Gloriosa superba. L has been a source of medicine right from ancient times. The tubers of this plant are sold in Indian herbal market as an important source of an alkaloid colchicine. Surface sterilized seeds of Gloriosa superba were soaked overnight in 1% GA3 on the next day seeds were planted on germinating media containing MS basal salts with 0.5 mg/l GA3 and 1.0 mg/l BA, 1% sucrose and 0.8% agar. 72.5% of seed germination was observed. The germinated seeds were transplanted on MS basal medium supplemented with 1.0 mg/l BAP, 0.05 mg/l GA3, 9.5 mg/l NAA and 6% sucrose which led to 90% tuber induction within 6 weeks of culture. Since there is a great demand of colchicine in the market, we have made an attempt to estimate the colchicine content in different parts of the plant like leaf, seed, pericarp, tuber and in vitro produced tuber using High Performance Thin Layer Chromatography, using a mixture of Ethyl acetate:Methanol (10:1.3 v/v) as mobile phase and precoated silica gel F254 TLC aluminium sheets as the stationary phase. The detection of spot was carried out at 350nm. The calibration curve was found to be linear between 100 to 600 ng/spot for colchicines. The results revealed that in vitro tuber had highest amount (0.14249%) of cochicine, followed by in vivo seed (0.10900%), tuber (0.05761%), leaves (0.46470%) and pericarp (0.04574%). The proposed method can be used to determine the colchicine content in Gloriosa superba

Amzad Basha Kolar, .

Ghouse Basha, M.

ePrints@Bangalore University

Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) 300 - 310   This article can be downloaded from www.ijpbs.net B - 300   Research Article                                                                                                                         Biotechnology       International Journal of Pharma and Bio Sciences ISSN 0975-6299  IN VITRO TUBERIZATION  AND QUANTITATIVE ANALYSIS OF COLCHICINE USING HPTLC IN GLORIOSA SUPERBA. L AN ENDANGERED MEDICINAL PLANT OF PACHAMALAI HILLS, A PART OF EASTERN GHATS,  TAMIL NADU.  AMZAD BASHA KOLAR*1 AND M. GHOUSE BASHA2                                  1Department of Molecular Biology, Bangalore University, Bangalore-560 056, India.                                              2P.G.  and Research Department of Botany, Jamal Mohamed College, Tiruchirappalli-620 020, India.  ABSTRACT  Gloriosa superba. L has been a source of medicine right from ancient times. The tubers of this plant are sold in Indian herbal market as an important source of an alkaloid colchicine. Surface sterilized seeds of Gloriosa superba were soaked overnight in 1% GA3 on the next day seeds were planted on germinating media containing MS basal salts with 0.5 mg/l GA3 and 1.0 mg/l BA, 1% sucrose and 0.8% agar. 72.5% of seed germination was observed. The germinated seeds were transplanted on MS basal medium supplemented with 1.0 mg/l BAP, 0.05 mg/l GA3, 9.5 mg/l NAA and 6% sucrose which led to 90% tuber induction within 6 weeks of culture. Since there is a great demand of colchicine in the market, we have made an attempt to estimate the colchicine content in different parts of the plant like leaf, seed, pericarp, tuber and in vitro produced tuber using High Performance Thin Layer Chromatography, using a mixture of Ethyl acetate:Methanol (10:1.3 v/v) as mobile phase and precoated silica gel F254  TLC aluminium sheets as the stationary phase. The detection of spot was carried out at 350nm. The calibration curve was found to be linear between 100 to 600 ng/spot for colchicines. The results revealed that in vitro tuber had highest amount (0.14249%) of cochicine, followed by in vivo seed (0.10900%), tuber (0.05761%), leaves (0.46470%) and pericarp (0.04574%). The proposed method can be used to determine the colchicine content in Gloriosa superba.  KEY WORDS: Gloriosa superba. L, Colchicine, HPTLC, In vitro tuberisation.                    *Corresponding author                                           AMZAD BASHA KOLAR  Department of Molecular Biology, Bangalore University, Bangalore-560 056, India.  Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) 300 - 310   This article can be downloaded from www.ijpbs.net B - 301   INTRODUCTION  Gloriosa superba (Liliaceae) is an indigenous medicinal plant commonly known as glory lily, distributed throughout India from 68.7° to 97.25° E and 8.4° N to 37.6° N and 0 to 2043 m.a.s.l. The plants of this species have proved highly adapted to extreme conditions, including temperatures ranging from 22°C to 32°C (Seemanti Ghosh et al., 2008), studies reveal that all parts of the plant, especially the tubers are extremely toxic due to the presence of a highly active alkaloid, colchicine. Apart from its action on mitosis, colchicine reduces the inflammation and relieves the pain associated with acute gout (Insel, 1996). Colchicine therapy diminishes the metabolic activity of