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Journal of Medicinal Plants Research Vol. 6(12), pp. 2443-2447, 30 March, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1509 ISSN 1996-0875 ©2012 <strong>Academic</strong> <strong>Journals</strong> Full Length Research Paper Betalains from stem callus cultures of Zaleya decandra L. N. Burm. f. - A medicinal herb M. Radfar, M. S. Sudarshana and M. H. Niranjan* Medicinal Plant Tissue Culture Laboratory, Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore, 570006 Karnataka, India. Accepted 1 December, 2011 The plant synthesizes various medicinal important compounds; similarly in vitro derived callus also synthesizes the compounds. Hence, callus can also be used as an alternative to whole plant for the production of secondary metabolites. A preliminary experiment was conducted using stem explants of Zaleya decandra (Aizoaceae) on MS, B5 and Whites media containing different concentrations of auxins and cytokinins in order to test the best suitable medium for callogenic potentiality. Murashige and Skoog (MS) medium elicited a better response than other media used. The callus formation and the nature of callus varies depending upon the nature of the growth regulators used. The various concentrations and combinations of dichlorophenoxy acetic acid (2,4-D), 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin (Kn), naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), indole-3butyric acid (IBA) and coconut milk were tested. Of these, combination of 2,4-D (1.0 mg/L) and TDZ (2.0 mg/L) was found to be the best in inducing pigmented callus. As the concentrations of 2,4-D and TDZ increased upto 5 mgL -1 , callogenic potentiality of the explants decreased. Phenotype color ranged from white/green through yellow, mezenda, violet red to red representing different types of pigments. The pigment when subjected to laboratory test using a spectrophotometer confirmed as betalains. Key words: Callus, betalain, growth regulators, medicinal plant, Zaleya decandra. INTRODUCTION Natural products have been a major source of new drugs (Vuorela et al., 2004). The use of medicinal plants to treat human diseases has its roots in pre-historical times. Medicinal plants are used by 80% of the world population as the only available medicines especially in developing countries (Hashim et al., 2010). Unfortunately, we know very little or still unaware of a number of herbs whose potential is yet to be fully exploited (Kamboj, 2000). In the last few decades, the traditional system of medicine has become the interest of many scientists as a tool to *Corresponding author. E-mail: niranmhniran@gmail.com. Abbreviations: BAP, 6-benzylaminopurine; NAA, naphthaleneacetic acid; MS, Murashige and Skoog; 2, 4-D, dichlorophenoxy acetic acid; Kn, kinetin; CW, coconut water; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; TDZ, thidiazuron; FW, fresh weight; OD, optical density; DF, dilution factor; LCMS, liquid chromatography- mass spectrometry; NMR, nuclear magnetic resonance improve the global health issues. At the same time, the needs for the basic scientific investigation of medicinal plants using indigenous medical systems have become more significant and in the world of technology, the importance of it is much more highlighted. Zaleya decandra L. is a prostrate weed belonging to the family Aizoaceae. It is distributed in the tropical and subtropical regions of the world and southern parts of India. The root is used for the treatment of hepatitis, asthma and orchitis, and also, the decoction of the root bark is credited with properties of aperients. The juice of the leaves is dropped into the nostrils to relieve partial headache (Nadkarni, 1996; Kirtikar and Basu, 1991). Z. decandra callus culture was considered as a prospective producer of betalains with high radical scavenging activity. Betalains comprise a class of nitrogen containing plant pigments found in the cell sap of plants belonging to ten families of Caryophyllales (Benson and Laudermilk, 1957; Johri and Bhattacharyya, 1998). Biotechnological approaches, specifically plant tissue culture plays a vital role in search for alternatives to
2444 J. Med. Plants Res. production of desirable medicinal compounds from plants (Ramachandra and Ravishankar, 2002). On a global scale, medicinal plants are mainly used as crude drugs and extracts. Several of the more potent and active substances are employed as isolated compounds, including many alkaloids such as morphine (pain killer), codeine (antitussive), papaverine (phosphordiesterase inhibitor) and various types of cardiac glycosides (heart insufficiency) (Wink et al., 2005). The capacity for plant cell, tissue, and organ cultures to produce and accumulate many of the same valuable chemical compounds as the parent plant in nature has been recognized almost since the inception of in vitro technology. The strong and growing demand in today’s market place for natural, renewable products has refocused attention on in vitro plant materials as potential factories for secondary phytochemical products, and has paved the way for new research exploring secondary product expression in vitro. Plant-produced secondary compounds have been incorporated into a wide range of commercial and industrial applications, and fortuitously, in many cases, rigorously controlled plant in vitro cultures can generate the same valuable natural products. Plants and plant cell cultures have served as resources for flavors, aromas and fragrances, biobased fuels and plastics, enzymes, preservatives, cosmetics (cosmeceuticals), natural pigments, and bioactive compounds (Karuppusamy, 2009). In the present investigation, this paper describes the isolation of the secondary metabolite (betalain pigment) from the callus mass of Z. decandra by altering various growth hormones and adjuvants. MATERIALS AND METHODS Z. decandra L. were collected from in and around Mysore University campus, Karnataka and maintained as stock plants in the Departmental garden. Stem explants were utilized for investigation. Explants were thoroughly washed in running tap water for 30 min followed by 5% (v/v) liquid detergent laboline for 5 min. Then washed in distilled water and followed by surface sterilization in 0.1% (w/v) mercuric chloride solution for 2 to 3 min for stem explants. After surface disinfection the material was thoroughly washed in sterile distilled water 4 times. Then stem explants were aseptically cut into pieces of required sizes and they were inoculated on MS medium supplemented with different concentrations and combinations of auxins and cytokinins. All the media contained 3% (w/v) sucrose. All the cultures were maintained at the temperature of 25±2°C under 16 h photoperiod and maintained for the callus formation. For realizing the pigments the following experiment has been done. The weight of violet red coloured callus obtained has been measured. 862 mg of fresh weight (FW) of callus having violet red colour was aseptically removed from the culture flask, macerated and extracted the pigment with cold water (temperature
