THE ROLE OF 2, 4 –D AND NAA IN CALLUS INDUCTION OF ACHYRANTHES ASPERA AND ITS SECONDARY METABOLITE STUDIES

Jamonline / 2(3); 2012 / 232–243 Lakshmi Bhavani A et al

All rights reserved© 2011 www.jamonline.in 232

Research Article

Journal of Atoms and Molecules An International Online JournalAn International Online JournalAn International Online JournalAn International Online Journal ISSN ISSN ISSN ISSN –––– 2277 2277 2277 2277 –––– 1247124712471247

THE ROLE OF 2, 4 –D AND NAA IN CALLUS INDUCTION OF ACHYRANTHES ASPERA

AND ITS SECONDARY METABOLITE STUDIES

J.Senthilmanickam1, A.Lakshmi Bhavani1*, K.Venkatramlingam 2, G.Chandra2 1PG and Research Department of Biotechnology, Sengunthar Arts and Science College,

Tiruchengode 637205, Nammakal Dist. Tamilnadu, India. 2 Dept. of Zoology, Govt. Arts and Science College, Salem-636007, Tamilnadu, India

Received on: 28-02-2012 Revised on: 04-05-2012 Accepted on: 14–05–2012

Abstract:

In the present investigation callogenic and secondary metabolite studies of Achyranthes aspera

were carried out, stems were used as explants, callus was induced by using two different plant

growth hormones 2,4-D (Dichlorophenoxyacetic acid) and NAA (1-Naphthalene acetic acid) in

different concentration and in combinations supplemented in the MS Medium ( Murashige and

Skoog). The effect of growth hormones (2, 4-D and NAA) in callus induction was studied at the

second and fourth week of culture. During the second week of growth, media supplemented with

the growth hormones 2,4-D and NAA separately showed best response at a concentration of

2.0mg/L and 4.0mg/L respectively. With a combination of both hormones in the media, best

response was seen at the concentration of 2.0+2.0mg/L of 2, 4-D and NAA. Similar results were

observed in the fourth week of culture. Under the morphological character study of the callus

induced by the hormones, A different observation was made with the color of the calli ranging from

large brown to brownish yellow. The presence of steroids, alkaloids and sugars in the callus was

also analyzed by performing thin layer chromatography using various spray reagents accordingly

Key Words: Achyranthes aspera, 2, 4-D, NAA, callus, alkaloids, steroids, sugars, TLC.

Introduction:

Plants have been used in traditional medicine

for several thousand years. The use of

traditional medicine in most developing

countries is a normative basis for the

* Corresponding author

Lakshmi Bhavani A,

Email: [email protected]

Tel: +91 – 9003641450



maintenance of good health as quoted by

Lucy (Hoareau and Edger Dasila, 1999). The

secondary metabolites of the plants are the

major sources of pharmaceutical, food

additives and fragrances. (Danhanakar et al.,

2000). Medicinal plants have been used as an

exemplary source for centuries as an

alternative remedy for treating human

diseases because they contain numerous

active constituents of immense therapeutic

value (Nostro et al., 2000). In India, herbal

medicines have been the basis of treatment

and cure for various disease and physiological

conditional in traditional methods practiced

such as ayurveda, unani and siddha (Perumal

samy et al., 1998). Asima Chakrabarty and

Adelheid Brantner, (2002) reported the

methanolic extracts of Achyranthes aspera

leaves are valuable antitumor promoter in

cancer. It contains ecdysterone,

dihydroxyketone, alkaloids, saponins and free

tannins. Gokhale et al (2002) showed anti

inflammatory activities of Achyranthes

aspera, in swiss albino mice and inbred wistar

rats. This is due to presence of alkaloid,

saponins and oleanolic acid.

Achyranthes aspera Linn is an abundant

indigenous herb in India and also indigenous

medicinal plant of Asia, South America and

Africa. Commonly used by traditional healer

for the treatment of fever, especially malarial

fever, dysentery, asthma, hypotension and

diabetes. The dried aerial parts are taken

orally in case of diabetes, powder made from

the dried plant is given orally against

whooping cough, decoction of the plant is

used as laxative. Roots of Achyranthes are

used as medicine for diarrhea and cold and the

leaves are used against hypoglycemic activity

and asthma (Rina Chakrabarti and Rao

Vasudeva, 2006). Plant tissue culture method

is used for regeneration and propagation of

plants from unorganized callus tissue derived

from different explants by dedifferentiation

induced by exogenous growth regulators.

Plant regeneration from calli is possible by

denovo organogenesis or somatic

embryogenesis. Callus culture also facilitate

the amplification of limiting plant material.

