Screening the elite chemotypes of Gloriosa superba L. in India for the production of anticancer colchicine: simultaneous microwave-assisted extraction and HPTLC studies.

BACKGROUND: Gloriosa superba L. (Colchicaceae) is a high-value medicinal plant indigenous to Africa and Southeast Asia. Its therapeutic benefits are well-established in traditional medicines including Ayurveda. It is well known for its natural bioactive compound colchicine which exhibits a wide range of pharmacological activities i.e. rheumatism, gout and was also introduced into clinical practices. The increasing demand as well as its illegal harvesting has brought this valuable plant under threatened category. METHODS: The present investigation describes a microwave assisted extraction (MAE) strategy coupled with a densitometric-high performance thin layer chromatographic (HPTLC) methodology for the analysis of colchicine from 32 different populations of G. superba. A Box-Behnken statistical design (3 level factor) has been employed to optimize MAE, in which power of microwave, time of irradiation, aqueous ethanol and pH were used as independent variables whereas colchicine was used as the dependent variables. Chromatography was carried out on Silica gel 60 F254 TLC plates with toluene: methanol, 85:15 (v/v) being used as solvent system. Densitometric measurement was performed at λ=254 nm following post-derivatization (10% methanolic sulphuric acid). RESULTS: Optimal conditions for extraction to obtain the maximum colchicine yield was found to be 7.51 mg g- 1 which was very close to be predicted response 7.48 mg g- 1 by maintaining microwave power (460 W), irradiation time (6.4 min), aqueous ethanol-30, pH -3. Colchicine content ranged between 2.12-7.58 mg g- 1 among 32 G. superba populations in which only three chemotypes viz. GS- 1, GS- 3, and GS- 2 collected from West Bengal and Sikkim, respectively exhibited maximum yield of colchicine. CONCLUSION: Therefore, this newly developed optimized MAE coupled with HPTLC densitometry methodology not only quantifies colchicine in order to identify elite chemotypes of G. superba, but it also encourages in selecting high yielding populations of the plants for industrial use and economic boost for the farmers. This validated, simple and reproducible HPTLC protocol is being used for the first time to estimate colchicine from natural populations of G. superba obtained from 32 different geographical regions of India.

Discovery and engineering of colchicine alkaloid biosynthesis.

Few complete pathways have been established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture1. A lead example is colchicine, a US Food and Drug Administration (FDA)-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species2-5. Here we use a combination of transcriptomics, metabolic logic and pathway reconstitution to elucidate a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome or genetic tools in the native host. We uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine6. Notably, we identified a non-canonical cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16 enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine alkaloids and provides insights into the unique chemistry that plants use to generate complex, bioactive metabolites from simple amino acids.

Microsome-mediated transformation of O-methylandrocymbine to demecolcine and colchicine.

Microsomal preparations from immature seeds of Colchicum autumnale L. catalyse the ring expansion reaction of O-methylandrocymbine to demecolcine in the presence of NADPH and O2. In addition evidence is given for further transformation of demecolcine to colchicine in the presence of acetyl-CoA and NADPH.