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2. REVIEW OF LITERATURE
2.1. Survey of Medicinal Plants
India is a veritable emporium of medicinal plants and is bestowed with rich
natural wealth due to its diverse ecological conditions. Indian forests are source of a large
proportion of the world’s recognized medicinal plants and constitute an enormous
potential source of useful plant derived chemicals. Jain and De (1966) reported the use of
some medicinal plants in the treatment of various ailments, used by various tribes of
Puruliya. Trichodesma indicum fresh root is rubbed on body in the treatment of swellings
and the plant is used in rheumatism and dermal infection (Jain, 1991). Literate people
were found to be less knowledgeable on the use of medicinal plants as compared to
illiterate ones due to modernization (Wester and Yongvanit, 1995). Urgenia indica bulb,
Tribulus terrestris leaves, Tinospora cordifolia leaves and stem, Solanum xanthocarpum
fruit, Sida cordifolia seeds, Daemia extensa, Boerhaavia diffusa, Bacopa monnieri and
Acalypha indica plants respectively used for digestion, jaundice, inflammation,
aphrodisiac, rheumatism, jaundice, blood purification, cough and skin diseases
(Jain,1996).
Bhattacharjee (1998) reported that Xanthium strumarium fruit cures small pox and
Oxalis corniculata plant cures stomach trouble. Ram and Saha (1998) carried out
preliminary studies on medicinal plants, used against dysentery at Ranchi District of
Bihar. They identified Achyranthes aspera, Aloe barbadensis, Aristolochia bracteata,
Chenopodium album and Urginea indica plants of therapeutic value. Gupta et al. (1999)
surveyed medicinal plants at Amarkantak forest, Madhya Pradesh and reported thirty nine
different plants used for various ailments.
Sen and Pradhan (1999) enumerated 24 medicinal plants in Bargarh district of
Orissa such as Alstonia scholaris, Celastrus paniculatus, Codia macleodii, Embelia ribes,
Mesua nagassarium, Operculina tarpethum, Oroxylum indicum, Pueraria tuberosa,
Symphorema polyandrum etc. used for various diseases. The leaf extract of Abutilon
indicum, leaf of Azadirachta indica, leaf of Pergularia daemia, leaf of Hygrophila
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auriculata, leaf of Lawsonia inermis, root of Phyllanthus fraternus and Tinospora
cordifolia stem are used for curing jaundice (Sen et al.,2000). Giday (2001) reported that
Achyranthes aspera, Calotropis procera, Kedrostis foetidissima and Withania somnifera
are used respectively for skin wounds, haemorrhoids, chest pain and typhoid.
Mitaliya et al. (2001) enumerated 21 plant species which have medicinal value for
bark due to certain phytochemicals. A few important plants such as Acacia nilotica
(wounds), Aegle marmelos (cough), Holoptelea integrifolia, Zizyphus mauritiana and
Moringa oleifera are given in wounds, cough, diarrhoea and rheumatism respectively.
Mucuna monosperma, Argemone mexicana, Acalypha indica, Datura metel, Fritillaria
cirrhosa and Hyoscyamus niger used for asthma were reported by Jha (2001), available at
Chhotanagpur, Jharkhand.
Shukla et al.(2001) studied 51 wild plant species (Alangium lamarkii, Diospyros
melanoxylon, Feronia elephantum, Mucuna pruriens, Semecarpus anacardium etc.)
which provide food and vegetable to inhabiting tribals of Gond, Baiga, Bhaina, Bhumia,
Binjhwar, Khirwar, Kanwar, Korwa, Uraon etc.Traditional phytotheraphy of Maikal
range and plateau of Pendra District, bilaspur was studied by Shukla et al. (2001) and
reported that Abrus precatorius, Adhatoda vasica, Cleome viscosa and Tephrosia
purpurea respectively are used for treating whoopingcough, asthma, headache and joints
pain.
Many informants agreed that they kept their medicinal plant knowledge secret
(Gedif and Hahn, 2002). Auti et al. (2004) enumerated 50 weeds of medicinal value from
shrirampur tahsil. Das and Devi (2004) reported Cajanus cajan, Cissampelos pareira,
Cyperus rotundus, Streblus asper and Terminalia chebula as effective medicinal plants
against jaundice, used by Bodo tribes of Assam. Nadanakunjidam (2004) reported 301
plants of medicinal value from Attapadi hills, Western Ghats.
Rajaram (2004) studied 31 plants used by Tribals of Moolagangai and Gobanari
of Coimbatore District, TamilNadu, India. Rajaram (2004) surveyed Velliankadu and
Paalamalai of Coimbatore District, TamilNadu and reported 30 medicinal plants curing
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various ailments. Ageratum conizoides and Acacia nilotica are used in traditional
medicine for dressing wounds and treating diarrhoea respectively (Geyid et al., 2005).
Kadavul and Sujesh (2005) surveyed a few medicinal plants such as
Andrographis paniculata, Albizzia malabarica, Bacopa monnieri, Merremia tridentata,
Mucuna pruriens, Oxalis corniculata, Hemidesmus indicus, Gloriosa superba,
Tylophora indica and Tinospora cordifolia which cure respectively jaundice, bronchitis,
epilepsy, leucorrhoea, rheumatism, antidote, urinary hindrance, expulsion of placenta,
insanity, bronchial asthma and leprosy at Vatakara Taluk, Kerala State. Bondya and
Sharma (2005) reported that many medicinal plants were depleted and 4 plants had
completely lost in the region of Bharagora block of Jharkhand and its adjacent border
areas of West Bengal and Orissa.
Dhiman (2005) discussed the problems of medicinal plant wealth upon the
phytogeography of Uttaranchal and reported that many plant species either have gone
endangered or are on the verge of extinction. Patel et al. (2005) identified 52 species of
ethnomedicinal value present in Mukteshwar village of Banaskantha District, north
Gurajat used by tribals and local people.
A survey has been carried out in N.R. Pura taluk, Karnataka for documenting
rural area people knowledge about the usage of plants, against dental problems (Ocimum
sanctum, Eucalyptus globulus, Calotropis procera, Ricinus communis, Azadirachta
indica and Emblica officinalis) and cardiovascular disorders (Centella asiatica, Withania
somnifera, Rauwolfia serpentina, Carica papaya, Aegle marmelos and Murraya konigii)
(Prakasha and krishnappa, 2006). Boopathi (2006) reported 15 medicinal plants from the
Anchetty forest of Dharmapuri District and a few important plants are Derris indica,
Justicia adhatoda, Ficus bengalensis and Eclipta prostrata.
