Development of a multianalytical strategy for detection of frauds in Coleus forskohlii supplements.

A new multianalytical methodology based on gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometry (MS) has been proposed to evaluate frauds affecting the composition of Coleus forskohlii root supplements (FKS). After optimization and validation of chromatographic methods, 24 FKS were analyzed. Forskolin, their main bioactive component, was only found in 50% of the FKS evaluated (in the 0.032-17.1% range), with 27% of these supplements showing concentrations of this bioactive lower than those declared in their labels. Application of this methodology also proved to be successful for the detection of frauds regarding the replacement of C. forskohlii by other vegetable sources (green tea, soy leaves and a plant of the Berberidaceae family) in 17% of supplements analyzed. A study on stability of forskolin under accelerated conditions allowed to rule out its degradation as responsible for the lack of this bioactive or other natural constituents in 25% of FKS evaluated. It can be concluded that the multianalytical methodology here developed is an advantageous alternative to address the wide diversity of frauds affecting these supplements.

Study on Chemotypic Variability of Coleus forskohlii Briq., Samples Collected from Different Phytogeographical Locations of India and Evaluation of Its Inhibitory Potential.

Coleus forskohlii Briq. is an important medicinal herb, endowed with a wide range of medicinal properties against the variety of ailments. Seven germplasm of C. forskohlii collected from different phyto-geographical locations and identification of elite chemotype was performed with the help of high performance thin layer chromatography. Data of soil analysis correlated with the bioactive compounds and inhibitory potential of the species. Quantification of forskolin and its isomer (iso-forskolin) content were done in all the collected samples of C. forskohlii, which revealed a wide range of variations, varying from 1.15-0.004% and 0.0091 to 0.1077% per dry weights basic, respectively. Variation in the bioactive content may be due to the soil nature and environmental factors. Soil analysis of collected samples demonstrated that there is significant variation in available NPK and micronutrient content and may be reasoned for existing chemotypic variability. In vitro biological activity (antioxidant and antidiabetic) analyses were performed, which reveals that germplasms have a high amount of forskolin and iso-forskolin, both show more activity. The aim of this study was to elucidate the effect of elicitors and precursors on the production of bioactive compounds and identification of best elite germplasm among the populations, to provide basic lead to the industry for commercial exploitability including its location-specific commercial cultivation.

Coleus forskohlii: advancements and prospects of in vitro biotechnology.

Coleus forskohlii syn. Plectranthus barbatus is a popular medicinal plant belonging to the family Lamiaceae and order Lamiales. The leaf and root extracts can be utilized for the treatment of various ailments like bronchitis, asthma, hay fever, angina and abdominal disorders. The major metabolite that is found exclusively in the cork cells of the root in C. forskohlii is forskolin, which is used commercially for the treatment of glaucoma, asthma and several heart ailments. The essential oil extracted from the tubers of the plant also exhibits anti-microbial properties. The present review recounts the existing reports on biotechnological approaches like direct, indirect organogenesis and somatic embryogenesis for mass propagation of plantlets; the amelioration of forskolin production through cell suspension and genetic transformation as well as slow growth storage for germplasm conservation. Additionally, the unexplored arenas and the prospective novel approaches are also addressed in this review that can be utilized in designing new experiments in near future on this plant.

Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis.

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (CH 1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between VH -CH 1 and VL -CL was improved by the CH 1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91-62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.

Potential benefits of triethylamine as n-electron donor in the estimation of forskolin by electronic absorption and emission spectroscopy.

Diterpenoid forskolin was isolated from Coleus forskolii. The electronic absorption and emission studies of forskolin were investigated in various solvents with an aim to improve its detection limits. The two chromophores present in the diterpenoid are not conjugated leading to the poor absorption and emission of UV light. The absorption and fluorescence spectra were solvent specific. In the presence of a monodentate ligand, triethylamine the detection of forskolin is improved by 3.63 times in ethanol with the fluorescence method and 3.36 times in DMSO by the absorption spectral method. The longer wavelength absorption maximum is blue shifted while the lower energy fluorescence maximum is red shifted in the presence of triethylamine. From the wavelength of fluorescence maxima of the exciplex formed between excited forskolin and triethylamine it is concluded that the order of reactivity of hydroxyl groups in the excited state forskolin is in the reverse order to that of the order of the reactivity of hydroxyl groups in its ground state.

