Chapter 2. Review of Literature 2.1 Medicinal...

Review of Literature

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Chapter 2. Review of Literature

2.1 Medicinal plants

Traditionally, all most of the drugs are derivatives of herbes. The wealthy

knowledge in ancient medicine from plants has directed to the intense interest by

drug manufacturers to use this resource for research and development programs for

discovering novel drugs (Krishnaraju et al., 2005). In addition, the active principles

of many medicinal plants constitute an important part of human diet.

Due to the increase in deforestation, land degradation, human and livestock

population, many species or population of plant species are now threatened with

extinction. Therefore, a proper documentation of useful plants with their current

status and ethnopharmacological knowledge is urgently needed. Effort should also

be made to employ appropriate conservation measures for preservation and

sustainable uses of these species (Vijayan et al., 2004; Jan et al., 2009)

Many pharmaceutical products like laxatives, blood thinners, antibiotics and

antimalarial medications contain ingredients from plants. In addition active

ingredients like Taxol, vincristine, and morphine have been isolated from foxglove,

periwinkle, and opium poppy, respectively (Hassan et al., 2012).

2.2 Asteraceae family

The Asteraceae family is one of the largest families with 1,100 presently accepted

genera and 25,000 species (Heywood et al., 1977). The family has 12 subfamilies

and 43 tribes which are distributed worldwide (Panero and Funk, 2008).

Asteraceae presumes almost every life-form: herbs, succulents, epiphytes or

shrubs. The majority members are evergreen shrubs or subshrubs or perennial

rhizomatous herbs; annual and biennial herbs to shrubs are also frequent.

Asteraceae is a "natural" family with well established limits and a basic uniformity

of inflorescence imposed on all members by the common possession of characters

such as the aggregation of the flowers into multiple capitula (Zareh et al., 2005).

There was some controversy concerning the family age, biogeographical

considerations and origin. All these controversies were answered by Barreda et al.,


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(2010) who found a fossil flower of Asteraceae family plant that was nearly 50

million years old in north western Patagonia, southern South America. This

discovery provided important information about the age and diversity of the

subtropical flora.

2.3 Ancient medicinal uses of Asteraceae plants

Plants as sources of novel compounds have sustained to play a major role in the

protection of health since earliest times (Nair et al., 2005). There is no doubt that

several Indians and others across the globe have experienced the benefits of

Ayurvedic treatments. The diagnostic treatment procedures of Ayurveda are

unique and are still valid today as being its foundational principles of

panchamahabhutha (five basic elements of nature), tridosha (three humours) and

prakrithi (individual constitution) (Venkatasubramanian, 2007).

A bunch of information on folk medicine has been traditionally passed down from

generation to generation. In ancient days Asteraceae family plants were commonly

used in the treatment of common diseases. It is pointless to say that this folk

information requires proper verification through experiments and clinical

investigation in order to determine and establish their efficacy in therapeutics.

Several researchers have systematically investigated Asteraceae plants for their

utility in therapeutics. The plants have proven for their healing properties.


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Table 2.1. List of important plants of Asteraceae family with their therapeutic uses

Botanical Traditional Pharmacological References Name Uses Activity

Ageratum conyzoides Fresh cuts and sores Antioxidant Patel, 2012;

Piles, Wound healing Wound healing, Dash and Murthy, 2011;

Anus prolapse Antidiabetic, Nyunai et al., 2010;

Antiseptic Upadhye et al., 1986.

Anaphalis neelgerriana Antiseptic, Antioxidant Vijaylakshmi et al., 2009.

Ringworms

Arctium lappa Throat complaints Cytotoxic Magdalena et al., 2012.

Artemisia absinthium Jaundice Anthelmintic, Tariq et al., 2009;

Cytotoxic Magdalena et al., 2012;

Jan et al., 2009.

Artemisia herba-alba Analgesic, Hypertension, Ziyyat et al., 1997;

Stomach disorders, Diabetes, Tahraoui et al., 2007;

Wormicide Antibacterial,

Analgesic, Laid et al., 2008.

Antispasmodic

Artemisia mongolica Worm infection Insecticidal Liu et al., 2010.

Blumea lacera Boils, Wounds, Antimalarial Sahu, 1984;

Blisters Islam et al., 2012.


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Blumea membranacea Body pain Antioxidant,

Analgesic, Roy et al., 2012.

Cytotoxic

Calendula officinalis Wound healing Cytotoxic Magdalena et al.,2012.

Gnaphalium luteo Breast lactation Sahu, 1984.

Helianthus annuus Urinary diseases Antimalarial Islam et al., 2012;

Antisporulant Deepak et al., 2007.

Launaea nudicaulis Headache Antimicrobial Rashid et al., 2000.

Parthenium hysterophorus Antimalarial Islam et al., 2012.

Siegesbeckia orientalis Toothache Antimalarial Islam et al., 2012.

Sphaeranthus indicus Diarrhoea, Antihyperlipidemic Pande and Dubey, 2009;

Urinary diseases Wound healing, Jha et al., 2009;

Anxiolytic Ambavade et al., 2006.

Spilanthus acmella Blood purifier, Insecticidal Rahmatullah et al., 2010;

Colic, Headache, Anticancer

Gastrointestinal antibacterial Hossan et al., 2010.

disorders

Spilanthus calva Dry cough Insecticidal Dolui and Debnath, 2010;

Throat complaints Antimicrobial Vyas et al., 2009;

Cold Antimutagenic Sukumaran et al., 1995.


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Spilanthes paniculata Toothache Jaundice Srivastava et al., 2010;

Worm infection Skin disease Zheng and Xing, 2009;

Constipation Kala, 2005.

Tagetes minuta Insecticidal, Prosperity, Larvicidal Macedo et al., 1997.

Stomachic, Muscular pain,

Boil, Scorpion bite

Tridax procumbens Minor injuries Blood clotting, Boil, Upadhye et al.,1986;

Wound healing, Patel, 2012.

Xanthium strumarium Scrofula, herpes, Insecticidal Sahu, 1984;

Urinary diseases Blood clotting Patel, 2012.

2.4 Ethnobotanical and Phytochemical information on the ten selected species

2.4.1 Artimisia cina

Artemisia cina O. Berg, (wormseed) is a member of Asteraceae family, belongs to

the large genus Artemisia that comprises of above 300 species. Artemisia cina is a

shrubby aromatic plant, a xerophyte, growing in semi-desert areas where extremes

of temperature, both high and low, prevail. It is a resident of the steppe-areas east

of the Caspic Sea, in Afghanistan and in the Southern Ural region. This species

prefers a saline sandy soil. It slightly remembers camphor in characteristics. The

size of the plant ranges from small 6 to 8 inch mounds to upright stems and

branches getting up to 10 feet in height. Plants of this genus are remarkably

resistant to several Armillaria fungus, they are rarely troubled by browsing deer

and have numerous traditional medicinal uses.


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2.4.2 Artimisia vulgaris

Artemisia vulgaris. L. (Mugwort) is a native of temperate Europe, Asia and

northern Africa commonly regarded as an unwanted garden weed. It commonly

grows on nitrogenous soils, like weedy and uncultivated areas, such as waste

places, landscapes and roadsides (Barney et al., 2005). It is an herbaceous

perennial plant growing 1-2 m tall, with a woody root. The leaves are 5-20 cm

long, dark green, pinnate, with dense white hairs on the underside and have a

distinctive aroma. It has been known as an edible plant as flavoring agent to season

fat, meat, fish and also as a folk medicine resource (Lee et al., 1998).