Rajendran et al. (2006) stated that Adhatoda zeylanica, Andrographis paniculata,
Tridax procumbens, Phyllanthus amarus, Phyllanthus emblica, Leucus aspera, Gloriosa
superba, Ficus bengalensis, Moringa oleifera, Cardiospermum halicacabum, Solanum
nigrum, Vitex negundo and Tribulus terrestris are used to treat asthma, skin diseases,
8
wounds, jaundice, dysentery, tonsillitis, rheumatism, fever, poisonousbites, rheumatism,
ulcer, headache and jaundice respectively. Medico-botanical studies in the villages of
Cuddalore District were carried out by Karuppaiah and Sekar (2006). They reported 28
plant species such as Abutilon indicum, Cassia auriculata, Cardiospermum halicacabum,
Argemone mexicana, Cyperus rotundus, Cleome viscosa, Coccinia grandis, Lippia
nodiflora etc. which cure dreadful diseases.
Muthu et al. (2006) studied the medicinal plants used by traditional healers in
Kancheepuram District of Tamil Nadu and stated that Andrographis paniculata, Ricinus
communis, Azadirachta indica, Ficus bengalensis, Trianthema portulacastrum,
Wattakaka volubilis, Pongamia pinnata and Cardiospermum halicacabum are used to
treat diabetes, stomachache, small pox, skin diseases, heeltracks, asthma, rheumatic pain,
wounds and throat infection respectively. Rajaram (2006) reported 27 plants of medicinal
importance from the villages such as Boothikuppam, Kallamballi, Allimaayaru,
Nadukaanji, Iruttupallam, Kambathumedu, Gandhavayil and Koothamandi of Coimbatore
District.
Wondimu et al. (2007) reported that Carica papaya, Cissus quadrangularis,
Dichrostachys cinerea, Melia azedarach and Xanthium strumarium are used to treat
respectively wounds, toothache, snake bite, diarrhoea and fungal disease on skin. Singh et
al. (2008) listed out many medicinal plants used in skin diseases. Aloe vera, Calotropis
procera, Datura stramonium, Oxalis corniculata, Solanum nigrum and Withania
somnifera are used to cure respectively cough, spasm, asthma, urinary inflammations,
spasm and small pox and cancer (AlQuran, 2009). Kosalge et al. (2009) reported that
Gond tribes of Gadchiroli District used Boerhaavia diffusa, Cassia tora, Euphorbia hirta,
Evolvulus alsinoides, Plumbago zeylanica, Sida acuta and Tribulus terrestris
respectively to treat scorpion bite, eye sores, rheumatism, snake bite, wound infection,
dysentery and wounds.
Suresh and Kujur (2009) studied the therapeutic values of some medicinal plants
of Jamshedpur, Jharkhand, India such as Abutilon indicum for fever, Oxalis corniculata
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for indigestion, Clitoria ternatea for constipation, Rauwolfia serpentina for blood
pressure, Tinospora cordifolia for cancer and Commelina benghalensis for leprosy.
Zheng and Xing (2009) studied the medicinal plants of Hainan island, China and
reported that Achyranthes aspera, Adiantum caudatum, Cassia tora, Cassia occidentalis,
Celosia argentea, Dodonaea viscosa, Euphorbia hirta, Phyllanthus emblica, Ricinus
communis, Sida acuta, Tamarindus indica and Vitex negundo are used respectively for
pyogenic infection with abscess, nail separation, gastrointestinal disorders, cold,
rheumatoid arthritis, rheumatoid arthritis, skin diseases, cough, ear diseases, sore throat,
cough and nose bleed. The leaves are the usually used part for the treatment of diseases
as they contain more active principle (Jain et al., 2009).
2.2. Macro and Microscopical Studies
The evolutionary relationships of plant species are studied by the presence or
absence of crystals (Franceschi and Horner, 1980). High concentrations of calcium can
interfere with many cell processes like calcium dependent signaling and micro skeletal
dynamics (Webb,1999). The distribution and shapes of crystals have been used as
taxonomic characters for a number of plant families(Flores, 2001). Ogundipe and Wujek
(2004) reported that the leaf anatomical characters are useful in distinguishing the
species. Ajayi et al. (2011) studied the leaf anatomy of Rytigynia species and reported
that the leaves were glandular, hypostomatic and stomata were paracytic types and
deposits of calcium oxalate crystals were found in the leaves.
2.3. Physico-Chemical Analysis
2.3.1. Organoleptic characters, Moisture and Ash content
Extraction of compounds in different solvents is helpful in the evaluation of drugs
(Miller, 1973). Pruthi (1980) reported that the quality of any powdered drug has been
assessed by the water soluble ash content. Trease and Evans (1983) determined that high
water content in the plants indicates the presence of large amount of mucilage or starch
and more chances of microbial degradation. Adulteration can be determined by increased
ash content in the plant sample. Deshmukh and Beal (1984) reported that organoleptic
10
parameters are helpful in the identification of raw materials. Khatoon et al. (2006) have
revealed the extractive value profile in many medicinal plants.
2.3.2. Mineral Studies
The minerals are essential for a number of metabolic processes like blood
coagulation, muscle contraction and enzyme action. Calcium is necessary for the
formation and growth of bones and teeth. Calcium is essential for neuromuscular
mechanism (Chaudhari and Gokhale, 1991). Deficiency of calcium leads to
hypocalcaemia, rickets and osteoporosis. Osmotic pressure, irritability of muscle,
permeability of the cell, heart beat and glycone absorption are being maintained by
sodium (Dandiya and Sharma, 1996). Potassium plays a crucial role in nerve impulses,
cardiac function, maintenance of osmotic pressure, protein synthesis and muscular
activity. Zinc is necessary for wound healing, normal growth and reproduction
(Murugesh, 1998). Magnesium is a constituent of bones, dental enamel and dentin and
effective in treating depression. Manganese is essential for normal bone structure and
functioning of central nervous system (Deb, 2004). Sanchez (2005) observed 1.4%
nitrogen and 0.11% phosphorus in the fresh leaves of Faramea occidentalis, a Rubiaceae
member. Thirumurugan et al. (2008) reported that Mallotus philippinensis fruit wall
contain sodium, potassium, magnesium, manganese, calcium, zinc, iron, copper and
cobalt. Osman (2010) reported that the leaves of Olea europaea contain nitrogen,
phosphorus, potassium, iron, manganese, calcium, zinc and copper. Nitrogen,
phosphorus, sulphur, sodium, potassium, calcium, lithium, iron, manganese, zinc and
copper were present in the leaves of Pavetta indica (Prasad and Bisht, 2011).
2.3.3. Heavy metal Analysis
Jordan (1975) documented that increasing concentration of cadmium inhibited the
plant growth, internal water deficit in the shoot system and resulted in poor development
of root system. Burnzynski (1988) reported that the young seedlings or two week old
plants placed in lead chloride showed a significant decrease in transpiration and uptake of
water. Moreno et al. (1993) reported that the contents of cadmium, zinc, lead and copper
11
were higher in areas of greatest industrial activity. Heavy metals occur in soil, water and
plants are readily mobilized by human activities that include mining and discarding
industrialized waste materials in natural ecosystems that include forests (Larison et al.,
2000). Heavy metals have been implicated in forest decline because their deposition
pattern is correlated with forest loss (Ravindran et al., 2005).