Forskolin-free cAMP assay for Gi-coupled receptors.

G protein-coupled receptors (GPCRs) represent the most successful receptor family for treating human diseases. Many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays. Measurement of intracellular cyclic AMP (cAMP) levels is a validated strategy for measuring GPCRs activation. However, agonist ligands for Gi-coupled receptors are difficult to track because inducers such as forskolin (FSK) must be used and are sources of variations and errors. We developed a method based on the GloSensor system, a kinetic assay that consists in a luciferase fused with cAMP binding domain. As a proof of concept, we selected the succinate receptor 1 (SUCNR1 or GPR91) which could be an attractive drug target. It has never been validated as such because very few ligands have been described. Following analyses of SUCNR1 signaling pathways, we show that the GloSensor system allows real time, FSK-free detection of an agonist effect. This FSK-free agonist signal was confirmed on other Gi-coupled receptors such as CXCR4. In a test screening on SUCNR1, we compared the results obtained with a FSK vs FSK-free protocol and were able to identify agonists with both methods but with fewer false positives when measuring the basal levels. In this report, we validate a cAMP-inducer free method for the detection of Gi-coupled receptors agonists compatible with high-throughput screening. This method will facilitate the study and screening of Gi-coupled receptors for active ligands.

Synergy between Glomus fasciculatum and a beneficial Pseudomonas in reducing root diseases and improving yield and forskolin content in Coleus forskohlii Briq. under organic field conditions.

Root rot and wilt, caused by a complex involving Fusarium chlamydosporum (Frag. and Cif.) and Ralstonia solanacearum (Smith), are serious diseases affecting the cultivation of Coleus forskohlii, a crop with economic potential as a source of the medicinal compound forskolin. The present 2-year field experiments were conducted with two bioinoculants (a native Pseudomonas monteilii strain and the exotic arbuscular mycorrhizal (AM) fungus Glomus fasciculatum) alone and in combination under organic field conditions in order to evaluate their potential in controlling root rot and wilt. Combined inoculation of P. monteilii with G. fasciculatum significantly increased plant height, plant spread, and number of branches; reduced disease incidence; and increased tuber dry mass of C. forskohlii, compared to vermicompost controls not receiving any bioinoculants. Increase in tuber yields was accompanied by an increase in plant N, P, and K uptake. Co-inoculation of P. monteilii with G. fasciculatum significantly improved the percent AM root colonization and spore numbers retrieved from soil. This suggests P. monteilii to be a mycorrhiza helper bacterium which could be useful in organic agriculture. The forskolin content of tubers was significantly increased by the inoculation treatments of P. monteilii, G. fasciculatum, and P. monteilii + G. fasciculatum.

A rapid HPLC with evaporative light scattering method for quantification of forskolin in multi-herbal weight-loss solid oral dosage forms.

A rapid reverse-phase HPLC method with evaporative light scattering detection (ELSD) was developed for the determination of forskolin in weight loss multi-herbals products. The analysis was performed by water-acetonitrile gradient elution at a temperature of 40 degrees C and a flow rate of 1.0 mL/min. The evaporator tube temperature of ELSD was set at 35 degrees C, and with the nebulizing gas flow-rate (pressure) of 3.0 bar. The method was validated for linearity, accuracy, precision and limits of detection (LOD) and quantification (LOQ). Good linear relationships were obtained with correlation coefficients exceeding 0.9995. The average recovery of forskolin ranged from 99.4% to 100.4% with RSDs below 3%. The percent relative standard deviations (%RSD) of intra- and inter-day precision varied by less than 2.1%. LOD and LOQ were 0.95 microg/ml and 3.21 microg/ml, respectively. The validated ELSD method permits a shorter determination time without compromising accuracy and demonstrates that it can be used for quantification of forskolin incorporated in multi-herbal solid oral dosage forms.

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in Escherichia coli: characterization of the recombinant peptide and assessment of its effects on cellular signaling pathways in Y-organs.

