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1| P a g e International Standard Serial Number (ISSN): 2319-8141

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International Journal of Universal Pharmacy and Bio Sciences 5(3): May-June 2016

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.966***

ICV 6.16***

Pharmaceutical Sciences RESEARCH ARTICLE …………!!!

FREE RADICAL SCAVENGING ACTIVITY AND PHYTOCHEMICAL

INVESTIGATION OF ACHYRANTHES SPLENDENS SEEDS

Gill N.S*, AroraRashmi, KausharAnmol, KaurManpreet

Department of Pharmaceutical Chemistry, Rayat Institute of Pharmacy, Railmajra, SBS Nagar,

Pb. 144533.

KEYWORDS:

Achyranthes splendens,

Antioxidant activity,

Hydrogen peroxide.

For Correspondence:

Gill N.S.*

Address:

Department of

Pharmaceutical

Chemistry, Rayat

Institute of Pharmacy,

Railmajra, SBS Nagar,

Pb. 144533.

ABSTRACT

Achyranthes splendens is commonly called a Puthakanda and it is

also known as Ewa Hinahina also called Round chaff flower, is a

species of flowering plant in the pigweed family, Amaranthaceae

that is native to United state Hawaiian’. It is natural habitats are dry

forest, low shrublands, and shandy shores. It is threatened by

habitat loss. The present study was designed to investigate the

Antioxidant property of A. splendens seeds. The dried coarse

powder of A.splendens was exhaustively extracted with Methanol

and the resulting crude extracted was assayed for Antioxidant

activity. The extract showed strong Antioxidant property which

suggests the use of plant for therapeutic purpose, supporting

tradition claims.

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INTRODUCTION:

Achyranthes splendens is also called a Round chaff leaves and in Hindi it’s called a Puthakanda

belonging to Family Amarantheceae. It is found in Africa, Uganda, Kenya and Asia. Achyranthes

splendensis a small shrub ranging in a height from 2-6 feet tall. The oval leaves are ¾ to 4 ½ inches

long and ½ to 2 ½ inches wide. Pairs of leaves are arranged opposite each other on the stem. The

flower spikes to the end of branches of splendens[1, 2].

Indian system of medicines (Ayurveda,

Unani, and Siddha) has developed over a long period of time. Ayurveda is one of the oldest

medication systems of Disease prevention in the World and is called in its complete form under the

name Maharishi Ayurved[3].

The World Health Organization has approved its efficacy [4].

Fig 1:- Plant of A.splendens Fig 2:- Powder form of A.splendens

This medication Systems usually use plants or plant products for treatment of several diseases. A

number of Indian medicinal plants have been used extensively in the traditional medicinal system

for the Treatment of numerous diseases. A. splendensis an indigenous medicinal plant of Asia,

South America and Africa, it Throughout the India belonging to the family Amaranthaceae. The

plants are known for various medicinal properties and used widely for the treatment of different

diseases in Humans. In the recent time, A. splendens reported to have array of medicinal

compounds and Medicinal properties. A. splendens is used for the treatment of fever, dysentery,

asthma, Hypertension and diabetics. A. splendens seeds were reported to contain emetic and

hydrophobic Properties. A. splendens have application in infantile diarrhea and Cold. The dried

leaves of A. Splendens are reported to cure asthma [5-8].

A. splendens reported to possess wound

healing Activity, immune stimulatory properties, parricidal activity, antibacterial activity and

Antifungal activity. Roots of A. splendens reported to possess antioxidant activity and anti

inflammatory Properties [9-15].

Oxidation is a natural metabolic process in cell, some time resulting

in the formation of free radicals such as hydrogen peroxide ( H2O2), hypochlorous acid (HOCl) and



ozone (O3) [16].

Free radicals are also generated through cigarette smoke, automobile exhaust,

radiation, pesticides and air pollution etc [17].

Free radicals play a crucial role in normal aging and

neurodegenerative disorders [18].

Free radicals damage the cell membranes, proteins, fats and cause

heavy damage to the genetic material in the cell, this oxidative damage increase with age. Damaged

protein and enzymes can result in premature wrinkling, aging and even cancerous growths on the

damaged skin. [19],

Natural oxidants namely phenols in medicinal and dietary plants can prevent the

Oxidative damage caused by free radicals. Plants produce a variety of antioxidants Compounds to

control the oxidative stress caused by sunbeam and oxygen, so they represent a source of

compounds with antioxidant activity.

