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Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods

CHAPTER 3

MATERIALS AND METHODS

3.1 Collection of Plant Material

The targeted plant species in present investigation was carried out on the

biochemical studies of six medicinally important plants viz. Argemone mexicana

Linn., Asparagus racemosus., Cyperus rotundus Linn., Melia azedarach Linn.,

Tagetes erecta Linn. and Tinospora cordifolia. Medicinal plants were collected

separately during the spring, summer and winter seasons from Sh. Kapur Chand

Kulish Smrti Udhyaan, Jaipur, Rajasthan . Plants were identified by comparing with

those available in the Herbarium, Department of Botany, University of Rajasthan,

Jaipur, India.

3.2 Enumeration of the Targeted Plant Species

The plant species were identified using standard literature and authenticated

voucher specimens. Morphological and Ecological information gathered from

physical/field observation, Chemical Composition and published literature. Ethano-

botanical and Pharmaceutical uses were compiled consulting published and

unpublished records.

3.2.1 Argemone Mexicana Linn

Classification of Argemone Mexicana Linn.

Family : Papaveraceae

Genus : Argemone

Species : A.mexicana

Botanical name : A.mexicana L.

Common name : Srigalkanta, Shialkanta (Sanskrit and Bengali),

Satyanashi (Hindi), Mexican prickly poppy (English),

Brahmadandicettu (Telugu), Ponnummuttai (Tamil),

Darudi, Daruri (Marathi and Gujarati).

Materials and Methods 91

(a) Leaves (c) Root

(b) Stem (d) Seeds

Plate 1: Argemone mexicana Linn (a) Leaves; (b) Stem; (c) Root; (d) Seeds.

HABIT & HABITAT

That plant adapted for a very broad range of habitats and tends to grow best

in soil of low fertility. It is a very common annual weed in agricultural and

wastelands found all parts of India where it is popular as ‘Satyanashi’ (Hindi). It is

everywhere by road-sides on various soil types and fields in India. Argemone is a

coarse ‘erect herb’ with prickly stems and milk sap. Leaves are irregularly pinnate

lobed, serrate, glaucous, edges crisped-undulate and oblong-oblanceolate. Flowers

are cremish yellow, 6 cm broad (Sambamurthy, 2005); or ‘annual herb’ with stems


2.5-10 cm diameter long, branched, sparsely to covered with prickles. Roots are sub-

cylindrical and long capsules having blackish brown oblong to roughly ellipsoid, 3-

4.2 cm long (Pullaiah, 1997), seeds numerous, 1.3-1.7 mm in diameter. Argemone

mexicana possess light yellow coloured fat oil. Argemone oil is raw tasted and softly

has nauseous odour it can be simply saponified (Millspaugh, 1974).

DISTRIBUTION

Argemone mexicana L. (Papaveraceae) found in Mexico and now has widely

naturalized in the United States, India, Bangladesh and Ethiopia. It occurs as

wasteland weed in almost every part of India (Mukherjee and Namahata, 1990; Das

and Misra, 1987). It mainly grows at sea level, though also found at an elevation of

2,900 m in Tanzania. In Mexico, the seeds have been used as an antidote to snake

poisoning (Bhattacharjee et al., 2006). It is an herbaceous plant with latex, which

has naturalized extensively in many tropical and subtropical regions although it’s a

native of tropical America (Osho and Adetunji, 2010).

CHEMICAL COMPOSITION

A.mexicana is reported to posses alkaloids (Nakkady and Shamma, 1988),

amino acids (Dinda and Bandyopadhyay, 1986), phenolics (Harborne and

Williams,1983) and fatty acids (Gunstone et al.,1977). A. mexicana posses

isoquinoline alkaloids such as berberine, cheilanthifoline, muramine, scoulerine,

cryptopine, thalifone, sanguinarine, protopine, and benzylisoquinoline alkaloids

(Chang et al.,2003;Nakkady and Shamma, 1988). Alkaloids such as berberine,

Benzophenanthridines, protopine, tetrahydroberberine and have been isolated from

the plant (Kenneth and Bentley, 2001). Seed oil or else known as Argemone oil

reported to contain sanguinarine and dihydrosanguinarine. It also possesses myristic,

palmitic, oleic and linoleic acids (Mukherjee and Namhata, 1990).

ETHANOBOTANICAL USES

A.mexicana is uses in India as traditional medicine for ophthalmia, dropsy,

jaundice, scabies and cutaneous affections (Chopra et al., 1956). Plant parts are used

in skin diseases and demulcent, diuretic, expectorant and emetic; the seeds and seed

oil are use therapy for dysentery, ulcers, asthma and other intestinal affections


(Chopra et al., 1956). Leaves and seeds are application maintaining normal

cholesterol level and blood circulation in human body (Savithramma et al., 2007);

Flowers are use as expectorant and also in the action of whooping cough

(Brahmachari et al., 2010). Plant seeds are use as digestive, laxative and purgative

while its latex is used against conjunctivitis (Agra et al., 2008).

PHARMACEUTICAL USES

Antimicrobial activity (Bhattacharjee et al.,2006); Antibacterial activity

(Bhattacharjee et al.,2010); Antidiabetic activity (Rout et al.,2011); Antioxidant

activity (Bhardwaj et al.,2011; Dash and Murthy,2011); Antihelmintic activity

(Sakthivadivel and Thilagavathy,2003); Anticancer activity (Gacche et al.,2011);

Hepatoprotective activity (Das et al.,2009); Larvicidal activity (Willcox et

al.,2007).

