Chemodiversity and molecular variability in the natural populations (India) of Gloriosa superba (L.) and correlation with eco- geographical factors for the identification of elite chemotype(s).

Gloriosa superba L. has economic significance due to colchicine, a bioactive compound used for gout. In present study metabolic and molecular variability in natural population of species was analyzed and correlated with edaphic and climatic factors. Thirty populations (wild) of G. superba were mapped from 10 different eco-regions of India at an elevation range of 10-1526 m, having no morphotypic variations. The two known biologically active alkaloids colchicine (ranged from 0.015-0.516%) and gloriosine (0.19-0.44%) were significantly varied (p < 0.05) among populations, leading to the identification of four elite chemotypes. Molecular variability from ISSR data divides the population in different sub clusters at intra-specific level, presenting the high similarity percentage with bootstrap value of 66-100%. Principal component analysis (PCA) revealed that elite chemotypes are related to temperature, precipitation and aridity gradient. The rhizospheric soil selenium was significantly correlated with colchicine content in G. superba.

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.

Saikosaponin b2 enhances the hepatotargeting effect of anticancer drugs through inhibition of multidrug resistance-associated drug transporters.

AIMS: Vinegar-baked Radix Bupleuri (VBRB) potentiates the activity of anticancer drugs in the liver by increasing their hepatic distribution. However, this phenomenon may be associated with drug transporters. We investigated the effect of saikosaponin b2 (SSb2; the main component of VBRB) on the activity and expression of different drug transporters in both normal cells and those that overexpress the transporter. MAIN METHODS: The activities of transporters were analyzed by concentration of their cellular substrates. Concentrations of colchicine (substrate of Pgp and MRP1) and cisplatin (substrate of OCT2 and MRP2) were determined by high-performance liquid chromatography (HPLC). The concentration of rhodamine B was determined by flow cytometry. The expression of transporter gene and protein were determined by qRT-PCR and Western blotting analysis. KEY FINDINGS: SSb2 increased colchicine efflux in HEK293 cells by primarily increasing Mrp1 activity, independent of gene and protein expression. SSb2 enhanced Mrp2 function and increased cisplatin efflux in BRL3A cells by upregulating Mrp2 gene expression, with a marginal effect on Pgp in normal cells. SSb2 increased OCT2 activity in OCT2-HEK293 cells by increasing the expression of OCT2 protein and mRNA; however, SSb2 inhibited MRP2 activity in MRP2-HEK293 cells by decreasing MRP2 protein expression, and decreased Pgp and MRP1 activity in Pgp- and MRP1-HEK293 cells. SIGNIFICANCE: SSb2 might potentially be the key active component of VBRB that enhances the hepatotargeting of anticancer drugs through the inhibition of multidrug resistance-associated drug transporters (Pgp, MRP1, and MRP2) in an environment-dependent manner.

MOF-5(Zn)-Fe2O4 nanocomposite based magnetic solid-phase microextraction followed by HPLC-UV for efficient enrichment of colchicine in root of colchicium extracts and plasma samples.

In present work, facile method is developed for determination of colchicine in human plasma sample, autumn and spring root of colchicium extracts by ultrasound assisted dispersive magnetic solid phase microextraction followed by HPLC-UV method (UAD-MSPME-HPLC-UV). Magnetic (Fe2O4-nanoparticles) metal organic framework-5, (MOF-5(Zn)-Fe2O4NPs) was synthesized by dispersing MOF-5 and Fe(NO3)3.9H2O in ethylene glycol (as capping agent) and NaOH (pH adjustment agent) by hydrothermal method. The prepared sorbent was characterized via XRD and SEM analysis and applied as magnetic solid phase in UAD-MSPME-HPLC-UV method. In this method, colchicine molecules were sorbed on MOF-5(Zn)-Fe2O4NPs sorbent by various mechanisms like ion exchange, hydrogen bonding and electrostatic, á´¨-á´¨, hard-hard and dipole-ion interaction followed by exposing sonication waves as incremental mass transfer agent and then the sorbent was separated from the sample matrix by an external magnetic fields. Subsequently, accumulated colchicine were eluted by small volume of desorption organic solvent. Influence of operational variables such as MOF-5(Zn)-Fe2O4NPs mass, volume of extracting solvent and sonication time on response property (recovery) were studied and optimized by central composite design (CCD) combined with desirability function (DF) approach. Under optimum condition, the method has wide linear calibration rang (0.5-1700ngmL-1) with reasonable detection limit (0.13ngmL-1) and R2=0.9971. Finally, the UAD-MSPME-HPLC-UV method was successfully applied for determination of colchicine autumn and spring root of colchicium extracts and plasma samples.