2.3.4. Vitamin Studies
Vitamins E and B and minerals such as manganese, copper, selenium and zinc are
nonenzymatic antioxidants (Cadenas and Packer, 2001). Jacab and Sotoudeh (2002)
found that the vitamins are reducing the damage caused by free radicals and check
degenerative disease. Vitamin B12 is the largest and most complex of all the vitamins.
Cobalamin is the term used to refer to a group of cobalt containing compounds
(Watanabe, 2007). Srilakshmi (2008) reported that decarboxylation, deamination,
transamination, nicotinicacid formation and haemoglobin synthesis are carried out by
vitamin B6 and vitamins E and C have antioxidant properties. 2.3.5. Phytochemistry
Farnsworth and Morris (1976) opined that many secondary metabolites cannot be
synthesized on a commercial basis. Phytochemical screening of Strumpfia maritima
flowering tops revealed the presence of sterols, tannins, polyphenols and flavonoid
glycosides (Hsu et al. 1981). Mothes et al. (1985) reported that Berberidaceae,
Leguminosae, Boraginaceae, Apocynaceae, Asclepiadaceae, Liliaceae, Gnetaceae,
Ranunculaceae, Rubiaceae, Solanaceae and Rutaceae members contain rich alkaloid in
their plant parts. Nagakura et al. (1993) reported that Cephaelis ipecacuanha stem,
leaves, flowers and fruits contain isoquinoline alkaloids with varying amounts. Indole
alkaloids, the chemotaxonomic markers, iridoids, triterpenes and anthraquinones are
remarkably distinctive in Rubiaceae family (Young et al., 1996). Phenolics are of great
importance as cellular support materials and form an integral part of cell wall structure,
with which the plants become adapted to terrestrial life by building rigid organs (Dey and
Harborne, 1997). Terpene content exhibits antimicrobial activity (Toyota and Asakawa,
1999).
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Niranjan et al. (2000) reported that Anthocephalus cadamba bark contains
triterpenes, terpenoid glycosides, saponins and indole alkaloids. Antioxidants such as
polyphenols play an important role in adsorbing and neutralizing free radicals (Anderson
et al., 2001). Both enzymatic and non-enzymatic antioxidant system scavenge and
deactivate excessive free radicals, helping to prevent cell damage (Cadenas and Packer,
2001). Hamerski et al. (2003) reported that Randia dumetorum contains phenolics.
Galium aparine, a Rubiaceae member contains tannins, phenolicacids, flavonoids and
iridoid glycosides in leaves and stem (Vanwyk and Wink, 2004). Lopes et al. (2004)
reported that leaves of Chiococca braquiata, a Rubiaceae member contain flavonoid.
Heitzman et al. (2005) reported that Uncaria species contains alkaloids, triterpenes,
glycosides, flavonoids and Coumarins.
Rubiaceae species are known to be used as bioproducers of alkaloids, tannins,
saponins, steroids, fatty acids, fatty alcohols, terpenes and flavonoids. Some species are
important in traditional medicine (Mongrand et al., 2005). Djeridane et al. (2006)
reported high total phenolics in Saharan plants. Herbal products have played an important
role in the development of drugs for various diseases. The secondary metabolites from
natural source are good candidates for they are perceived to exhibit more similarities to
drugs and show more biological friendliness than totally synthetic drugs (Shoeb, 2006).
Abere et al. (2007) evaluated Mitracarpus scaber leaves as they contain alkaloids,
tannins, cardiac glycosides and saponins. Phenolic phytochemicals are secondary
metabolites of plant origin which constitute one of the most abundant groups of natural
metabolites and are synthesized by plants in order to protect themselves from biological
and environmental stresses. Phenolic compounds possess high antioxidant activity.
Daniel (2008) studied quinoline alkaloids in the bark of Cinchona officinalis,
Cinchona calisaya, Cinchona succirubra, Cinchona ledgeriana and Remijia pedunculata.
He also reported that the leaves and stem bark of Gardenia gummifera contain flavones
such as sitosterol, gardenin and erythrodiol. Gardenia resinifera resin contains flavones.
Rubia cordifolia contains two anthraquinones, purpurin and xanthopurpurin which are
helpful in curing skin diseases and improving complexion. Kannan et al. (2009) reported
alkaloids, cardiac glycosides, tannins, flavonoids, phenols and trace amount of steroids in
13
Rubia cordifolia. The chemical constituents present in Paederia foetida are iridoid
glycosides, sitosterol, stigmasterol alkaloids, carbohydrates, protein, aminoacid and
volatile oil (Kumar et al., 2009).
Singh et al. (2010) reported mannitol and saponins from the fruits of Randia
spinosa. Rubiaceae family is considered as one of the biggest families among the
angiosperms, comprising around 637 genera and approximently 10,700 species, which
are of great importance to the food, ornamental and pharmaeceutical industries (Karou et
al., 2011).
2.3.6. HPTLC (High Performance Thin Layer Chromatography) Analysis
Laus and Teppner (1996) determined qualitatively by HPTLC method, the indole
and oxyindole alkaloids in the different parts of Uncaria rhynchophylla.
A rapid and simple high performance thin layer chromatography (HPTLC)
method has been developed for the simultaneous quantitative estimation of the
biologically active diterpenoids - 14 - decoxy - 11, 12-didehydroandrographolide,
andrographolide, neoandrographolide and andrographiside in Andrographis paniculata.
The assay combines the isolation and separation of andrographolide derivatives on silica
gel 60 F254 HPTLC plates with spot visualization and scanning at 540nm. Methanol was
found to be the most appropriate solvent for the exhaustive extraction of andrographolide
derivatives (Saxena et al., 2000). Sasikumar et al. (2009) reported that Pandanus
odoratissimus root on UV derivatization showed brown colour zone which confirms the
presence of polyphenol by HPTLC method.
Singh (2010) studied the phytochemicals of Sonchus oleraceus leaves and
Citrullus colocynthis root and reported that quercetin has antiinflammatory, antioxidant
and anticancer properties and isolation and identification by HPTLC method can be used
further for qualitative and quantitative analysis of flavonoids in medicinally important
plant. Fractionation methodologies were developed and standardized for isolation of
components rich in polysaccharides, anthraquinones and alkaloids. The TLC studies
confirmed the presence of scopoletin, an important marker in the identification of
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Morinda fruits. This study can serve as a guideline for laying down specifications for
dried Morinda fruits as well as fruit powder so as to prevent the adulteration of the raw
material (Nayak and Mengi, 2010). Choudhury and Choudhury (2011) reported that
Lasianthus lucidus leaf contain alkaloids, tannins, glycosides, anthraquinones, fixed oil,
saponins and flavonoids.
2.3.7. GC-MS Analysis
The chemical composition of the volatile oil constituent from Pulicaria odora L.
roots has been analysed by GC-MS. Twenty-seven components were identified, thymol
(47.83%) and its derivative isobutyrate (30.05%) are the main constituents in the oil and
the oil exhibited a significant antibacterial activity (Hanbali et al., 2005).