3.2.2 Asparagus racemosus.

Classification of Asparagus racemosus.

Family : Liliaceae

Genus : Asparagus

Species : A. racemosus.

Botanical name : A. racemosus

Common name : Satavari, Satamuli (Sanskrit and Bengali), Satavar

(Hindi), Wild asparagus (English), Satavari (Telugu),

Satavali (Tamil), Satavarmul, Satawari (Marathi and

Gujarati).


(a) Leaves (c) Root

(b) Stem (d) Seeds

Plate 2: Asparagus racemosus (a) Leaves; (b) Stem; (c) Root; (d) Seeds.

HABIT & HABITAT

Asparagus racemosus is a mounting plant which cultivate in tiny forest areas

throughout India especially in Northern India. It is a slender tall climbing under

shrub, extensively scandent; much branched growing to 1-2 m in height. Stems are

woody terrete with whitish grey or brown color. The young stems are very smooth,

brittle and delicate. The leaves are like pine needles, small, uniform, reduced to

minute chaffy scales and spines. The spines are 5-13mm long, recurved or rarely

straight (Sala, 2002; Kirtikar and Basu,1996; Wealth of india, 1995; Rajpal, 2002).

Cladodes are 1.3-2.5cm long, curved in tufts of 2-6, more or less acicular, falcate,

finely acuminate and glabrous (Rajpal,2002; Chatteijee and Pakrashi,2001; Kirtikar


and Basu,1996; Wealth of india,1995) . Flowers white perfumed, fascicled, simple

or twigged racemes 2.5-5cm long on the naked nodes of the main shoots or in the

axils of the thorns. Pedicles are 5mm long, jointed in the middle. Stamens are long

as the perianth (Rajpal, 2002 and Sala, 2002). Fruits are spherical or obscurely 3-

lobed. Berries are red pulpy, 5-6mm in diameter and become purplish black when

riped, seeds with hard and brittle testa (Sala, 2002).The adventitious roots arising at

a point become fleshy, tuberous succulent, fascicled at the stems base. They borne in

a compact bunch, spindle shaped, swollen in the middle for food storage and

tapering at ends (Rajpal, 2002; Sala, 2002 and Wealth of india,1995).The roots are

30cm to a meter or more in length, l-2cm thick, silvery white or light ash-colored

externally and white internally.

DISTRIBUTION

Asparagus racemosus is a significant medicinal plant of subtropical, tropical

and arid region of western Rajasthan, India. The Asparagus genus consists of 300

species around the world. The genus is medicinally significant the occurrence of

steroidal sapogenins and saponins in a variety of plant parts. In India out of the

twenty two Asparagus species verified; in which Asparagus racemosus is one of

plant usually used in conventional medicine. (Hussain et al., 2011). In India the crop

is grown in Rajasthan, Tamil Nadu, Kerala, Andhra Pradesh, Maharashtra, Gujarat,

Karnataka and northern states. It grows successfully in black cotton soil mixed with

river sand sunny position. The plant found throughout the subtropical and tropical

regions of India, including Andaman Islands and ascending up to 300m-1500m in

the Himalaya from eastwards Kashmir and in Ceylon (Bopana and Saxena,2007;

Yelavan et al.,2007 and Chatteijee and Pakrashi,2001).


It contain carbohydrate 3.2%, Protein 22%, fat 6.2%, vitamin C 0.04%,

vitamin B 0.36% and traces of vitamin A (Yelavan et al.,2007 ).The roots contain

four major steroidal saponins Shatavarins I-IV. The major glycoside is Shatavarin I

with three glucose and rhamnose moieties connected to Shatavarin IV and

sarsasapogenin is a glycoside of sarsapogenin having one molecule of glucose and

two molecules of rhamnose has been isolated from the roots (Chatteijee and


Pakrashi,2001; The Wealth of india,1988; Ravikumar et al.,1987). It also contains

9,10-dihydrophenanthren derivative racemosol (Sekine,1997); 4,6-dihydroxy-2-O-

(2-hydroxyisobutyl) benzaldehyde, undecanylceta-noate, (Chatteijee and Pakrashi,

2001).Five new steroidal saponins, shatavarins VI-X (Patricia,2008)

and isoflavone8-Methoxy-5,6,4trihydroxyisoflavone7-O-β-D-gluco-pyranoside,

asparagamine A (polycyclic alkaloid) has been isolated from the roots (Saxena and

Chourasia,2001). Flowers contain quercetin, rutin and hyperoside. The stem bark

contains cyaniding-3-monogiucoside, cyaniding-3,5-di-glucoside (Chatteijee and

Pakrashi, 2001). The leaves contains diosgenin, quercetin-3-glucuronide.The fruits

contain glycosides of quercetin, rutin and hyperoside, cyanidin-3-galactoside,

cyanidin-3-glucorhamnoside, sarsapogenin, two spirostanolic and furostanolic

saponins, stigmasterol and their glucosides (Yelavan et al.,2007; Bopana and

Saxena,2007; Chatteijee and Pakrashi, 2001 ).Three steroidal saponins, racemosides

A, B, C has been isolated from the fruits (Mandal et al.,2006).