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.

Chemical investigation of Iphigenia stellata blatter by GC-MS.

GC-MS analysis of Iphigenia stellata Blatter in methanol extract revealed the presence of twenty chemical compounds in corm, nine in capsule wall, seven in leaves and six in seeds. Amongst the different phytochemicals identified 2-furan-carboxaldehyde-5-(hydroxymethyl) (38% in corms), glycerine (35.4% in seeds) and n-Hexadecanoic acid (31.5% in leaves, 28% in capsule wall) were significant. Iphigenia stellata is known for the presence of colchicine. However, in the methanol extract it was not detected.

Colchicine poisoning: a case report and review of literature.

Colchicine, a natural pseudo-alkaloid found in plants such as the Colchicum autumnale and Gloriosa superba has tremendous medicinal properties, but if misused by an unqualified person can result in fatal consequences. We report a case of colchicine poisoning in an adult man as a result of consumption of the herb G. superba by a 50-year-old man and review the literature.

[Study on correlation between effective ingredients of dogwood fruit from genuine producing regions and traits].

OBJECTIVE: In order to provide a theoretical foundation for the medically effective ingredient-based selection of elite cultivars in Comnus officinalis, a study has been conducted on the variation in medicinal effective ingredients of the fruit from the genuine producing areas, the correlation among effective medicinal ingredients and the correlation between effective ingredients and fruit shape as well as nutritional indexes. METHOD: The completely mature fruit was collected from the genuine producing areas Chunan county and Lin'an city of Zhejiang province. The contents of colchicine, ursolic acid and oleanolic acid were determined by HPLC, and vertical diameter/transversal diameter of the fruits, soluble solid matter and percentage of fresh flesh to the fruit were also measured. RESULT: (1) Ursolic acid, oleanolic acid, and colchicine in fruits ranged from 0.1010% to 0.4786%, 0.0149% to 0.1274% and 0.59% to 2.30%, respectively, and their RSD were 34.33%, 40.48% and 28.50%, respectively. (2) The correlation between effective ingredients and that between effective ingredients and fruit shape as well as nutritional indexes were as follows: the content of ursolic acid was significantly correlated with that of oleanolic acid with a correlation coefficient of 0.9796; both ursolic acid and oleanolic acid were in significantly negative correlation with soluble solid matter with a correlation coefficient of -0.5544 and -0.5118, respectively; colchicine was significantly associated with soluble solid matter with a correlation coefficient of 0.2412; colchicine, ursolic acid and oleanolic acid were in significantly negative correlation with the percentage of fresh flesh with a correlation coefficient of -0.2507, -0.2443 and -0.2406, respectively; three effective ingredients showed no correlation with the ratio of vertical diameter to transversal diameter of the fruit. CONCLUSION: There is a significant difference in effective ingredients among individual trees, which means that there is a big potential for selection of cultivars. Individual tree-based selection should be mainly adopted when effective ingredients are used as a main index in selection on the basis of the correlation among effective ingredients and that between effective ingredients and fruit shape as well as nutritional indexes, while ursolic acid could be combined with oleanolic acid to be used as an index and a preliminary screen could be conducted using soluble solid matter.

Sesquiterpene lactones from the roots of Ferula varia and their cytotoxic activity.

The ethyl acetate-soluble fraction from a MeOH extract of the roots of Ferula varia gave six new sesquiterpene lactones (1-6) and five known sesquiterpenes (7-11). Their structures were established on the basis of spectroscopic evidence. The cytotoxic activities of 1-11 were evaluated against selected human cancer cell lines. Compound 4 showed significant selective cytotoxicity against multidrug-resistant cancer cells (KB-C2). The cytotoxicities of compounds 1, 3, 5, 8, and 11 against KB-C2 cells were enhanced in the presence of nontoxic concentrations of colchicine.

Synthesis and hepatic transport of strongly fluorescent cholephilic dipyrrinones.