(Flick and Evans, 1983).The successful

establishment of cell line is capable of

producing high yields of secondary

compounds in cell suspension cultures. The

accumulation of secondary products in plant

cell culture depends on the composition of the

culture medium. In vitro grown plant cells and

tissues have been used extensively for the

production of secondary metabolites. Growth

of the cell in a totally controlled environment

of physical and chemical factors provides an

excellent system for studying changes in the

production of secondary metabolites.

Thin layer chromatography method was used

for qualitative investigation of various groups

of secondary metabolites. Alkaloids are eluted

with different polar solvents like methanol,

chloroform ammonium hydroxide. (Russel

Molyneux and Dale Gardner, 2002). The main



objective of this study was to initiate callus

from explants of Achyranthes aspera and

check for the presence of secondary

metabolites in the callus extract by TLC.

Materials and methods

Callus induction

Juvenile stems of Achyranthes aspera grown

in the herbal garden of Sengunthar Arts and

Science College, Tiruchengode, were used as

explants source. The explants were washed in

Tween-20 solution for 10min with vigorous

shaking, and then were washed 4 times with

sterile distilled water to remove the excess

Tween-20. The explants were then given a

sodium hypochlorite wash and then washed

with distilled water. Then further disinfection

of the explants were done with 0.1%HgCl2

for 2-5min, rinsed 5 times with sterile

distilled water and then inoculated and

cultured on MS media (Murashige and Skoog,

1962) containing the respective hormones 2,4-

D, (0.5,1.0,2.0,4.0mg/L) NAA

(0.5,1.0,2.0,4.0mg/L) and 2,4-D+NAA

(0.25+0.25, 0.50+0.50,1.0+1.0,2.0+2.0mg/L).

The pH of the medium was adjusted to 5.8

before autoclaving for 20min at 1210C and the

culture tubes were incubated at 25±20C with

55-60% relative humidity and continuous

light.

Analysis of secondary metabolites by TLC

Preparation of TLC Plates:

The adsorbent used for thin layer

chromatography was silica gel G. About 25 g

of silica gel G was taken in a glass mortar and

about 35 ml of the distilled water was added

to it. The mixture was then allowed to swell

for about 15 min. To this an additional 15 ml

of distilled water was added to it with stirring.

The suspension was then spread immediately

on glass plate.

Drying and activation of plates:

The freshly coated plates were then air dried

until the transparence of the layer had

disappeared. The plates were then stacked in a

drying rack and were heated in an oven for 30

min at 110° C. The activated plates were then

used.

Developer:

The developer used were the solvents in the

combination, Toluene: Ethyl acetate: Formic

acid = 3:3:0.5

Preparation of extract using Methanol:

The extracts were prepared using the solvent

methanol. Dry callus of A. aspera were

washed with sterile water and drained, 5 gm

of the dry callus was weighed and

homogenized with 50ml of methanol. The

mixture was transferred to sterile bottles and

kept at room temperature for 24 hours. The

extract was then extracted with a soxlet

apparatus. The mixture was then centrifuged

at 2000 rpm for 10min.The supernatant was

filtered through a sterile funnel containing

sterile whattman filter paper No.1.The filtrate

was stored in sterile bottles at 40C until use.



Thin Layer Chromatography

Following the spreading of silica gel and the

activation of plates, about 40µ1 of samples

were spotted about 1 cm above the bottom of

the plate and were allowed to dry. The glass

plate was kept in a beaker containing 10ml of

the developer (The samples were not allowed

to immerse in the developer).The plate was

allowed to develop for 5 min till the solvent

front reaches 1 cm below the top of the plate.

The solvent front was marked and allowed to

dry. Visualization reagents were used to stain

the glass plate to observe steroid alkaloids,

steroids, alkaloids and sugars.

Visualization reagents

Meyer reagent: (Mercury (II) chloride-

potassium iodide for steroid alkaloid)

Spray solution I: 13.55 gm mercury II

chloride and 49.8 gm potassium iodide was

dissolved separately each in 20 ml of water.

Both solutions were mixed and made up to

one liter with distilled water. Before spraying

a one part of 17 %Hydrochloric acid was

added to 10parts of this solution.

Spray solution II: 5 gm zinc chloride was

dissolved in 80 ml water; 15ml of 36%

hydrochloric acid was added to it.

Spray solution III: 15% Ammonia solution.

Procedure:

After spraying with solution I, the steroid

alkaloid appears as faint yellow spot. The

chromatogram is rinsed for 10 minutes with

water and, then sprayed with solution II and

subsequently with solution III.

Vanillin sulphuric acid: (For steroid)

Spray reagent: 1gm vanillin is dissolved in

100ml of 97% Sulphuric acid.

Procedure: Following the spraying of the

reagent the chromatogram is heated at 120°C

until the spots attain maximum color

intensity.