Four tocoperols, three sterols, amyrine, cycloartenol, actinidiolide and linolenic
acid were identified by GC-MS method in four Staphylea species (Lacikova et al., 2007).
GC-MS analysis of ethyl acetate extract of Goniothalamus umbrosus revealed the
existence of 1-butyl-2-cyclohexenol (46.84%), benzaldehyde (4.42%) and Globulol
(4.07%) (Abdelwahab et al., 2009).
GC-MS of the volatile components of the aerial part of Macfadyena unguis-cati
revealed the presence of 74 compounds, 52 (75.97%) of them were identified. The major
compound was n-decane (12.12%) followed by phytol (12.19%). The saponifiable
fraction of the petroleum ether extract contained 21 fatty acids, identified as methyl
esters. In the unsaponifiable fraction, 37 compounds (representing 93.26%) were
identified. β-amylin, squalene, β-sitosterol and 3α, 5-cyclo-ergosta-7, 22-dien-6-one were
the major compounds. The total ethanol was found to be the most potent as antipyretic,
followed by ethyl acetate extract. The ethanol extract, as well as the coumarin containing
fraction exhibited a significant analgesic activity (Aboutab et al., 2010). Mussaenda
frondosa plant contains 20 different chemical constituents (Gopalakrishnan and Vadivel,
2011) by GC-MS analysis. The ethanol extract of wood and bark of Pterocarpus
marsupium has been subjected to GC-MS analysis. Eight phytochemical constituents
have been identified in wood and bark respectively. The major chemical constituents are
15
3-0-methyl-d-glucose, n-Hexadecanoic acid, 1, 2- Benzene-dicarboxylic acid, diisooctyl
ester, tetradecanoic acid and 9, 12- Octadecadienoic acid (Z, Z) in wood. D-Friedoolean-
14-en-3-one and lupeal were the main constituents in the bark (Maruthupandian and
Mohan, 2011).
Rajeswari et al. (2011) reported that the leaves of Canthium dicoccum were
characterized by substantial levels of sesquiterpenoids, nitrogenous compounds,
aldehydes, terpinolene and phenol constituents possess antimicrobial, antitumour and
antioxidant properties. Ramalakshmi and Muthuchelian (2011) have reported 18 chemical
constituents from the ethanolic leaf extract of Tabebuia rosea by GC-MS analysis. The
extract of Tabebuia rosea was characterized by various types of active compounds such
as aromatic aldehydes, sugar, aromatic compounds, terpenoids, quinone, alkanes,
phenolics and flavonoid. A total of 29 compounds from leaves and roots of Premna
serratifolia have been identified by GC-MS technique (Singh et al., 2011). GC-MS
technology can be used in fire investigation, explosive investigation, identification of
unknown samples and to detect substances in luggage (Manjamalai et al., 2011).
Mothana et al. (2011) analysed the various components in Boswellia species by GC-MS
techniques.
2.4. Pharmacological Investigation
2.4.1. Antimicrobial Activity
Usually most of the gram negative bacteria are more resistant than gram positive
bacteria (Barberan, 1988). Several phytoconstituents like flavonoids, phenolics and
polyphenols, tannins, terpenoids and sesquiterpenes healing the wounds and are effective
antimicrobial substances against a wide range of microorganisms (Scortichini and
Piarossi, 1991). Escherichia coli causes septicemias and can infect the gallbladder,
meninges, surgical wounds, skin and the lungs (Black, 1996). Several flavonoids and
phenolic acids have exhibited interesting antiviral and antimicrobial properties both in
vitro and in vivo (Colombo and Bosisio, 1996).
Darokar et al. (1998) reported that Hamelia patens and Mussaenda frondosa of
Rubiaceae exhibited activity against E.coli and showed 4mm and 3mm zone of inhibition
16
respectively. Plants are a rich source of wide variety of secondary metabolites such as
tannins, terpenoids, alkaloids, flavonoids, phenols and essential oils which have been
found to have antimicrobial properties. Many natural products including pigments,
enzymes and bioactive components are soluble in water (Cowan, 1999). Antibiotics are
reported to have inhibitory effects on cell wall synthesis and nucleic acid production
(Hammer, 1999). Muller-Hinton agar medium appears to be the best medium to explicate
the antibacterial activity (Lin et al., 1999).
Caesalpinia digyna fruit extract inhibited the growth of Streptococcus aureus,
Candida albicans and E.coli due to the presence of gallic acid, tannin and lupeol in
plant and showed 200-800 µgmL-1 MIC (Rastogi and Mehrotra, 1999). Proteus mirabilis
causes wound infections and urinary tract infection in the elderly and young males
(Cheesebrough, 2000).
Houghton et al. (2000) have reported that the isolated aza anthraquinone alkaloid
from Mitracarpus scaber had been shown to exhibited activity against Staphylococcus
aureus and Bacillus subtilis. The crude protein extracts from the leaves of Rauwolfia
tetraphylla inhibited Aspergillus flavus after 72 hrs (Thapliyal et al., 2000). The presence
of bioactive substances have been reported to confer resistance to plants against bacteria,
fungi and pests (Srinivasan et al., 2001). Sesquiterpene lactones from Vernonia colorata
possessed high activity against gram positive and low activity against gram negative
species (Rabe et al., 2002).
Subramani et al. (2004) stated that phenolic acids and flavonoids are active
against fungi like Aspergillus niger, Curvularia lunata and Curvularia gloeosporioides.
Organic extracts are more active than aqueous extracts againt microorganisms due to the
better solubility of the active components in organic solvents (de Boer et al., 2005).
Mandal et al. (2007) evaluated the antimicrobial activity of Hyptis sp. and reported that
the leaf extract exhibited broad spectrum activity against Aspergillus sp. and Micrococcus
sp. The dichloromethane and methanol extract of Gonzalagunia rosea of Rubiaceae
displayed moderate and strong activities simultaneously against Candida albicans and
Fusarium solani respectively (Nino et al., 2007).
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Sanjay et al. (2007) reported that alcoholic and aqueous extracts of Anthocephalus
cadamba showed a significant antibacterial and antifungal activity against almost all the
organisms. Mahesh and Satish (2008) reported that Aspergillus flavus was inhibited by
(12mm zone of inhibition ) Acacia nilotica leaf extract. Odunbaku and Illusanya (2008)
reported that Morinda lucida leaf extract showed inhibitory activity against E.coli and
Pseudomonas aeruginosa.
Bohra and Bohra (2009) reported that fresh aqueous garlic bulb extract showed
more inhibition zone than the dried bulb. Duraipandiyan et al. (2009) reported that
Aspegillus niger and Candida albicans were inhibited by n-hexane extract of
Sphaeranthus indicus. The aqueous, alkaloid and methanol extract showed antimicrobial
activity against Salmonella typhi and Bacillus subtilis. The minimum inhibitory
concentration of extracts determined ranged from 3.12 to 6.25 mg/ml. The alkaloid
extract was found to be the most effective against all the tested micro organisms
(Parthasarathy et al. 2009).
