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Chapter 5 DISCUSSION

5.1 Collection and Maintenance of Live Plants in Greenhouse and Garden:

The targeted medicinal plants were collected from different parts of Assam

specially Houttuynia cordata, Eryngium foetidum and Polygonum microcephallum from

Sivasagar, Chenopodium album from bank of the river Brahmaputra near Sualkuchi

outskirts of Guwahati, Phyllanthus fraternus and Gynandropsis gynandra from natural

habitat of within the Gauhati University campus. The plants have been maintained at

Greenhouse as well as in the experimental garden.

5.2 Enumeration of the Targeted Plant Species incorporating Morphological and

Ecological information, Mode and purpose of Uses:

All the plant species viz., Phyllanthus fraternus, Polygonum microcephallum,

Houttuynia cordata, Eryngium foetidum, Chenopodium album and Gynandropsis

gynandra were identified using standard literature and authenticated voucher specimens.

Morphological and ecological information gathered from physical / field observation,

and published literature. Mode and purpose of uses were compiled consulting published

and unpublished records.

5.2.1 Ecology and Distribution:

Phyllanthus fraternus occurs as a weed in open ground, waste land, grossy scrub

and dry deciduous forest and usually grows well on humid, sandy soils, up to 1000m

altitude (Heyne, 1987). P. fraternus plants found in Kamrup, Assam and entire north

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eastern region. The species is mostly found in China and tropical locations ranging from

the Philippines to Cuba. In India it is found in the hotter parts extended from Punjab to

Assam.

Polygonum microcephallum is indigenous to the NE India i.e., Khasi Hills (up to

5,000 ft.); Naga Hills, Assam, N. C. Hills (Haflong), Upper Assam but not common in

distribution. Small population is occasionally found in household cultivation.

Houttuynia cordata plant grows well in moist to wet soil and even slightly

submerged in water in partial or full sun. Plants can become invasive in gardens and

difficult to eradicate. It is also cultivated in gardens. H. cordata is found naturally in

Naga Hills, Khasi Hills (up to 6,000 ft.), Manipur, Sylhet and plains of Assam.

Eryngium foetidum, the plants grow in Assam Plains, N. C. Hills and Khasi Hills

(up to 5,000 ft.), It is native to Mexico and South America, but is cultivated worldwide.

Chenopodium album, grow abundant in Assam and rest of the NE India. Its

native range is obscure due to extensive cultivation, but includes most of Europe. It is

introduced in Africa, Australasia, North America, and Oceania. Now it occurs almost

everywhere in soils rich in nitrogen.

Gynandropsis gynandra, found mostly in tropical and subtropical regions. It is

very common in abandoned fields, and old ruins in the plains of Assam and Bengal.

5.3 Treatment of Targeted Plants with various Fertilization regimes:

Wild seedlings were cultivated in greenhouse under control conditions. Initially

pots filled with sandy-loamy soil were used for growing of the plants. Treatments

consisted of four different fertilization regimes: (I) Full (100%) Hoagland solution; (ii)

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50% Hoagland solution; (iii) 20% Hoagland solution; and (iv) Control (irrigatied with

tap water).

In the present investigation, it is successfully established that the possibility of

cultivation of wild growing Phyllanthus fraternus, Polygonum microcephallum,

Eryngium foetidum, Houttuynia cordata, Chenopodium album and Gynandropsis

gynandra medicinal plants in green house as well as in the field. Cultivation has

revealed several advantages. First, cultivation reduces the possibility of incorrect

identification and adulteration. Second, cultivated plants can be irrigated and fertilized

to increase their growth rate thereby improving yield. Third, cultivated plants can be

grown in areas of similar climate and soil. It also showed better result in full (100%)

Hoagland solution.

Hassan et al., (2005) reported the fertilization- induced changes in growth

parameters and antioxidant activity of medicinal plants used in traditional Arab

Medicine. They have investigated four medicinal herbs Cichorium pumilum, Eryngium

creticum, Pistacia palaestina and Teucrium polium. They have the plants cultivated in

greenhouse to assess the effects of different fertilization regimes on their growth and

antioxidant activity.

Hassan et al., (2005) also reported that increase in the amount of fertilizer

caused a significant concentration-dependent increase in antioxidant activity of the

cultivated T. polium compared with the wild type. In contrast, increasing the amount of

fertilizer caused a significant concentration-dependent reduction in the antioxidant

activity of powders prepared from the cultivated E. creticum when compared with wild

plants. Significantly they have remarked that the cultivation success should not rely

solely on growth parameters but have the therapeutic potential. It can reduces or even

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eliminate the variation in content of the desired aromatic compounds as well as

providing information on the best time to harvest so that the content of the desired

aromatic compound(s) is maximal.

The germplasm of wild growing Phyllanthus fraternus, Polygonum

microcephallum, Eryngium foetidum, Houttuynia cordata, Chenopodium album and

Gynandropsis gynandra medicinal plants have the concern for conservation, sustainable

utilization, and development. These plants exhibited an increase in medicinal efficiency

as well as in growth rate under fertilization regimes.

