (white tea) (E:89%, C:87%) > F. vesiculosus (E:50%, C:25%) > G. aparine (E:58%,

C:7%) > R. centifolia (E:22%, C:41%) > C. sinensis (green tea) (E:10%: C:47%) > A.

archangelica (E:32%, C:17%) > I. verum (E:32%, C:6%) > Punica granatum (E:15%,

C:11%) (Thring et al., 2009).

Another study was conducted to investigate collagenase inhibitory activity of n-

butanol fraction of flaxseed and it showed the inhibition of collagenase (IC50 value: 78.80

μg/ml) (Kasote et al., 2013). A quinazolinedione alkaloid isolated from the fruits of Evodia

officinalis have been reported to have collagenase inhibitory activity (Jin et al., 2008).

Aucubin isolated from Eucommia ulmoides has been found to inhibit MMP-1 (Enikuomehin

et al., 1998). Recently, Cucumis sativus fruit has been found to possess in vitro inhibition of

hyaluronidase and elastase and collagenase, which suggested the potential of this plant as

anti-wrinkle (Nema et al., 2011).

Myrrh (guggulu) oleoresin from the Commiphora mukul tree is an important

component of antiarthritic drugs in Ayurvedic medicine. Triphala guggulu (TG) is a guggulu-

based formulation used for the treatment of arthritis. Sumantran et al., 2007 assessed the

chondroprotective potential of TG by examining its effects on the activities of pure

collagenase type 2 enzymes and observed TG as a potent inhibitor of enzyme. Plant-derived

inhibitors of hyaluronidase and collagenase type 2 include curcumin, quercetin and

29

aristolochic acid, which are potent inhibitors of snake venom hyaluronidase (Girish and

Kemparaju 2005). Polyphenols of blackberry fruits also inhibit hyaluronidase (Marquina et

al., 2002). Demeule et al., 2000 reported that green tea polyphenols show gelatinase

inhibitory activity. Ethanol extract of Eucalyptus globulus inhibits collagenase activity (Daiki

et al., 1999).

Chompoo et al., 2012 tested the aqueous extract of Alpinia zerumbet (rhizome) for

inhibition of collagenase enzyme and found that it have potent inhibitory effects.

Furthermore, 5, 6-dehydrokawain (DK), dihydro-5, 6-dehydrokawain (DDK) and 8(17), 12-

labdadiene-15, 16-dial (labdadiene), isolated from rhizome, were tested for enzyme inhibition

and observed that DK had stronger inhibitory activities against collagenase, (IC50= 24.93 ±

0.97) than DDK and labdadiene.

Kim et al., 2006 reported the inhibitory effects of phlorotannins in brown algae,

Ecklonia cava on MMP activities in cultured human cell lines. Moreover the extract did not

exert any cytotoxic effect even at 100 µg/ml, anticipating its potential use as a safe MMP

inhibitor.

Madhan et al., 2007 explored the Inhibitory effect of green tea polyphenols i.e.

catechin and epigallocatechin gallate on the action of collagenase against collagen. Catechin

and EGCG treated collagen exhibited 56% and 95% resistance, respectively, against

collagenolytic hydrolysis by collagenase. Whereas direct interaction of catechin and EGCG

with collagenase exhibited 70% and 88% inhibition of enzyme respectively, and the

inhibition was found to be concentration dependent.

Sin et al., 2005 examined the inhibitory activities of various flavonoids, including the

flavanones, flavones/ isoflavones and flavonols, on collagenase from C. histolyticum to

establish their therapeutic potential against skin inflammation and photoaging. In general, the

flavonols were stronger inhibitors than the flavones/isoflavones. Quercetin was the most

active flavonoid among those tested, and it showed an IC50 of 286 pM.

Tate et al., 2004 demonstrated that water extracts of Rubus occidentalis, Rubus

fruticosus and Muscadinia rotundifolia inhibit the activities of metalloproteinases 2 and 9.