Since it is used as an herbal remedy, deserved attention has been paid towards the

chemical compositions of its volatile oils (Blagojevica et al., 2006). It is known to

contain adenine, amyrin, artemisia ketone, borneol, cadinenol, coumarin, fernenol,

esculin, esculetin, inulin, muurolol, myrcene, nerol, molybdenum, quercetin,

scopoletin, β-sitosterol, spathulenol, stigmasterol, tauremisin, tetracosanol, thujone,

vulgarin, vulgarol, vulgarole and umbelliferone (Almeida et al., 2013). A number

of species of butterflies and moths feed on the leaves and flowers of A. vulgaris

and also there is belief that it can induce vivid dreams when it is placed below the

sleeping pillow.

2.4.3 Eclipta alba

Eclipta alba (L.) Hassk., (Bhringaraja) is an edible perennial shrub, spreads on

ground or partly ascending with its stem, leaves are small and succulent, plants are

found widely in moist tropical and subtropical regions. It prefers clayey soil with

plenty of moisture (Nahid and Agarwal, 2004). It is of great economic value both

for its protein as well as existence of essential amino acids (Behera and Patnaik,

1977). The earliest record of E. alba was in the Chinese Tang Medica of 659 AD.

Plant is bitter, hot, sharp and dry in taste and is used in Ayurveda "Siddha" for the

treatment of Kapha and Vata imbalances. It is one of the ten auspicious flowers

(Dasapushpam) in Ayueveda and said to be the best herb in Ayurveda to treat liver

cirrhosis and infectious hepatitis.


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Eclipta alba is a prominent medicinal plant with a renowned reputation, is widely

used in the history of many medicinal systems of the world. It has been bestowed

with the natural gift of tonic and has been traditionally used for hundreds of years

in Chinese and Indian medicines to eliminate numerous diseases. Earlier reports

show that the whole plant contains 0.078% nicotine on dry basis (Pal and

Narshimhan, 1943). In addition, it contains steroids, sterols, flavonoids, triterpenes,

hydrocarbons alkaloid ecliptine as well as coumarin. It is also known to contain

alkaloid ecliptine, wedelolactone (Wagner et al., 1986) and its derivatives

demethylewedelolactone -7-glucoside and nor-wedelolactone (Bhargava et al.,

1972).

2.4.4 Glossocardia bosvallea

Glossocardia bosvallea (L.f.) an diffuse or ascending (up to 9,000 feet) weed. It is

commonly called as Khadakshepu, Pittapapda or Parpata in Sanskrit and

distributed over the greater parts of India (Rajopadhye and Upadhye, 2012). It is

used as major constituent of many household and ayurvedic medicinal preparations

including Parpatadi-kwath, Parpatadi-arishta, and Parpatadi-arka (Upadhye

et al., 1988; Rajopadhye and Upadhye, 2012). Bhale. (2013) reported that

G. bosvallea has vesicular arbuscular mycorrhizal fungi colonized in its root

system.

2.4.5 Mikania micrantha

Mikania micrantha is a perennial creeper, roots easily at the nodes where fertility,

organic matter, soil wetness and humidity are high. It spreads so quickly that it is

also called “mile-a-minute weed”. Major constituents isolated from plants of this

genus are terpenes and essential oil compounds, which have large applications in

pharmaceutical and cosmetic industries (Santos et al., 2004). It kills other plants by

cutting out the light and smothering them. It was introduced to India during the

Second World War which is native to Central and South America (James, 2003;

Shao et al., 2005).


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2.4.6 Spilanthes uliginosa

Spilanthes uliginosa is a medicinal herbaceous pantropical plant that grows

abundantly in roadside, marshes, pastures, meadows, ditches and along streams

which prefers wet, loam and clay soils (Chung et al., 2007). Plants are typically

straight and nodal roots are absent on higher parts, whereas nodal roots are seen on

lower portions of stems (Jansen, 1985).

2.4.7 Vernonia cinerea

Vernonia cinerea is an annual herb, reported to have many medicinal properties. It

is one of the ten herbs that comprise the group of a reputed Ayurvedic medicine

‘Dasapushpam’. In India, it is found all over the country, ascending up to 2400 m

in the Himalaya, Khasi hills and hills of peninsular India (Ganesh et al., 2011). It is

slender, erect, annual, hispid herb commonly found on the roadsides, garden lands

and open forests growing up to 75 cm in height. It has cylindrical and sparingly

branched stem with simple and alternate leaves. Inflorescence is laxdivaricate,

corymbose, terminal cymose heads with pink-purple colored flowers. The studies

on the biological and chemical constituents resulted in various chemical

constituents with significant cytotoxic activities (Chen et al., 2006).


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Table 2.2. Ethnobotanical and Phytochemical information of ten selected species

Botanical Traditional Pharmacological References

Name Uses Activity

Artemisia cina Antihelmintic, Antioxidant,

Vermifuges, Antimalarial, Vicidomini, 2011;

Digestive stimulant. Antileukemic, Aryanti et al., 2001;

Immune response, Yasser et al., 2010;

Antihelminthic, Asanova et al., 2003;

Antiparasitic, Ramadan and Doaa

Antiinflammatory 2012.

Artimisia vulgaris Digestive, Insecticide, Hwang et al.,1985;

Expectorant, Antifertility, Shaik et al., 2014;

Purgative, Hepatoprotective, Gilani et al., 2005;

Women’s Estrogenic Activity, Lee et al., 1998;

complaints Antiviral, Bamunuarachchi et al.,

2013;

Foot care, Larvicidal Ikram et al., 2013 ;

Diuretic, Anticonvulsant, Almeida et al., 2013;

Stimulant. Antioxidant, Erel et al., 2012;

Nematicide, Costa et al., 2003.

Eclipta alba Antiageing, Hepatoprotective, Nahid and Agarwal. 2004;

Skin disorders, Lal et al., 2010;

Headache, Anti-diabetic Ananthi et al., 2003;

Rejuvenates Hair, Anti-inflammatory Aggarwal et al., 2009;

Teeth, bones, memory Anti-venomous Samy et al., 2008;

and hearing, Antiaggression Banji et al., 2010;

Hepatitis, Antileptospiral Prabhu et al., 2010;

Wormicide. Antioxidant Chandan et al., 2012;

Explicit memory Banji et al., 2007;

Hair color and growth Antimicrobial Karthikumar et al., 2007;


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DNA damaging Abdel et al., 1998;

Anticancer Khanna et al., 2009.

Glossocardia bosvallea Wound healing, Antibacterial, Mangesh et al., 2011;

Emmenagogue, Antimycotic, Pathak and Dixit, 1984;

Antialcoholic, Patil and Patil, 2006; Typhoid, Birasdar and Ghorband, 2010; Throat infection, Jagtap et al., 2013;

Gastric, Chronic fever Kanthale and Biradar, 2012.

Mikania micrantha Fever, Rheumatism, Allelopathic Lalmuanpuii and

Influenza, Respiratory Sahoo, 2011;

diseases Anti stress, Ittiyavirah and Sajid, 2013;

Antibacterial, Hajra et al., 2010;

Anti-inflammatory Amador et al., 2010.

Spilanthes uliginosa Sore throat,

Mouth ulcer, Antinociceptive, Ong et al., 2011;

Stomach ache, Analgesic, Koubemba et al., 2011;

Diabetis Cytotoxicity Soladoye et al., 2012.

Vernonia cinerea Wormicide, Antimicrobial, Latha et al., 2009;

Diuresis, Anti-inflammatory, Latha et al., 1998;

Pain, Antioxidant, Kumar and Kuttan, 2009;

Abortion Antidiarrhoeal, Ganesh et al., 2011;

Antibacterial, Gupta et al., 2003;

Antimalarial, Chea et al., 2006;

Ameliorative, Gokilaveni et al., 2006;

Hepatoprotective, Leelaprakash et al., 2011;

Antitumor, Sangeetha and Kumar,

Antipyretic 2011; Anand et al., 2011.