5.4 Antioxidant Activity:

Antioxidants are molecules, which can safely interact with free radicals and

terminate the chain reaction before vital molecules are damaged. Free radicals are major

factors leading to more than sixty different health problems including aging, cancer and

atherosclerosis. Antioxidant compounds like phenolic acids, polyphenols and flavonoids

scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit

the oxidative mechanism that lead to degenerative diseases. DPPH stable free radical

method is an easy, rapid and sensitive way to analyze the antioxidant activity of plant

extracts. Scavenging activity for free radicals of DPPH (2, 2-diphenyl-1-picrylhydrazyl)

has been widely used to evaluate the antioxidant activity of natural products from plant.

5.4.1 Assessment of Antioxidant capacities - The DPPH method:

A rapid, simple and inexpensive method to measure antioxidant capacity of

foods and medicinal plants involves the use of the free radical, 2,2-Diphenyl-1-

picrylhydrazyl (DPPH). DPPH is widely used to test the ability of compounds to act as

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free radical scavengers or hydrogen donors (Sanchez Moreno et al., 1998; Tominaga et

al., 2005; Ramteke et al., 2007) and to evaluate antioxidant activity of foods and

medicinal plants. It has also been used to quantify antioxidants in complex biological

systems in recent years. The DPPH method can be used for solid or liquid samples and

is not specific to any particular antioxidant component, but applies to the overall

antioxidant capacity of the sample. A measure of total antioxidant capacity helps

understand the functional properties of medicinal plant.

The DPPH method was evidently introduced nearly 50 years ago by Marsden

Blois, working at Stanford University (Blois, 1958). A simple method that has been

developed to determine the antioxidant activity of foods and medicinal plants utilizes

the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. The odd electron in the DPPH

free radical gives a strong absorption maximum at 517 nm and is purple in color. The

color turns from purple to yellow as the molar absorptivity of the DPPH radical at 517

nm reduces from 9660 to 1640 when the odd electron of DPPH radical becomes paired

with a hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-

H. The resulting decolorization is stoichiometric with respect to number of electrons

captured. (Aruna Prakash, Ph.D, Fred Rigenof and Eugene Miller, Ph. D. Antioxidant

Activity, Medallion Laboratories, Analytical Progress, 2000).

Antioxidant assay are based on measurement of loss of DPPH color at 517nm

after reacting with test compounds (Bondet et al., 1997). This assay is based on the

measurement of the reducing ability of antioxidant towards DPPH. The ability can be

evaluated by electron spin resonance (ESR) or by measuring the decrease of its

absorbance. The widely used discoloration assay was first reported by Brand-Williams

and co-workers, 1995. In the presence of an antioxidant, DPPH radical obtains one more

electron and the absorbance decreases (Koleva

Antioxidant assay are based on measurement of loss of DPPH color at 517nm after

reacting with test compounds was mo

2005).

Diphenylpicrylhydrazyl (Free Radical)

The DPPH test (Wanger, 1996) provided information on the reactivity of test

compounds with stable free radicals. As the odd electron of DPPH radicals paired with a

hydrogen from a free radical scavenging antioxidant to form the reduced DPPH

absorption vanishes and the resulting decolourization is stoictiometric with respect to

the number of electrons taken up (Oke

The hydrogen donation potential is known to be one of the various mechanisms for

measuring antioxidant activity. The color of methanolic DPPH changes from purple to

yellow, due to the formation of diphenylpicrylhydrazine (a stable diamagnetic

molecule), upon reduction by either the process of hydrogen radical or electron donation

(Jao and Ko, 2002; Oktay et al

DPPH method has been followed by several recent workers (Kim

al., 2002). Accordingly, antioxidant activity of

microcephallum, Houttuynia cordata, Eryngium foetidum, Chenopodium album, and

Gynandropsis gynandra were screened by DPPH method.

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electron and the absorbance decreases (Koleva et al., 2002; Pourmorad et al

Antioxidant assay are based on measurement of loss of DPPH color at 517nm after

reacting with test compounds was monitored by a spectrophotometer (Prior

Diphenylpicrylhydrazyl (Free Radical) Diphenylpicrylhydrazyl (Non-Radical)

The DPPH test (Wanger, 1996) provided information on the reactivity of test

mpounds with stable free radicals. As the odd electron of DPPH radicals paired with a

hydrogen from a free radical scavenging antioxidant to form the reduced DPPH

absorption vanishes and the resulting decolourization is stoictiometric with respect to

the number of electrons taken up (Oke and Hamburger, 2002; Iluvarasan et al

The hydrogen donation potential is known to be one of the various mechanisms for

measuring antioxidant activity. The color of methanolic DPPH changes from purple to

ow, due to the formation of diphenylpicrylhydrazine (a stable diamagnetic

molecule), upon reduction by either the process of hydrogen radical or electron donation

et al., 2003). The illustrious explanation of original Blois

thod has been followed by several recent workers (Kim et al., 2002; Zhu

, 2002). Accordingly, antioxidant activity of Phyllanthus fraternus, Polygonum

microcephallum, Houttuynia cordata, Eryngium foetidum, Chenopodium album, and

were screened by DPPH method.

et al., 2006).

Antioxidant assay are based on measurement of loss of DPPH color at 517nm after

nitored by a spectrophotometer (Prior et al.,

Radical)

The DPPH test (Wanger, 1996) provided information on the reactivity of test

mpounds with stable free radicals. As the odd electron of DPPH radicals paired with a

hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H the

absorption vanishes and the resulting decolourization is stoictiometric with respect to

et al., 2005).