GEOGRAPHICAL AREAS:

The Achyranthes splendens are invasive part of:

Fig 3:- Geographical Areas

ANTIOXIDANT ACTIVITY:

Oxidation is essential to many living organisms for the production of energy for Biological

process. However, oxygen centered free radicals and other reactive oxygen species (ROS), which

are continuously, produced in vivo, result in cell death and tissue damage. Antioxidants, free radical

scavengers prevent pathological conditions of human body namely ischemia, anemia, asthma,

arthritis inflammation, neuron degeneration, ageing process [20].

Some natural antioxidants are

ginger Root, gingko leaf, grape skin extract, guar gum seed, kudzu root, rosemary leaf, turmeric

root etc. Many synthetic antioxidants such as butylated hydroxyl anisole (BHA) and butylated

hydroxyl toluene (BHT) are very effective and are used for industrial process but they possess

potential health risk and toxic properties to human health. Literature survey revealed that chemical

constituents like flavanoids, triterpenoids, polyphenolic compounds and steroids are responsible for



antioxidant activity and these chemical constituents were reported in the methanol extract of aerial

parts of A.splendens DPPH (1, 1-diphenyl-2-picryl hydrazyl) and hydrogen peroxide is an easy,

rapid and sensitive method for the antioxidant screening of plant extracts by spectral photometry. In

the presence of an antioxidant, DPPH radical obtains one more electron and the absorbance

decreases [21].

TAXONOMICAL CLASSIFICATION:

Kingdom PLANTAE

Phylum TRACHEOPHYTA

Class MANGOLIOPSIDA

Order CARYOPHYLLALUS

Genus ACHYRANTHES

Species SPLENDENS

Family AMARANTHACEAE

DIFFERENT TYPE OS SPECIES:

Achyranthes arborescence, Achyranthes atollensis, Achyranthes bidentata, Achyranthes longifolia,

Achyranthes mutica, Achyranthes talbotii, Achyranthes aspera, Achyranthes indica and

Achyranthes splendens

TRADITIONAL USES:

Traditionally, the plant is used in asthma and cough. It is pungent, diuretic, purgative and laxative,

useful in edema, dropsy and piles, boils and eruptions of skin etc. Crushed plant is boiled in water

and is used in pneumonia. Infusion of the root is a mild astringent in bowel complaint [22].

It is useful

in hemorrhoids; leaves and seeds are emetic, hydrophobia, carminative, Resolve swelling, digestive

and expel phlegm. Ash of the plant is applied externally for ulcers and warts. The crushed leaves

rubbed on aching back to cure strained back [23].

PHYTOCHEMISTRY:

The photochemistry of A. splendens has revealed the presence of various active constituent which

are listed below

A.splendens contains triterpenoidsaponin which posses’ oleanolic acid as the aglycon[24,

25].Ecdysterone, an insect moulting hormone, and long chain alcohols are also found in A.

splendens[26].

Betain is obtained from the whole plant of A. splendens[27].

Fruit contain the two

constituents are saponin C and Saponin D. Leaves of A. splendens contain the volatile oils [28].



LITERATURE SURVEY

Achyranthes splendens is also known as a Round leaf chaff flower. It is a species of flowering

plant in the pigweed family, Amarantheacea, Its natural habitatare dry forest, low shrublands,

and sandy shores. It is threatened by habitat loss [29].

A. splendens are following species:

Achyranthes aspera: which is commonly known as Latjeera (Hindi) & Rough Chaff tree (English).

It is the erect, annual or perennial herb, 1-2m in height, often with a woody base, commonly found

as a weed of waysides, on roadsides [30, 31, and 32].

A.aspera contains a triterpenoids saponin which

possesses oleanolic acid as the aglycon, an insect moulting hormone, and long chain alcohols,

which are also found in the A. aspera[33].

This plant is used for the liver complaints, rheumatoid

arthritis, scabies and other skin diseases. It also possesses tranquillizer properties [34, 35].

Achyranthes atollensis:It belongs to to the family Amarantheous. It is a natural habitat was sandy

shores. It was become extinct due to habitats (residential, commercial and military) and it was last

seen in 1964 [36].