Vicoa indica Indigestion, Antifertility, Rao et al., 1996;

Dysentery, Antiviral, Chowdhury et al., 1990;

Cough, Jaundice, Allelopathic, Dhole et al., 2013;

Anti-inflammatory, Krishnaveni et al., 1997;

Antibacterial, Kesavan et al., 2006;


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Hypoglycemic Aditya et al., 2012;

Wedelia chinensis Hair growth, Cough, Antioxidant, Manjamalai and Berlin

Cephalalgia, Antifungal, 2012;

Skin diseases Anti-inflammatory, Manjamalai et al., 2011;

Antibacterial, Darah et al., 2013;

Anticancer, Tsai et al., 2009;

Antistress, Nitin and Khosa, 2009;

Central nervous system

depressant activity, Suresh et al., 2010;

Hepatoprotective Garima et al., 2009;

Wedelia trilobata Respiratory infections, Anti-diabetic, Kadea et al., 2010;

Pain Hepatoprotective, Murugaian et al., 2008;

Wound healing, Balekar et al., 2013;

Antimicrobial, Toppo et al., 2013;

Anti-inflammatory, Govindappa et al., 2011;

Antihelminthic, Tambe et al., 2006;

2.4.8 Vicoa indica

Vicoa indica is an erect annual herb, grows up to 1-3 ft tall. It is found throughout

the drier parts of India, ascending to an altitude of about 1800 m in the Himalayas.

The stem is cylindrical and branched in the upper part. Leaves are alternately

arranged with rounded ears at the base. The major phytochemicals reported to

contain are germacranolide (Dilip et al., 1998), Vicolides A, B, C and D, the

sesquiterpene lactones (Alam et al., 1992), Vicogenin (Balakrishna et al., 1995),

oleanane triperpenoids (Vasanth et al., 1992), n-alkanes and their derivatives

(Balakrishna et al., 1993).


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2.4.9 Wedelia chinensis

Wedielia chinensis is popularly called Pitabhringi (Sanskrit), Gargneri (Kannada)

and Pila-Bhangra (Hindi) (Nomani et al., 2013; Madhavan and Balu, 1995). It is a

tender, spreading and hairy herb with ascending rooting at the lower nodes. Leaves

are 2-4.5 cm in length, spathulate-oblanceolate, acute, trinerved and attenuate

margins flat or slightly inrolled. It grows on edges of paddy fields, in grassy fields,

open waste places and moist lowland depressions, and it is also common in coastal

areas. It is also noted to be cultivated in some places. It bears bright yellow flowers

and a light camphor-like odour and is used to relieve fever and to reduce cough and

phlegm (Nomani et al., 2013). The plant leaves contain isoflavonoids,

bisdesmoside wedelolactone and dimethyl wedelolactone which are potent

antihepatotoxic (Khare, 2007; Manjamalai et al., 2012). Leaf juice is used for

tattooing with its deep permanent blue black color. Root on pounding produces

a black dye with salts of iron.

2.4.10 Wedelia trilobata

Wedelia trilobata is native to tropics of Central America. It is evergreen perennial

herb which has very wide ecological tolerance range, but grows best in sunny areas

with well-drained, moist soil at low elevations. It grows up to 45 cm in height,

rooting at nodes and with a pair of lateral lobes (Hossain and Hassan, 2005).

Ascending stems bear daisy like orange and yellow flower throughout the year,

which is approximately 1 inch across (Keerthiga et al., 2012). It is spread by

people as an ornamental or groundcover planting in gardens and spreading into

surrounding areas by dumping garden waste (Song et al., 2009). It spreads

vegetatively and is difficult to control or eradicate. The IUCN has listed this

plant in its 100 of the world's worst invasive alien species (Lowe, 2000).

This species continues to be available as an ornamental and is therefore likely to

spread further. It is reported that numerous potential bioactive molecules such as

wedelolactone, diterpene, eudesmanolide lactones, luteolin, β-pinene, germacrene

D and phytol have been isolated from various parts of the plant (Block et al., 1998,

Taddei and Romero, 1999, Li et al., 2012).


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2.5 Endophytes

2.5.1 Historical perspective

The term “endophyte”, was first introduced by De Bary in 1866 (Rodrigues, 1996),

with reference to any organism present within plant tissues (Stone et al., 2000).

The word ‘endo-’ means inside and ‘phyte’ is derived from the Greek word phyto

meaning plant. Carroll (1986) projected endophytes as “mutualists, those fungi that

colonize aerial parts of living plant tissues and do not cause symptoms of disease”.

Petrini (1991) defined an expansion of definition to include “all organisms

inhabiting plant organs that at some time in their life can colonize internal plant

tissues without causing apparent harm to the host”. Wilson (1995) proposed that

“endophytes are fungi or bacteria which, for all or part of their life cycle, invade

the tissues of living plants and cause asymptomatic infections entirely within plant

tissues”. Bacon and White (2000) give a widely accepted definition of endophytes

as “microbes that colonize living, internal tissues of plants without causing any

immediate, overt negative effects”.

2.5.2 Endophytes from plants

Large numbers of endophytic microbes are associated with plants and have been

isolated from representatives of all major land plants (Taylor et al., 1999;

Saikkonen et al., 2000 and Photita et al., 2004). Several studies have revealed the

diversity of these endophytes, with an estimation of at least a million species in

plants (Huang et al., 2008). Endophytic fungi have been isolated from every tissue

of the plant including petioles, root, seed, bark, flower, leaves and twigs (Stone et

al., 2000; Rolston et al., 1986; Suryanarayanan and Vijaykrishna, 2001). Several

researchers have studied about endophyte occurrence, biology, evolution,

taxonomy and biotechnological applications (Kumar and Hyde, 2004; Arnold et

al., 2003; Saikkonen et al., 2004; Jeewon et al., 2004 and Tomita, 2003).

Some fungal endophyte species afford their host with prominent tolerance to

intense environmental conditions. For example, the grass species Dichanthelium

lanuginosum is able to survive high soil temperatures, when it is in association


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with fungal endophyte Curvularia protuberata (Redman et al., 2002), whereas,

Suryanarayanan et al. (1998) isolated endophytes from leaves of R. apiculata and

R. mucronata and observed that the diversity of endophytes is more in the wet

season. Fungal endophytes indirectly affect plant community dynamics by

influencing the host plant health. Recent studies indicate that endophytes also

protect the host against plant pathogens, nematodes and from insect damage

(Vega et al., 2008). Conversely, fungal endophytes are reported to decrease

photosynthetic efficiency and increase water loss from leaves under drought

conditions (Pinto et al., 2000; Arnold and Englebrecht, 2007).

Camacho et al. (1997) reported that the plant DNA can be frequently got

contaminated with DNA of associated symbiotic microbes. They provided proof to

their inference by demonstrating that the DNA sequence data reported for Picea

species by Smith and Klein (1994) actually came from associated fungus

Hormonema dematioides. This view of plant DNA contamination was further

supported by Saar et al. (2001) who detailed procedure for screening angiosperm

DNA for its purity.

Endophytes have been known to possess superior biosynthetic capabilities.

Maurette et al. (2009) isolated endophytic fungus Alternaria alternata from

Coffea arabica, and evaluated its antimicrobial, antioxidant and cytotoxic

activities. Hazalin et al. (2009) demonstrated cytotoxic activity of endophytic

Sporothrix sp. isolated from plants of Malaysia. Maksum et al. (2011) documented

endophytes from leaves of Garcinia mangostana which showed potential

antibacterial activity against S. aureus, B. subtilis, E. coli, P. aeruginosa, S. typhi

and M. luteus.