Chalcones and their analogs were extracted from the twigs of Dorstenia barteri and

investigated for their capacity to inhibit matrix metalloproteinase (MMP)-2 secretion from

brain tumor derived U87 glioblastoma cells (Ngameni et al., 2007). Among all tested

compounds, potent inhibitory activities were recorded for chalcones isobavachalcone,

paratocarpin C, stipulin and dorsmannin A and to a lesser extent for 4-hydroxylonchocarpin

and kanzonol C.

30

A study was undertaken to screen extracts from eight plants, native to Taiwan (Alnus

formosana, Diospyros discolor, Eriobotrya deflex, Machilus japonica, Pyrrosia polydactylis,

Pyrus taiwanensis, Vitis adstricta, Vitis thunbergii) for their potential to inhibit matrix

metalloproteinase-9 (MMP-9) activity. All of these extracts (except V. adstricta) were shown

to inhibit MMP-9 activity of WS-1 cell after ultraviolet B irradiation (Lee et al., 2009).

Many in vitro scientific studies have shown that plants possess the ability to inhibit

collagenase activity (Wang et al., 2006; Sumantran et al., 2007; Hsu and Chiang, 2009).

Some more studies done on plants to check their potential as MMP inhibitors are listed in

Table 2.3.

Table 2.3 List of some plants exhibiting inhibition of Matrixmetalloproteinases (MMPs)

Sr. No. Name of plants and

family

Part used Types of MMPs inhibited References

1 Aloe barbadensis Gel Inhibit stimulated granulocyte

MMPs

Barrantes and

Guinea 2003

2 Berberis aristata Berries Inhibited expression of

MMP-9

Kim et al., 2008

3 Calendula officinalis Flower Control the activity/secretion

of MMP-2 and

MMP-9

Yris et al., 2010

4 Camellia japonica Oil Induce type-1 procollagen

synthesis and inhibit

MMP-1 activity

Jung et al., 2007

5 Camellia sinensis Leaves Suppress overexpression of

MMP-2 and MMP-9

Li et al., 2009

6 Curculigo orchioides Rhizome Inhibited MMP-1 expression Lee et al., 2009b

7 Curcuma longa Rhizome Inhibited MMP-2 expression Sumiyoshi and

Kimura 2009

8 Curcuma xanthorrhiza Rhizome Inhibited MMP-1 expression Oh et al., 2009

9 Emblica officinalis Fruit Inhibited type-I collagen

collagenase

Takashi et al.,

2008

10 Fraxinus chinensis Seeds Decreased the MMP-1 mRNA

expression

Lee et al., 2007

11 Labisia pumila Root Inhibition of MMP-1 and

MMP-9 expression

Choi et al., 2010

12 Machilus thunbergii

Stem bark Strong inhibition of MMP-1 Moon and Jung

2006

13 Melothria heterophylla Root Inhibited MMP-1 activity Cho et al., 2006

14 Panax ginseng Root Prevent MMP-9 gene

induction and decrease

expression of MMP-1

Cho et al., 2009

15 Tapirira guianensis Leaves Gelatinases inhibition Longatti et al.,

2011

16 Terminalia chebula

Fruit MMP-2 enzyme

inhibition

Kim et al., 2010

17 Viola hondoensis Whole plant Inhibition of MMP-1

expression, at the both

mRNA and protein levels

Moon et al.,

2005

31

2.5 Phytochemical analysis

Medicinal plants are the richest bio-resource of drugs of traditional systems of

medicine, modern medicines, food supplements, folk medicines, nutraceuticals and

pharmaceutical intermediates for synthetic drugs (Tiwari et al., 2011). Plants have the ability

to synthesize a wide variety of chemical compounds that are used to perform important

biological functions, and to defend against attack from predators such as insects, fungi and

herbivorous mammals. Many of these phytochemicals have beneficial effects on long-term

health when consumed by humans, and can be used to effectively treat human diseases. It is

estimated that more than 25,000 terpenoids, 12,000 alkaloids and 8,000 phenolics have been

identified in plants, but a very large number still remain unknown and need to be identified

and quantified before their health benefits can be evaluated (Hollman and Arts, 2000).