The hydrogen donation potential is known to be one of the various mechanisms for

measuring antioxidant activity. The color of methanolic DPPH changes from purple to

ow, due to the formation of diphenylpicrylhydrazine (a stable diamagnetic

molecule), upon reduction by either the process of hydrogen radical or electron donation

2003). The illustrious explanation of original Blois

, 2002; Zhu et

Phyllanthus fraternus, Polygonum

microcephallum, Houttuynia cordata, Eryngium foetidum, Chenopodium album, and

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5.4.2 Solvent systems:

Regarding the solvent to be used, the method seems to work equally well with

methanol, neither of which seems to interfere with the reaction. The use of other solvent

systems, such as almost neat extracts in water or acetone, seems to give low values for

the extent of reduction (Guo et al., 2001). Methanol gives the maximum percentage

inhibition. Methanol as a solvent used for DPPH radical scavenging activity has been

reported extensibly since 2005.

Kumaran and Karunakaran (2005) investigated the in vitro antioxidant activities

of methanol extracts of five Phyllanthus species from India.

Aqil et al., (2006) screened the methanolic crude extracts of 12 traditionally

used medicinal plants for their antioxidant and free radical scavenging properties using

DPPH radicals. Iqbal et al., (2006) reported that the antioxidant properties of

methanolic extracts from leaves of Rhazya stricta. The methanolic extract exhibit the

highest antioxidant activity.

Gupta et al., (2007) evaluated the antioxidant and free radical scavenging

activity of methanolic extract of Plumeria acuminata leaves (Apocynaceae) in various

systems. DPPH radical scavenging assays were carried out to evaluate the antioxidant

potential of the extract. The antioxidant activity of methanolic extract increased in a

dose dependent manner. Jimoh et al., (2007) reported the antioxidant properties of the

methanol extracts from the leaves of Paullinia pinnata. Paullinia pinnata is an African

woody vine widely used in traditional medicine for the treatment of malaria. In vitro

antioxidant activities of the methanol extract of its leaves were evaluated using different

testing systems. Its scavenging activities on 1,1-diphenyl-2-picrylhydrazyl were

assessed. Mosquera et al., (2007) investigated the antioxidant activity of twenty five

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plants from Colombian biodiversity. The antioxidant activity of methanol extracts from

25 species belonging to the Asteraceae, Euphorbiaceae, Rubiaceae, and Solanaceae

families were evaluated by using the spectrophotometric 1,1-diphenyl-2-picrylhydrazyl

(DPPH) free radical-scavenging method. The strongest antioxidant activities were the

methanol extracts. Ozturk et al., (2007) investigated the antioxidant activity of stem and

root extracts of Rhubarb (Rheum ribes): An edible medicinal plant. The antioxidant

activity of chloroform and methanol extract of roots and stems of Rhubarb (Rheum ribes

L.), which are used for medicinal purposes and also its fresh stems and petioles are

consumed as vegetable, was studied. However, both methanol extracts exhibited higher

DPPH radical scavenging activity than the corresponding chloroform extracts,

moreover, methanol extract of the stems showed better activity than BHT.

Arokiyaraj et al., (2008) scrutinized the free radical scavenging activity and

HPTLC finger print of Pterocarpus santalinus L. – an in vitro study. The methanol

extract of Pterocarpus santalinus showed significant DPPH radical inhibition (83.4% at

25 mg/ml concentration). Khalaf et al., (2008) screened the antioxidant activity of

methanolic crude extracts of some commonly used medicinal plants. The methanolic

extracts exhibited antioxidant activity significantly. Li et al., (2008) reported the

antioxidant properties in vitro and total phenolic contents in methanol extracts from

medicinal plants. Manian et al., (2008) reported the antioxidant activity and free radical

scavenging potential of two different solvent extracts of Camellia sinensis (L.) O.

Kuntz, Ficus bengalensis L and Ficus racemosa L. Though all the extracts exhibited

dose dependent reducing power activity, methanol extracts of all the samples were

found to have more hydrogen donating ability. Mothana et al., (2008) antimicrobial,

antioxidant and cytotoxic activities and phytochemical screening of some Yemeni

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medicinal plants. The 16 selected plants were collected from different localities of

Yemen, dried and extracted with two different solvents (methanol and hot water) to

yield 34 crude extracts. Six methanolic extracts especially those of A. fruticosa,

Actiniopteris semiflabellata, D. viscosa, P. hadiensis, T. pungens and V. bottae showed

high free radical scavenging activity. Saha et al., (2008) determined the antioxidant

activity of the methanol extract of Ixora coccinea L. by DPPH. The extract showed

significant activities in all antioxidant assays compared to the standard antioxidant.

Sarikurkcu et al., (2008) studied on the antioxidant activity of the essential oil and

methanol extract of Marrubium globosum subsp. globosum (lamiaceae) by three

different chemical assays. Antioxidant activity of the polar sub-fraction of methanol

extract was superior to the all samples. Souria et al., (2008) tried to screening of thirteen

medicinal plant extracts for antioxidant activity. Antioxidant activity and radical

scavenging activity of methanolic extracts of selected plant materials, traditionally used

by Iranian population as folk remedies was evaluated. Teixeira et al., (2008)

investigated the seasonal variation, chemical composition and antioxidant activity of

Brazilian propolis (Populus nigra i.e.black poplar, Baccharis dracunculifolia

i.e.,

alecrim, Apis mellifera) samples. Methanolic extracts from Itapecerica and Paula

Candido exhibited pronounced scavenging activity towards DPPH, indistinguishable

from BHT activity.

