Jamonline / 2(3); 2012 / 232243 Research Article

Lakshmi Bhavani A et al

Journal of Atoms and Molecules

An International Online Journal
ISSN 2277 1247

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.Venkatramlingam2, G.Chandra2 1 PG 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 Abstract: In the present investigation callogenic and secondary metabolite studies of Achyranthes aspera Revised on: 04-05-2012 Accepted on: 14052012

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: * Corresponding author Lakshmi Bhavani A, Email: albhavani@gmail.com Tel: +91 9003641450 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

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Jamonline / 2(3); 2012 / 232243 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

Lakshmi Bhavani A et al 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

additives and fragrances. (Danhanakar et al., 2000). Medicinal plants have been used as an exemplary alternative source remedy for for centuries treating as an

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

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

methanolic extracts of Achyranthes aspera leaves are valuable antitumor promoter in cancer. It contains ecdysterone,

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 www.jamonline.in 233

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 All rights reserved 2011

Jamonline / 2(3); 2012 / 232243 objective of this study was to initiate callus from explants of Achyranthes aspera and check for the presence of secondary

Lakshmi Bhavani A et al 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

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

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

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

(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 2520C with 55-60% relative humidity and continuous light. Analysis of secondary metabolites by TLC Preparation of TLC Plates: The adsorbent used for thin layer

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.

chromatography was silica gel G. About 25 g All rights reserved 2011 www.jamonline.in 234

Jamonline / 2(3); 2012 / 232243 Thin Layer Chromatography Following the spreading of silica gel and the activation of plates, about 401 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) chloridepotassium 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

Lakshmi Bhavani A et al 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 120C 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

chromatogram is rinsed for 10 minutes with All rights reserved 2011 www.jamonline.in 235

Jamonline / 2(3); 2012 / 232243 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

Lakshmi Bhavani A et al 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

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

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 www.jamonline.in 236

(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 All rights reserved 2011

Jamonline / 2(3); 2012 / 232243 + NAA (0.25+0.25, 0.50+0.50mg/L) and 100% response at high concentration of 2, 4D + NAA (1.0+1.0, 2.0+2.0mg/L) was observed (Table-2). In the observation for morphological separation

Lakshmi Bhavani A et al 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

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 2,4D 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 advantagesmany samples can be analyzed

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 Mayers 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 www.jamonline.in 237

simultaneously and quickly, and multiple All rights reserved 2011

Jamonline / 2(3); 2012 / 232243 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 Concentration S. no. Hormones Mg/L 0.5 1.0 1. 2,4-D 2.0 4.0 0.5 1.0 2. NAA 2.0 4.0 0.25+0.25 0.50+0.50 3. 2,4-D+NAA 1.0+1.0 2.0+2.0 5 5 5 5 5 5 5 5 5 5

Lakshmi Bhavani A et al 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. No. of culture vessels 5 5 Culture vessel with callus 1 1 2 2 1 1 2 3 1 2 2 3 % of callusing

20 20 40 40 20 20 40 60 20 40 40 60

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

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Lakshmi Bhavani A et al

Concentration S. no. Hormones Mg/L

No. of culture vessels

Culture vessel with callus 2 3 4 4 1 1 3 3 3 3 5 5 40 60 80 80 20 20 60 60 60 60 100 100 % of callusing

0.5 1.0 1. 2,4-D 2.0 4.0 0.5 1.0 2. NAA 2.0 4.0 0.25+0.25 0.50+0.50 3. 2,4-D+NAA 1.0+1.0 2.0+2.0

5 5 5 5 5 5 5 5 5 5 5 5

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

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Lakshmi Bhavani A et al

Concentration S. no. Hormones Mg/L Callus size

Morphological character

0.5 1.0 1. 2,4-D 2.0 4.0 0.5 1.0 2. NAA 2.0 4.0 0.25+0.25 0.50+0.50 3. 2,4-D+NAA 1.0+1.0 2.0+2.0

Small Medium Medium Large Medium Medium Medium Large Large Large Large Large

Brownish white Brownish yellow Brownish yellow Brown Brownish white Brownish yellow Brownish green Yellowish green 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.

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Lakshmi Bhavani A et al

Presence of Alkaloids (Yellow colour)

Presence of Alkaloid (Orange colour)

TLC Plate Figure 1 : Identification of alkaloids by Mayers Reagent

TLC Plate Figure 3 : Identification of alkaloid by Dragendroff reagent

Presence of Steroid (Green Colour) Presence of Steroid alkaloids (Dark Yellow colour) Presence of Sugar (Red colour)

TLC Plate Figure 2: Identification of Steroid type alkaloids by Vanillin sulphuric acid spray solution All rights reserved 2011

TLC Plate Figure 4: Identification of Steroids and Sugars by Anisaldehyde Sulphuric acid spray solution

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