Efficacy of An Aqueous Morinda citrifolia Fruit Extract-Phytosome Gel in Treating Oral Inflammatory Ulcer in Rabbit Model.

BACKGROUND: Oral inflammatory ulcers are one of the common complaints of patients attending out-patient clinics. Previous in vivo studies had shown that an Aqueous M. citrifolia Fruit Extract (AMFE) possessed anti-inflammatory and ulcer healing activities. Therefore, a standardized topical bioadhesive gel containing AMFE-phytosome was developed and determined for its oral ulcer healing efficacy in a rabbit model. METHODS: The AMFE phytosome (AMFE-P) was prepared by a complexation method with the required amount of AMFE: Phosphatidylcholine: Tween 80 to weigh ratio of 2:1:0.2. Poloxamer 407 was used as a gelling agent. The oral ulcer was induced in male New Zealand white rabbits by topical application of acetic acid. Each test compound was applied to the ulcer for 10 days beginning on the second day after the ulcer induction. Complete ulcer healing on the specimen obtained on day 12 was observed histologically using the histological scoring protocol. RESULTS: The optimized gel containing AMFE-P equivalent to AMFE 10%w/w (10%AMFE-P gel) showed the best bioadhesive gel quality, a smooth and homogeneous texture with an optimum viscosity and pH range used in human oral cavity, a good physical and chemical stability and the highest percentage cumulative release of total phenolic and scopoletin content. It was found that a daily application of 10% AMFE-P gel exerted a superior ulcer healing efficacy and a significantly rapid ulcer healing process than a twice daily application of topical gel containing AMFE 10%w/w or chlorhexidine 0.2%. CONCLUSION: These findings demonstrated that 10% AMFE-P gel has potential as a safe and effective alternative therapeutic agent for oral ulcers.

Phytochemical investigation of Hyoscyamus albus.

The work presented in this paper illustrates the isolation and structure elucidation of secondary metabolites of Hyoscyamus albus. Two new natural source and three known compounds were isolated from the Hyoscyamus albus. Among the isolated compounds, grivilloside H (1) and betulaplatoside (2) were isolated for the first time while scopolamine (3), ß-sitosterol (4) and stigmasterol (5) have been reported previously from the same plant. The structures of all the isolated compounds were established by using modern spectroscopic technique (UV, IR, NMR, and EI-MS) and by comparing with those available in literature.

Comparison of two hyoscyamine 6ß-hydroxylases in engineering scopolamine biosynthesis in root cultures of Scopolia lurida.

Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6ß-hydroxylase genes of Hyoscyamus niger (HnH6H) and S. lurida (SlH6H) were cloned and respectively overexpressed in hairy root cultures of S. lurida, to compare their effects on promoting the production of TAs, especially the high-value scopolamine. Root cultures with SlH6H/HnH6H overexpression were confirmed by PCR and real-time quantitative PCR, suggesting that the enzymatic steps defined by H6H were strongly elevated at the transcriptional level. Tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were analyzed by HPLC. Scopolamine and anisodamine contents were remarkably elevated in the root cultures overexpressing SlH6H/HnH6H, whereas that of hyoscyamine was more or less reduced, when compared with those of the control. These results also indicated that SlH6H and HnH6H promoted anisodamine production at similar levels in S. lurida root cultures. More importantly, HnH6H-overexpressing root cultures had more scopolamine in them that did SlH6H-overexpressing root cultures. This study not only provides a feasible way of overexpressing H6H to produce high-value scopolamine in engineered root cultures of S. lurida but also found that HnH6H was better than SlH6H for engineering scopolamine production.

Identification of Datura Species Involved in a Food-Poisoning Case Using LC-MS/MS and DNA Barcording.

A food-poisoning case due to eating the roots of Datura occurred in Kawasaki City, Japan in 2014. The Datura plant was mistakenly collected instead of burdock in a domestic garden. The roots of these plants are quite similar to each other. We presumed that the specimen was the root of Datura, but it was difficult to classify it only from the morphology. Using LC-MS/MS, we detected atropine and scopolamine from the remaining plant specimen. Therefore, we applied the DNA barcoding method. The results showed that the specimen was classified into Solanaceae family, but not Asteraceae family. Thus, the specimen was confirmed to be Datura species based on both chemical and genetic analyses.