A new class of highly fluorescent (phi(F) 0.3-0.8) low molecular weight water-soluble cholephilic compounds has been synthesized in two steps from dipyrrinones. The dipyrrinone nitrogens are first bridged by reaction with 1,1'-carbonyldiimidazole to form an N,N'-carbonyldipyrrinone (3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidine-3,5-dione) nucleus, and a sulfonic acid group is then introduced at C(8) by reaction with concd H(2)SO(4). The resulting sulfonated N,N'-carbonyl-bridged dipyrrinones ("sulfoglows") are isolated as their sodium salts. When the alkyl substituents of the lactam ring are lengthened from ethyl to decyl, sulfoglows become increasingly lipophilic while maintaining water solubility. Low molecular weight sulfoglows were rapidly excreted intact in both bile and urine after intravenous infusion into rats, but higher molecular weight sulfoglows were excreted more selectively in bile. Hepatobiliary excretion of sulfoglows was partially, but not completely, blocked in mutant rats deficient in the multidrug-resistance associated transport protein Mrp2 (ABCC2). These observations point to the feasibility of developing simple sulfoglows with clinical diagnostic potential that are normally excreted in bile but appear in urine when hepatic elimination is impaired by cholestatic liver disease.

Fast determination of colchicine by TLC-densitometry from pharmaceuticals and vegetal extracts.

Colchicine, (S)-N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo-(a(-heptalen-7-yl)-acetamide, is the main alkaloid contained in Colchicum autumnale (meadow saffron). There are known colorimetric, spectrophotometric, volumetric, potentiometric, voltametric, gravimetric and various chromatographic methods for quantitative determination of colchicine, each of them presenting a series of advantages and disadvantages. As an alternative, we proposed the use of a densitometric determination for colchicine allowing the determination of this alkaloid from pharmaceutical products, as well from seeds of meadow saffron. The total alkaloid extract was separated by Thin-Layer Chromatography using Silicagel 60F(254) layers and a mixture of chloroform:acetone:diethylamine (5:4:1) as mobile phase. The same conditions were used for the determination from pharmaceutical products. Densitometric measurements were carried out at the absorption maximum (350 nm) of colchicine, the determinations being made by reflectance and by fluorescence. The peaks were optimized regarding to their area and shape by varying four scanning parameters (slit width and height, number of measurements and scanning speed). We established the calibration plot using pure colchicine in the range 50-600 ng mL(-1). The proposed method could be widely used in the pharmaceutical industry for the quick and accurate quantitative determination of colchicine because it eliminates the interferences given by other bioactive or degradation compounds. The method was characterized by validation parameters (linearity, accuracy, fidelity, sensitivity) and it was established its performances in comparison with an HPLC method and an official quantitative determination from the Romanian Pharmacopoeia X edition respectively.

Determination of colchicine in Colchicum steveni and C. hierosolymitanum (Colchicaceae): comparison between two analytical methods.

The amounts of colchicine present in two Jordanian species of Colchicum, namely, C. steveni Kunth and C. hierosolymitanum Feibrun (Colchicaceae), have been determined. An HPLC-UV (photodiode array) method employing gradient elution was developed and the results compared with those obtained using a simple TLC-spectrophotometric method. The levels of colchicine as measured by these methods were not significantly different (p < 0.05) indicating that the spectrophotometric method is an acceptable alternative to HPLC. With respect to C. steveni, the leaves contained the largest amount of colchicine (0.204/100 g), whilst in C. hierosolymitanum corms showed the highest colchicine content (0.126/100 g). As a source of colchicine, the two investigated species showed levels comparable with those found in C. autumnale, the traditional source of colchicine.

Determination of colchicine content in Colchicum hierosolymitanum and Colchicum tunicatum under cultivation.

Corms of Colchicum hierosolymitanum and Colchicum tunicatum were collected, identified and planted under field condition. We report here and for the first time the presence of colchicine in an appreciable amount in both species. The effect of different NPK fertilizer levels on colchicine content of the two Colchicum species at different growth stages were evaluated by HPLC. Results indicated that increasing NPK fertilizer levels significantly improve colchicine content in different plant parts and stages. The highest colchicine content observed in corms was at maturity stage 0.766 mg/g and 0.688 mg/g dry weight with C. hierosolimitanum and C. tunicatum, respectively.

Evaluation of commercial ginkgo and echinacea dietary supplements for colchicine using liquid chromatography-tandem mass spectrometry.