leukocytes, resulting in reduced phagocytosis of urate microcrystals, thereby interrupting the cycle of new crystal deposition (Seegmiller         et al.,1962). In ayurveda and Yunani systems of medicine, the tuber is well known due to its pungent, bitter, acrid, heating, anthelmintic, laxative, alexiteric and abortifacient nature. It is widely used in the treatment of ulcers, leprosy, piles, inflammations, abdominal pains, intestinal worms, thirst, bruises, infertility and skin problem (Kirtkar and Basu, 1935). Glory lily is among some of the modern medicine’s most important plant actually facing local extinction. The Pachamalai hills are part of Eastern Ghats, Tamil Nadu, known for the abundance of medicinal plant wealth. The tribal people of this place are called as ‘Malayalis’. They depend only on medicinal plants for all ailments, and the over exploitation of medicinal plants has lead to endangeredness of the most valuable plants. Due to this reason Gloriosa superba is getting endangered in Pachamalai hills (Soosairaj et al., 2007). Various factors like excessive collection of the plant by people, susceptibility towards many insects and poor seed germination have lead this plant to become endangered (Sivakumar and Krishnamurthy, 2004).  The use of in vitro cultures enables the selection of uniform individuals for studying the morphogenetic aspects of tuber formation process under controlled conditions (Ulloa  al., 1997). The present study deals with the in vitro tuberization of Gloriosa superba through in vitro regeneration techniques. We have attempted to compare the colchicine content in different parts of the in vivo plant with the in vitro produced tuber through High Performance Thin Layer Chromatography (HPTLC), in order to establish the significance of tissue culture technique.  MATERIALS AND METHODS  Collection of plant material The plant materials (Gloriosa superb) were collected from Pachamalai hills, a part of Eastern Ghats, Tamil Nadu, during harvest season of November (Fig 1A). The seeds were used as explants for induction of rhizome production through in vitro techniques. And other parts were shade dried, powdered and used for HPTLC analysis.    In vitro tuberization The seeds of Gloriosa superba were washed with 5% Teepol (detergent) for 10 min and kept in running tap water for 30 min. they were surface sterilized by immersing in 70% for 2min, followed by 0.1% mercuric chloride for 3 min for the purpose of breaking the dormancy of the seeds. Seeds were rinsed with sterilized distilled water several times under asceptic conditions and immersed in 1% GA3 for overnight in asceptic condition. On the next day, seeds were washed five times with sterile distilled water and planted on seed germinating medium containing MS basal medium (Murashige and Skoog, 1962) with GA3, BA, 1% sucrose and 0.8% Agar. The germinated seeds were transplanted on corm-producing medium containing MS basal salts BAP, GA3 and NAA.  Quantification of colchicines in G.superba samples by HPTLC technique 1.0g of the powdered plant material was dissolved in 10 ml of methanol individually and kept for about 15 minutes at a temperature of 30°C and filtered, made upto the volume with methanol. 10mg of standard colchicine extrapure (Sigma-Aldrich, St.Louis, USA) of purity (98% w/v) was dissolved in 10ml of methanol by sonication. Specific quantity of the sample and standard was applied with the help Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) 300 - 310   This article can be downloaded from www.ijpbs.net B - 302   of Linomat IV as bands on the Pre-coated silica gel F254 TLC aluminium sheets and developed with mobile phase- Ethyl acetate : Methanol (10:1.3), upto about 7.5cm. The developed chromoplate was dried by a hair-dryer. The plate was scanned at 350nm by using the Camag TLC plate Scanner III. The content of colchicine was calculated by comparing the peak areas of sample and standard spots winCATS Planar Chromatography Manager program (Ver. 1.4.3) (Wagner and Bladt, 1996).  RESULTS  The seeds of Gloriosa superba are very much prone to insect attack and having very less viability for germination. The seeds were collected from wild plants of Pachamalai hills (Fig. 1B & C), surface sterilized and planted on Seed germinating medium consisting of MS basal salts with GA3, BA, 1% sucrose and 0.8% agar. Highest germination percentage (72.5%) was observed when the medium containing MS basal salts supplemented with 0.5 mg/l GA3 and 1.0 mg/l BAP. (Fig. 1E) (Table-1).The germinated seeds were transferred in the corm-producing medium containing MS basal salts with BAP, GA3 and NAA. Maximum percentage (90%) of in vitro tuberization (3.5cm length) and rooting were observed when the MS medium fortified with 1.0mg/l BAP, 0.05mg/l GA3 and 9.3mg/l NAA with 6% of sucrose. 