Huda-faujan et al., (2009) reported the antioxidant activity of plants methanolic

extracts containing phenolic compounds. Methanolic extracts of C. caudatus showed the

highest antioxidant effect. Sahgal et al., (2009) examined the in vitro antioxidant and

xanthine oxidase inhibitory activities of methanolic Swietenia mahagoni (SMCM seed)

extracts. Zahin et al., (2009) Methanolic extracts of Plumbago zeylanica (Root), Acorus

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calamus (Rhizome), Hemidesmus indicus (Stem) and Holarrhena antidysenterica

(Bark), used in Ayurvedic medicines for number of ailments were evaluated for their

antioxidant activity.

Asgarirad et al., (2010) studied the in vitro antioxidant analysis of Achillea

tenuifolia. The potency of radical scavenging effect of methanol extract was about 3.5

times greater than synthetic antioxidant BHT. Doss et al., (2010) studied the antioxidant

activity of methanolic seed extracts of Canavalia ensiformis and Canavalia gladiata by

DPPH free radical scavenging assay. The highest radical scavenging effect was

observed in Canavalia gladiate. The antioxidant potency of the freeze-dried methanol

extract of O. sintenisii bulbs and aerial parts were investigated by Ebrahimzadeh et al.,

(2010). Eldeen et al., (2010) reported in vitro antibacterial, antioxidant, total phenolic

contents and anti-HIV-1 reverse transcriptase activities of extracts of seven Phyllanthus

sp. Eighty percent methanol extracts obtained from seven Phyllanthus sp. were

evaluated. Esmaeili and Sonboli (2010) studied the antioxidant, free radical scavenging

activities of Salvia brachyantha and its protective effect against oxidative cardiac cell

injury. Methanolic extract of S. brachyantha could increase the endogenous antioxidant

enzymes in cells, and where such increased cellular defences could provide protection

against oxidative cell injury. Patil et al., (2010) reported the evaluation of in vitro

antioxidant activity of herbage of aromatic plants, Vetiver zizanioides Grass, Ailanthus

malabarica and Schinus terebinthifolius leaves. Methanolic extracts exhibited free

radical scavenging activity maximum being of Ailanthus and Schinus leaves (IC50

5ug/ml) a value lower than the positive control namely α-tocopherol (IC50 12µg/ml).

Kumar et al., (2010) investigated the in vitro antioxidant potential of various extracts of

whole plant of Bridelia scandens (Roxb) wild. IC50 value was found that methanolic

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extract of Bridelia scandens is more effective in hydroxyl radical scavenging activity

than that of petroleum ether and ethyl acetate extracts. Debasish et al., (2010)

investigated the antioxidant (in vitro) activity of methanolic extract of leaves of

Hygrophila difformis. Antioxidant activity of methanolic extract was assessed by

different in vitro model of measuring antioxidant profile i.e. DPPH method. Hazra et al.,

(2010) reported the comperative study of the antioxidant and reactive oxygen species

scavenging properties in the extracts of the fruits of Terminalia chebula, Terminalia

belerica and Emblica officinalis. The 70% methanol extracts were studied for in vitro

total antioxidant activity by DPPH method along with phenolic and flavonoid contents

and reducing power. Soman et al., (2010) evaluated the in vitro antioxidant activity of

herbage of aromatic plants. Methanolic extracts exhibited free radical scavenging

activity maximum being of Ailanthus and Schinus leaves (IC50 5µg/ml) a value lower

than the positive control namely α-tocopherol (IC50 12µg/ml). Peteros and Uy (2010)

reported the antioxidant activity of crude methanol extracts of four Philippine medicinal

plants namely Brucea amarissima (Lour.) Merr. Bark, Intsia bijuga (Coebr.) O. Kuntze,

Laportea meyeniana Warb, and Pipturus arborescens (Link) C.B. Rob leaves.

Vaghasiya and Chanda (2010) evaluated antibacterial and antioxidant activity of

different solvent (petroleum ether, chloroform, ethyl acetate, acetone and methanol)

extracts of Mangifera indica L. seeds. Maximum antibacterial activity was shown by

methanol extract followed by acetone extract. Acetone (IC50=11µg mL-1

) and methanol

(IC50=12µg mL-1

) extract also showed DPPH scavenging activity which was

comparable with that of standard ascorbic acid (IC50= 11.4µg mL-1

).

Ghimire et al., (2011) screened the total phenols, flavonoids, and free radical

scavenging activity of the methanolic crude extracts of 24 commonly used medicinal

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plants from Jhapa and Illam District of Nepal. A. vulgaris, F. lacor, M. philippensis,

Trachyspermum ammi, and Amomum subulatum showed strong activity against DPPH.