Achyranthes arborescence: It is the commonly called a Chaff. Chaff Tree is a branched soft-

wooded tree growing to 9m tall. The leaves are like yellow-green to dark green, elliptic to slightly

lance-shaped, 5–8 cm long and 2–3.5 cm broad. The flowers are like white or pale yellow, with a

pale amber colour, and 20–90 flowers per inflorescence [37].

The flowers are enclosed by shining

pink colour, sharp-pointed bracts [38].

Achyranthes bidentata: English name: ox knee) [39]

is a species of flowering plant in the amaranth

family, Amarantheceae. It was found in India, Nepal, China, and Japan. Anti-inflammatory activity

is involved. In Nepal, root juice is used for toothache. Its seeds have been used for substitute for

cereal grains in famine years [40].

In Mizoram, the plant is used externally in the treatment of leech

bites, India and a decoction as a diuretic [41].

Achyranthes bidentata is used in Traditional Chinese

medicines. It inhibits collagen-induced arthritis and acute oedema. Most significantly, two novel

triterpene glycosides separated from the plant have been shown to be 1,000 times more potent. An

important blood group antigen that inhibits excess recruitment of neutrophils to injured tissues. This

property has made it useful for treating arthritis; when too many white blood cells are recruited to

an injured joint the neutrophils attack and destroy healthy tissues, leading to conditions such as

septic shock and rheumatoid arthritis [42].

Achyranthes mutica:It is also known as Blunt chaff flower is a species of Amarantheceae family. It

is endemic shrubs. Natural habitat is dry forest and subtropical or tropical dry shrubland. It is also

threatened by habitat loss[43].



Achyranthes talbotii:Itisa species of plant Amarantheous. It is found in Cameroon and Nigeria. Its

natural habitat is subtropical or tropical moist low land forest it is threatened by habitat loss. The

main constituent is triterpenoids saponin which possesses the oleanolic acid [44, 33].

MATERIALS AND METHODS:

Collection of plant material:

The seeds of A.splendens were collected from the Backyard of Rayat Institute of Pharmacy,

Railmajra, Ropar, and Punjab during the month of March and further authenticated by Guru Nanak

Dev University (GNDU), Amritsar (Punjab). The plant material was dried under the shade at room

temperature than make a powder in mixing at below 40ºC temperature. The powder was sieved for

removing the dust particles.

Preparation of extract and fractions:

The powdered fruit material was extracted by simple maceration for 72 hours using methanol as

solvent at room temperature and was defatted using hexane. The solvent was filtered off using

muslin cloth and the residue macerated again with the fresh solvent [45].

Both the solvents were

combined and filtered using whattmann filter paper and concentrated under reduced pressure and

the concentrated extract was stored in refrigerator throughout the investigation.

Drugs and chemical:

Ascorbic acid, methanol, Hydrogen peroxide, Potassium dihydrogen phosphate, sodium chloride

and disodium hydrogen phosphate

Instruments:

Digital weighing machine (Kern), UV spectrophotometer, pH meter (Control dynamic pH meter),

Micro Pipette (Riviera).

Phytochemical screening of the extract:

Phytochemical screening was carried out for various constituents such as: flavanoids, tannins,

alkaloids, terpenoids, carbohydrates, anthraquinone glycosides, coumarin glycosides, proteins

according to standard procedure [46].

Test for alkaloids

1. Dragendroff’s test: Dragendroff’s reagent (potassium bismuth iodide solution) was added to the

extract, reddish brown precipitates confirm the presence of alkaloids.

2. Hager’s reagent test: Hager’s reagent (picric acid solution) was added to the extract, yellow

colored precipitates confirm the presence of alkaloids.

3. Mayer’s reagent test: Mayer’s reagent (potassium iodide solution) was added to the extract,

cream-colored precipitates confirm the presence of alkaloids.



4. Wagner’s reagent test: Wagner’s reagent (solution of iodine in KI) was added to the extract,

reddish brown precipitates confirm the presence of alkaloids.

Test for sterols

1. Moleschott’s test: A few mg of the extract was heated with 1mL mixture of sulphuric acid

and water (5:1) red violet color confirms the presence of sterols.

2. Hess’s test: A few mg of the extract was dissolved in 1mL of chloroform and equal volume

of sulphuric acid, a blood red color confirms the presence of sterols.

Test for flavanoids

1. Aqueous sodium hydroxide test: Aqueous sodium hydroxide was added to the extract, a red

or purple color confirms the presence of flavanoids.

2. Concentrated nitric acid test: Concentrated nitric acid was added to the extract, crimson or

magenta color confirms the presence of flavanoids.