Raviraja (2005) studied five medicinal plant species of Western Ghats, Karnataka

and 18 endophytic fungal species were isolated from bark and leaf segments of

which Curvularia sp. and Fusarium oxysporum were the most dominant

organisms. Nalini et al. (2005) during an investigation of fungal endophytes of

C. magna, isolated 96 endophytic fungal species from bark and twig segments,

majority of which were Deuteromycetes followed by Zygomycetes and


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Ascomycetes. Fusarium verticilloides, Nigrospora oryzae and Verticillium sp. were

other dominant endophytes recorded. Tejesvi et al. (2006) studied the endophytic

assemblage of inner bark of some of the important medicinal trees, like

Azadirachta indica, Butea monosperma, Crataeva magna, Holarrhena

antidysenterica, Terminalia arjuna and T. chebula and recorded a total of 48

endophytic species of which mitosporic fungi represented a major group followed

by Ascomycetes and Sterile mycelia.

Chaetomium, Fusarium, Myrothecium, Pestalotiopsis, Trichoderma and

Verticillium were the dominant endophytes recorded by them. Chunying et al.

(2009) investigated the diversity of root endophytes in Rhododendron fortunei

from four habitats of subtropical forests of China. Arnold et al. (2003)

demonstrated the capacity of diverse, horizontally transmitted endophytes, which

limit pathogen damage in a tropical tree, Theobroma cacao. Conn et al. (2008)

isolated endophytic Actinobacteria from healthy wheat tissue, which are capable of

suppressing wheat fungal pathogens both in vitro and in plant. The identified

endophyte had the ability to activate key genes in the systemic acquired resistance

or the jasmonate/ethylene pathways in Arabidopsis thaliana.

2.6 Techniques for studying endophytes

2.6.1 Isolation of endophytes

The diversity of microbes in atmosphere is greater compared to laboratory

collections (Courtois et al., 2003). Only 1% of the living organisms have been

cultured and characterized in a laboratory setting. Kellenberger, 2001 revels that

around 107 bacteria were counted in 1 g of soil, but only 0.1% was culturable in

laboratory. Handelsman et al., 1998 tells that remaining 99.9% of the microbial

population may represent novel genetic diversity. Approximately 72,000 fungus

were described, but the number of fungi that exist in nature is predicted to be as

high as 1.5 million (Bull et al., 2000).


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Basic things required to isolate endophytes, selection of source of interest with

unique characteristics. Small portion of tissues are cut from the plant. Until

isolation procedures are accomplished, the materials are stored at 4 °C. Plant

materials are systematically surface sterilized by immersion in 70% ethanol for 1

min, 5% sodium hypochlorite solution for 5 min and sterile distilled water for 1

min two times under a laminar flow hood until dried (Wiyakrutta et al., 2004).

Ethanol substantially improves the efficacy of surface sterilization, however

triplicates are maintained to eliminate contamination (Hua et al., 2006). After 2–4

weeks of incubation, colonies of endophytes could be seen, from which hyphal tips

are inoculated to newly prepared PDA plates to get pure cultures (Strobel, 2002a).

Due to the lack of sporulation several endophytes cannot be identified. Several

methods have been standardized to support the sporulation on plating media, like

treating under near-ultraviolet at 8ºC or fluorescent light with a 12 h dark-light

cycles, or incubation at 4ºC in darkness, etc. Culturing nonsporulating endophytes

in natural media or on chemically defined media such as Czapek’s agar, corn meal

agar, V8 agar, potato carrot agar, Banana leaf agar and peptone dextrose agar may

also induce sporulation. Culturing the fungus on host substrate may also induce

sporulation (Guo et al., 1998; Taylor et al., 1999; Frohlich et al., 2000).

Non-sporulating fungi are commonly termed as ‘mycelia sterilia’ and grouped as

‘morphospecies’ based on resemblance in cultural characteristics (Bills, 1996;

Umali et al., 1999). However, grouping mycelia sterilia into morphospecies, will

not reflect species phylogeny, because morphospecies are not real taxonomic

entities (Guo et al., 2003). To resolve this problem, many alternative methods have

been developed to categorize these fungi to genus level and also to evaluate the

validity of the current morphospecies concept (Lacap et al., 2003; Guo et al.,

2003). Huang et al. (2009) inoculated each isolate of fungal strain on PDA, PCA,

and WA to achieve optimum conditions for sporulation, whereas, Marquez et al.

(2008) used sterilized pieces of host plant Ammophila or Elymus leaves in water

agar, to induce sporulation in non sporulating isolates in PDA. Only a selected

group of strains can be grown on isolation media which suppress majority of

population (Connon and Giovanni 2002).


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Different concentrations, sterilization times and volumes were tested to different

thickness and texture of sample. Larran et al. (2002) determined the efficacy of

surface sterilization by subjecting to different treatments such as dipping the

samples successively in 70% ethanol for 1 min, 50 or 100% sodium hypochlorite

for 1.5, 3, 5 and 10 min, followed by washing twice in sterilized distilled water or

surface sterilization by immersing consecutively in 90% ethanol for 1 min,

washing in sterilized distilled water, then dipping in 30% hydrogen peroxide

solution for 1.5, 3, 5 and 10 min, and rinsing twice in sterilized distilled water.

Marciano et al. (2005) isolated endophytic fungi from resistant, healthy susceptible

and diseased plants of cacao. Thirty branches were randomly sampled from each

tree. After surface sterilization, the samples were cut into 3-4 mm pieces and

placed onto PDA containing 50 µg/ml tetracycline. Fungal growth was counted and

each morphologically different hyphal tip was pure cultured.

Suryanarayanan and Vijaykrishna, (2001) screened for endophytes from lamina

segments cut from mid portion of healthy mature leaves, petiole segments and

segments of aerial root tissues of F. benghalensis. They selected the root segment

for screening from the subapical portion about 5 mm behind the growing tip for

better inoculum. They sterilized the tissues by dipping the segments in 75%

ethanol for 1 min, immersed in 4% NaOCl for 3 min and then rinsed with 75%

ethanol for 0.5 min.

2.6.2 Identification of endophytes

Identification of endophytes is based on colonisation, sterilants, culture conditions

and media. Fungi that protrude out of plated samples can be identified

morphologically or by molecular methods. Sequencing of ribosomal DNA and

internal transcribed spacers (ITS) were applied to identify fungal taxa.

Suryanarayanan et al. (1998) proposed that sterile mycelia should not be identified

as any group and should be coded based on culture characteristics. Lacap et al.

(2003) reports that the sterile forms with different culture characteristics should be

assumed to represent different taxonomic entities.


27

2.6.2.1 Conventional technique

Larran et al. (2002) identified the fungal endophytes from PDF plates, but when

that was not possible, fungi were subcultured on 2% malt extract agar (MEA) and

incubated with a 12 h photoperiod under fluorescent and near ultraviolet lighting.

They made direct microscopic observations, microculture techniques and

micrometrical measurements and all the fungi taxa were identified.

Guo et al. (2008) identified the C. fusiforme endophytic fungal strain from

Scapania verrucosa by mechanism and characteristics of spore and spore

production. Huang et al. (2009) identified 108 endophytes and classified them into

27 different morphological taxa based on colony, hyphae, spores and reproductive

structures. In addition to these morphological characterizations they carried out

molecular analyses to confirm the identification.

2.6.2.2 Molecular technique

Identification of fungi using morphological characteristics has been the order until

now. With initiation of various molecular tools such as polymerase chain reaction,

DNA hybridization, restriction enzyme analysis (RFLP), amplification using

random primers (RAPD), amplification of microsatellite DNA, electrophoretic

karyotyping, sequencing techniques and taxonomic characterization of species

have become much easier (Lucero et al., 2011).