Phytochemical constituents such as alkaloids, flavonoids, tannins, phenols, saponins, and

several other aromatic compounds are secondary metabolites of plants that serve a defense

mechanism against predation by many microorganisms, insects and other herbivores (Bonjar

et al., 2004).

Phenols: Phenols are aromatic chemical compounds with weakly acidic properties

and characterized by a hydroxyl group attached to aromatic ring. Presence of phenols is

considered to be potentially toxic to the growth and development of pathogens (Okwu and

Okwu, 2004).

Flavonoids: Flavonoids are 15-carbon compounds generally distributed throughout

the plant kingdom. They are known to be synthesized by plants in response to microbial

infection and have been found to be effective against a wide array of microorganisms

(Harborne, 1973).

Alkaloids: Alkaloids comprise the largest class of secondary plant substances which

contain one or more nitrogen atoms (Harborne, 1973). They are often toxic to humans and

many have dramatic physiological activities, hence they are widely used in medicine for the

development of drugs (Okwu, 2005).

Saponins: Saponins are glycosides of both triterpines and steroids that are

characterized by their bitter taste, foaming property, haemolytic effect on red blood cells and

cholesterol binding properties (Okwu, 2005).

Phytochemical studies have attracted the attention of plant scientists due to the

development of new and sophisticated techniques. These techniques played a significant role

in giving the solution to systematic problems on the one hand and in the search for additional

resources of raw materials for pharmaceutical industry on the other hand (Iqbal, 2012).

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One of the most popular methods of studying phytochemical composition is Gas

Chromatography and Mass Spectroscopy (GC-MS), which is used for the quantitative

estimation of phytochemicals and allows the identification of the specific natural compounds

found in a plant extracts by comparing their relative retention times and indices and their

mass spectra. It also finds application in other areas such as the medical, environmental,

chemical engineering and law enforcement fields etc.

It is a prevalent technique for the analysis of phytocompounds because it has very

good separation ability, which can produce a chemical fingerprint of high quality.

Furthermore, with the coupled mass spectroscopy and the corresponding mass spectra

database, the qualitative and relatively quantitative composition information of the herb

investigated could be provided by GC-MS, which will be extremely useful for elucidating the

relationship between chemical constituents in herbal medicine and its pharmacology in

further research (Jothy et al., 2013). Thus individual components or compounds in the soluble

extracts or oil samples can be studied or identified by utilizing the GC-MS (Akande, 2012).

This technique is very useful for the detection of biological volatile organic compounds and

corresponding volatile profile characteristics (Zhang et al., 2009).

2.6 Molecular docking

The need for a rapid search for molecules that may bind to targets of biological

interest is of crucial importance in the drug discovery process. One way of achieving this is

the in silico screening of compound collections to identify a subset of compounds that

contains relatively many hits against the target, compared to a random selection from the

collection. If a three-dimensional (3D) structure of the target is available, then „Docking

program‟ can be used to place computer-generated representations of a small molecule into a

target structure in a variety of positions, conformations and orientations (Lengauer and Rarey,

1996). Three basic tasks are accomplished by docking program: (1) characterization of the

binding site; (2) positioning of the ligand into the binding site (orienting); and (3) evaluating

the strength of interaction for a specific ligand-receptor complex (“scoring”). Thus, docking

program generates a pose after docking and energetically most favorable pose is identified by

its scoring. Scoring is done for all molecules in the collection, which are then rank-ordered by

their scores. This rank-ordered list is then used to select those compounds that are predicted

to be most active. Assuming that both the poses and the associated affinity scores have been

predicted with reasonable accuracy, this selection will contain a relatively large proportion of

active molecules, i.e. it will be „enriched‟ with actives compared to a random selection.

