Review of Literature
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
Name
Uses
Activity
Ageratum conyzoides
Anaphalis neelgerriana
References
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.
Antioxidant
Vijaylakshmi et al., 2009.
Antiseptic,
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
Gnaphalium luteo
Breast lactation
Helianthus annuus
Urinary diseases
Cytotoxic
Magdalena et al.,2012.
Sahu, 1984.
Antimalarial
Islam et al., 2012;
Antisporulant
Deepak et al., 2007.
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.
Blood purifier,
Insecticidal
Rahmatullah et al., 2010;
Colic, Headache,
Anticancer
Gastrointestinal
antibacterial
Hossan et al., 2010.
Dry cough
Insecticidal
Dolui and Debnath, 2010;
Throat complaints
Antimicrobial
Vyas et al., 2009;
Cold
Antimutagenic
Sukumaran et al., 1995.
Launaea nudicaulis
Spilanthus acmella
Headache
disorders
Spilanthus calva
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Spilanthes paniculata
Toothache
Jaundice
Srivastava et al., 2010;
Worm infection
Skin disease
Zheng and Xing, 2009;
Constipation
Tagetes minuta
Kala, 2005.
Insecticidal, Prosperity,
Larvicidal
Macedo et al., 1997.
Blood clotting, Boil,
Upadhye et al.,1986;
Wound healing,
Patel, 2012.
Scrofula, herpes,
Insecticidal
Sahu, 1984;
Urinary diseases
Blood clotting
Patel, 2012.
Stomachic, Muscular pain,
Boil, Scorpion bite
Tridax procumbens
Xanthium strumarium
Minor injuries
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
Name
Artemisia cina
Artimisia vulgaris
Traditional
Pharmacological
Uses
References
Activity
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.
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.,
Foot care,
Larvicidal
Ikram et al., 2013 ;
Diuretic,
Anticonvulsant,
Almeida et al., 2013;
Stimulant.
Antioxidant,
Erel et al., 2012;
Nematicide,
Costa et al., 2003.
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;
Antimicrobial
Karthikumar et al., 2007;
2013;
Eclipta alba
Antiageing,
Hair color and growth
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Glossocardia bosvallea
Mikania micrantha
DNA damaging
Abdel et al., 1998;
Anticancer
Khanna et al., 2009.
Wound healing,
Antibacterial,
Mangesh et al., 2011;
Emmenagogue,
Antimycotic,
Pathak and Dixit, 1984;
Antialcoholic,
Typhoid,
Throat infection,
Patil and Patil, 2006;
Birasdar and Ghorband, 2010;
Jagtap et al., 2013;
Gastric, Chronic fever
Kanthale and Biradar, 2012.
Fever, Rheumatism,
Allelopathic
Influenza, Respiratory
diseases
Spilanthes uliginosa
Vernonia cinerea
Vicoa indica
Lalmuanpuii and
Sahoo, 2011;
Anti stress,
Ittiyavirah and Sajid, 2013;
Antibacterial,
Hajra et al., 2010;
Anti-inflammatory
Amador et al., 2010.
Mouth ulcer,
Antinociceptive,
Ong et al., 2011;
Stomach ache,
Analgesic,
Koubemba et al., 2011;
Diabetis
Cytotoxicity
Soladoye et al., 2012.
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.
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;
Sore throat,
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Wedelia chinensis
Hypoglycemic
Aditya et al., 2012;
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
Wedelia trilobata
depressant activity,
Suresh et al., 2010;
Hepatoprotective
Garima et al., 2009;
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.
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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.
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Review of Literature
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
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Review of Literature
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
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Review of Literature
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.
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Review of Literature
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
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Review of Literature
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).
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Review of Literature
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.
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Review of Literature
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.
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Review of Literature
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).
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Review of Literature
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 αglucosidase are the key enzymes involved in the enzymatic breakdown of complex
carbohydrates. By inhibition of these enzymes, type 2 diabetes-associated postprandial hyperglycemia can be targeted (Patrick et al., 2005).
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Review of Literature
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 sterptozotocinnicotinamide 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
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Review of Literature
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.
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Review of Literature
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
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Review of Literature
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
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Review of Literature
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).
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Review of Literature
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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Review of Literature
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.
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