A neuropeptide, molt-inhibiting hormone (MIH), negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in Escherichia coli. Bacteria were transformed with an expression plasmid containing a cDNA insert encoding MIH. After induction of protein synthesis, recombinant MIH (recMIH) was detected in the insoluble fraction of cell lysates. The insoluble recMIH was refolded and purified by reversed-phase high performance liquid chromatography (RP-HPLC). The refolded peptide was MIH-immunoreactive and comigrated with native MIH on RP-HPLC. Mass and CD spectral analyses showed the mass number and secondary structure of the recombinant peptide were as predicted for MIH. Bioassays showed recMIH dose-dependently suppresses ecdysteroid synthesis by Y-organs. The combined results suggest that recMIH is properly folded. In subsequent experiments, recMIH was used to assess cellular signaling pathways linked to MIH-mediated suppression of ecdysteroidogenesis. Incubation of Y-organs with recMIH produced an increase in intracellular cGMP content, but had no effect on intracellular cAMP. Further, a cGMP analog significantly suppressed ecdysteroid production, but neither cAMP analogs nor an activator of adenylyl cyclase had a detectable effect on ecdysteroidogenesis. The results are consistent with the hypothesis that MIH-induced suppression of ecdysteroidogenesis in Y-organs of C. sapidus is mediated by a cGMP second messenger. We anticipate recMIH will be a useful tool for additional studies of the cellular actions and physiological functions of MIH.

Structural and spectral assignment of three forskolin-like diterpenoids isolated from Plectranthus ornatus.

Three labdane diterpenoids were isolated from an acetone extract of Plectranthus ornatus. Their structures, closely related to that of forskolin, were determined by NMR studies. Unambiguous and complete assignments of the 1H and 13C NMR chemical shifts for these substances are presented. The assignments are based on 2D shift-correlated [1H, 1H-COSY, 1H, 13C-gHSQC-1J (C,H), 1H, 13C-gHMBC-(n)J (C,H) (n = 2 and 3)] and NOE experiments.

Quantitative analysis of forskolin in Coleus forskohlii (Lamiaceae) by reversed-phase liquid chromatography.

A rapid method was developed for the evaluation of forskolin in Coleus forskohlii Briq. (Lamiaceae). Forskolin was quantitated in the root and stem of dried C. forskohlii and in 17 market products by reversed-phase liquid chromatography (LC) with a photodiode array detector at 210 nm. The temperature was held constant at 30 degrees C, and the retention time of forskolin was approximately 6.8 min. The samples were extracted with acetonitrile by sonication. The precision of the method was confirmed by a standard deviation < 5.0% (n = 3), and forskolin recovery was 99.1%. Limit of detection was 1.5 microg/mL, and the response was linear through zero from 6.3 to 630 microg/mL with a correlation coefficient (R2) of 0.9998. Identity of the marker compound was confirmed by an LC/mass spectrometry experiment. The method was successful in the qualitative and quantitative evaluation of the marker compound in C. forskohlii plant material and in market products claiming to contain C. forskohlii.

Rapid analysis of small samples containing forskolin using monoclonal antibodies.

The effective range of the competitive ELISA test for detection of forskolin content in clonally propagated plant organs of Coleus forskohlii using monoclonal antibodies extends from 5ng to 5 micrograms. A correlation between the forskolin accumulation and the growth rate was investigated using the clonally propagated shoots. An increase of forskolin content was noted, beginning at week 6. Flowers, rachises, leaves, stems, tuberous roots, and roots were analyzed. Tuberous roots and the stem base contained higher amounts of forskolin than other organs. The forskolin content in the stem decreased gradually towards the top of the shoot.

Effects of subarachnoid hemorrhage on vascular responses to calcitonin gene-related peptide and its related second messengers.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and a primary signaling molecule in neurovascular communication. In the present study, the authors examined cerebrovascular responses to CGRP and its related second messenger systems during cerebral vasospasm induced by subarachnoid hemorrhage (SAH). Tension measurements were performed in vitro on ring strips of basilar arteries obtained from rabbits subjected to artificial SAH and from control (non-SAH) animals. In vessels from SAH animals, which were preconstricted with serotonin, the vasorelaxant response to CGRP was attenuated. Because it has been suggested that vasodilation elicited by CGRP is mediated by cyclic 3',5'-adenosine monophosphate (cAMP) and/or cyclic 3',5'-guanosine monophosphate (cGMP), the vascular effects of directly activating these second messenger systems were also examined. The relaxant effect of forskolin, which activates adenylate cyclase directly, was slightly enhanced after SAH. In contrast, the relaxant effect of nitroglycerin (GTN), which activates soluble guanylate cyclase directly, was unchanged after SAH. The attenuation of CGRP-induced vasorelaxation could be the result of a modification in its ability to stimulate the production of second messengers. Experiments testing the capacity of CGRP to elevate cAMP levels showed no significant differences between vessels from non-SAH and SAH animals. Similarly, the resting levels of cAMP and the forskolin-induced elevations of cAMP did not differ between non-SAH and SAH animals. In contrast, cGMP levels were lower in resting and CGRP-treated vessels from SAH animals than in those from non-SAH animals. No significant differences in the levels of cGMP were observed between non-SAH and SAH vessels treated with GTN. This study indicates that CGRP-induced vasodilation is attenuated during vasospasm in a rabbit model of SAH. The findings also demonstrate that vasodilatory responses mediated by cAMP and cGMP are intact, although the levels of cGMP in SAH vessels are reduced. Together, these observations suggest that an attenuation in the capacity of vessels to dilate in response to CGRP occurs during cerebral vasospasm, and this change in CGRP vasoactivity is a result of modifications prior to, or independent of, the elevation of cyclic nucleotide second messengers.

