The presence of Colchicine in Gloriosa superba was confirmed by Thin Layer Chromatography and High Pressure Liquid Chromatography ( HPLC ) in which a topographic point co-occuring with the reliable sample of Cochicine in Rf value ( Colchicine 0.70 ) appeared. The maximal sum of Colchicine was found in flower ( 20.7 mg/gdw ) and minimal sum was found in root ( 7.4 mg/gdw ) .The In vitro surveies showed the maximal sum of Colchicine in 6 hebdomad old civilizations ( 22.6 mg/gdw ) ) and minimal sum in 2 hebdomads old civilizations ( 15.3 mg/gdw ) .
The workss of G. superba are stamp, tuberous rooted deciduous perennials, adapted to summer rainfall with a hibernating dry season. G. superba grows in sandy-loam dirt in the assorted deciduous woods in cheery places. It is really tolerant of nutrient-poor dirts. It occurs in midst, wood borders and boundaries of cultivated countries in warm states up to a tallness of 2530 m. It is besides widely grown as an cosmetic works in cool temperate states under glass or in conservatories ( Neuwinger, 1994 ) . G. superba A is an emerging industrial medicative harvest in South India for its high colchicines content. Due to over-exploitation and jobs faced during field cultivation, this species is now on the brink of extinction ( Siva Kumar and Krishnamurthy, 2002 ) . Colchicine is an alkaloid of Colchicum autumnale.
Colchicine was isolated in 1820 by Pelletier and Caventou. It is besides present in Gloriosa superba ( Glory Lily ) ( Gooneratne, 1966 ; Nagaratnam et Al, 1973 Thakur et Al, 1975 ; Finnie et al,1991 ) Isolation of colchicine from G. superba vary in the alkaloid degrees of workss grown in vivo.
Material and methods
Collection of works stuffs
G. superba was collected ( July-August, 2009 ) from botanical garden of Dr. Y. S. Parmar University of Horticulture & A ; Forestry, Nauni, Solan ( Himachal Pradesh ) . The gathered workss were shade dried and finely powdered. Different works was extracted with changeless agitation for 48 h. The infusions were filtered utilizing Whatman filter paper ( no. 1 ) and so concentrated in vacuo at 40 A°C utilizing a Rotary evaporator and stored at 4A°C. ( Harborn,1984, Harborn and Harborn1998 )
Constitution of tissue civilization
For in vitro surveies terminal shoot tips, root nodes with individual subsidiary bud, dormant and non hibernating rootstock were used as explants. The explants were washed with running tap H2O pre soaked in 0.1 % liquid detergent for about 30 min, so the explants were surface sterilized with 0.1 % ( w/v ) mercuric chloride for 3 min. followed by two to three rinses of sterile distilled H2O.
The basal medium contained MS ( Murashige and Skoog, 1962 ) salts, B5 ( Gamborg et al. , 1968 ) vitamins, 3 % saccharose and 0.9 % agar, Basal medium was supplemented with assorted concentrations and combinations of growing regulators such as 2,4-D ( 2,4-dichlorophenoxy acetic Acid ) , BAP ( 6-benzylamino purine ) , NAA ( Naphthalene acetic acid ) , Kinetin, IBA ( Indole butyric acetic acid ) and IAA ( Indole acetic acid ) . The medium was adjusted to pH 5.8 with NaOH/HCl and dispensed in civilization tubings and conelike flasks of 100 milliliter capacity. The media was sterilized by autoclaving at 1.063 Kg/cmA? force per unit area for 20 proceedingss.
Colchicine was extracted from different works parts of G. superba ( root, root, foliage, and flower ) ( 2, 4, 6 and 8 hebdomads old every bit good as tissue samples ) utilizing a standard method ( Hayashi et al, 1988 ) . The samples were air dried and powdered, individually. 20 g stuff extracted for 6 hour in a Soxhlet extractor with methyl alcohol. The infusion was diluted with distilled H2O, partitioned against crude oil quintessence and eventually the aqueous stage ( incorporating colchicine ) was extracted with trichloromethane. The trichloromethane infusion was so evaporated.
High-performance liquid chromatography ( HPLC )
The designation of colchicine was done by comparing the keeping clip of the sample with that of the reliable colchicine ( Sigma ) by High-performance liquid chromatography. The instrument was used an 1100 LC system ( Hewlett-Packard, Waldbronn, Germany ) . A C18 column ( 250A-4.6 millimeter ) was used as the stationary stage. The nomadic stage consisted of a mixture of methyl alcohol and 0.1 % acetic acid solution ( 40: 60 ) with a flow-rate of 1.0 ml min-1. The wavelength selected for UV sensing was 254 nanometer ( Sivakumar et al, 2004 ) .
Result and treatment
The qualitative and quantitative appraisal of identified Alkaloid ( Colchicine ) from G. superba in vivo and in vitro has been presented in ( Table, 1,2 ) The presence of Colchicine was confirmed by thin bed chromatography in which a topographic point co-occuring with the reliable sample of Colchicine in Rf value ( Colchicine 0.70 ) appeared. The developed home bases when air dried and visualized under UV visible radiation and by exposure to ammonia exhausts clearly showed the presence of Colchicine in works parts every bit good as in tissues. Designation of stray compound was established by military policeman ( 153-1570C ) and characteristic HPLC extremums, which were superimposable to respective reliable sample ( Fig.1 )
The maximal sum of colchicine was found in flower ( 20.7mg/gdw ) and minimal sum was found in root ( 7.4mg/gdw ) . The In vitro surveies showed the maximal sum of colchicine in 6 hebdomad old civilizations ( 22.6 mg/gdw ) and minimal sum was found in the 2 hebdomad old civilizations ( 15.3 mg/gdw ) ( Table, 1,2 )
Table 1. Colchicine content ( mg/gdw ) in assorted works parts of G. superba
Table. 2. Growth indices ( GI ) and Colchicine content ( mg/gdw ) in vitro tissue civilizations of G. superba
Age of the tissue
( in hebdomads )
( GI )
( mg/gdw )