Phytochemical analysis of Gloriosa superba L. using GC-MS from five different ecotypes of Tamil Nadu State, India

Gloriosa superba L is an important medicinal plant and its seeds, tubers are used for medicine. To investigate the phyto-components of Gloriosa superba L were collected from various habitats of Tamil Nadu state, India. In the present study, the phyto-components from the tubers of Gloriosa superba L cultivars from Sirumalai (GA1) Mulanoor (GA2), Thuraiyur (GA3), Konganapuram (GA4) and Vedaranyan (GA5) were extracted with ethanol and the composition of chemicals and its concentration in the tubers were determined by Gas Chromatography – Mass spectrometry (GC-MS) analysis.Among the phyto-components GA1 showed 15 phyto-components, GA2 showed 13 phyto-components, GA3 showed 8 phyto-components, GA4 showed 14 phyto-components and GA5 showed 13 phyto-components. GA1, GA2, GA4 and GA5 ecotypes possessed higher phyto-components. Colchichine an important alkaloid of Gloriosa superba L was found in GA2, GA3, GA4 and GA5 accessions in good concentration. The results reveals that the geographical origin and climatic condition of a accession caused polymorphisms in the accumulation of phyto-components, its composition and morphological traits in Gloriosa Superba L originating from different ecotypes of Tamil Nadu state.


INTRODUCTION
Gloriosa Superba L. belongs to the family Colchicaceae. This plant is popularly known by other terminologies such as "Glory Lily", Kalihari, Ognisikha etc., due to the presence of wavy edged yellow and red flowers. G. superba is a perennial climbing glabrous herb with tuberous root. All the parts of the plant especially tuber is poisonous. The plant species contain high amount of Colchicine, which is a toxic alkaloid. It also contains alkaloid Gloriocine. The seeds of Gloriosa superba L is an important source for Colchicine extraction. The plant almost distributed throughout India and also found in forests of Tamil Nadu.
In some Districts, farmers cultivate the plant species for its seeds. Tuber is a rich source of alkaloid colchicine [1]. Most of the medicinal plants were the best source to get a variety of new herbal products [2]. In current scenario WHO estimated 80% of the people from all over the world are interested towards traditional medicine. Medicinal plants are commonly known as people's friend, providing the food, fuel and medicine [3]. Medicinal plant resources are major sources of Indian traditional and modern medicine [4&5].
The tubers are commonly used for the treatment of inflammation, ulcers, bleeding piles, white discharge, scrofula, skin diseases, leprosy, indigestion, helminthes, snake bites, intermittent fever, baldness and debility [6&7]. Most of the research findings are plant oriented products towards identifying the chemical compounds especially secondary metabolites [8].
Mostly medicinal plants are warrantly being screened for their biological and pharmacological activities such as anti-diabetic, antioxidant, antimicrobial, laxative and anti cancer activities [9][10][11][12][13][14]. India was sourced with its rich traditional knowledge and has heritage of herbal products and medicines and enhanced its richness with varying biodiversity [15]. The present investigation was carried out to evident the presence of phyto-components with varying quality and quantity in the samples of Gloriosa superba L collected from five different accessions of Tamil Nadu State, India.

Plant Collection
The five ecotypes of Gloriosa superba L cultivated in the places such as Sirumalai (GA1), Mulanoor (GA2), Thuraiyur (GA3), Konganapuram (GA4) and Vedaranyam (GA5) belongs to the districts such as Dindigul, Tiruppur, Trichy, Salem and Nagapatinam respectively ( Figure 1). They were indentified and authenticated by the Botanical Survey of India, (Southern Circle), Coimbatore, Tamil Nadu, India. The plants were deposited in the department of Botany, Arignar Anna Government Arts College, Namakkal, Tamil Nadu, India.

Preparation of Powder and Extract
One kilogram of tubers was shade dried and powdered. The powder extracted with ethanol for 8 hours by using Soxhlet apparatus. After 8 hours, the extract was filtered through muslin cloth, evaporated under reduced pressure condition and vacuum dried to get the viscous residue. Ethanolic extract of Gloriosa superba L was used for GC-MS analysis. 2µl of the ethanolic leaf extract of Gloriosa superba L employed for GC-MS. Yield of extract was calculated by yield (g/100g)=(W1x100)/W2, where W1 is the weight of the extract residue obtained after solvent removal and W2 is the weight of tuber taken.

GC-MS Analysis
GC-MS analysis on the ethanolic extract of Gloriosa superba L was carried out in the Indian Institute for Crop Processing Technology (IICPT), Thanjavur. GC clarus 500 perkin Elmer system comprising a Aoc -20i auto sampler and gas chromatography interfaced to a pass petometer (GC-MS) instrument was used, applying following conditions [16].

Column
Elite-1 silica capillary column (30 mm x 0.25mm ID x M µM df composed of 100% Dimethyl poly siloxane), operating in electron impact mode at 70ev was used.

Carrier Gas
Helium (99.99%) generally used as carrier gas at a constant flow of 1ml/min and an injection volume of 0.5 µl was employed (split ratio of 10:1) at injector temperature 250ºC and ion source temperature 280ºC. The oven temperature generally programmed from 110ºC, with an increase of 10ºC/min, to 200ºC, then 5ºC/min up to 280ºC, finally ending with a 9 min isothermal at 280ºC.

Spectra
Mass spectra were taken at 70eV with a scan interval of 0.5 seconds and fragments from 45 to 450 Da. GC total running time is 36 minutes. The plant extract was dissolved in methanol and filtered with polymeric solid phase extraction (SPE) column and analyzed in for different components.

Component Identification
Here interpretation on mass spectrum GC-MS was conducted by using database of National Standard and Technology (NIST) library, having more than 62,000 patterns. The spectrum of unknown component was compared with the spectrum of the known components stored in the NIST library [17]. The compound name, molecular weight and structure of the components for the test materials were ascertained.

DISCUSSION
Our study reveals that Gloriosa superba L tuber contains various phyto-chemicals including alkaloids, steroids, triterpenoids, saponins and tannins. The results supported similar findings observed by Listsea glutinosa [18]. GC-MS analysis of drugs revealed that the presence of various phyto-chemicals, including alkaloids and other observed structured compounds in GC-MS analysis in our findings [19].
Medicinal plant derived phyto-chemicals are an important source of medicinal plants and for eco friendly applications [20]. Plant extracts and phyto-components are active against various diseases [21]. In the present investigation, methanol extracts of tuber Gloriosa superba L from five different ecotypes such as Sirumalai (GA1), Mulanoor(GA2), Thuraiyur (GA3), Konganapuram (GA4) and Vedaranyam (GA5) contain various phyto-components. The results are obtained in accordance with the previous reports.
The analysis indicates that domesticated plant of Gloriosa superba L morphologically as well as physiologically divergent from their ancestor wild ecotypes. The present investigation reveals a comparative analysis of the phyto-components of five different accessions of Gloriosa superba L from Tamil Nadu state, India by using GC-MS method. Most of the accessions possess higher phyto-components in the tuber of Gloriosa superba L.

CONCLUSION
The present investigation on the phyto-components of Gloriosa superba L accessions may be utilized as an application oriented tool to find out a new source of natural antioxidants, pharmaceutical applications in a quantitative and qualitative approach.