50% of in vitro tuberization with small rhizome (1.5cm) and 25 roots were observed when the MS medium supplemented with 1.25mg/l BAP, 0.05mg/l GA3 and 9.4mg/l NAA was used in the culture medium. It was inferred that when BAP and NAA concentration was increased, tuberization was decreased and root induction was increased (Table-2).  In vitro tubers (Fig 1F&G), in vivo leaf, seed, tuber (Fig. 1D), and pericarp were thoroughly washed, shade-dried, and powdered to use in the estimation of colchicine content through HPTLC. Different compositions of mobile phase for HPTLC analysis were tested in order to obtain high resolution, symmetrical and reproducible peaks for colchicine. The desired resolution of compound was achieved by using Ethyl acetate: Methanol (10:1.3) as the mobile phase. On this system, separation was good and peaks of colchicines were well defined. The scanning wavelength of 350nm was found to be optimal for high sensitivity for colchicine spots. During the development of the HPTLC method, it was observed that a pre-saturation of TLC chamber with mobile phase for at least 1 hour was required to obtain a good separation. Estimation of the colchicine was carried out by comparing the peak area of sample with the standard. The samples were subjected to HPTLC quantification and the results obtained were tabulated (Fig 2 to 7). Colchicine content was quantified in raw material of G.superba and the values in percentages were found to be AB1 (0.14249%), AB2 (0.05761%), AB3 (0.04574), AB4 (0.04647%) and AB5 (0.10900%). The maximum colchicine content was seen in the AB1 i.e, in vitro tuber of G. superba L. and followed by in vivo seeds, tuber, leaf and pericarp. An interesting feature was the presence of a reasonably high quantity of colchicine in the pericarp which is normally discarded after harvesting of seeds (Table-3).  Table 1 Effect of growth hormones on in vitro seed germination of Gloriosa superba.  Growth Hormone (mg/l) No. of seeds inoculated/ tube No. of seeds germinated (Mean ± S.D) Percentage of seed germination BA GA3 0.5 0.5 4 1.1±0.95 27.5 1.0 0.5 4 2.9±0.94 72.5 1.5 0.5 4 1.3±1.15 32.0 2.0 0.5 4 0.9±0.7 22.0 2.5 0.5 4 No germination -     Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) 300 - 310   This article can be downloaded from www.ijpbs.net B - 303   Table 2 Effect of growth regulators on in vitro tuberization when incorporated in  MS medium supplemented with 6% sucrose  BAP (mg/l) GA3 (mg/l) NAA (mg/l) Percentage of tuberization Length of rhizome (Mean± S.D) No. of roots formed in rhizomes 0.50 0.05 9.1 25 1.2±0.1 3-4 0.75 0.05 9.2 65 2.5±0.7 9-11 1.00 0.05 9.3 90 3.5±0.3 10-15 1.25 0.05 9.4 50 1.5±0.5 20-25 1.50 0.05 9.5 No tuberization - -  Table 3 Quantification of colchicine from different samples  S.No. Samples Sample ID Peak area Amount detected in the sample (%) 1. In vitro tuber AB1 15127.0 0.14249 2. In vivo tuber AB2 9175.0 0.05761 3. In vivo pericarp AB3 7284.8 0.04574 4. In vivo leaf AB4 7400.5 0.04647 5. In vivo seeds AB5 17360.0 0.10900 Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) 300 - 310   This article can be downloaded from www.ijpbs.net B - 304    Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) 300 - 310   This article can be downloaded from www.ijpbs.net B - 305    Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) 300 - 310   This article can be downloaded from www.ijpbs.net B - 306    Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) 300 - 310   This article can be downloaded from www.ijpbs.net B - 307     Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) 300 - 310   This article can be downloaded from www.ijpbs.net B - 308     DISCUSSION  The seeds of Gloriosa superba have poor seed germination and susceptibility towards pests. Hence, the standardization of in vitro germination of seeds is a boon for mass propagation of this plant. In the present study highest percentage of seed germination was observed when the MS medium fortified with 0.5 mg/l GA3 and 1.0 mg/l BAP. Our results correlate with Sivakumar et al., (2003) who achieved over 40% of seed germination in Gloriosa superba when MS medium fortified with 1.44µM GA3 and 4.44µM BA. The germinated seeds were transferred to corm-producing media containing BAP, GA3 and NAA. In vitro tuberization was achieved after four weeks. Higher concentrations of NAA induced rooting in the shootlets probably due to “carry over” effect of BAP in these shoots which were continuously subcultured on medium supplemented with BAP. 1.0mg/l BAP lead to the proliferation of multiple shoots and 9.3mg/l NAA induced prolific rooting of the shoots. When BAP and NAA at this concentration were used together, formation of tubers occurred. BAP alone suppressed rooting and NAA alone did not favour proliferation of shoots. Incorporation of GA3 (0.05 mg/l) along with optimal concentration of BAP and NAA enhanced the percentage of cultures showing tuber formation. GA3 acted in synergism with BAP and NAA in the process of in vitro tuberization. BAP and NAA have shown to influence in vitro rhizome formation which is evident by the work of Shimasaki and Uemoto (1991) in the orchid Cymbidium goeringii. While, in vitro corms were produced in Gloriosa superba L. using MS medium supplemented with 17.22µM 2iP, 9.52µM ADS and 10µM ANL from dormant corm buds as explants. For corm production from non dormant corm bud explants, MS medium supplemented with 9.28µM Kin, 2.72µM ADS, and 10µM ANL were used (Sivakumar et al., 2003).  Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) 300 - 310   This article can be downloaded from www.ijpbs.net B - 309   Estimation of colchicine by HPTLC reveals that the in vitro tuber produced maximum amount of colchicine followed by the in vivo seeds, tuber, leaf and pericarp. The present work highlights the importance of in vitro culture of this plant for the quantitative analysis of the phytoconstituent. The study also suggests that the biochemical mechanisms used to produce the large array of compounds are not adversely affected by in vitro propagation. Our observations are supported by the work of Lakshmi et al., (1991) who has reported the maximum occurrence of colchicine in seeds of Gloriosa superba. Shah et al., (2006) reported that HPTLC can be used to determine the drug content of marketed formulations. The development and validation of a HPTLC method for the simultaneous estimation of Atorvastatin, Calcium and Ezetimibe was carried out by using a mixture of Chloroform: benzene: methanol: acetic acid (6.0:3.0:1.0:0.1 v/v/v/v) as the mobile phase (Chaudhari et al., 2006).  Three different methods of extraction of colchicine was studied and the concentration was quantified using high performance liquid chromatography (HPLC) in six different species of Gloriosa (Bharathi et al., 2006). Therefore, the data derived from these experiments have provided the chemical basis for the wide use of this plant as therapeutic agent for treating various ailments. Furthermore, this data may be handy in probing biochemistry of this plant in the future. The significance of the study is to highlight the importance of in vitro tuberization in Gloriosa superba. In vitro tubers have several advantages, such as disease free after transplanting to soil, no dormancy period, year-round cultivation is possible. The colchicine content is also high comparing to the tubers grown in in vivo conditions. The production of   in vitro tubers takes short period comparing with in vivo tubers. The study also suggests the industries to employ in vitro techniques for the mass propagation and cultivation of this highly medicinal and economically valuable plant for abundant colchicine production.  ABBREVIATIONS   HPTLC- High Performance Thin Layer Chromatography, MS-Murashige and Skoog (1962), BA- N6-benzyladenine, NAA- α-Naphthalene acetic acid, GA3- Gibberellic acid, BAP- Benzyl amino purine,    ACKNOWLEDGEMENT  Authors are thankful to the Department of Science and Technology, New Delhi, for providing the DST FIST-Sponsored program to our Department. Authors are grateful to the University Grants Commission, New Delhi, for providing financial support through UGC Major Research Project.  REFERENCES  1. Bharathi P, Philomina D and Chakkaravarthi S, Estimation of colchicine in six different species of Gloriosa grown in vivo, Indian. J. Pharm. Sci. 68(6): 806-809, (2006). 2. Chaudhari B G, Patel N M, Shah P B and Modi K P, Development and validation of a HPTLC method for the simultaneous estimation of Atorvastatin Calcium and Ezetimibe, Indian. J. Pharm. Sci, 68(6): 793-796, (2006). 3. Insel PA, Analgesic antipyretic and anti-inflammatory agents and drugs employed in the treatment of gout: colchicine. In: Harman JG, Limrid LE  and Molinoff  RW, (eds) Goodman and Gilman’s the Pharmacological Basis of Therapeutics. Edn 9th, New York, Ny: Mc Graw-Hill. pp: 647-649, (1996). 4. Kirtikar K  and Basu L, Indian Medicinal Plants, Vol. III 2nd Ed. International Book Distributor, Allahabad, India, pp2060-2067, (1935).  Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) 300 - 310   This article can be downloaded from www.ijpbs.net B - 310   5. Lakshmi S R, Arambewela, Kumudini M A N and Ranathunga J,  Studies on Gloriosa superba grown in Sri Lanka, J. Natn. Sci. Coun. Sri Lanka, 19(2): 177-180, (1991).  6. 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In Vitro Tuberization and Quantitative Analysis of Colchicine Using Hptlc in Gloriosa Superba . L an Endangered Medicinal Plant of Pachamalai Hills, a Part of Eastern Ghats, Tamil Nadu.

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In Vitro Tuberization and Quantitative Analysis of Colchicine Using Hptlc in Gloriosa Superba . L an Endangered Medicinal Plant of Pachamalai Hills, a Part of Eastern Ghats, Tamil Nadu.

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