Dragendroff reagent: (For alkaloids)

Stock solution: 2.6 gm bismuth carbonate and

7 gms sodium iodide was boiled with 25 ml

glacial acetic acid for a few minutes, and after

12 hours the precipitated sodium acetate is

filtered, then 20 ml of the red brown filtrate is

mixed with 80 ml ethyl acetate and 0.5 ml

water is added and then stored in a dark

bottle.

Spray solution: 10 ml stock solution was

mixed with 100 ml glacial acetic acid and 240

ml ethyl acetate.

Anisaldehyde – Sulphuric acid for Sugars,

Steroid, Terpenes.

Spray solutions: This solution is prepared

freshly before use; 0.5ml solution of

Anisaldehyde is added to 50ml glacial acetic

acid and then 1ml of 97%sulphuric acid is

added to it. Following the spray the plates are

heated to 100-105° C until maximum

visualization of spot. The background may be

brightened by the water vapours. The plant



constituents, Phenols, Terpenes, Sugar and

Steroid turn Violet, Blue, Red, Grey or Green.

Modified spray solution: For visualization of

sugars, 0.5 ml Anisaldehyde, 9 ml ethanol,

0.5 ml of 95% Sulphuric acid and 0.1ml acetic

acid is mixed freshly before use.

Toluene: Ethyl acetate: formic acid (3:3:0.5

v/v/v) has been identified as the best solvent

system since maximum separation of the

chemical constituent was seen.

Results and Discussion

Callus induction

In the present investigation, two observations

were made to obtain a fine callus initiation

response from stem explants of Achyranthes

aspera. Callus induction by different

hormones supplemented in the MS medium

was studied The effect of the two growth

hormones, 2, 4-D and NAA supplemented in

the media individually as well as in

combination at different concentrations on

callus induction from stem explants of

Achyranthes aspera was carried out. The

results showed that callus response increases

gradually with increasing concentration of the

growth hormones. Similar pattern of work

was carried out by Zenk (1978). The effect of

2, 4-D in combination with BAP

(Benzylaminopurine), IAA (Indole Acetic

acid), IBA (4- Indol- 3-butyric acid) using

leaf explants of Achyranthes aspera was

studied by Sarakayani Mohammed Zia and

Sadia Sarwar (2008). The increasing use of

plant extracts in the food, cosmetic and

pharmaceutical industries suggests that in

order to derive active compounds, systematic

study of medicinal plant is very important

(Ncube et al., 2008)

Minimum number of callus was observed in

the two week old culture containing 2, 4-D

and NAA separately in the concentration of

0.5,1.0,2.0,4.0mg/L. A low percentage of

callusing was recorded and callus response

was also less significant in the medium with

less concentration of hormones. At the same

time, increased callus response was observed

in the tube containing high concentration of 2,

4-D and NAA (2.0+2.0mg/L) separately and

also in tubes with media containing NAA

individually at a concentration of 4.0mg/L

(Table-1).

Similarly in the four weeks old culture, a

better callus response was observed in all

culture vessels. Percentage of callusing

increased gradually with increasing

concentration of 2,4-D. Maximum 80% of

callus response was recorded at 2.0 and

4.0mg/L of 2,4-D and minimum of 40%

callusing appeared at concentration of

0.50mg/L of 2,4-D. a light decreasing level of

callus response was observed in NAA

containing tubes. Maximum response of 60%

was recorded in culture vessel containing

NAA and minimum of 20% response was

observed at low concentration of NAA. A

high significant response was obtained in the

callus response at low concentration of 2, 4-D



+ NAA (0.25+0.25, 0.50+0.50mg/L) and

100% response at high concentration of 2, 4-

D + NAA (1.0+1.0, 2.0+2.0mg/L) was

observed (Table-2).

In the observation for morphological

character of callus with the two week and

four week old culture small brownish color

callus was noticed at a concentration of 2,4-D,

(0.50mg/L). Similarly, moderate, brownish

yellow, large and brown color callus appeared

in the concentration of 1.0, 2.0mg/L and

4.0mg/L of 2,4-D respectively.

A different morphological character was

observed in NAA containing culture vessel.

Moderate sized, brownish white, brownish

yellow and brownish green color callus was

observed in the concentration of 0.50, 1.0,

2.0mg/L of NAA respectively. At high

concentration of NAA (4.0mg/L), large and

yellowish green color callus had appeared.

Similar result of callus induction by NAA in

other genera was reported by Mischenko and

Fedoreyev (1999). But in combination of 2,4-

D with NAA, large greenish brown, yellowish

green color callus appeared at different

concentration (0.25+0.25, 0.50+0.50, 1.0+1.0,

2.0+2.0mg/L), (Table-3).