Therefore docking is useful for predicting the preferred orientation, strength and type of

33

signal produced when two molecules bound to each other to form a stable complex, thus

playing an important role in the rational design of drugs (Kitchen et al., 2004). Success has

been reported for numerous high throughput docking studies using X-ray receptor structure.

Filikov et al., 2000 identified lead compounds that disrupt HIV-1 TAR- TAT binding,

an interaction necessary for viral replication while Olson et al., 1998 identified HIV protease

inhibitors. Powers et al., 2002 identified a noncovalent inhibitor of AmpC β-lactamase.

Perola et al., 2000 screened a chemical database and identified lead compounds that act as

inhibitor of farnesyl transferase. Tondi et al., 1999 discovered novel competitive inhibitors of

thymidylate synthase. Hopkins et al., 2000 discovered inhibitors of kinesin activity from

structure-based computer screening. In silico screening of Angiotensin-I Converting Enzyme

(ACE) inhibitors from Hibiscus sabdariffa (Roselle) chemical compounds was done by

Yuliana et al., 2013. The results showed that Hibiscetin, Hibiscetin 3-glucoside, and

delphinidin 3-sambubioside showed potential as inhibitors of Angiotensin-I Converting

Enzyme. Molecular Docking and ADMET predictions showed that curcumin can be a potent

inhibitor of Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (pfSAHH)

enzyme with ability to modulate the target in comparatively smaller dose (Singh et al., 2013).

Therefore, curcumin is likely to become a good lead molecule for the development of

effective drug against malaria. Applications and benefits of computational techniques have

been reviewed and demonstrated in growing number of publications and supported by

examples of drugs derived from the in silico approach (Alvarez and Shoichet, 2005;

Congreve et al., 2005; Keri et al., 2006; Kubinyi, 2006).

Structure-based drug design computational techniques have emerged as a very

effective and low-cost strategy to improve the rate of success at any stage of the drug

discovery pipeline mainly using the protein-ligand docking (Williams et al., 2005). The

docking procedure responsible for fitting ligand and receptor together in 3D-space is

attracting much attention, and there are a growing number of software packages (AutoDock,

GLIDE, SLIDE, GOLD etc.) available to enable this important process in drug design

(Shaikh et al., 2007). Many docking programs exist, but no single program has yet emerged

that outperforms all others in all cases. Generally programs do an adequate job searching

conformational space and generating correct ligand poses, but the scoring functions need

improvement. Some of the problem areas are target flexibility and low correlation between

calculated and observed binding affinities etc. But, because of the biological

and pharmaceutical significance of molecular docking, considerable efforts have been

directed towards improving the methods used to predict docking.

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Both computational and experimental techniques have important roles in drug

discovery and development and represent complementary approaches. In silico docking

entails:

1. Use of computing power to streamline drug discovery and development process

2. Leverage of chemical and biological information about ligands and/or targets to

identify and optimize new drugs.

3. Design of in silico filters to eliminate compounds with undesirable properties (poor

activity and/or poor Absorption, Distribution, Metabolism, Excretion and Toxicity,

ADMET) and select the most promising candidates.

The classical herbal bioprospection is identification of herbal medicinal plants based

on its ethnopharmacological importance, as testified in ancient literature or otherwise in

clinical literature of various countries. This process is time consuming, tedious, generally

observation or experience based, and might lack scientifically evident and validated proofs

(Mary et al., 2012). Evolution of new techniques of deploying dynamic search protocols,

priority indexing, systemic categorization and cross-verification could be referred to as an in

silico bioprospection tool (Thakur et al., 2013). The parallel research efforts globally on both

plants and their costituents provides enormous web based data that requires to be filtered

systematically towards a logical conclusion for further in vitro and in vivo validation. This

study has provided an insight into a systematic analysis of phytocompounds to obtain a

logical output for ascertaining a desired biological activity.