Ehsan et al., (2011) investigated the phenolics and flavonoids profiling and antioxidant

activity of three varieties of Labisia pumila Benth (alata, pumila, lanceolata), Malaysian

indigenous medicinal herb. Antioxidant activities of methanolic extract of three

varieties of Labisia pumila were determined by DPPH. Ali et al., (2011) investigated

the effects of solvent (methanol, acetone and chloroform) type on phenolics and

flavonoids content and antioxidant activities in two varieties of young ginger (Zingiber

officinale Roscoe) extracts. For routine screening of young ginger varieties with higher

antioxidant activity, methanol was recommended. Nabavi et al., (2011) measured the

antioxidant activities of methanol extract of walnut green husks (Juglans regia L.) from

11 regions of Iran with the different geographical and climatic conditions. The

antioxidant activities were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH). Singh

et al., (2011) estimated the total phenolic and flavonoid content, and to evaluate in vitro

antioxidant activity of methanolic fruit extract of Garcinia Indica. The raw, dry fruit

powder was extracted with 99.9% of methanol.

5.4.3 DPPH Radical Scavenging Activity of Targeted Plants:

The methanolic extract of targeted plants, Phyllanthus fraternus, Polygonum

microcephallum, Houttuynia cordata, Eryngium foetidum, Chenopodium album, and

Gynandropsis gynandra were used for the DPPH radical scavenging assay.

Plant extracts from Phyllanthus fraternus, Polygonum microcephallum,

Eryngium foetidum, Houttuynia cordata, Chenopodium album and Gynandropsis

gynandra the medicinal plants were investigated for antioxidant activities. Catechin, a

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major phenolic constituent was employed as the reference compound in this experiment.

Free radical scavenging activities of total crude extracts of Phyllanthus fraternus,

Polygonum microcephallum, Eryngium foetidum, Houttuynia cordata, Chenopodium

album and Gynandropsis gynandra were quantitatively determined by using a DPPH

assay at 517nm with Double Beam UV-VIS Spectrophotometer, Spectrascan Uv-2600

(Chemito). Antioxidant properties of Phyllanthus fraternus, Polygonum

microcephallum, Eryngium foetidum, Houttuynia cordata, Chenopodium album and

Gynandropsis gynandra exhibited strong activity on scavenging DPPH radicals in

methanolic extracts. Inhibition (%) of Phyllanthus fraternus, 62.89 ± 0.027 (wild plant)

and 60.29 ± 0.022 (cultivated plant); Polygonum microcephallum, 68.04 ± 0.022 (wild

plant) and 66.34 ± 0.012 (cultivated plant); Eryngium foetidum, 69.62 ± 0.007 (wild

plant) and 66.25 ± 0.042 (cultivated plant); Chenopodium album, 50.08 ± 0.015 (wild

plant) and 50.02 ± 0.003 (cultivated plant); Gynandropsis gynandra, 29.30 ± 0.031

(wild plant) and 27.22 ± 0.004 (cultivated plant); and Houttuynia cordata 23.64 ± 0.021

(wild plant) and 23.31 ± 0.002 (cultivated plant).

The IC50 value of the methanolic extract of aerial parts of Eryngium foetidum,

51.32±0.152a

in 10µg/ml, Phyllanthus fraternus was 56.12 ± 0.050a

and Houttuynia

cordata, 50.30±0.032a in 20µg/ml, Polygonum microcephallum,57.45±0.001

a and

Gynandropsis gynandra, 61.57±0.035a in 30µg/ml and Chenopodium album,

69.95±0.007a where as catechin 50.51 ± 0.022

a at 20 µg/ml.

On the basis of presented results, the investigated plant extract of Phyllanthus

fraternus, Polygonum microcephallum, Houttuynia cordata, Eryngium foetidum,

Chenopodium album, and Gynandropsis gynandra are can be considerd as good source

of antioxidants. This study could be beneficial for the development of herbal extracts for

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pharmaceutical application or food supplements in order to promote growth of live

stock and to increase the nutritional value of different foods and diet.

5.5 Biochemical Evaluation:

Polyphenols are the major plant compounds with antioxidant activity. Typical

phenolics that possess anti-oxidant activity are known to be mainly phenolic acids and

flavonoids (Demiray et al., 2009). It is reported that the phenolics are responsible for

the variation in the antioxidant activity of the plant (Luo et al., 2004). They exhibit anti-

oxidant activity by inactivating lipid free radicals or preventing decomposition of hydro

peroxides into free radicals (Pokorny 2001; Pitchaon et al., 2007).

Phenolic compounds and flavonoids are widely distributed in plants. Phenolic

compounds are secondary metabolites that are derivatives of the pentose phosphate,

shikimate and phenylpropanoid pathways in plants (Randhir et al., 2004). Phenolics

have been reported to have a capacity to scavenge free radicals. They are commonly

found in both edible and non-edible plants and have multiple biological effects

including antioxidant activity. The antioxidant activity of phenolics is mainly due to

their radox properties (Rice et al., 1996, Rice et al., 1996, Kahkonen et al., 1999;

Valenzuela et al., 2003, Hsu, 2006). Phenols, the aromatic compounds with hydroxyl

groups are said to offer resistance to diseases and pest in plants (Sadasivam and

Manickam, 1991). Plant phenolics are a major group of compounds acting as primary

antioxidants or free radical scavengers (Ayoola et al., 2008).