Chunying et al. (2009) provided new ericoid mycorrhizal material for the world

resource by conforming 15 Ascomycetes and two Basidiomycetes from R. fortunei

on the basis of results obtained from internal transcribed spacer-restriction

fragment length polymorphism (ITS-RFLP) analysis. Phongpaichit et al. (2007)

identified 15 endophytic fungal isolates based on internal transcribed spacer rRNA

sequence analysis. They reported that they belong to following nine genera:

Aspergillus, Botryosphaeria, Curvularia, Fusicoccum, Guignardia, Muscodor,

Penicillium, Pestalotiopsis, and Phomopsis sp.


28

Collado et al. (2006) described a new Coelomycete, Morinia longiappendiculata

sp. By comparing ITS rDNA region and many other genes of the M.

longiappendiculata and M. pestalozzioides isolates. Promputtha et al. (2005)

identified non sporulating endophytes from Magnolia liliifera using ribosomal

DNA fingerprinting. Rungjindamai et al. (2008) employed molecular techniques to

characterize the endophytic basidiomycete assemblage isolated from the oil palm.

Partial large subunit (LSU) of nuclear ribosomal DNA was selected for a

preliminary experiment so as to characterize their higher taxonomic placement.

The internal transcribed spacer (ITS) was further generated in order to define and

confirm their lower taxonomic position. Guo et al. (2000) identified 19 endophytic

morphospecies from Livistona chinensis using phylogenetic analysis of the ITS

and 5.8S regions.

Totally 103 endophytic species were identified from Ammophila or Elymus using

nucleotide sequence of ITS1-5.8S rRNA-ITS2 regions (Marquez et al., 2008).

Marciano et al., (2005) identified endophytic fungal community of Cacao by

morphological traits and by rDNA sequencing. They characterized the organism by

amplifying ITS region in a 50 µl final Polymerase chain reaction.

2.6.3. Microbial fermentation

Extraction from natural sources presents disadvantages such as dependency on

climate, seasonal and possible ecological problems in extraction, thus aiming at

innovative approaches for extraction of secondary metabolites. Microbial

production through fermentation or biotransformation has advantage for the

extraction of secondary metabolites from natural sources as fungal metabolites can

be produced by mycelial fermentation. So far very limited studies have been

documented on fermentation process for metabolite production (Kostecki et al.,

1999).

Kusari et al. (2009) established methodology using shake-flask fermentation

conditions for characterization of plant Camptotheca acuminata, which produced

camptothecin, 9-methoxy camptothecin and 10-hydroxy camptothecin in rich


29

mycological medium. Filip et al. (2003) used submerged cultures for endophytic

fungus H. dematioides in Yeast malt glucose (YMG) medium in a 20 L fermenter

to extract a new cytotoxic compound hormonemate. Hestbjerg et al. (2002)

attempted production of trichothecenes and other secondary metabolites by

Fusarium culmorum and Fusarium equiseti on common potato sucrose agar, yeast

extract sucrose agar and Soil Organic Matter Agar.

Endophytic Fusarium redolens is a high producer of the antibiotic compound

beauvericin. Xu et al. (2009) tried enhanced beauvericin production with

integrated fermentation in situ adsorption with a polymeric resin in mycelial liquid

culture of F. redolens. Justo et al. (2007) clarifies that the production of anticancer

indolizidine alkaloid from M. anisopliae will be maximum when oatmeal extract

supplemented with 1.8 g/L DL-lysine was fermented in shake flask at 26 ºC with

200 rpm. Guo et al., (2008) extracted bioactive potentials from endophytic fungus

Chaetomium fusiforme by fermenting in potato dextrose broth. Wiyakrutta et al.

(2004) adopted stationary condition of fermentation at 250C for 21 days in a

1000 mL Erlenmeyer flask containing 200 mL of malt Czapek broth or yeast

extract sucrose broth.

2.6.4 Secondary metabolites of endophytes

Natural products play an important role in the discovery of leads for the

improvement of drugs in the treatment of human diseases. Much of nature remains

to be explored. Over the last few years, substantial knowledge has been

accumulated on the biology of endophytic microorganisms. Historically, secondary

metabolites have a remarkable impact on society (Fox and Howlett, 2008).

Strobel (2002b) reported that the fungal endophytes residing within the plants

produce metabolites similar to or with more activity than that of their respective

host. Discovery of Penicillin led to later discoveries of potent antibiotics isolated

from microbes (Pinner et al., 1996). Taxol, billion dollar anticancer drug,

previously obtained from Pacific Yew tree (Taxus brevifolia), is now obtained

from its endophytic fungus, T. andreanae (Stierle et al., 1993). The quantitative


30

and qualitative aspects secondary metabolite typically starts late in the growth of

the microbe, often upon entering the stationary phase (Bu’Lock. 1961; Knight et

al., 2003).

An antitumor pentacyclicquinoline alkaloid, camptothecin was isolated by Kusari

et al. (2009) from the wood of Camptotheca acuminate. This naturally occurring

antitumor compound was reported initially by Wall et al., (1966), which targets the

intranuclear enzyme DNA topoisomerase I, which is required for the relaxation of

DNA during molecular procedures, namely, DNA replication and transcription

(Hua et al., 2003). Camptothecin also hinders the synthesis of RNA (Bendixen et

al., 1990). Natural hormonemate was purified by fermentation of an endophytic

fungus Hormonema dematioides from Pinus species (Filip et al., 2003). Three

compounds were isolated by spectroscopic techniques from endophytic fungal

strain Aquilaria malaccensis (Shoeb et al., 2010).

Aspergillus terreus produces Butyrolactone I, but this same compound from other

organism acts as antibiotic and virulence factor production (Calvo et al., 2001;

Davies et al., 1998; Schimmel et al., 1998). John et al. (1999) identified two

known secondary metabolites and four new diterpenes named oidiolactones A-F,

and the antibiotic cladosporin was isolated from the fungus Oidiodendron truncate.

Five cadinane sesquiterpenes derivatives were isolated from Phomopsis cassiae,

which was isolated from Cassia spectabilis and 3,11,12-trihydroxycadaleneas, one

among the five derivatives was revealed as the most active antifungal compound

against C. sphaerospermum and C. cladsporioides (Silva et al., 2006).

Altersetin, a new alkaloid isolated from endophytic Alternaria sp., showed

antibacterial activity against several pathogenic Gram positive bacteria (Hellwig

et al., 2002). Muscodoralbus, an endophytic fungus of tropical tree species,

produces many volatile organic compounds including tetrohydofuran, 2-methyl

furan, 2-butanone and aciphyllene, which have antibiotic activities (Atmosukarto

et al., 2005). However, significant numbers of interesting natural bioactive

compounds have been reported during the last few decades. Thus, many

researchers have focused on the secondary metabolites produced by fungal

endophytes.


31

2.7 Bioassays for therapeutic targets

2.7.1 Antioxidant

Considering the importance of polyherbal formulations, Jain and Agrawal, (2008)

developed mono and polyherbal tablet formulations namely, Withatab

(from Withania somnifera), Asparatab (from Asparagus racemosus), Centab

(from Centella asiatica) and Ascenwit (from combination formulations of

W. somnifera, A. racemosus and C. asiatica) which have potent antioxidant

activity. The antioxidant defense machinery possesses variety of enzymatic and

non-enzymatic molecules in its system (Gill and Tuteja, 2010). Oyawoye et al.