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Journal of Medicinal Plants Researc
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Editors Prof. Akah Peter Achunike E
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Electronic submission of manuscript
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Fees and Charges: Authors are requi
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Table of Contents: Volume 6 Number
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Figure 1. Chuanxiong Rhizoma (http:
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Table 1. Identification of main pea
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Table 3. Pharmacokinetic parameters
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active component study, many invest
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and 1185 kg ha -1 in the first site
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Table 1. Recommendation for use fer
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Sadanandan AK, Hamza S (1996c). Stu
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and it has been traced to South Ame
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Table 3. Results of phytochemical a
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(a) (b) (c) Figure 1. (a) Normal ce
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emedies in the south of Brazil. J.
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known fatty acids and terpenoids we
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Table 1. Renal function tests of ra
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Al-Radahe et al. 2271 Figure 3.nnnn
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namely disruption of the vascular e
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Zayachkivska OS, Konturek SJ, Drozd
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β-carotene were purchased from Sig
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Figure 1. Effect of ethanol concent
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Table 3. Climatic and soil factors
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content of polysaccharides. Phospha
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Serge et al. 2285 Table 1. Relative
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Percentage inhibition % of inhibiti
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Table 1. Result of phytochemical sc
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Table 3. Result of thin layer chrom
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Table 2. MIC of a compound 1 from c
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Zirihi et al. 2301 Figure 2. Termin
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Figure 5. T. catappa Linn. (Combret
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Figure 8. A. fumigatus: Aspect of c
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Figure 12. Sensitivity of A. fumiga
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Noormi et al. 2311 Table 1. Callus
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Table 2. Contd. NoC=No callus. 4.0
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Figure 2. Contd. at 2.0 mg/L yellow
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Table 1. Biochemical parameters in
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Figure 3. Bcl-xL reactions in inter
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ACKNOWLEDGMENTS The authors would l
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Figure 1. The first page of the boo
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Figure 3. The most widely included
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Figure 4. Lithographic print “St.
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Table 1. List of plants included in
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Table 1. Contd. Nedelcheva 2333 Cin
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Table 1. Contd. Nedelcheva 2335 Rhe
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14 7 1 15 1 35 17 16 Imported plant
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Academy of Science Publishing House
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et al., 1996; van Wyk and Gericke,
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DPPH inhibition (%) DPPH % inhibiti
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% Mortality Concentration (µg/ml)
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information on plants used for the
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Table 1. Levels of independent vari
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Arbutin (mg/g) Co-solvent Figure 1.
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Arbutin (mg/g) Extraction pressure
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Table 3. Arbuitn content of Asian p
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Arbutin (mg/g) Extraction pressure
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Arbutin (mg/g) Extraction temperatu
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Table 1. Chemical composition of es
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Darjazi 2369 Table 2. Statistical a
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Table 3. Correlation matrix (number
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Table 1. Precision test. extraction
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Figure 2. Effect of different alcoh
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Table 6. Results of ANOVA. Nie et a
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2500 J. Med. Plants Res. Flowering
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2524 J. Med. Plants Res. ACKNOWLEDG
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UPCOMING CONFERENCES 15th European
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