Asparagus racemosus have antioxidant activities. Asparagus racemosus

(100mg/kg BW) given orally for 15 days defense antioxidant in methanolic extract,

that is increase significantly enzymes catalase, superoxidase dimutase and ascorbic

acid, whereas a significantly decrease in lipid peroxidation. The antioxidant

activities was found due to presence of isoflavons specially racemosol, racemofuran

and asparagamine-A (Wiboonpun et al., 2004). It is best known as a female

rejuvanitive and also used sexual weakness, inability, spermatorrhea, swelling of

sexual organs and helpful for dysentery, stomach ulcers, hyperacidity and bronchial

infections (Saxena and Bopana, 2009).Almost all plant parts are use by the Indian

traditional system of medicine such as ayurveda, siddha and yunani for the cure of

many diseases in human being. Herb is used for female rejuvenation and female

problems including infertility, leucorrhea, amenorrhea, dysmenorrhoea, menstrual

disorders, endometriosis, menopausal symptoms, menorrhagia, miscarriage or

habitual abortion, pelvic inflammatory disease, sexual debility, arthritis, headache,

toothache, stomachache and peptic ulcers (Yelavan et al., 2007). Roots are uses as

confectionary (Yelavan et al., 2007 ). It is applied in various skin diseases boiled


among some bland oil and is chiefly use in the formation of medicated oils,

prescribed for nervous disorder and rheumatic complaints (The Wealth of india,

1988).Which is boiled in milk and taken to relieve bilious dyspepsia and diarrhoea

and to promote appetite. It is Bitter, emollient, diuretic, dyspepsia, constipating,

ophthalmic, galactagogue, anodyne, aphrodisiac, rejuvenating, tuberculosis,

bronchitis, diarrhoea, antispasmodic, dysentery, stomachic, cough, carminative,

appetizer and general debility (Nadkarni,2002; Sala,2002; Chatteijee and

Pakrashi,2001). Leaves part of A. racemosus with butter-fat useful with assistance to

boils, small pox and to prevent confluence (Chatteijee and Pakrashi, 2001).Tubers

are sweet in taste eaten for nutritional value as a food but not as part of a meal. In

dyspepsia fresh root juice is given along with honey as a demulcent and with milk

is useful in gonorrhea (Nadkarni, 2002).

PHARMACEUTICAL USES

Antibacterial activity (Narayanan et al., 2011);Antidepressant activity (Singh

et al., 2009); Antiparkinsonian activity (Parihar and Hemnani, 2004);

Antineoplastic activity (Rao ,1981); Antitussive activity (Mandal et al., 2000);

Antioxidant activity (Visavadiya and Narasimhacharya, 2009); Antidiarrhoeal

activity (Venkatesan et al., 2005); Anti-diabetic activity (Hannan et al.,2011);

Diuretic activity (Kumar et al., 2010); Enzymes inhibitory activity (Meena et al.,

2011); Immunomodulatory activity (Gautam et al., 2009).

3.2.3 Cyperus rotundus Linn

Classification of Cyperus rotundus Linn.

Family : Cyperaceae

Genus : Cyperus

Species : C.rotundus.

Botanical name : C.rotundus.Linn.

Common name : Armoda, Motha (Sanskrit and Bengali), Mutha

(Hindi), Nut Grass (English), Tunga Mustalu

(Telugu), Korai (Tamil), Nagarmotha, Motha

(Marathi and Gujarati).


(a) Leaves (b) Rhizome

Plate 3: Cyperus rotundus Linn (a) Leaves; (b) Rhizome

HABIT & HABITAT

It is also known as purple nut edge or nut grass is a general perennial weed

with slender, crusty creeping rhizomes, globular at the base and arising from the

tubers which are about 1-4 cm long. The tubers are exterior black in colour and

inside reddish white with a typical odour. The leaf sheaths are slender and vertical

culms or stems are 10-35 cm tall. The flowers are bisexual each among three

stamens. The stems grow 35-40 cm tall and nut is oblong-ovate, three angled,

yellow in colour and black when ripe. Rhizomes &Tubers that are cultivate

downhill or parallel form tubers or chains of tubers. Mature individual tubers are

dark red-brown, 12 mm thick and between 10-32 mm long. (Andrews, 1940; Smith

and Fick, 1937). Under good conditions, an each tuber can manufacture 99 tubers in

90 days (Rao, 1968).

DISTRIBUTION

It is apparently native to India, but it has been introduced around the World

(Holm et al., 1977). This genus infrequently happens in more temperate regions.

Cyperus rotundus is indigenous to India, but are now found in subtropical, tropical

and arid regions (Pooley et al., 1998). The genus Cyperus includes common weeds

found in upland and paddy fields in temperature to tropical regions. C.rotundus also

found in subtropical, tropical and arid regions from Asia, South Africa, South


America, Arabia, Persia, Afghanistan, Pakistan, Tropical Africa, Madagascar,

SriLanka, China etc. (Gordon, 1995 and Pooley et al., 1998).