Thin layer chromatography

Thin-layer chromatography continues to be an

important method for qualitative analysis of

steroids because of its inherent advantages-

many samples can be analyzed

simultaneously and quickly, and multiple

separation techniques and detection

procedures can be applied. (Bhawani et al.,

2010). Achyranthes aspera contains

alkaloids, flavanoids, saponins, steroids and

terpenoids (Niranjan Sutar et al., 2011). Out

of which the water soluble alkaloids possess

anti-inflammatory activity. For the analysis of

secondary metabolite, fresh dried callus were

subjected to extraction in a solvent apparatus

with methanol and this sample was used for

chromatography studies. The present study

investigates the presence of secondary

metabolites in the callus from stem explants

of Achyranthes aspera by thin layer

chromatography. The presence or absence of

various secondary metabolites are confirmed

using different visualization reagents viz

Mayer’s reagent, Vanillin sulphuric acid

solution, Dragendroff reagent, Anisaldehyde

sulphuric acid solution (Fig 1, 2, 3&4).

Similar work was studied by various workers

Russell Molyneux and Dale Gardner (2002)

and Peter Houghton (2002). The current

studies reported that steroid alkaloids and

sugars were found in the callus of

Achyranthes aspera and other compounds like

phenols and terpenes were not found in the

callus.

Russell Molyneux and Dale Gardner (2002)

used thin layer chromatography method for

qualitative investigation of various groups of

secondary metabolites. Alkaloids are eluted

with different polar solvents like methanol,

chloroform and ammonium hydroxide. Thus



appropriate level of 2,4-D and NAA are able

to induce the formation of calli from stem

explants of Achyranthes aspera and this callus

effectively produces alkaloids of steroid type

and sugars which has been revealed by the

work carried out.. The result may further be

exploited for production of certain secondary

metabolites by callus culture and may also be

useful for other tissue culture technique. In

future, a modern analytical method like

HPLC, HPTLC can be employed to identify

the presence of other secondary metabolites

and specific type of alkaloid in the callus of

Achyranthes aspera.

Acknowledgements

The authors are obliged to the management of

Sengunthar Arts & Science College,

Tiruchengode for providing the facilities

without restriction in the laboratory.

S. no. Hormones Concentration

Mg/L

No. of

culture

vessels

Culture

vessel with

callus

% of

callusing

1. 2,4-D

0.5

1.0

2.0

4.0

5

5

5

5

1

1

2

2

20

20

40

40

2. NAA

0.5

1.0

2.0

4.0

5

5

5

5

1

1

2

3

20

20

40

60

3. 2,4-D+NAA

0.25+0.25

0.50+0.50

1.0+1.0

2.0+2.0

5

5

5

5

1

2

2

3

20

40

40

60

Table-1 Effect of plant growth hormones 2, 4-D and NAA on second week old culture of

Achyranthes aspera.




Mg/L

No. of

culture

vessels

Culture

vessel

with

callus

% of

callusing

1. 2,4-D

0.5

1.0

2.0

4.0

5

5

5

5

2

3

4

4

40

60

80

80

2. NAA

0.5

1.0

2.0

4.0

5

5

5

5

1

1

3

3

20

20

60

60

3. 2,4-D+NAA

0.25+0.25

0.50+0.50

1.0+1.0

2.0+2.0

5

5

5

5

3

3

5

5

60

60

100

100

Table-2 Effect of plant growth hormones 2, 4-D and NAA on fourth week old culture of

Achyranthes aspera.




Mg/L Callus size

Morphological

character

1. 2,4-D

0.5

1.0

2.0

4.0

Small

Medium

Medium

Large

Brownish white

Brownish yellow

Brownish yellow

Brown

2. NAA

0.5

1.0

2.0

4.0

Medium

Medium

Medium

Large

Brownish white

Brownish yellow

Brownish green

Yellowish green

3. 2,4-D+NAA

0.25+0.25

0.50+0.50

1.0+1.0

2.0+2.0

Large

Large

Large

Large

Yellowish green

Greenish brown

Yellowish green

Yellowish green

Table-3 Callogenic response of Achyranthes aspera at different concentration and combination

of 2, 4-D and NAA.



Presence of

Alkaloids

(Yellow colour)

TLC Plate Figure 1 : Identification of alkaloids by Mayer’s Reagent

Presence of

Steroid

alkaloids (Dark

Yellow colour)

TLC Plate Figure 2: Identification of Steroid type alkaloids by Vanillin sulphuric acid spray solution

Presence of

Alkaloid

(Orange colour)

TLC Plate Figure 3 : Identification of alkaloid by Dragendroff reagent Presence of Steroid (Green Colour) Presence of Sugar (Red colour) TLC Plate Figure 4: Identification of Steroids and Sugars by Anisaldehyde – Sulphuric acid spray solution



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Documents

THE ROLE OF 2, 4 –D AND NAA IN CALLUS INDUCTION OF ACHYRANTHES ASPERA AND ITS SECONDARY METABOLITE STUDIES