(2003) investigated relationship between free radicals and reactive oxygen species

with respect to ovulation, fertilization and conception in female reproductive

functions using ferric reducing antioxidant power (FRAP) assay. They found that

in 303 follicular aspirates, 71.9% yielded oocytes, 77.5% fertilized and 79.3% of

embryos survived until the time of embryo transfer. Quercetin was isolated from

leaves of P. guajava which exhibited significant scavenging activity providing

scientific basis for the use of this plant in traditional medicine (Tachakittirungrod

et al., 2007).

Liu et al. (2010) isolated exopolysaccharides from endophytic bacterium

Paenibacillus polymyxa and they were evaluated for antioxidant activity by in vitro

and in vivo methods. Their results suggested that exopolysaccharides possessed

potent antioxidant activity and they can be explored as novel natural antioxidant

molecules. Verma et al. (2010) evaluated the antioxidant properties of

Ficus glomerata fruits in CCl4 induced toxic rats. Among different solvent

extracts, ethyl acetate fraction showed potent antioxidant activity by increasing

GSH, CAT, SOD and decreasing LPO.

Fritz et al. (2003) conducted an antioxidant in vivo study in human system by

determining the urinary excretion of secondary lipid peroxidation products. They

subjected ten healthy women in age group of 18-35 to consume self-selected diet

and avoid legumes, whole grains and isoflavone containing foods. They analyzed


32

urine samples from 24-h collections at the end of each diet period for the presence

of lipophilic aldehydes and related carbonyl compounds by HPLC method.

Gupta et al. (2010) examined anti-oxidative potential of a well-known medicinal

plant Rhodiola imbricata root aqueous extract in rats. Administration of single

dose of aqueous extract of root significantly restricted rise in blood

malondialdehyde, increased blood, reduced glutathione and superoxide dismutase

(SOD) activity. Multiple dose treatment of the extract further restricted the

increase in LDH and increased blood, liver and muscle GSH and GST levels.

Alia et al. (2003) evaluated the effects of diet rich fiber from grapes on antioxidant

(glutathione) concentration and antioxidant enzyme activity in liver homogenates

and in plasma. Oxidative stress was tested in vivo in rats which were induced acute

oxidative stress by acetaminophen.

2.7.2 Hepatoprotective

Liver is the key organ of our body which is involved in immune system, nutrient

supply and in all biochemical pathways. Oxidative damage by toxic chemicals,

drugs etc., is critically involved in various types of hepatic injuries, as a result

inflict liver diseases remain one of the serious health problems. The supervision of

liver disorders is still a major challenge to the modern medication.

Shankar et al. (2008) studied the capacity of Commiphora berryi in offering

protection against CCl4 induced hepatotoxicity by detecting increased AST, ALT,

ALP and bilirubin in Albino Wistar rats. Premna serratifolia is a plant traditionally

used in Ayurvedic system of medicine, the alcoholic extract of P. serratifolia

leaves possesses hepatoprotective activity against CCl4 induced hepato-toxicity in

rats. The extent of protection offered was measured by biochemical parameters

such as serum glutamate oxalate transaminase, serum glutamate pyruvate

transaminase, ALP, bilirubin and total protein (Vadivu et al., 2009).


33

Hepatoprotectivity against ethanol in primary cultures of rat hepatocyte were

estimated using in vitro and in vivo models, using Thunbergia laurifolia extracts.

In the in vitro study, ethanol decreased the percent MTT nearly by half, which was

brought back to normal. In in vivo study, liver cells were recovered by bringing

HTg, ALT and AST back to normal by aqueous extract of T. laurifolia

(Pramyothin et al., 2005). Bacopa monnieri is a succulent herb used in Ayurveda

for the treatment of epilepsy and insanity. Ethanol extract of B. monnieri produced

significant hepatoprotective effect by decreasing the activity of serum enzymes and

bilirubin when assayed in Wistar albino rats (Ghosh et al., 2007).

Gujrati et al. (2007) investigated the hepatoprotective activity Tylophora indica

leaves against ethanol induced hepototoxicity. Both alcohol and aqueous extracts

significantly disallowed the physical, biochemical, histological and functional

changes induced by ethanol in the liver. Their result indicated that alcoholic extract

exhibited greater hepatoprotective activity than the aqueous extract. Methanolic

extract of Careya arborea (Barringtoniaceae) known as Padmaka in the

Ayurvedic system of medicine was tested for its hepatoprotective activity.

Animals inoculated with Ehrlich Ascites carcinoma possess significant alteration in

the levels of hepatoprotective parameters which was reversed by the methanol

extract of C. arborea (Senthilkumar et al., 2008). Naaz et al. (2007) evaluated the

hepatoprotective activity of P. amarus on aflatoxin B1-induced liver damage in

mice. The ethanolic extract of P. amarus showed hepatoprotective effect by

lowering the content of thiobarbituric acid reactive substances and enhancing the

glutathione level. Histopathological analyses of liver samples also confirmed the

hepatoprotective value which was comparable to the standard antioxidants.

Kamble et al. (2008) developed polyherbal formulations F1 and F2 from dried

aqueous extracts of Acacia catechu, Allium sativum and alcoholic extracts of

A. paniculata, A. indica, B. diffusa, C. longa, E. alba, E. officinalis, L. echinata,

P. kurroa and P. amarus. Both the formulations at 400 mg/kg dose showed

significant hepotoprotective activity in experimental liver damage.


34

2.7.3 DNA protection

Reactive oxygen species are toxicants generated mainly by mitochondrial

respiration, biochemical metabolism within cells and by other environmental

pollutants. Hydrogen peroxide (H2O2) which is freely diffusible within the cell is

one of the important compounds which produce oxidative stress. It can be

generated from superoxide dismutase or can arise spontaneously. In the presence

of metal ions such as copper or iron, H2O2 undergoes Fenton’s reaction producing

hydroxyl radical which causes oxidative induced braking in DNA strands to yield

its open circular forms (Santos et al., 2003).

Verma et al. (2010) found that ethylacetate fractions of Ficus glomerata fruits

effectively protected the super coiled pBR322 DNA from oxidative damage.

Janzowski et al. (2003) investigated the induction of oxidative DNA damage in

mammalian cells as a result of glutathione depletion induced by food related

α,β-Unsaturated carbonyl compounds. In addition to direct DNA breakage, these

compounds stimulated oxidative purine modifications in mammalian cells. Arsenic

is a naturally occurring inorganic compound which accumulates in skin and

increases risk of skin cancer by generating 8-hydroxyl-2’- deoxyguanine (reactive

oxygen) and 3-nitrotyrosine (nitrogen species) in human keratinocytes (Ding et al.,

2005).

Rajkumar et al. (2010) evaluated the ability of methanolic and aqueous extracts of

Bergenia ciliata to protect DNA against H2O2 oxidative damage. Many researchers

have found the effectiveness of various medicinal plants like Opuntiaficus indica,

Polygonum aviculare, T. zeylanicus, Cichori umintybus and Azadirachta indica in

protecting plasmid DNA against the hydroxyl radicals (Lee et al., 2002; Tharakan

et al., 2005; Ilaiyaraja and Khanum, 2010; Ghimeray et al., 2009). Fabiani et al.

(2008) observed that phenolics play an important role in the anticarcinogenic

properties of olive oil. Their results suggested that at concentrations of 1 mM/L

there was 93% and 89% protection to promyelocytic leukemia cells and to human

peripheral blood mononuclear cells, respectively.


35

2.7.4 Antimicrobial

Antimicrobial compound kills or inhibits the growth of microbes such as bacteria

or fungus. A wide range of medicinal plants make richest contribution as

antimicrobial agents. Medicinal plants generally produce many secondary

metabolites which constitute rich sources of natural pesticides, microbicides and

many pharmaceutical drugs (Bobbarala et al., 2009). Useful antimicrobial

phytochemicals from medicinal plants are divided into several categories like

phenolics, polyphenols, terpenoids, essential oils, alkaloids, lectins, polypeptides

and many other compounds (Cowan, 1999).