Soneja et al. (2009) reported that the ethanol fruit extract of Mitragyna sp. did not
exhibit any antibacterial potential against Staphylococcus aureus, Bacillus subtilis, E.coli
and P.aeruginosa. Kumar et al. (2010) reported that the alcoholic extract exhibited a
significant antibacterial and antifungal activity as compared to the standard drug
tetracycline. Basavaraja et al. (2011) stated that Mussaenda frondosa bark extract
exhibited almost equipotent antibacterial activity as compared with that of standard drug
and exhibited potent antifungal activity as compared with that of standard clotrimazole.
Bhardwaj (2011) reported that aqueous extracts of Indian medicinal plants highly
active against Alternaria brassicae. Karou et al. (2011) stated that more than 60 species
of Rubiaceae are used for medicinal indication including eczema, oedema, cough and
hepatitis. Many plants exhibited antimicrobial activities as they contain indole alkaloid,
terpenoids and anthraquinones especially in Nauclea, Morinda, Mitragyna and
Crossopteryx species.
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2.4.2. Wound Healing Activity
Baboir (1978) investigated that the free radical scavenging enzymes (FRSE) are a
cytoprotective enzymal group that has an essential role in the reduction, deactivation and
removal of ROS (reactive oxygen species) as well as in the regulation of the wound
healing process. These cells through their characteristic respiratory burst activity produce
free radicals. Smith (1985) stated that any agent which accelerates the process is a
promoter of wound healing.
Wound healing involves different phases such as contraction, epithelialization,
granulation and collagenation (Purna et al., 1995). Leaf infusion of Tarenna asiatica is
given orally in plough injuries (Sudarsanam et al., 1995). Wound healing is an anabolic
process that requires both energy and nutritive substances. It is reported that serum
albumin level of 3.5g or more is necessary for proper healing (Hanna and Giacopelli,
1997) and protein is essential for collagen synthesis on wound site. Fibroblasts produce a
variety of substances essential for wound repair including glycosaminoglycans and
collagen (Stadelmann et al. 1998). Nayak (1999) reported that Ixora coccinea flowers
healing the wounds in rats. Participation of various inflammatory cells such as
macrophages and neutrophils is extremely crucial to the repair process (Rasik et
al.,1999). Sidhu et al. (1999) reported that repair of injured tissues occurs as a sequence of
events which include inflammation, proliferation and migration of different cell types.
Wound related non-phagocytic cells also generate free-radicals by involving non-
phagocytic NADPH oxidase mechanism (Griendling, 2000). Phenolic acids from
Chromolaena sp. (Phan et al., 2001), β-sitosterol from Aloe vera (Krishnan, 2006) and
proanthocyanidins resveratrol from Vitis vinifera (Khanna et al., 2002) are few important
plant derived wound healing compounds which were tested in animal model. The fresh
leaf is used for the treatment of wounds and also as a poultice for broken bones and
sprains (Ross, 2001).
Several antioxidants such as ascorbic acid and catalase were found to heal the
wounds. The presence of β-carotene, flavonol glycosides and iridoid glycosides in
19
Morinda citrifolia leaf extract is decreasing the level of lipid peroxide in treated wounds
(Sang et al. 2001). Diallo et al. (2002) stated that polysaccharides are partly responsible
for the process of wound healing. Any drug that inhibits lipid peroxidation is believed to
increase the viability of collagen fibres, increasing the circulation, preventing the cell
damage and promoting the DNA synthesis (Getie et al., 2002). Essential oils obtained
from the various parts of the plant have a long history of use for treating wounds, skin
abrasions, excoriations, skin infections and other topical health problems (Kerr, 2002).
Wound healing involves coagulation, inflammation, formation of granulation
tissue, matrix formation, remodeling of connective tissue, collagenization and acquisition
of wound strength (Reddy et al., 2002). Beloz et al. (2003) stated that Hamelia patens
increased the breaking strength of wounds by double incision wound healing assay.
Triterpenoid is reported to possess an ability to increase the collagen content, which is
one of the important factors promoting wound healing (Joshi et al., 2003). 70% of the
wound healing Ayurvedic drugs are of the plant origin, 20% of mineral origin and the
remaining 10% consisting of animal products and the drugs stated to be effective in
different conditions such as wounds, ulcers, abscess and erysipelas (Biswas and
Mukherjee, 2003). Somava et al. (2003) reported that triterpenoids are known to promote
wound healing process due to their antioxidant activity.
Topical applications of compounds with free-radicals scavenging properties in
patients have shown to improve significantly wound healing and protect tissues from
oxidative damage (Thiem and Grosslinka, 2003). Mohideen et al. (2003) reported that
aqueous and ethanol extracts of leaves of Canthium parviflorum applied topically on
excision wounds in rats showed significant healing process as evidenced by increased
rate of wound contraction. Gulcin et al. (2004) reported that saponins have antioxidant
activity and promote wound healing.
Leaves are mostly used in wound healing as they are active in photosynthesis and
production of metabolites (Ghorbani, 2005). Manjunatha et al. (2005) have reported that
an aqueous and methanol leaf extracts of Vernonia arborea promoted high rate of wound
contraction on 18th day (100% in methanol extract), skin breaking strength (494.41±4.3,
20
463.74±3.63) in the wistar rats. A combination of antioxidants comprising vitamin E,
sodium pyruvate and fatty acids have been employed for enhancing healing in normal
and immunocompromised wounds (Soneja et al., 2005).
Wound healing processs is categorized into inflammatory phase, proliferated
phase and remodeling phase which ultimately determines the strength and appearance of
the healed tissue (Sumitra et al., 2005). They also reported that wound healing is
promoted by triterpene, alkaloid, flavonoids, and biomolecules. Sanjay et al. (2007)
evaluated the Anthocephalus cadamba plant extract on excision and incision wound
model (rat) and reported that the plant extract has potent wound healing capacity since it
contracts the wound and increased the tensile strength.
Sussman (2007) reported that haemorrheologics, retinoids, phenytoin, vitamin A,
vitamin C, zinc and some growth factors are the drugs which are having the potential of
improving the healing of wounds. Rasal et al. (2008) investigated the effect of the
aqueous extract of Morinda citrifolia leaves on experimental wounds and reported that
the extract accelerated the healing process of wounds.
Ixora coccinea, Morinda pubescens, Vitex altissima, Scoparia dulcis, Euphorbia
hirta and Cleome viscosa were studied in animal models for wound healing and anti
inflammatory activity on the basis of their use in traditional medicine and these plants can
be used to formulate drugs in pharmaceutical companies (Ayyanar and Ignacimuthu,
2009). Chopda and Mahajan (2009) reported that Anthocephalus cadamba, Borreria
hispida, Chasalia chartacea, Morinda citrifolia, Oldenlandia biflora, Pavetta indica,
Randia dumetorum, Rubia cordifolia and Tarenna asiatica plants of Rubiaceae are used
for wound healing in human.
