Article

Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilanthes acmella Murr.

Department of Biotechnology, Indian Institute of Technology - Guwahati, Guwahati 781039, Assam, India
Industrial Crops and Products (Impact Factor: 2.47). 03/2012; 36(1):321–328. DOI: 10.1016/j.indcrop.2011.10.029

ABSTRACT The study revealed, for the first time, accumulation of spilanthol, an antiseptic alkylamide, in in vitro cultures of Spilanthes acmella Murr., a medicinal plant of immense commercial value. To achieve this, in vitro shoots were regenerated via direct organogenesis from leaf-disc explants of Spilanthes. Shoots were induced in the presence of N6-benzylaminopurine (BAP) alone or in combination with either α-naphthalene acetic acid (NAA) or Indole-3-acetic acid (IAA) in Murashige and Skoog medium. The best treatment for shoot regeneration was MS + BAP (5.0 μM) + IAA (5.0 μM), which promoted adventitious shoot proliferation in >82% cultures with an average of 5.3 shoots per explant. Regenerated shoots rooted spontaneously with a frequency of 100% on half strength MS medium (major salts reduced to half strength) containing 50 g l−1 sucrose. The plantlets were acclimatized successfully with 90% survival rate. Additionally, ploidy stability of the regenerated plants was assessed by flow cytometry which showed that all investigated plants had the similar ploidy as that of the mother plant. For spilanthol identification, peaks eluted from HPLC were analyzed by mass spectrometry with its characteristic fragmentation pattern. For quantification studies, calibration curve was generated, which revealed a higher amount of spilanthol content (3294.36 ± 12.4 μg/g DW) in the leaves of in vitro plants compare to those of in vivo plants (2703.66 ± 9.6 μg/g DW of spilanthol). An efficient multiplication frequency, ploidy stability and enhanced spilanthol accumulation ensure the efficacy of the protocol developed for this industrially important medicinal plant.

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    ABSTRACT: The present study describes an efficient and reproducible protocol for micropropagation of S. acmella. Shoot tips taken from 3 week-old aseptic seedlings were cultured on Murashige and Skoog (MS) semi-solid medium supplemented with different concentrations of TDZ. Among various concentrations, 0.25 μM TDZ was found to be optimum for shoot regeneration as it induced a maximum of 30.0 shoots per explant however with retarded growth (1.0 cm). Among different volumes of culture media, 15 ml liquid culture medium favored best response wherein a maximum of 80.2 shoots per explant with an average shoot length of 7.0 cm were induced after 6 week of subculturing. Successful in vitro rooting was induced on 2.5 μM NAA containing half-strength MS medium. Almost 96% rooted plants successfully transferred and acclimatized ex vitro under green house conditions. Morphological and physiological parameters compared with the in vivo-grown seedlings of the same age appeared to be ‘normal’ in respect to the fundamental characteristics examined. Keywords:
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    ABSTRACT: Plants have been extensively used for medicinal purposes and human disease management since early ages, due to presence of valuable chemical identities. Present study was carried out to identify chemical identities and the cytotoxicity of Spilanthes paniculata Wall. ex DC. which belongs to family Asteraceae. This plant is commonly known as 'Tooth-ache plant' due to the presence of anesthetic properties. A mother stock has been maintained for experiments. Leaves obtained from green-house grown plants and in vitro callus cultures of 3-weeks-old were used in this study. In vitro callus induction was optimized with leaf disc explants grown on MS medium supplemented with 0.15 mg/L of NAA and 1.50 mg/L of BA. GC-MS analysis of ethyl acetate extracts of leaf and callus samples confirmed the existence of the key chemical compound spilanthol in both samples, with significantly higher amount in fresh leaves. Cytotoxicity tests confirmed the toxicity of hexane extracts of leaf and callus samples against late third/ early fourth instar larvae of Aedes aegypti and nuaplii of Artemia salina (100% mortality with 800 µg/mL). Leaf extracts were found to be highly toxic (70% mortality with 100 ppm for A. aegypti and 100% mortality with 800 µg/mL for nuaplii of A. salina) to both test organisms than callus extract.
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