Moses et al. (2013) demonstrated the control of Gram-positive bacterium S. aureus

from Kenyan traditional herbs like, C. spinarum, U. dioica, W. ugandensis,

S. didymobotrya, P. peruviana, B. pilosa, L. nepetifolia and T. asiatica. Whereas

Gram-negative E. coli was controlled only by dichloromethane and ethanol

extracts of L. nepetifolia. Kurdekar et al. (2012) made an attempt to find

antimicrobial activity of five medicinal plants against human pathogenic bacterial

strains namely, S. aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and

Proteus vulgaris and two fungal strains Candida albicans and Aspergillus niger by

comparing two different extraction methods. Palombo, (2009) discussed about the

plant extracts that inhibited the growth of oral pathogens like, Porphyromonas

gingivalis, Actinobacillus, Prevotella and Fusobacterium, thereby reducing the

symptoms of oral diseases.

Tamarix ramosissima is one of the traditional herbal medicinal plants, which is

known for the treatment of rheumatism and jaundice, was screened for its

antibacterial and antifungal activity. Butanol and ethyl acetate fractions of

T. ramosissima exhibited antifungal activity against human pathogen A. niger at

concentration of 400 g/ml. Antibacterial activity against C. diphtheria (25 g/ml)

and P. mirabilis (100 g/ml) was observed in ethyl acetate and tannin fractions

whereas butanolic fraction inhibited S. typhi and C. diphtheria at concentration of

100 g/ml (Sultanova et al., 2001).


36

Laorpaksa et al. (2008) isolated 54 endophytic bacteria which demonstrated

antimicrobial active zone of inhibition against B. cereus, S. aureus, P. aeruginosa,

E. coli and C. albicans. Wiyakrutta et al. (2004) isolated 92 endophytic fungi

which inhibited Mycobacterium tuberculosis as determined by microplate Alamar

blue assay and six isolates inhibited Plasmodium falciparum when tested by

hypoxanthine incorporation method. Subbalakshmi and Sitaram, (1998)

demonstrated the mechanism of antimicrobial action of Indolicidin. It was isolated

from cytoplasmic granules of bovine neutrophils, which affects the synthesis of

RNA and protein by inhibiting the DNA synthesis in E. coli cells. Indolicidin also

exhibits activity against many other Gram-positive and Gram-negative bacteria as

well as fungi.

2.7.5 Antidiabetic

Diabetes is a serious illness with complex metabolic condition which is growing

terrifically all over the world. Even though under considerable progress in

understanding and management of diabetes, the complications of this disease are

increasing every year. Presently available therapeutic choices for the treatment of

diabetes include dietary modification, use of insulin or oral hypoglycaemic drugs

such as sulfonylureas, biguanides and thiazolidinediones (Gad et al., 2006).

Traditional medicines and herbal extracts which are characteristically rich in

phenolic compounds are known to interact with proteins and can inhibit the

enzymatic activity of glycoside trimming α-glucosidase and α-amylase enzymes to

control post-prandial hyperglycemia. Pancreatic α-amylase and intestinal α-

glucosidase are the key enzymes involved in the enzymatic breakdown of complex

carbohydrates. By inhibition of these enzymes, type 2 diabetes-associated post-

prandial hyperglycemia can be targeted (Patrick et al., 2005).


37

Gad et al. (2006) demonstrated that Fenugreek and Balanites extracts inhibit

α-amylase activity in in vitro model and suppress starch digestion and absorption

in dose-dependent in vivo rat model. Klein et al. (2007) studied antidiabetic

activity of L. speciosa (Banaba) by identifying Penta-O-galloyl-glucopyranose

(PGG) as the most potent gallotannin. They also stated that obesity leads to

hyperinsulinemia which eventually leads to hyperglycemia.

The leaf extract of T. indica was able to inhibit 90% of α-amylase activity (Funke

and Melzig, 2006). Bhat et al., (2008) tested α-amylase inhibitory property in six

ethno-botanically known plants in which the chloroform extract of O. tenuflorum,

Bougainvillea spectabilis, Murraya koenigii and Syzygium cumini demonstrated

significant inhibition. Nickavar and Yousefian (2009) investigated α-amylase

inhibitory effects of some plants using an in vitro model and reported a favourable

α-amylase enzyme inhibitory activity. Karthic et al. (2008) purified the compounds

betulinic acid and 3574-tetrahydroxy flavanone from seed extracts of S. cumini

which showed maximum inhibitory activity of α-amylase.

Ravenala madagascariensis has been extensively used in conventional system of

medicine. Priyadarsini et al. (2010) demonstrated significant inhibitory effect of

ethanolic and aqueous extracts of R. madagascariensis on glucose diffusion

in vitro and in vivo antidiabetic activity. Barik et al. (2008) evaluated the aqueous

root extract of Ichnocarpus frutescens for its antidiabetic activity in sterptozotocin-

nicotinamide induced type 2 diabetes in rats. Whereas Dhanabal et al. (2008)

investigated that ethanol extract of Clerodendron phlomoidis exhibited significant

hypoglycemic activity at 200 mg/kg dose in alloxan-induced diabetic rats.

2.7.6 Antihypertension

Natural compounds are accepted as significant sources of ACE inhibitors.

A peptide from Bothrops jararaca was the base for the development of ACE

inhibitor captopril (Hansen et al., 1995, Serra et al., 2005). There are several

methods as well as many enzyme sources and substrates for in vitro screening of

ACE inhibitors. Amaranth hypochondriacus is a striking source of vegetal protein


38

because of its nutritional value. Identification of two ACE inhibitor tetrapeptides

from A. hypochondriacus, which was experimentally validated by in vitro assay

showed potent ACE inhibitory activity (Vecchi et al., 2009).

ACE inhibitory peptides were isolated from Brachionus rotundiformis and the

hydrolysates were prepared by Alcalase, papain, etc. From which Alcalase

hydrolysate showed the higher inhibitory activity when compared to all other

hydrolysates. Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-Thr-Gly-Glu-Ala-Met, was

the amino acid sequence of the purified peptide with a molecular weight of 1538

Da (Lee et al., 2009). Fagopyrumes culentum is an valuable source of ACE

inhibitor. A tripeptide, Gly-Pro-Pro, isolated from this plant showed IC50 value of

6.25 µg protein/ml (Ma et al., 2006). Balasuriya and Rupasinghe, (2011)

investigated the ACE inhibitory property of fruit flavonoids in apple skin extract.

The isolated compounds showed a concentration dependant enzyme inhibition.

2.7.7 Cytotoxicity

In recent years, pharmaceutical companies perform cell based assays for high

throughput secondary chemical cytotoxicity screening. Improved simplicity of

similar methods and correlation with in vivo toxicity records have contributed to

the use of cell based assays as alternatives for testing animals in toxicology

laboratories. There are a variety of ways to measure cell viability or cytotoxicity

in vitro. Development of in vitro MTT tetrazolium reduction assay and the

handiness of the 96 well plate reader cemented the way toward streamlining the

mechanical measurement of cellular growth and survival. Consequently, several

diverse tetrazolium and indicator dyes have been introduced for the additional

improvement of cell viability assays (Riss and Moravec, 2004).

Mosmann, (1983) developed a quantitative colorimetric assay to detect living cells

in mammalian cell. This method was therefore, used to measure cytotoxicity,

proliferation and activation of cell. Vichai and Kirtikara. (2006) developed a

protocol using Sulforhodamine B to determine the cell density based on the

measurement of cellular protein content. This simple, efficient and cost effective

method was optimized to screen toxicity of large number of compounds within few

hours in 96 well formats.