C.rotundus exposed the existence of flavonoids, alkaloids, starch, tannins,

glycosides, sitosterol, furochromones, monoterpenes, sesquiterpenes, fatty oil

containing a neutral waxy substance, linolenic, myristic and stearic acids. (Harborne,

1982; Ranjani and Prince, 2012). Plants have been examined for their biological

actions and antioxidant principles (Saleem et al., 2001).The chief compounds

separated from essential oil and extracts of C.rotundus rhizome are α-cyperone, α-

rotunol, β-pinene,, β-cyperone, β-rotunol, β-selinene, Cyperene, Calcium,

Camphene, Cyperenone, Copaene, Cyperol, Cyperolone, Cyperotundone D-

copadiene, D-epoxyguaiene, D-fructose, D-glucose, Flavonoids, γ-cymene,

Kobusone, Isocyperol, Isokobusone, Limonene, Linoleic-acid, Linolenic-acid,

Manganese, Magnesium, C. rotunduskone, Myristic-acid, Oleanolic-acid, Oleanolic-

acid-3-oneohesperidoside, Polyphenols, Patchoulenone, Oleic-acid, P-cymol, Pectin,

Rotundene, Rotundenol, Rotundone, Selinatriene, Sitosterol, Stearic-acid, Sugeonol,

Sugetriol (Khan et al., 2011; Sonwa and Konig,2001; Jeong et al., 2000 ).


In ancient medicine in India rhizomes of C. rotundus have been used for

dysentery, vomiting, fever, purities, pain and various blood disorders (Kirtikar et al.,

1944). C. rotundus plants have a traditionally use good remedy for dyspepsia and

useful for metabolic disorders and psychotic diseases in dietary management.

C.rotundus used in cure of Nausea and vomiting, dyspepsia, colic, fever, dysentery,

diarrhoea, cough, antispasmodic, bronchitis, intestinal parasites, malaria, renal and

vesical calculi, skin diseases, antitussive, wounds, cold, carminative, amenorrhoea,

diuretic, stomachic and dysmenorrhoea (Mehrotra et al., 2007;Williamson and

Hooper,2002). Deficient lactation, loss of memory, infertility, insect bites, dysuria,

food poisoning, cervical cancer and menstrual disorders and the aromatic oils are

made of perfumes or splash. (Manila, 1998).C.rotundus oil is evidence for notable

antibacterial activity and anti-mutagenic activity (Kilani et al., 2005).


PHARMACEUTICAL USES

It have a various pharmacological activities like as Anti-inflammatory

(Sundaram et al.,2008), Antipyretic (Gupta et al.,1971), Analgesic (Gupta et al.,

1971), Antiseptic, Antiemetic (Singh et al.,1970), Anticonvulsant (Pal et al.,2009),

Antimalarial (Thebtaranonth and Thebtaranonth,1995), Antidiarrhoeal (Uddin et al.,

2006),Anticancer (Mazzi and Soliman, 2009), Anti-obesity (Bambhole, 1988),

Gastroprotective (Zhu et al.,1997), Hypotensive and Tranquilizing (Singh et al.,

1970), Antimicrobial (Zeid et al.,2008), Hepatoprotective (Kumar and Mishra,

2005 ), Wound healing activity (Puratchikody et al.,2006).

3.2.4 Melia azedarach Linn

Classification of Melia azedarach Linn

Family : Meliaceae

Genus : Melia

Species : M.azedarach

Botanical name : M.azedarach.Linn.

Common name : Mahanimbah, Mahanim (Sanskrit and Bengali),

Bakayan (Hindi), Persian lilac (English), Turakavepa

(Telugu), Malaivempu (Tamil), Bakana-nimb, Bakam

limbodo (Marathi and Gujarati).


(a) Leaves (c) Root

(b) Bark (d) Seeds

Plate 4: Melia azedarach Linn (a) Leaves; (b) Bark; (c) Root; (d) Seeds

HABIT & HABITAT

M. azedarach is a shrub or small evergreen or medium-sized deciduous tree,

upto 5 feet girth and 6–25 m high. Bark dark grey or blackish with shallow

longitudinal furrows somewhat reticulate fissures, but smooth or young stems. Blaze

1-12 cm, coarsely fibrous, pale yellowish with darker parallel lines. Young shoots

clothed at first with stellate hairs but rapidly becoming glabrous. Leaves alternately

arranged, pinnately compound, 40 cm long, composed of 8-18 short-petiolate

narrow-ovate, curved, pointed, crowded towards the end of branches. Leaflets, 3-10

cm long and 1-4 cm large arranged in alternate pairs glabrous on both surfaces, base

often oblique, acuminate, serrate or gashed, sometimes lobed. Flowers 1-1.5cm


long, fragrant, in long peduncled axillary panicles 3-9 cm length, puberulous at first.

Petals lilac, staminal tube, 0.5-1cm long, very conspicuous, purple, slightly ribbed

outside. Fruits ellipsoid-globose 4-seeded drupes 1-1.5cm long, 3-6 celled, yellow

and plump when ripe, become wrinkled and remaining on the tree long after

ripening. Which grown in October-December. Seeds are dirty brown, 1cm in length

and 4-5 mm in width, taste bitter, odour characteristic and one in each. (African

pharmacopoeia, 1985; Manila, 1998)

DISTRIBUTION

Melia azedarach is commonly known as Maha neem tree and tree found in

India and most similar to Neem. It has become naturalized to tropical and warm

temperate regions of the India and is planted in similar climates around the world. It

is inhabitant to upper Burmah region (Nahak and Sahu 2010). It grows wild in the

Sub-Himalayan tract up to 1700 m, Rohilkhand, Dehradun and Saharanpur forests. It

is commonly cultivate almost throughout India, Rajasthan, Bihar, Orissa, West

Bengal, Punjab, Konkan and Deccan and in most districts of south India, as an

ornamental avenue tree. The various parts of the tree possess the same therapeutic

significance as neem tree. It is widespread and grow naturally in most of the tropical

and subtropical regions (Ramya et al., 2009). It has a wide natural distribution 11

extending from China, India and Japan to Indonesia, Tropical South America,

Northern Australia, Africa, North America and Southern Europe. (Nikoletta, 2010;

Chiffelle et al., 2009).