Karodi et al. (2009) have reported that Rubia cordifolia extract on wound
contracting ability, wound closure, decrease in surface area of wound and tissue
regeneration at the wound site was significant in treated mice. Sheeba et al. (2009)
evaluated the methanol crude extract of Cassia occidentalis leaves and a pure compound
chrysophanol isolated from it and reported that chrysophanol was found to possess
21
significant wound healing and tensile strength (381.04±0.83) property and Cassia
occidentalis can be a good source of wound healing compound.
Ezike et al. (2010) studied the beneficial effects of the stem bark of Prosopis
africana in wound care and reported that methanol extract of the stem bark of Prosopis
africana significantly reduced bleeding, clotting, coagulation and the epithelialization
period of excision wounds in rats. Joshi et al. (2010) reported that the aqueous extract of
Mussaenda frondosa leaves significantly increased the wound breaking strength in the
incision wound model. Paul et al. (2010) demonstrated that chloroform and methanol
extracts of Mimosa pudica root significantly healing the wounds and increasing the
tensile strength of skin (545.16±12.3) in wistar rats.
Nirmala and Karthiyayini (2011) stated that the alcoholic and aqueous extracts
ointment of Achillea millefolium produced complete healing on 18th (97.9%) and 20th day
(98.85%) by 10% w/w extract ointment respectively and the tensile strength was
528.28±38.4g in alcoholic extract ointment. Patil and Joshi (2011) stated that Mussaenda
frondosa leaves significantly contracted the wounds on day 16 and showed 293g tensile
strength.
Senthil et al. (2011) observed a significant wound healing activity of Guazuma
ulmifolia alcoholic extract on albino wistar rat and reported that terpenoid, tannins,
saponins, alkaloids and flavonoids are known to promote wound healing process due to
their antioxidant and antimicrobial activities. Suruse et al. (2011) reported that alcoholic
extract of tubers of Arisaema leschenaultii showed better wound healing potential (99.6%
on 18th day) than control group. In the incision wound model a significant increase in
breaking strength was observed. They also suggested that the tubers may be useful in the
management of abnormal healing and hypertropic scars.
2.4.3. Antioxidant Activity
Oxygen derived free radicals and their products are known to play an important
role in the pathogenesis of chronic inflammatory disorders. The reduction in superoxide
dismutase activity increases the toxic effects of oxygen which lead to severe cellular
22
damage (Fridovich, 1978). Danh et al. (1983) reported that GSH-PX is responsible for
decomposition of H2O2. Catalase prevents chromosomal aberration caused by
hypoxanthine/xanthine oxidase in Chinese hamster cells (Iwata et al., 1984). Reactive
oxygen species excess has a harmful effect on the peroxidation of the membrane lipids,
aggression to tissue proteins and membranes on damage to DNA and enzymes (Husain et
al., 1987).
Glutathione peroxidase (GPx) is a general name of enzyme family with
peroxidase activity whose main biological role is to protect the organism from oxidative
damage. The biochemical function of glutathione peroxidase is to reduce lipid
hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to
water (Meister, 1988). Robak and Glyglewski (1988) reported that flavonoids are
effective antioxidants mainly because they scavenge superoxide anions. Biological
membrane lipids are susceptible to peroxidative attack (Cheesman, 1993). The toxicity of
nitric oxide increases greatly when it reacts with superoxide radical, forming the highly
reactive peroxynitrite (Huie and Padmaja, 1993). Glutathione peroxidase eliminates H2O2
(Quinlan et al., 1994).
Overproduction of ROS and for inadequate antioxidant defense can easily affect
and persuade oxidative damage to various biomolecules including proteins, lipids,
lipoproteins and DNA (Farber, 1994). Metal Chelating agents play a vital role in the
stabilization of fatty acids against rancidity (Yen and Duh, 1994). Reduced glutathione is
a major low molecular weight scavenger of free radicals in the cytoplasm and an
important inhibitor of free radical mediated lipid peroxidation (Halliwell, 1995). The
antioxidants are known to mediate their effect by directly reducing the ROS, quenching
them and/or chelating the catalytic metal ions (Robak and Marcinkiewicz, 1995).
Flavonoids are a group of polyphenolic compounds with known properties, which
include free radical scavenging, inhibition of hydrolytic and oxidative enzymes and anti-
inflammatory action (Frankel, 1995). Catalase is a H2O2 scavenging enzyme with
optimate activity at high H2O2 concentrations (Kinnula et al., 1995). Cook and Samman
23
(1996) reported that phenolic compounds are very important plant constituents because
their hydroxyl groups confer scavenging ability.
Superoxide dismutase and catalase are antioxidant enzymes, involved in direct
elimination of reactive oxygen metabolites (Halliwell and Gutteridge, 1997). The levels
of reactive oxygen species are controlled by antioxidant enzymes, such as superoxide
dismutase and glutathione peroxidase. Serum malondialdehyde level as a marker for lipid
peroxidation is used as an indicator for oxidative damage in cells and tissues (Mano et
al., 1997). Rubia cordifolia has been reported to possess a significant antioxidant activity
in vitro studies (Tripathi et al., 1998).
Quercetin, a ubiquitous bioactive flavonoid, can inhibit the proliferation of cancer
cells. The antitumour effects of plant flavonoids have been reported to induce cell growth
inhibition and apoptosis in a variety of cancer cells (Dicarlo et al., 1999). Duh et al.
(1999) stated metal chelating capacity is significant in Chrysanthemum species, since it
reduced the concentration of the transition metal that catalyses lipid peroxidation. A
strong relationship between total phenolic content and antioxidant activity has been
reported by Kahkonen et al. (1999).
The antioxidant system is composed of superoxide dismutase which converts the
superoxide radical into hydrogen peroxide and catalase and glutathione peroxidase
(GPx), both of which detoxify the hydrogen peroxide (Halliwell, 2001). The enzymatic
and non-enzymatic antioxidant defense systems allow the scavenging of reactive oxygen
and nitrogen species. Catalase, glutathione peroxidase and superoxide dismutase need a
small concentration of minerals to show their optimal enzymatic activity. Ascorbate, β-
carotene and minerals generally do not accumulate in the body.
The body’s antioxidant system is influenced by dietary intake of non-enzymatic
antioxidants such as manganese, copper, selenium, zinc, beta-carotenes, Vitamin C,
Vitamin E, taurine, hypotaurine and B Vitamins. Glutathione, a tripeptide is produced by
the cell and plays a crucial role in maintaining the normal balance between oxidation and
antioxidation. Both enzymatic and non-enzymatic antioxidant systems scavenge and
24
deactivate the excessive free radicals, helping to prevent cell damage (Cadenas and
Packer, 2001).
Triterpenoids from Boswellia regia have been shown to have anti-elastase activity
(Melzig et al., 2001). Sang et al. (2001) stated that lipid peroxide level is decreased in
Morinda citrifolia leaf extract treated animal because of the presence of β-carotene,
flavonolglycosides and iridoid glycosides which have antioxidant activity.