In response to concerns that commercial dietary supplements containing Ginkgo biloba (ginkgo) and Echinacea purpurea, Echinacea angustifolia, or Echinacea pallida (echinacea) might be contaminated with colchicine, a highly selective and sensitive assay was developed for colchicine that is based on high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). The method utilizes reversed-phase HPLC separation of compounds in a methanolic extract of the dietary supplement or botanical sample followed by positive ion electrospray ionization with collision-induced dissociation and multiple reaction monitoring of three characteristic fragmentation pathways of the protonated molecule of colchicine, m/z 400 --> 358, 400 --> 326, and 400 --> 310. The minimal detectable concentration of colchicine using this assay was 10 pg on-column, which is equivalent to 20 ppb colchicine in a 0.5 g ginkgo leaf sample. The method was validated by analyzing 0.5 g samples spiked with colchicine and determining the recovery. A total of 26 commercial ginkgo and echinacea dietary supplements were purchased from pharmacies in Chicago, IL, and analyzed for colchicine. In contrast to a recent report, no colchicine was detected in any of the samples. In addition, authenticated ginkgo leaves were collected, assayed, and found to contain no colchicine, which is consistent with the botanical literature. On the basis of the results obtained using this new LC-MS-MS assay, which is more sensitive and more selective than previously published methods for colchicine, we find no cause for concern regarding colchicine contamination of ginkgo or echinacea dietary supplements.

Evaluation of commercial Ginkgo biloba dietary supplements for the presence of colchicine by high-performance liquid chromatography.

A high-performance liquid chromatographic method with photodiode array detection was developed for the detection of the presence of colchicine in commercial ginkgo products. The method is based on the baseline separation of constituents in ginkgo samples plus reference colchicine. The minimal detectable concentration of colchicine is 1.0 ng on column in the current assay. By analysis of retention time and UV profile of suspect peaks in the sample with those of reference colchicine, none of the nine commercial ginkgo products analyzed contained colchicine.

A new glycoside, 3-O-demethylcolchicine-3-O-alpha-d-glucopyranoside, from Gloriosa superba seeds.

A new colchicine glycoside, 3-O-demethylcolchicine-3-O-alpha-D-glucopyranoside, has been isolated from Gloriosa superba seeds. The assigned structure has been corroborated by spectroscopic data and enzymatic hydrolysis.

Colchicine poisoning by accidental ingestion of meadow saffron (Colchicum autumnale): pathological and medicolegal aspects.

Although intoxications with colchicine, the alkaloid of Colchicum autumnale (meadow saffron), are well known, in most cases the intoxications are evoked by oral or parenteral preparations traditionally used as medication against gout. The accidental ingestion of Colchicum autumnale, on the other hand, is a rare event and has to our knowledge only twice been described in detail. We report a further case in which two persons confused this highly poisonous plant with wild garlic (Allium ursinum), a popular spice in the Central European cuisine. While one person merely complained about a 3-day episode of nausea, vomiting and watery diarrhea, the second person died of multi-organ system derangements 48 h after the ingestion of the colchicum leaves. At autopsy hemorrhagic lung oedema, hypocellular bonemarrow, centrilobular fatty necrosis of the liver and necrosis of the proximal convoluted tubuli of the kidneys were observed. A colchicine concentration of 7.5 micrograms/ml was found in the bile whereas no substance was detected in the postmortem blood.

Tissue distribution and milk transfer of colchicine in a lactating sheep following a single dose intake.

The present communication describes the use of a quantitative thin-layer-chromatography method for the determination of colchicine in milk of a lactating sheep following a 10 mg single oral intake of the alkaloid extracted from the leaves of Colchicum autumnale. The concentration of the alkaloid in the leaves was 0,18% by weight. Unchanged colchicine can be detected in milk with the described method within the first 6 hours following the intake, whereas the maximal concentration of the alkaloid is reached 9 hours after the administration. Almost 40 hours following the intake the detection of the alkaloid in milk is not more possible. Postmortal determinations of the tissue levels of the alkaloid revealed major concentrations in the bone marrow, whereas only trace amounts were identified in heart and muscular tissue.

[Synthesis of new fluorescent reagent and its application in solution fluorescence analysis].

In our laboratory we have designed and synthesized a new fluorescent CGE(N), which has a fluo-rigen and can react with active hydrogen in chemical compounds. We have studied its application in solution fluorescence, thin-layer fluorescence and solid fluorescence analysis. The results have indicated that CGE (N) is a good reagent in analysis. And the example of its application in solution fluorescence analysis is given in this paper.