The M. azedarach includes various organic molecules such as flavonoids,

terpenoids, steroids, acids and anthraquinones. A selection of compounds detected in

M. azedarach leaf extract contain stigmasterol, Kampherol,Quercetin (Flavonoids),

Campesterol (phytosterols), β-sitosterol, Pyhtol (Diterpene), 3-Methyldecane,

Heptadecane (alkane hydrocarbon), hexadecanic acid, β-Carotene, Pentadecanoic

acid (n-alkanoic acids), tocopherol (Vitamin-E) and saqulene, 1 -Eicosanol

(triterpene), 3, 5, 11, 15- Tetramethyl-2-hexadecen-1 -ol (Terpene alcohol) (Sen and

Batra,2012). Root bark contains terpenoids such as Azedarachtin-A, Azedarachtin-B

(Kumar et al., 2003). Seeds contain β-sitosterol, β-D-glucopyranose, liminoid


glycoside viz. 6,11-diacetoxy-7-oxo-14β-epoxymeliacin (1, 5-diene-3-O-beta-D-

glucopyranoside) and meliacarpin, melianol meliacin, meliartenin vanillin,

hydroxyl-3-methoxcinnamaldehyde and (±) pinoresinol (Carpinella et al., 2005)

Limonene (0.68%) acts as natural food flavors also use in fragrances and

aromatherapy (Duetz et al., 2003).


The exuded gum obtained from its trunk is considered useful in spleen

enlargement, its wood extract is prescribed internally in asthma (Dhiman, 2003),

decoction of bark is use in paroxysmal fever to relieve thirst, nausea, vomiting and

general debility, loss of appetite and skin diseases (Sharma et al.,2001). Leaf juice is

anthelmintic, diuretic and emmenagouge, expectorant, vermifuge and their

decoction is astringent and stomachic (Warrier et al., 1995; Dhiman, 2003; Sharma

et al., 2001). Leaves have been used keep with stored food as a natural insecticide

but they are highly poisonous must not be eaten.Leaves are applied in the form of

poultice to relieve nerves headache and to cure the eruption on the scalp; they are

used topically in leprosy, scrofula and other skin diseases (Nadkarni, 1954). Flowers

are astringent, deobstruent, refrigerent, emmenagouge, diuretic, resolvent,

stomachic, alexi-pharmic and anodyne (Warrier et al., 1995; Sharma et al., 2001;

Zhou et al., 2005). Fruits are considered tonic, internally in indigestion, colic and

intestinal cathartic. Which are anthelmintic, emmolient and purgative (Rani et al.,

1999). Seeds are bitter, expectorent, anthelmintic and aphrodiasic, and are useful in

helminthiasis, typhoid fever, pain in the pelvic region, uropathy, vitiated conditions

of vata and scrofula (Warrier et al., 1995). They are prescribed in rheumatism; oil

obtained from seeds is applied locally in skin diseases (Dhiman, 2003). Barks are

bitter, astringent, mildly thermogenic, anodyne, depurative, vulnerary, antiseptic,

constipating, expectorant, urinary astringent, anthelmintic and bitter tonic in low

doses. They are useful in headache, leprosy, leucoderma, skin diseases, wounds,

ulcers, piles, worm infestation, cough, asthma, ammenorrhoea, dysmenorrhoea,

diabetes, abnormal urethral discharge, chronic, intermittent fevers, vomiting and

post labour pain in uterus (Warrier et al., 1995; Sharma et al., 2001).


PHARMACEUTICAL USES

M. azedarach possesses Antioxidant activity (Munir et al.,2012),Antiviral

activity (Wachsman et al., 1998), Antifungal activity (Carpinella et al., 1999),

Antibacterial activity (Carpinella et al., 1999; Khan et al., 2001), Antimalarial

activity (Ofulla et al., 1995), Antipyretic activity (Sultana et al.,2013), Antifertility

activity (Choudhary et al., 1990; Roop, 2005), Anthelmintic activity (Pervez et al.,

1994),Cytotoxic activity (Itokawa and Qiao, 1995; Zhou et al., 2004),

Immunomodulatory activity (Benencia et al., 1997), Insecticidal activity (Rani et al.,

1999), Wound healing activity (Vidya et al.,2012).

3.2.5 Tagetes erecta Linn

Classification of Tagetes erecta Linn.

Family : Asteraceae

Genus : Tagetes

Species : T.erecta

Botanical name : T.erecta.Linn.

Common name : Sthulapuspha, Gaenda (Sanskrit and Bengali), Genda

(Hindi), Marigold (English) Bantichettu (Telugu),

Tulukkasamandi (Tamil), Jhenduphool, guljharo



(a) Leaves (c) Root

(b) Stem (d) Flower

Plate 5: Tagetes erecta Linn (a) Leaves; (b) Stem; (c) Root; (d) Flower

HABIT & HABITAT

Tagetes erecta L. commonly known as marigold is cultivate as an

ornamental plant. Flowers are use in garlands for religious and community points in

Eastern Countries. It is commonly known as aromatic annual herb native to Mexico

extensively cultivated as a border annual in gardens all over India. It is a garden

plant and consent a muscularly aromatic essential oil (Tagetes oil) used for the


compositing of better grade perfumes. A stout branching herb 60 cm tall leaves

strong scented pinnately dissected segments 1-5 cm long oblong or lanceolate,

serrate; flower heads solitsry, clawed, sometimes 2 lipped or quilled, involucres

campanulaye achenes 6-7 mm long, pappus scaly, bright yellow flowers and

beautifully dissected foliage (The Wealth of India., 1992).