Kumaraguruparan et al. (2002) reported that elevated level of serum lipid peroxide in
breast carcinoma may be due to defective antioxidant system.
Beal (2002) studied that reactive oxygen species can cause oxidation of the side
chains of lysine, proline, arginine and threonine residues by binding ferrous ion and
copper state. The reducing power of bioactive components is associated with antioxidant
activity (Siddhuraju et al., 2002). SOD and CAT can counteract the deleterious actions of
reactive oxygen species and protect tissues against cellular and molecular damage (Singh
et al., 2003). The antioxidant compounds are anthocyanin, carotenoids, ascorbic acid,
tannins, benzoic acid and mallic acid (Lugasi and Hovari, 2003).
The balance between the formation and removal of lipid peroxides determines the
peroxide level in cells. This balance can be disturbed if cellular defences are decreased or
if there is a significant increase in peroxidative reactions (Karbownik and Lewnski,
2003). Joharapurkar et al. (2003) reported that ethanolic extract of Rubia cordifolia has
antioxidant activity. Flavonoids show antioxidant activity and their effects on human
nutrition and healths are considerable. The mechanisms of action of flavonoids are
through scavenging or chelating process. The oxygen radiant scavenging activity of
glutathione directly facilitates ROS neutralization and the repair of ROS induced damage
(Pastore et al., 2003).
Galium aparine, a Rubiaceae member contains tannins, phenolic acids, flavonoids
and iridoid glycosides in leaves and stem (Vanwyk and Wink, 2004). As plants produce a
huge amount of antioxidants they can represent a source of new compounds with
antioxidant activities (Bassman, 2004). Flavonoids, phenols, tannins and terpenoids in the
25
plants exhibit antioxidant activity (Aderogba et al., 2005). Phenolic constituents found in
vegetables have antioxidant activity and play an important role in the adsorption or
neutralization of free radicals (Basile et al., 2005).
Polyphenol isolated from Diospyros sp. leaf showed anti-collagenolytic and anti-
elastase activity. This activity was thought to be due to the flavonoids present in the
polyphenol extract (An et al., 2005). Berberis vulgaris fruit extract is scavenging the
DPPH radicals (Motalleb et al., 2005). DPPH assay measures the ability of antioxidants
to scavenge free radicals which is not directly associated with the real oxidative
degradation or effects of transient metals (Roginsky and Lissi, 2005).
Oxidative stress can cause damage to lipids, proteins, carbohydrates and nucleic
acids (Pryor et al., 2006). Vitamin E, a potent chain breaking lipid soluble antioxidant
reacts with lipid peroxyl radicals eventually terminating the peroxidation chain reaction
and thereby reducing oxidative damage (Stambullian et al., 2007). Anthocephalus
cadamba plant extract possesses potent antioxidant activity by inhibiting lipid
peroxidation and increasing the superoxide dismutase and catalase activity (Sanjay et al.,
2007).
Rajneesh et al. (2008) have observed an increased level of lipid peroxidation in
the plasma of patients of breast cancer. Dutra et al. (2008) reported that the essential oil
of Pterodon emarginatus seeds containing phenol content, showed DPPH scavenging
activities with IC50 value 163.22.
Rasal et al. (2008) reported that antioxidants such as ascorbic acid and catalase
improve the dermal healing process in rat and the catalase detoxifies hydrogen peroxide
which can inflict severe damage to regenerating cells. Free radical scavenging capacities
of the essential oil of Thymus fallax measured in DPPH and essential oil has strong
antioxidant activity. IC50 value of Thymus fallax essential oil was found to be 215mg/ml,
while IC50 value for Butylated hydroxy toluene (BHT) was found to be 10.5 mg/ml (Goze
et al., 2009) Mothana et al. (2009) reported that 1000μg Acacia pennivenia methanol
extract exhibited 94% free radical scavenging effect. The methanol extract of the
26
Mitragyna leaf exhibited a significant dose dependent inhibition (IC50) at a concentration
of 37.08mg/ml (Parthasarathy et al., 2009).
2.4.4. Skeletal Muscle Relaxant Activity
The decrease in motor activity gives an indication of the level of excitability of
the CNS and this decrease maybe related to sedation resulting from depression of CNS
and the observed CNS depressant activity maybe due to flavonoids, tannins, saponins,
steroids in the plant extract (Masur et al., 1971). Parthenin, one of the major components
of methanolic extract shows depolarizing neuromuscular junction blocking effect (Porsolt
et al., 1978).
Rakotonirina et al. (2001) reported that rotarod test mainly used to screen
centrally acting muscle relaxants. Diazepam adversely affects the respiratory, digestive
and immune system of body and the chronic treatment with Diazepam often proved more
harmful in the longer run (Dhawan et al., 2003). Chattopadhyay et al. (2003) reported
that triterpenoids produced CNS depressant action.
Hosseinzadeh and Asl (2003) reported that Carbenoxolone (400mg/kg) showed
muscle relaxant activity and a decline in motor coordination. The muscle relaxant activity
in animals could be due to the interaction of isoflavonoids with the benzodiazepine
receptor complex in brain (Trofimiuk et al., 2005). Isoflavonoids and their metabolites
from Pueraria lobata showed muscle relaxant activity in animal models (Yasuda et al.,
2005).
The ethanolic extract (150mg/kg) of Pachyrrhizus erosus seeds reduced the time
(199sec.) spent by the animals on revolving rod and the low dose of drug (75mg/kg) did
not show any significant effect (Abid et al., 2006). Kumar et al. (2008) reported that the
methanol extract of Careya arborea at 200mg/kg significantly (80%) reduced the motor
coordination.
The skeletal muscle relaxation activity (21sec.) by rotarod method showed more
relaxation in methanolic extract of Leucas longifolia at 400mg/kg (Pranit et al., 2010).
27
Rathor and Ram (2010) reported that Churna prepared from plants exhibited a marked
reduction in motor coordination in mice after an oral administration of 20mg/kg and the
mean reduction time after drug administration was 130.05±6.42 seconds.
Ganatra et al. (2011) revealed that the methanolic leaf extract (200mg/kg) of
Hibiscus rosa-sinensis reduced the time of fall (17sec.) when compared with control thus
showing skeletal muscle relaxant effect. The methanolic leaf extract of Parthenium
hysterophorus significantly reduced the fall off time (motor coordination) at 30 min
duration and the activity is due to the presence of different chemical compounds present
in the extract (Jha et al., 2011). Kumar et al. (2011) reported that Rumex nepalensis leaf
extract at a dose of 400mg/kg showed highly significant skeletal muscle relaxant activity
at 30 min of duration.
Deoliveira et al. (2012) detected that Octacosanol treated mice did not show any
significant motor performance alterations with the dose of 10mg/kg (232.02±39.03) as
compared to saline treated animals (205.33±37.95) in the rota rod test. The positive
control Diazepam, the most widely used benzodiazepine derivative, causes sedation and
inhibits locomotor activity and reduced the motor performance time of mice after 30min
of treatment (66.25±15.39s).
