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.

Improved growth and colchicine concentration in Gloriosa superba on mycorrhizal inoculation supplemented with phosphorus-fertilizer.

BACKGROUND: Gloriosa superba produces an array of alkaloids including colchicine, a compound of interest in the treatment of various diseases. The tuber of Gloriosa superba is a rich source of colchicine which has shown anti-gout, anti-inflammatory, and anti-tumor activity. However, this promising compound remains expensive and Gloriosa superba is such a good source in global scale. Increase in yield of naturally occurring colchicine is an important area of investigation. MATERIALS AND METHODS: The effects of inoculation by four arbuscular mycorrhizal (AM), fungi, Glomus mossae, Glomus fasciculatum, Gigaspora margarita and Gigaspora gilmorei either alone or supplemented with P-fertilizer, on colchicine concentration in Gloriosa superba were studied. The concentration of colchicine was determined by high-performance thin layer chromatography. RESULTS: The four fungi significantly increased concentration of colchicine in the herb. Although there was significant increase in concentration of colchicine in non-mycorrhizal P-fertilized plants as compared to control, the extent of the increase was less compared to mycorrhizal plants grown with or without P-fertilization. This suggests that the increase in colchicine concentration may not be entirely attributed to enhanced P-nutrition and improved growth. Among the four AM fungi Glomus mossae was found to be best. The total colchicine content of plant (mg / plant) was significantly high in plants inoculated with Glomus mossae and 25 mg kg(-1)phosphorus fertilizer (348.9 mg /plant) while the control contain least colchicine (177.87 mg / plant). CONCLUSION: The study suggests a potential role of AM fungi in improving the concentration of colchicine in Gloriosa superba tuber.

Biochemical characterization and antitumor study of L-glutaminase from Bacillus cereus MTCC 1305.

L-Glutaminase (E.C.3.5.2.1) extracellularly produced by Bacillus cereus MTCC 1305 was purified to apparent homogeneity with a fine band. The molecular weight of native enzyme and its subunit were found to be approximately 140 and 35 kDa, respectively, which indicates its homotetrameric nature. The substrate specificity test of this enzyme showed its specificity for L-glutamine. The purified enzyme showed maximum activity at optimum pH 7.5 and temperature 35 °C. The enzyme retained stability up to 50 and 20 % even after treatment at 50 and 55 °C, respectively, for 30 min. Monovalent cations (Na(+), K(+)) and phosphate ion activated the enzyme activity, while divalent cations (Mg(2+), Mn(2+), Zn(2+), Pb(2+), Ca(2+), Co(2+), Hg(2+), Cd(2+), Cu(2+)) inhibited its activity. Reducing agents (cysteine, glutathione, dithiothreitol, L-ascorbic acid, and ß-mercaptoethanol) stimulated its activity, whereas thiol-binding agents (iodoacetamide, p-chloromercuribenzoic acid) resulted in the inhibition of this enzyme. Kinetic parameters, K m, V max, K cat, of purified enzyme were found to be 6.25 mM, 100 µmol/min/mg protein and 2.22 × 10(2) M(-1)s(-1), respectively. The gradual inhibition in growth of hepatocellular carcinoma (Hep-G2) cell lines was found with IC50 value of 82.27 µg/ml in the presence of different doses of L-glutaminase (10-100 µg/ml).

Extractive fermentation for enhanced production of alkaline phosphatase from Bacillus licheniformis MTCC 1483 using aqueous two-phase systems.

A study was made to find out maximum partitioning of Bacillus licheniformis alkaline phosphatase in different ATPSs composed of different molecular weight of PEG X (X=2000, 4000, 6000) with salts (magnesium sulphate, sodium sulphate, sodium citrate) and polymers (dextran 40, dextran T500). Physicochemical factors such as effect of system pH, system temperature and production media were evaluated for partitioning of alkaline phosphatase. PEG 4000 [9.0% (w/v)] and dextran T500 [9.6% (w/v)] were selected as most suitable system components for alkaline phosphatase production by B. licheniformis based on greater partition coefficient (k=5.23). The two-phase system produced fewer enzymes than the homogeneous fermentation (control) in early stage of fermentation, but after 72 h the enzyme produced in the control system was less than that in the ATPS. Total alkaline phosphatase yield in ATPS fermentation was 3907.01 U/ml and in homogeneous fermentation 2856.50 U/ml.

Optimization of nutrients for gellan gum production by Sphingomonas paucimobilis ATCC-31461 in molasses based medium using response surface methodology.

A molasses based medium for the production of gellan by Sphingomonas paucimobilis ATCC-31461 was developed. Placket-Burman design criterion was applied to study the effect of various nutrient supplements on gellan production using molasses. Among the 20 variables tested, molasses, tryptone, casaminoacid, disodium hydrogen orthophosphate and manganese chloride showed significant effect on gellan production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Placket-Burman design. Most suitable medium composition for production of gellan was (g/l): molasses-112.5; tryptone-1; casaminoacid-1; disodium hydrogen orthophosphate-1; manganese chloride-0.947 and the optimum gellan production was 13.814 g/l.

Improvement in production and quality of gellan gum by Sphingomonas paucimobilis under high dissolved oxygen tension levels.

The effect of agitation rate and dissolved oxygen tension (DOT) on growth and gellan production by Sphingomonas paucimobilis was studied. Higher cell growth of 5.4 g l(-1) was obtained at 700 rpm but maximum gellan (15 g l(-1)) was produced at 500 rpm. DOT levels above 20% had no effect on cell growth but gellan yield was increased to 23 g l(-1 )with increase in DOT level to 100%. Higher DOT levels improved the viscosity and molecular weight of the polymer with change in acetate and glycerate content of the polymer.