DISTRIBUTION

The genus Tagetes ( derived from Etruscan Tages) originated in North and

South America and widely cultivated in other Asian countries like Bhutan, China,

Nepal, India etc become cultivated to tropical and warm temperate regions of the

India (Abdul Halim,2007). The present area cultivation of marigold is 28,825

hectares with a production of more than 2.0 metric tons in India (Anonymous,

2004). The marigold carotenoid is done in Cochin, Hyderabad, Tamil Nadu and

Bangalore (Karnatka). Regularly is exported to Japan, Turkey, Italy, Spain, Mexico,

Peru, USA, Canada, Australia and Africa. Tagetes erecta flowers are under

commercial cultivation for premium prices. Different varieties of African marigold

vary in plant height and spread, flower size, quality and yield (Jawaharlal, 2004).

The flowers are globular in shape. Colour shades vary from light yellow to creamy,

bright yellow,white and deep orange. Generally varieties can be found in both

domesticated and naturalized habitats in the form of local landraces, modern

varieties as well as wild strains (Turner and Nesom, 1993).


T.erecta content of numerous secondary metabolites and the most important

compounds are essential oils, terpenes thiophenes, flavonoids, polyphenols and

carotenoids (Marotti et al., 2004; Xu et al., 2011; Hadden et al., 1999).The plant T.

erecta has been shown to contain glucoside of quercetagetin, quercetin,

quercetagetin, phenolics, syringic acid, 3,5 methyl-dihydroxy-4-methoxy benzoate,

thienyl and ethyl gallate (Farjana et al., 2009; Ghani,1998). the major constituents is

one of lutein and the main pigment of Tagetes erecta i.e. xanthophylls or

oxycarotenoid having 2 cyclic end groups (one β and one α-ionone ring) and the

basic C-40 isoprenoid structure common to all carotenoids. The steam distillation of

fresh leaves offer 0.3% of essential oil with a strong, sweet lasting odor and contains


d-limonene, ocimene, l-linalyl acetate, linalool, tagetone, lutein (The Wealth of

India, 1992).


Plants contain different parts mainly flower part is used in folk medicine to

therapy different ailments. The leaves are useful against ulcers, piles, muscular pain,

ear ache kidney troubles and wounds. The flower is useful in fevers, epileptic fits,

astringent, carminative, stomachic, scabies and liver complaints and is also use in

eye diseases (Kiranmai and Ibrahim, 2012). Plant infusion is used against

rheumatism, cold, vermifuge diuretic, carminative and bronchitis. An extract of the

root has possessed laxative properties. The leaves extracts is use in ear ache. Their

infusion is prescribed as a vermifuge, diuretic and carminative. It is a garden plant

and consent a muscularly aromatic essential oil (Tagetes oil) used for the

compositing of better grade perfumes. The florets are use in the treatment of eye

diseases and ulcers. The flower heads are much employed at religious ceremonies

for decorative purposes (The Wealth of India, 1992).

PHARMACEUTICAL USES

Tagetes erecta comprise Anti-bacterial activity (Kiranmai and Ibrahim,

2012; Rhama and Madhavan, 2011), Anti-oxidant activity (Chivde et al., 2011),

Analgesic activity (Bashir and Gilani, 2008), Anti-hyperlipidemic activity

(Raghuveer et al., 2011), Anti-wrinkle activity (Maity et al., 2011), Insecticidal

activity (Farjana et al., 2009), Larvicidal activity (Marcia et al., 2011) and wound

healing activity (Chatterjee et al., 2011and Kiranmai et al., 2011).


3.2.5 Tinospora cordifolia

Classification of Tinospora cordifolia.

Family : Menispermeaceae.

Genus : Tinospora

Species : T.cordifolia

Botanical name : T.cordifolia.

Common name : Guduchi, Gulancha (Sanskrit and Bengali), Giloya

(Hindi), Indian tinospora (English), Tippatiga

(Telugu), Shindilakodi (Tamil), Shindilakodi, Galo


(a) Leaves (c) Root

(b) Stem (d) Seeds

Plate 6: Tinospora cordifolia (a) Leaves; (b) Stem; (c) Root; (d) Seeds.


HABIT & HABITAT

The leaves are membranous, exstipulate, long petioled approximately 15 cm,

round, heart shaped, twisted partially and half way round. Lamina is ovate, 10-20

cm long, 7 nerved and deeply cordate at the base and membranous (Raghunathan

and Sharma, 1969); the flower is yellow or greenish yellow or racemose panicles.