2.4.5. Antispasmodic Activity
Armitage et al. (1961) have shown that promethazine gave good protection
against histamine aerosols and poor protection against acetylcholine aerosols. Broadbent
and Bain (1964) reported that histamine antagonists can be conveniently assayed by their
ability to protect animals against lethal effects of histamine induced bronchospasm.
Histamine, acetylcholine, leukotrienes and prostaglandins mediators directly cause acute
bronchoconstriction (Bosquet et al., 2000). Secondary metabolities can regulate the
disturbances of gastro intestinal tract such as inhibition of guinea pig intestinal peristalsis
by the flavonoids, quercetin, naringenin, apigenin and genistein (Gharzouli and Holzer,
2004). Aqueous extract of radish seeds which tested positive for terpenes, flavonoids,
28
phenols, alkaloids and saponins showed spasmogenic effect in isolated rabbit jejunum
and ileum (Ghayur et al., 2005).
Goze et al. (2009) reported that Thymus fallax essential oil has inhibited both
amplitude and frequency of Spontaneous contraction. The extract of Helichrysum
plicatum significantly inhibited the histamine, barium and potassium ions induced
contractions in a concentration dependent manner (Bigovic et al., 2010). Polyherbal
formulation prepared from petroleum ether leaf extracts of Solanum xanthocarpum,
Murraya konigii, Aegle marmelos and Caesalpinia bondac protected the wistar rats to a
significant extent from the development of asphyxia produced by histamine aerosol
(Parmar et al., 2010). Kumar et al. (2010) studied the effect of methanolic extract of stem
barks of Ailanthus excelsa and reported that the extract has significantly prolonged the
latent period of convulsions followed by exposure to histamine aerosol at the dose of 400
mg /kg and showed maximum protection of 59.4 % at 4th hour.
Cimanga et al. (2010) reported that Morinda morindoides leaf extract possesses
spasmogenic and spasmolytic properties. Ninave et al. (2011) have observed that the
ethanolic extract of Randia dumetorum (110. 25, 220.50 and 441 mg/kg p.o) significantly
prolonged the latent period of convulsions following exposure to histamine aerosol at 1st
and 4th hour. Chauhan et al. (2012) evaluated the histamine induced bronchospasm of
alcoholic extract of Clitoria ternatea roots in experimental animals and reported that
Clitoria ternatea shows 47.45% protection against histamine induced broncho
constriction in rats.
2.5. In vitro Regeneration Technique
Multiple shoot formation and their elongation from excised apical vegetative
shoots of a 40 year old tree of Mitragyna parvifolia was achieved in MS medium
supplemented with Benzyl adenine. The in vitro regenerated shoots are rooted when
cultured on modified medium containing low inorganic salts and auxin. Regeneration by
this method was suitable for mass propagation of this plant (Roy et al., 1988). Huetteman
and Preece (1993) reported that Thidiazuron is known to be effective for woody plant
29
tissue culture which can stimulate shoot proliferation in many recalcitrant species such as
Cercis canadensis var.alba, muscadine grape and Quercus suber.
Stem cuttings of Ixora coccinea cultured on woody plant medium containing 2.5
µm BA produced axillary shoots which branched repeatedly, yielding an average of 27
shoots per explant after 6 weeks in culture (Lakshmanan et al., 1997). Leaves of
Gardenia Jasminoides cultured on MS medium supplemented with Thidiazuron (10 µm
TDZ) and indole acetic acid produced calli and when these calli were transformed to the
MS medium containing Benzylaminopurine (BAP), Thidiazuron and Zeatin, longest
shoots were produced and highest root number, longest roots and highest rooting
percentage occurred at the highest concentration of auxin used (AlJuboory et al., 1998).
Handique and Bhattacharjee (2000) revealed that the lower concentration of BAP + KIN
(kinetin) favours good response in shoot proliferation of wood apple.
The efficacy of NAA (Naphthalene aceticacid) at lower concentrations in vitro
rooting has been reported in Verbascum thapsus (Turker et al., 2001). Noreen et al.
(2001) reported that callus initiation was significantly higher in MS medium having 2, 4
D at 3mg/L on the 8th day in Ixora chinensis, a Rubiaceae species. Stella and Braga
(2002) studied Rudgea jasminoides callus developed from the petiole explants on basal
MS medium supplemented with picloram alone or in combination with kinetin and
reported that the highest frequency of callus formation was observed in solid medium
containing 2.22 µm kinetin and 2.07 µm picloram. Chaudhuri et al. (2004) recorded less
number, stunted and hypertrophied roots in Tylophora indica at higher concentrations of
IBA and NAA.
2mg 2,4-D in both MS and WP (Woody Plant) media was found to be ideal for
callus initiation in Morinda citrifolia and calli from MS/WP media were further sub
cultured on media (MS/WP) containing 1mg BA, 4mg IBA that resulted in the formation
of a single shoot in 45 days (Selvaraj et al., 2006). Subramani et al. (2007) clearly
indicated that it is the hormonal combinations which are very vital for the in vitro
response, medium with BAP alone for shoot initiation, kinetin along with BAP for
multiple shoot formation.
30
Venkateshwarlu (2007) observed callus and shoot bud induction from shoot tip
explants of Psidium guajava and leaf explants of Zizyphus mauritiana on MS medium
supplemented with BAP + KIN. Dasilva et al. (2008) reported that Alibertia edulis can be
cultured on MS medium containing Benzyladenine (BA) alone or in combination with
IBA to induce more shoot proliferation and the presence of growth regulators did have a
positive effect on the shoot elongation and higher concentration had an inhibitory effect
on shoot bud formation.
Poornima and Shivamurthi (2008) developed a protocol for multiple shoot
induction in Adenocalymma allicea through nodal culture on basal woody plant medium
incorporated with activated charcoal to remove polyphenols. The leaf explants of Tragia
involucrata induced proliferated mass of callus on MS medium supplemented with 2, 4-D
and Kinetin. Luxuriant mass of callus was achieved by sub culturing the calli on MS
medium supplemented with BAP (3.0 and 5.0 mg/l) alone or in combinations with NAA
(Dharmendra and Sudarshana, 2010).
Alam et al. (2010) investigated that the nodal explants of Paederia foetida, a
widely used medicinal vine of Rubiaceae showed an average of 2.04 shoots per explant,
cultured on MS medium supplemented with BAP at 1.0 mg/l. with an average length of
1.6 cm and maximum rooting was obtained in MS + IBA (0.3mg/l) after 3 weeks with an
average of 4.2 roots/shoot.
A perusal of the available information on medicinal plants of Mookanur hill and
studies on Tarenna asiatica are scanty. Hence, an attempt has been made to survey the
medicinal plants in the hill and to investigate the pharmacognostical, pharmacological
and conservation studies on Tarenna asiatica.