39

Brasileiro et al. (2006) investigated cytotoxicity of ethanol extracts of 32 medicinal

plants by Brine shrimp lethality test (BST). Out of these, ten extracts from,

C. pisonis, C. nardus, E. alba, E. bulbosa, E. foetidium, E. tirucalli,

M. hirsutissima, M. charantia, S. microglossa and P. ornatus presented brine

shrimp toxicity. Kumar and Das, (1996) resorted to a calorimetric assay using

MTT to evaluate the cytotoxic activity of chicken intestinal intraepithelial

lymphocytes. These lymphocytes were found to exhibit natural killer cell like

cytotoxicity. Shaikh et al. (2009) examined the cytotoxic effect of 16 Bangladeshi

medicinal plants from their hexane, dichloromethane, methanol and water extracts

against using MTT assay. The extracts of H. auriculata and H. tiliaceous and water

extract of L. indica showed no toxicity against mouse fibroblasts.

2.8 Purification of potent bioactive compound

Traditional medicinal species are of immense importance to the health of

individuals and communities. Many of these native medicinal plants are utilized as

important components of diets. The medicinal value of these species lies in

chemicals that produce a distinct physiological action on the body. The most

important of these bioactive chemical constituents contributing to the protective

effects of plants are alkaloids, tannins, saponins, steroid, terpenoid, flavonoids,

phenolic compounds, etc. (Edeoga et al., 2005). The isolation and purification of

active principle of plants depend much on the structure, stability and quantity. A

variety of techniques can be used for the isolation and purification of natural

compounds. These techniques include column chromatography, thin layer

chromatography, high performance liquid chromatography (HPLC) and Nuclear

magnetic resonance (NMR) (Sultan et al., 2011).

Essential oils of medicinal plants are potent natural products used as such for many

areas, including in pharmaceuticals, perfumes, cosmetics, aromatherapy, spices and

nutrition. They can be obtained by hydro distillation, steam distillation, microwave

irradiation, hydro diffusion, CO2 extraction and by many other mechanical and

thermochemical reactions. Chemical analysis of essential oils is generally

performed quantitatively and qualitatively by GC and GC/MS, analysis


40

respectively. Compounds which are not easily separated by GC and structurally

similar molecules like stereo-isomeric compounds are analyzed by NMR spectra

(Lahlou, 2004). Essential oil from inflorescence of S. calva was investigated by

GC and GC/MS. Compound could be identified by comparing with National

Institute of Standards and Technology Chemistry web book data of the peaks with

those reported in literature (Begum et al., 2008).

Reverse phase HPLC method was developed for identification of pentacyclic

triterpenoids namely, ursolic and betulinic acid from the methanol extract of

V. negundo leaves. Compound analysis can be done with acetonitrile: methanol

(80:20) as isocratic elution mode with UV detection at 210nm. The peaks of

ursolic and betulinic acid could be confirmed by LC-MS (Taralkar and

Chattopadhyay, 2012). Mycotoxins are low molecular secondary metabolites

subsequently produced by filamentous fungi which are not necessary for growth or

development. Mycotoxins released by the fungus are connected to certain health

disorders and elicit acute toxic, mutagenic, teratogenic, carcinogenic and

sometimes estrogenic properties (Brzonkalik et al., 2011). Altenusin, alternariol,

3'-hydroxyalternariol monomethyl ether, and alternariol monomethyl ether were

separated and identified by liquid chromatograpy-solid phase extraction-nuclear

magnetic resonance spectroscopy from A. alternata. The structure was confirmed

by high-resolution mass spectrometry (De Souza et al., 2013).

Sultan et al. (2011) isolated seven polyesters from unidentified endophytic fungi

from roots of Callophyllum ferrugineum. These polyester compounds were isolated

based on dereplication process using HPLC and chemically characterized by mass

spectroscopy and CapNMR. Three compounds from the endophytic fungi from

A. malaccensis were isolated by column chromatography, HPLC and elucidated by

UV, IR and NMR spectra (Shoeb et al., 2010).

Amna et al. (2006) isolated a compound Camptothecin from E. infrequens from

the bark of Nothapodytes foetida plant. They applied gradient reverse phase HPLC

method with diode array and MS/MS detection for quantification of the compound.

Cytotoxic compound namely, Norcardiatones A and other two new 2-pyranone


41

derivatives Norcardiatones B and C were isolated from Nocardiopsis sp. Their

structures were elucidated by spectroscopic and mass-spectrometric analyses,

including 1D-,2D-NMR and HR Q-TOFMS (Lin et al., 2010).


42

2.9 Summary of Review of Literature

Medicinal plants have limitless ability to produce diverse range of bioactive

molecules which are considered as multi-component drugs and are thought to

bestow a wide range of beneficial effects. They have been used therapeutically for

centuries, because of their historical place in medicine, they have a better safety

profile than synthetic drugs. Even today, in most of the rural areas, people are

depending on local traditional healing systems for their primary health care due to

the poverty and lack of access to modern medicines (Ayyanar et al., 2012).

Asteraceae originated in South America and reached every continent except

Antarctica (Funk et al., 2005). Asteraceae taxa have good dispersal capacity and

the family managed to reach far off territories by means of long-distance dispersal

(Munoz and Munoz, 2007). Barnadesioideae, one of the subfamilies of Asteraceae

has been identified as the basal branch of the family. Asteraceae family has

remarkably great economic and ecological importance as vegetables (lettuce,

artichokes, endive), sources of oil (sunflower, safflower), insecticides (pyrethrum),

and garden ornamentals (chrysanthemum, dahlia, marigold, and many others)

(Jansen and Palmer, 1987 and Katinas et al., 2007).

Asteraceae family plants are rich sources of phytochemical compounds like

Alkaloids, Saponin, Tannin, Flavonoids, Lignans, etc., and they are helpful in the

treatment of many disorders (Patel, 2012). These plants also act as excellent hosts

for diverse endophytic fungal species which presumably occur in a mutualistic

association (Huang et al., 2009, Gu et al., 2007 and Margareth et al., 2009). Even

though there are lots of compounds isolated from this family, there is less evidence

of pure antioxidant or antihypertensive compounds from some plants. Also there

are lots of plants where there are no records of endophytic studies in some plants.

In this regards, an ethnomedical approach, oral or written information on the

medicinal use of a plant forms the basis for selection. Information from

conventional medical systems, herbals and folklore can be acquired from diverse

sources, such as books, herbals, review articles and databases. Fabricant and


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Farnsworth (2001) described four standard approaches for selecting plants:

(1) Random selection followed by chemical screening, (2) Random selection

followed by one or more biologic assays, (3) Follow-up of biologic activity reports

and (4) Follow-up of ethnomedical or traditional medicinal uses of plants.

Primarily, phytochemical constituents of plant secondary metabolites like

alkaloids, flavonoids, phenolics, etc., are very popular approaches for selection of

plants because they can be tested easily. Beside, all available plant parts are

selected, irrespective of previous knowledge and experience. Another approach is

by exploiting the vast resource of published reports on its activity.

On the basis of ethnopharmacological reports indicating their traditional use in the

treatment of many diseases the following medicinal plants were selected for the

study: Artemisia cina, Artemisia vulgaris, Eclipta alba, Glossocardia bosvallea,

Mikania micrantha, Spilanthes uliginosa, Vernonia cinerea, Vicoa indica,

Wedelia chinensis and Wedelia trilobata.

Chapter 2. Review of Literature 2.1 Medicinal...

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