Petals are also 6, smaller than sepals, free and membranous (Kirtikar and Basu,

1975); the stems are long filiform, fleshy aerial roots beginning the branches. The

bark is creamy white to grey in colour and deeply left spirally (Khosa and Prasad

1971); Aerial roots are tetra to penta arch primary structure. However, cortex of root

is divided in to outer thick walled and inner parenchymatous zone (Aiyer and

Kolammal M 1963); The fruits of Giloy are like peas which are seen in winter

season in India (Wagner and Birkhauser, 1999; Warrier et al., 1996).They are

orange-red in colour, fleshy, aggregate of 1-3 and ovoid, smooth, drupelets on thick

stalk with a sub terminal style scars (Nadkarni, 1976)

DISTRIBUTION

Tinospora cordifolia is a climbing shrub, leaves heart shape, glabrous,

deciduous. It is found all over tropical and semi arid zone of India, also found in

China, Bangladesh, Myanmar, Srilanka, South Asia, Indonesia, Phillipians and

Thailand. T. cordifolia prefers extensive range of soil, acid to alkaline and it requires

moderate level of soil moisture, mounting to an elevation of 300m-1200m in

worldwide (Sharma et al., 2010).


A variety of constituents have been isolated from T. cordifolia. Different

classes belong to such as alkaloids, steroids, phenolic, diterpenoid lactones,

polysaccharides, sesquiterpenoid, aliphatic compounds and glycosides. Rich in

protein (11.2%) of leaves parts and also rich in phosphorus and calcium (Singh et

al.,2003).The presence of palmatine, magnoflorine, berberine, tembetarine,

tinosporin, tinocordifolin (Kumar et al., 2000; Padhya 1986; Qudrat-I-Khuda et

al.,1964) from its stems, choline, isocolumbin, palmatine, tetra hydropal-matine,

magnoflorine from its roots sirosterol, sitosterol and 2, 3, 14, 20, 22-25

hexahydroxyl-5-cholest-7-en-6-one from its aerial parts (Hanauman et al.,1986) and


18- norclerodane, diterpene-O-glucoside from its stem wood (Khan et al., 1990)

have been reported.


T. cordifolia is use in tribal region and different plant parts as folk medicine

use in the country. Treatment of gout and ulcer use as Leaves. The stem is bitter

stomachic, diuretic, stimulates bile secretion, enriches the blood, cures jaundice,

bronchitis and skin diseases (Treadway, 1998). Stem as an infusion use to drunk as

a vermifuge, jaundice, against intestinal worms; Stem as decoction use for washing

sore eyes and syphilitic sores , antipyretic, antimalarial, Stimulate the immune

system(Nagarkatti et al.,1994). Starch use for chronic diarrhoea obtained from stem

and some form of chronic dysentery. In diabetes mellitus juices is useful and also

helpful in vaginal and urethral discharges, reduce fevers and enlarged spleen

(Stanely et al., 2001; Kirtikar Basu, 1918). Dried fruit with ghee or honey are use as

tonic and treatment of jaundice and rheumatism. Bark is Anti-allergic, Anti-malarial

Anti-spasmodic, Anti-leprotic (Ikram et al.,1987 and Nayampalli et al., 1986).

PHARMACEUTICAL USES

Analgesic activity (Hossain et al., 2009); Anti-stress activity(Singh et al.,

2003); Anti-oxidant activity (Stanley et al., 1999; and Desai et al., 2012);

Antipyretic (Jeyachandran et al., 2003;Gupta and Viswanathan, 1956); Antidote to

snake bite (Nadkarni, 1976); Anti-asthmatic (Spelman, 2001); Hepatoprotective

activity (Kavitha et al.,2011); Anti- neoplastic property activity (Dikshit et al.,

2000); Neuropharmacological activities. (Hossain et al., 2009); Anti-fertility activity

(Gupta and Sharma, 2003); Antidiabetic and Antihyperlipidemic activity (Selvaraj et

al., 2012); Immunomodulatory activity (Aher and Wahi, 2010).

3.3. Plant Collection and Identification

The targeted plant of the six plant species Argemone mexicana Linn.,

Asparagus racemosus, Cyperus rotundus Linn., Melia azedarach Linn., Tagetes

erecta Linn. and Tinospora cordifolia were collected separately during the spring,

summer and winter season. These plants were used as a source material for

experiment. The plant species were identified using standard literature and


authenticated voucher specimens available in the Herbarium, Department of Botany,

University of Rajasthan, Jaipur, India.

3.4 Preliminary Qualitative Phytochemical Screening

Preliminary qualitative phytochemical screening was done with method of

Harbone, (1998). The preliminary qualitative phytochemical analysis was carried

out in crude dry powder various parts of targeted six plants.

3.4.1 Phenols

Extracts were treated with 3-4 drops of ferric chloride solution (Ferric

Chloride Test). Formation of bluish black colour indicates the presence of phenols.

3.4.2 Alkaloids

The each plant of the crude dry powder of methanolic extract was evaporated

to dryness in a boiling water bath. The residue was dissolved in 2N HC1. The

mixture was filtered and the filtrate was divided into 3 equal portions. One portion

was treated with a few drops of Mayer’s reagent (Potassium Mercuric Iodide)

formation of a creamish yellow coloured precipitate indicates the presence of

alkaloids; one portion was treated with equal amount of Dragendroff’s reagent

(solution of Potassium Bismuth Iodide) formation of orange red precipitate

indicates the presence of alkaloids and the other portion was treated with equal

amount of Wagner’s reagent (Iodine in Potassium Iodide) formation of brown

precipitate indicates the presence of alkaloids(Salehi-Surmaghi et al., 1992).

3.4.3 Flavonoids

Extracts were treated with few drops of sodium hydroxide solution

(Alkaline Reagent test).Formation of intense yellow color, which become colourless

on addition of dilute acid indicates the presence of flavonoids and also extracts were

treated with few drops of lead acetate solution (Lead acetate Test). Formation of

yellow colour indicates the presence of flavonoids.


