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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,

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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

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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

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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.