Flavonoids are naturally occurring polyphenolic compounds with a C6-C3-C6

backbone. These groups of plant pigments are found in fruits, vegetables, grains, herbs

bark, roots, stems, flowers, tea and wine. According to Pratt (1992) phenolic

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compounds are found abundantly in all parts of the plant. Over 5000 different

flavonoids have been identified in plant materials (Harborne et al., 2000; Humadi and

Istudor, 2008). Polyphenolic compounds of flavonoids with known properties, which

include free radical scavenging, inhibition of hydrolytic and oxidative enzymes and

anti-inflammatory action (Frankel, 1995). Polyphenols (tocopherols, phenolics, phenolic

acids and flavonoids) from medicinal and aromatic plant possess a high antioxidant

potential due to their hydroxyl groups and protect more efficiently against free radical

related diseases such as atherosclerosis (Fuhrman et al., 2001; Kris-Etherton et al.,

2002; Vaya et al., 2003). Flavonoids are “the most common group of polyphenolic

compounds in the human diet and are found ubiquitously in plants” (Spencer, 2008).

Flavonoids are widely distributed in plants fulfilling many functions. Flavonoid gives

colour of the flower which attracts pollinator animals. They also protect plants from

attacks by microbes and insects. Flavonoids have been referred to as “nature’s

biological response modifiers” because of strong experimental evidence of their

inherent ability to modify the body’s reaction to allergens, viruses and carcinogens.

They show anti-allergic, anti-inflammatory (Yamamoto and Gaynor, 2008),

antimicrobial (Cushnie and Lamb, 2005), anti-neoplastic, anti-viral, anti-thrombic

(Ayoola et al., 2008) and anti-cancer activity.

Flavonoids, exhibit a wide range of biological effects, including antioxidant

activity, antibacterial, antiviral, anti-inflammatory, antiallergic, anti-thrombotic and the

ability to lower the risk of coronary heart diseases (Simonetti et al., 2001), anti-

mutagenicity, anti-canceroginicity and anti-aging, among others; originate from this

property (Huang et al., 1992; Cook and Summan, 1996).

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Natural antioxidants especially phenolics and flavonoids from fruits, vegetables,

spices, herbs, tea and wine are already exploited commercially either as nutritional

supplements (Schuler, 1990, Patel et al., 2010).

Aqil et al.,(2006) screened the methanolic crude extracts of 12 traditionally used

Indian medicinal plants for their antioxidant and free radical scavenging properties

using α-tocopherol and butylated hydroxy toluene (BHT) as standard antioxidants.

Phytochemical analysis of plant extracts indicated the presence of major

phytocompounds phenolics and flavonoids ranged from 28.66 to 169.67 mg/g.

Pourmorad et al., (2006) investigated phenol and flavonoid contents of some selected

Iranian medicinal plants were varied from 24.1 ± 1 to 289.5 ± 5 mg g-1 in and 25.15 ±

0.8 and 78.3 ± 4.5 mg g-1

respectively.

Zahin et al., (2009) evaluated the antioxidant activity methanolic extracts of

Plumbago zeylanica (Root), Acorus calamus (Rhizome), Hemidesmus indicus (Stem)

and Holarrhena antidysenterica (Bark), used in Ayurvedic medicines. Phytochemical

analysis revealed the presence of major phytocompounds, phenolics was found in the

range of 59.50 to 109.0 mg/g of plant extracts, which correlated with antioxidant

activity.

Andarwulan et al., (2010) screened for flavonoid and antioxidant activity

content from 11 vegetables of Indonesian origin. The flavonoid content of the

vegetables studied was ranged from 0.3 to 143 mg/100 g. found in Sauropus

androgynus (L) Merr. C. caudatus had the greatest total phenols among the vegetables

analysed.

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Nahak and Sahu (2011) evaluated the phytochemical and Antioxidant activity of

Piper cubeba and Piper nigrum. ). Piper cubeba showed the highest phenolic content

i.e. 123.1±0.05 (µg/g) in comparison to Piper nigrum with 62.3±0.08 (µg/g).

Singh et al., (2011) estimated the total phenolic and flavonoid content of

Garcinia Indica, 0.348 mg/g and 137.27µg/g.

Toda (2005) investigated the polyphenols content of Houttuynia cordata was

1.14%. Pawinwongchai and Chanprasert (2011) studied total flavonoid of H.cordata

ethanolic extract was 224.90±2.80 mg QE/g dried H.cordata while total flavonoid of

H.cordata water extract was 130.79±4.89 mg QE/g dried H.cordata.

The biochemical evaluation was done for analyzing primary and secondary

metabolites. Phyllanthus fraternus, Polygonum microcephallum, Houttuynia cordata,

Eryngium foetidum, Chenopodium album, and Gynandropsis gynandra extracts were

determined by biochemical tests for total phenol and flavonoid content.

5.5.1 Total Phenol and Flavonoid of the methanolic extracts:

Bioactive components and antioxidant properties of the medicinal plants have

been the area of research to justify the claims of traditional healers. The present study

showed that Phyllanthus fraternus, Polygonum microcephallum, Eryngium foetidum,

Houttuynia cordata, Chenopodium album and Gynandropsis gynandra the medicinal

plants were the good source of antioxidant substances. The high potential of phenolics

to scavenge free radicals may be due to many phenolic hydroxyl groups present in the

plant cells (Sewa et al., 1999). It has been recognized that flavonoids show significant

antioxidant action on human health and fitness. The flavonoids act through scavenging

or chelating process (Kessler et al., 2003).