3.4.4 Steroids

Liebermann-Burchard reaction was performed to assess the presence of

steroids. A chloroform solution of the crude dry powder of each plant was treated

with acetic anhydride and a few drops of concentrated H2SO4 were added down the

sides of the test tube. A blue green ring indicated the presence of terpenoids.

3.4.5 Tannins

The each plant of the crude dry powder of water extract was treated with

alcoholic FeCl3 reagent. Blue colour indicated the presence of tannins (Segelman et

al., 1969).

3.4.6 Cardiac Glycosides

Keller-kiliani test was performed to assess the presence of cardiac

glycosides. The crude dry powder of each plant was treated with 1mL of FeCl3

reagent (mixture of 1 volume of 5% FeCl3 solution and 99 volumes of glacial acetic

acid). To this solution a few drops of concentrated H2SO4 was added. Appearance of

greenish blue color within a few minutes indicated the presence of cardiac

glycosides (Ajaiyeobu, 2002).

3.5 Quantitative Phytochemical Screening

3.5.1 Extraction

Ten gram of air-dried powder was taken in 100 mL of petroleum ether in a

conical flask, plugged with cotton wool and then kept on a rotary shaker at 190-220

rpm for 24 h. After 24 h, the supernatant was discarded and petroleum ether was

evaporated from the powder. This dry powder was then taken in 100 mL of solvent

(methanol) in a conical flask, plugged with cotton wool and then kept on a rotary

shaker at 190-220 rpm for 24 h. After 24 h, the extracts were centrifuged at 5000

RPM for 10 min, the supernatant was collected, solvents were evaporated and the

dry extract was stored at 4°C in airtight bottles. The extraction was done at least

three times for each plant and the mean values of extractive yields are presented

(Vaghasiya and Chanda, 2007).


3.5.2 Total Phenol Determination

Total phenolic content of the extracts was determined by Folin Ciocalteu

reagent method (Mc Donald et al., 2001) with some modifications. Plant extract

(1 mL) was mixed with Ciocalteu reagent (0.1mL, 1N) and allowed to stand for 15

min. Then 5mL of saturated Na2CO3 was added. The mixtures were allowed to stand

for 30 min at room temperature and the total phenols were determined

spectrophotometrically at 760 nm. Gallic acid was used as a standard. Total phenol

values are expressed in terms of gallic acid equivalent (mg g-1 of extracted

compound).

3.5.3 Total Flavonoid Determination

Aluminium chloride colorimetric method (Chang et al., 2002) with some

modifications was used to determine flavonoid content. Plant extract (l mL) in

methanol was mixed with 1ml of methanol, 0.5 mL aluminium chloride (1.2%) and

0.5 mL potassium acetate (120 mM). The mixture was allowed to stand for 30 min

at room temperature; then the absorbance was measured at 415 nm. Quercetin was

used as standard. Flavonoid content is expressed in terms of quercetin equivalent

(mg g-1 of extracted compound).

3.6 Determination of Antioxidant and Free Radical Scavenging Properties

3.6.1 DPPH Assay

10 gm of each of the powdered plant samples of were taken for the

antioxidant activity and extracted with 100 ml of methanol for 3 days. The extracts

obtained from each of the plant materials were filtered separately and concentrated

by vacuum evaporation. Radical scavenging activity of plant extracts against stable

DPPH (2,2-diphenyl-2-picrylhydrazyl hydrate) was determined spectrophoto-

metrically. When DPPH reacts with an antioxidant compound, which can donate

hydrogen, it is reduced. The solution of DPPH in methanol (6x10-5M) was prepared

freshly, before UV measurements. 3.9 ml of DPPH is added in different concentr-

ation of extracts to measure IC50 value (Brand-Williams et al., 1995). The samples

were kept in the dark for 15 min at room temperature and then the decrease in

absorption was measured. Absorption of blank sample containing the same amount

of methanol and DPPH solution was prepared and measured in every experiment.


The changes in colour (from deep violet to light yellow) were measured at 517 nm

on a UV visible light spectrophotometer. The experiment was carried out in

triplicate. Radical scavenging activity was calculated by the following formula:

Inhibition (%) = [(Absorbance Control –Absorbance Sample)/Absorbance Control]

× 100

3.6.2 FRAP Assay

The determination of the total antioxidant activity in the extract is a modified

method (Benzie and Strain, 1996).The stock solutions included 300mM acetate

buffer (3.1g C2H3NaO2. 3H2O and 16 ml C2H4O2), pH 3.6,10 mM TPTZ (2,4,6-

tripyridyl-s-triazine)solution in 40mM HCl and 20 mMFeCl3.6H2O solution. The

fresh working solution was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ

and 2.5ml FeCl3.6H2O.The temperature of the solution was raised to 37°C before

use. Plant extracts (150µl) were allowed to react with 2850 µl of the FRAP solution

for 30 min in the dark condition. Readings of the colored product (ferrous tripyridyl

triazine complex) were taken at 593 nm. The standard curve was linear between 200

and 1000µMFeSO4. Results are expressed in µMFe(II)/g dry mass and compared

with that of ascorbic acid and quercetin.

3.7 Statistical Analysis

Each of the measurements was carried out in triplicate and the results are

expressed as Mean±SEM.

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