Test for tannins

Ferric chloride test: A few mg of the extract was dissolved in 2ml of distilled water and filtered. To

the filtrate ferric chloride solution was added. A bluish green/brownish green color confirms the

presence of tannins.

Test for carbohydrates

Molish test: A few mg of the extract was taken in 50% v/w ethanol. Two drops of 20% solution of

β-naphtha in ethanol was added and about 1mL of concentrated sulphuric acid was allowed to flow

down the sides of the test tube. A red or violet colored ring at the junction of two layers confirms

the presence of carbohydrates.

Test for anthraquinone glycosides

Brontrager’s test: A small amount of the extract was mixed in ether and after filtration aqueous

ammonia or caustic soda was added. Appearance of pink, red or violet color in aqueous layer after

shaking, confirms the presence of the anthraquinone glycoside.

Hydrogen peroxide (H2O2) free radical scavenging method:

Hydrogen peroxide (H2O2) free radical activity was determined according to the particular method

[47].The methanol fruit extract of Achyranthes splendens (MEAS) solution (1.0 ml in various

concentrations (100-200 μg mL-1

) was mixed with 2.4 ml of 0.1 M phosphate buffer (pH 7.4), and

then 0.6 ml of a 43 mm solution of H2O2 in the same buffer were added. After 10 min the

absorbance values of the reaction mixtures were recorded against a blank solution containing

phosphate buffer without H2O2 at 230 nm using a spectrophotometer. For each concentration, a

separate blank sample was used for background subtraction. Ascorbic acid was used as a standard



and mixture without sample was taken as a control. All tests were done in triplicate. The percentage

scavenging of H2O2 was calculated as:

Percentage scavenging of H2O2 = [(Ao−A1)/Ao] ×100

Where:

Ao is the absorbance of the control

A1 is the absorbance of the extract/standard

RESULTS AND DISCUSSION:

Plant Authentication:

Achyranthes splendens was collected in the month of March 2016 from the backyard of Rayat

Institute of Pharmacy, Railmajra, Ropar (Punjab). It was authentified in the Guru Nanak Dev

University (GNDU), Amritsar (Punjab).

Phytochemical screening:

The phytochemical screening of Powder extracts of Achyranthessplendens indicates the presence

of triterpenoid, coumarin glycoside, and phenol compound phytoconstituent.

Table no. 1. Phytochemical screening of A.Splendens extract

(-) indicates absence, (+) indicates minor presence (++) indicates high presence of the

Corresponding constituent.

Free radical scavenging activity by H2O2:

Antioxidant potential of methanol powder extract of Achyranthes splendens was evaluated by

Hydrogen peroxide (H2O2) radical scavenging activity. The reduction capability of Hydrogen

peroxide (H2O2) radical was determined by the decrease in its absorbance at 517 nm. Maximum

Plant constituent/Test Methanol extract

Alkaloids ++

Sterols +

Flavanoids ++

Tannins +

Carbohydrates +

Anthraquinone glycoside -



free radical scavenging activity of methanol power extract of Achyranthes splendens was shown at

a dose of 300 μg/ml is 70±0.65 by Hydrogen peroxide (H2O2) model as shown in Table. Ascorbic

acid was used as standard to compare the free radical scavenging activity of methanol powder

extract of Achyranthes splendens.

Table no. 2. Percentage inhibition of hydrogen peroxide (H2O2) and Ascorbic acid

Value are mean of Triplicate experiments and represented as Mean±SEM

Fig 5:- H2O2 and Ascorbic acid scavenging activity of extract

CONCLUSION:

The present investigation was for evaluation of Methanol extract of Achyranthessplendens for the

antioxidant activity and it justifies the traditional use of this plant in the treatment of various types

cancer. Further study has to be required.

S. No. Concentration Absorbance Methanol extract Ascorbic acid

1. 100 0.097 20±0.35 25±0.45

2. 200 0.072 50±0.47 65±0.52

3. 300 0.053 70±0.65 75±0.60

P a g e | 1 0 International Standard Serial Number (ISSN): 2319-8141


ACKNOWLEDGMENTS:

The authors are highly thankful to Professor A Banerjee and all faculty members of Rayat Institute

of Pharmacy for their encouragement and support. We are also grateful to Rayat and Bahra

Educational and Research Trust for their unconditional help to carry out this project.

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