Production of monoclonal antibodies and enzyme immunoassay for typical adenylate cyclase activator, Forskolin.

The ratio of hapten to bovine serum albumin in an antigen conjugate was exactly determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against forskolin was produced by fusing splenocytes immunized with a forskolin-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.57%. A very small cross-reaction appeared with 1-deoxy, 9-deoxy and 1,9-dideoxy forskolin derivatives. The full measuring range of the assay extends from 6 ng to 200 ng of forskolin. The competitive ELISA assay used for this analysis was found to be more sensitive than TLC (10 micrograms), GLC (30 ng) and HPLC (1 microgram) methods.

Characterization of [3H]forskolin binding sites in the human caudate nucleus.

[3H]Forskolin binds to a site in rat and bovine brain probably reflecting the coupling of the alpha subunit of the stimulatory guanine nucleotide binding protein with adenylate cyclase. The purpose of this study was to pharmacologically characterize high-affinity binding of [3H]forskolin in the human caudate nucleus. The dissociation constant (Kd) of [3H]forskolin binding was 102 nM and the Bmax was 628 fmol/mg of protein. Guanylyl-5'-imidodiphosphate significantly increased the number (Bmax) of [3H]forskolin binding sites in the caudate nucleus (EC50 = 3.1 +/- 0.05 microM). [3H]Forskolin binding in the human caudate may provide insights into the coupling of a stimulatory guanine nucleotide binding protein with adenylate cyclase.

Localization of the binding domain of the inhibitory ligand forskolin in the glucose transporter GLUT-4 by photolabeling, proteolytic cleavage and a site-specific antiserum.

The binding domain of forskolin in the adipocyte/muscle-type glucose transporter (GLUT-4) was localized with the aid of the photoreactive derivative, [125I]IAPS-forskolin (3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin). Plasma membranes from insulin-treated rat adipocytes containing predominantly the GLUT-4 isoform were irradiated with UV light in the presence of [125I]IAPS-forskolin. The covalently labeled glucose transporters were isolated by immunoprecipitation with specific antiserum and partially digested with trypsin and elastase. The fragments were separated by gel electrophoresis, transferred on to nitrocellulose membranes, and identified by direct autoradiography and by immunoassay with antiserum against a peptide sequence corresponding to the C-terminus of GLUT-4. Digestion with a high-purity grade trypsin generated two photolabeled fragments with apparent molecular weights of 21 and 16 kDa. Since the antiserum detected two fragments with identical electrophoretic mobility, both labeled fragments appeared to contain the intact C-terminus of GLUT-4. In contrast, digestion with elastase generated only one photolabeled fragment with intact C-terminus at 21 kDa, and a smaller unlabeled fragment with intact C-terminus at 15 kDa. A less pure trypsin preparation generated two labeled (21 and 17 kDa) and one unlabeled (15 kDa) fragment with intact C-terminus. These data suggest that the site of covalent binding of IAPS-forskolin in the GLUT-4 is located within a region of 1-6 kDa defined by the difference between the unlabeled C-terminal fragment (15 kDa) and the labeled fragments (21, 17 and 16 kDa). Based on a tentative allocation of the fragments to the sequence of the GLUT-4, it is suggested that the covalent binding site of IAPS-forskolin is located between the membrane spanning helices 7-9, possibly in the proximity of helix 9.