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Plant phenolics of Phyllanthus fraternus, Polygonum microcephallum, Eryngium

foetidum, Houttuynia cordata, Chenopodium album and Gynandropsis gynandra the

medicinal plants were investigated. Total phenolic content were measured by Folin

Ciocalteu reagent in terms of catechol equivalent content. Phyllanthus fraternus,

28mg±0.035 (wild) and 31±0.029 (cultivated); Polygonum microcephallum, 15±0.012

(wild) and 12±0.112 (cultivated); Eryngium foetidum,64±0.021 (wild) and 69±0.053

(cultivated); Houttuynia cordata, 58±0.043 (wild) and 63±0.014 (cultivated);

Chenopodium album, 49±0.020 (wild) and 48±0.022 (cultivated) and Gynandropsis

gynandra, 23±0.031 (wild) and 37±0.031 (cultivated).

The total flavonoid content of Phyllanthus fraternus, 3.7±0.062 (wild) and

3.2±0.078 (cultivated); Polygonum microcephallum, 6.2±0.024 (wild) and 6.9±0.019

(cultivated); Eryngium foetidum, 3.4±0.079 (wild) and 7.6±0.016 (cultivated);

Houttuynia cordata, 3.8±0.060 (wild) and 4.5±0.044 (cultivated); Chenopodium album,

2.3±0.132 (wild) and 3.6±0.064 (cultivated) and Gynandropsis gynandra, 8.3±0.014

(wild) and 3.2±0.081 (cultivated) were investigated.

5.6 Analysis of Hydroxyl radical-induced DNA strand scission:

The protective effect of methanolic plant extract of targated plants Phyllanthus

fraternus, Polygonum microcephallum, Eryngium foetidum, Houttuynia cordata,

Chenopodium album and Gynandropsis gynandra on ФX174 RF1 DNA cleavage by

hydroxyl radicals, generated from UV irradiated H2O2 were investigated.

Keum et al., (2000) studied antioxidant and anti-tumor promoting activities of

the methanol extract of heat-processed ginseng. The methanolic extract of heat-

processed neoginseng (designated as `NGMe') showed protection against strand scission

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in ФX174 supercoiled DNA induced by UV photolysis of H2O2. Keum et al. (2000)

have demonstrated that a methanolic extract of heat-processed ginseng can completely

protect the DNA strand scission at a dosage of 33000µg/ml.

Chang et al., (2001) studied the antioxidant activity of extracts from Acacia

confusa bark and heartwood. The heartwood extracts protected ФX174 supercoiled

DNA against strand scission induced by ultraviolet photolysis of H2O2, and it reduced

the amounts of intracellular hydrogen peroxide. The heartwood ethanolic extract

extracts of Acacia confuse showed complete protection of DNA strand scission at a

dosage of 1000µg/ml.

Chang et al., (2002) were evaluated several Korean medicinal plants for free

radical scavenging capacities and antioxidant activities using commonly accepted

assays. Among plant extracts, the root bark of Morus alba and the leaf of Saururus

chinensis showed stronger also showed a protective effect on DNA damage caused by

hydroxyl radicals generated from UV-induced photolysis of hydrogen peroxide.

Russo et al., (2003) studied the free radical scavenging capacity and protective

effect of Bacopa monniera L. on DNA damage. Bacopa monniera L. (family

Scrophulariaceae) (BM) is an Ayurvedic medicine, clinically used for memory

enhancing, epilepsy, insomnia and as a mild sedative. It showed a dose-dependent free

radical scavenging capacity and a protective effect on DNA cleavage. These results

were confirmed by a significant protective effect on H2O2 induced cytoxicity and DNA

damage in human non-immortalized fibroblasts.

Wang et al., (2003) examined the antioxidant activity of extracts from

Calocedrus formosana leaf, bark, and heartwood. The heartwood extract, at a dose of

approximately 500 µg/ml and 1000 µg/ml apparently completely prevented the ФX174

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supercoiled DNA cleavage induced by ultraviolet photolysis of H2O2, as judged by

agarose gel electrophoresis.

Wang et al., (2003) evaluated antioxidant properties and phytochemical

characteristics of extracts from Lactuca indica. The hot water extract of L. indica, at a

dosage of 5000µg/ml dramatically inhibit DNA strand cleavage under the same UV/

H2O2 condition.

Shyur et al., (2005) reported antioxidant properties of extracts from medicinal

plants popularly used in Taiwan. They observed that, at a dose of 1000 µg/ml, Ludwigia

octovalvis and Bombax malabaricum exhibited significant protection on ФX174

supercoiled DNA against strand cleavage induced by UV irradiated H2O2.

Hsu, (2006) evaluated the antioxidant activity of extract from Polygonum

aviculare. L. Free radicals induce numerous diseases by lipid peroxidation, protein

peroxidation, and DNA damage. Polygonum aviculare L. extract has DNA protective

effect in hydroxyl radical-induced DNA strand scission assays. Polygonum aviculare L.

extract decreased the DNA strand scission induced by both H2O2 and UV radiation. It

shows a dose-dependent protection of DNA under oxidative stress. The higher the

concentration of Polygonum aviculare L. extracts, the better the DNA protection. There

is almost complete protection at a dose of 1000 µg/ml.

Hsu, et al., (2007) investigated antioxidant activity of extract from Polygonum

cuspidatum. Numerous diseases are induced by free radicals via lipid peroxidation,

protein peroxidation and DNA damage. Polygonum cuspidatum extract has DNA

protective effect in hydroxyl radical-induced DNA strand scission assays. Polygonum

cuspidatum showed nearly complete protection at a dose of 5000 µg/ml.

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Chen et al., (2011) studied on DNA damage protection and 5-lipoxygenase

inhibiting activity of areca (Areca catechu L.) inflorescence extracts. DNA damage

caused by free radical is associated with mutation-based health impairment. The results

showed that the boiling water extract significantly restrain the oxidative damage of

pBR322 plasmid DNA induced by free radical, and synergize with trolox.

Guleria et al., (2011) evaluated antioxidant activity and protective effect against

plasmid DNA strand scission of leaf, bark, and heartwood extracts from Acacia catechu.

The ethyl acetate and acetone fractions of heartwood significantly protected pBR322

supercoiled plasmid DNA against strand scission induced by hydroxyl radicals in a

Fenton’s reaction mixture.

Kumar et al., (2012) studied on the antioxidant/genoprotective Activity of

extracts of Koelreuteria paniculata Laxm. The KPE extract and KPF fraction both

showed DNA protective effect in Calf thymus/pUC18 DNA protection studies.

A detectable effect on the prevention of DNA cleavage was found at the

Chenopodium album plant extract dosages between 1µg/ml, 10µg/ml, 100µg/ml,

500µg/ml, 1000µg/ml, 5000µg/ml and 10000µg/ml, with significant protection

observed at 1000-10000µg/ml of plant extract. At a dose of 1000µg/ml, 5000µg/ml and

10000µg/ml the supercoiled ФX174 RF1DNA was virtually totally protected, in

comparison to native DNA and DNA attached by hydroxyl radicals.

In Eryngium foetidum plant extract showed significant protection of DNA

cleavage dosages in 5000µg/ml and 10000µg/ml. At a dose of 5000µg/ml and

10000µg/ml the supercoiled ФX174 RF1DNA was completely protected, in comparison

to native DNA and DNA attached by hydroxyl radicals.

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Phyllanthus fraternus plant extract prevent the DNA cleavage at the dosages

between 100µg/ml, 500µg/ml, 1000µg/ml, 5000µg/ml and 10000µg/ml. The most

significant protection observed at 1000-10000µg/ml of plant extract. At a dose of

1000µg/ml, 5000µg/ml and 10000µg/ml the supercoiled ФX174 RF1DNA was

completely protected, in comparison to native DNA.

The Polygonum microcephallum plant extract showed best protection of DNA

cleavage at the dosages between 1µg/ml, 10µg/ml, 100µg/ml, 500µg/ml, 1000µg/ml,

5000µg/ml and 10000µg/ml. The most significant protection observed at 1000-

10000µg/ml of plant extract. At a dose of 1000µg/ml, 5000µg/ml and 10000µg/ml the

supercoiled ФX174 RF1DNA was totally protected, in comparison to native DNA and

DNA attached by hydroxyl radicals.

The prevention effect of DNA cleavage was found at the Houttuynia cordata

plant extract dosages between 1000µg/ml, 5000µg/ml and 10000µg/ml. The protection

observed at a dose of 5000µg/ml and 10000µg/ml of plant extract where the supercoiled

ФX174 RF1DNA was protected, in comparison to native DNA and DNA attached by

hydroxyl radicals.

The effect on the prevention of DNA cleavage was found at the Gynandropsis

gynandra plant extract at a dose of 5000µg/ml and 10000µg/ml, the supercoiled ФX174

RF1DNA was protected, in comparison to native DNA and DNA attached by hydroxyl

radicals.

The effect on the prevention of DNA cleavage was observed by Keum et al.,

(2000) in Neo-Ginseng at a dosage of 33000µg/ml, Chang et al., (2001) in Acacia

confusaa at a dosage of 1000µg/ml, Wang et al., (2003) in Calocedrus formosana, at a

dosage of 500 µg/ml and 1000 µg/ml, Wang et al., (2003) in Lactuca indica, , at a

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dosage of 5000µg/ml, Shyur et al., (2005) in Ludwigia octovalvis and Bombax

malabaricum at a dosage of 1000 µg/ml, Hsu, (2006), in Polygonum aviculare at a dose

of 1000µg/ml. and Hsu, et al., (2007) in Polygonum cuspidatum at a dose of 5000

µg/ml.

The Polygonum microcephallum, Chenopodium album and Phyllanthus

fraternus plant extract showed best protection of DNA cleavage at the dosage of

100µg/ml, 500µg/ml, 1000µg/ml, 5000µg/ml and 10000µg/ml while Houttuynia

cordata, Eryngium foetidum and Gynandropsis gynandra have showed equal and better

effect on the prevention of DNA cleavage at a dosage of 5000µg/ml and 10000µg/ml.