Simultaneous determination of atropine and scopolamine in buckwheat and related products using modified QuEChERS and liquid chromatography tandem mass spectrometry.

A method was developed for the determination of atropine and scopolamine in buckwheat and related products. A modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction procedure was evaluated. Dispersive solid phase extraction (d-SPE) was studied as clean-up step, using graphitized black carbon (GBC) and primary secondary amine (PSA). The extract was diluted with water (50:50, v/v) prior to chromatographic analysis. The method was validated and recoveries (except chia samples spiked at 10µg/kg) ranged from 75% to 92%. Intra and inter-day precision was lower than or equal to 17%. The limit of quantification of atropine and scopolamine was 0.4 and 2µg/kg, respectively. Eight types of samples (buckwheat, wheat, soy, buckwheat flour, buckwheat noodle, amaranth grain, chia seeds and peeled millet) were analyzed. Target compounds were not found above the detection limits of the method, but three transformation products of scopolamine (norscopine, hydroscopolamine and dihydroxyscopolamine) were putative identified in the tested samples using high resolution mass spectrometry (Exactive-Orbitrap).

Isolation of atropine and scopolamine from plant material using liquid-liquid extraction and EXtrelut® columns.

Tropane alkaloids are toxic secondary metabolites produced by Solanaceae plants. Among them, plants from Datura genus produce significant amounts of scopolamine and hyoscyamine; the latter undergoes racemization to atropine during isolation. Because of their biological importance, toxic properties and commonly reported food and animal feed contamination by different Datura sp. organs, there is a constant need for reliable methods for the analysis of tropane alkaloids in many matrices. In the current study, three extraction and sample-clean up procedures for the determination of scopolamine and atropine in plant material were compared in terms of their effectiveness and repeatability. Standard liquid-liquid extraction (LLE) and EXtrelut® NT 3 columns were used for the sample clean-up. Combined ultrasound-assisted extraction and 24h static extraction using ethyl acetate, followed by multiple LLE steps was found the most effective separation method among tested. However, absolute extraction recovery was relatively low and reached 45-67% for atropine and 52-73% for scopolamine, depending on the compound concentration. The same method was also the most effective one for the isolation of target compounds from Datura stramonium leaves. EXtrelut® columns, on the other hand, displayed relatively low effectiveness in isolating atropine and scopolamine from such a complex matrix and hence could not be recommended. The most effective method was also applied to the extraction of alkaloids from roots and stems of D. stramonium. Quantitative analyses were performed using validated method based on gas chromatography with flame ionization detector (GC-FID). Based on the results, the importance of the proper selection of internal standards in the analysis of tropane alkaloids was stressed out.

Efficient biosynthesis of rare natural product scopolamine using E. coli cells expressing a S14P/K97A mutant of hyoscyamine 6ß-hydroxylase AaH6H.

Hyoscyamine 6ß-hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6ß-hydroxyhyoscyamine to form scopolamine, a valuable natural alkaloid. In this study, random mutagenesis and site-directed saturation mutagenesis were used to enhance the hydroxylation activity of H6H from Anisodus acutangulus (AaH6H). A double mutant, AaH6HM1 (S14P/K97A), showed a 3.4-fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity was also improved by 2.3 times. After 34h cultivation of Escherichia coli cells harboring Aah6hm1 in a 5-L bioreactor with a working volume of 3L, scopolamine was produced via a single-enzyme-mediated two-step transformation from 500mgL(-1) (-)-hyoscyamine in 97% conversion, and 1.068g of the product were isolated, corresponding to a space-time yield of 251mgL(-1)d(-1). This study shows that the protein engineering of some key enzymes is a promising and effective way for improving the production of rare natural products such as scopolamine.

Preparative separation of atropine and scopolamine from Daturae metelis Flos using pH-zone-refining counter-current chromatography with counter-rotation and dual-mode elution procedure.

To isolate atropine and scopolamine from Daturae metelis Flos, three different elution modes have been applied in pH-zone-refining counter-current chromatography. These separations were performed with a two-phase solvent system composed of ethyl acetate/n-butanol/water (4:1:5 v/v) with 0.50% triethylamine in the organic phase and 0.15% hydrochloric acid in the aqueous phase. As a result, the best separation was obtained by counter-rotation and dual-mode elution procedure. In this new separation mode, the mobile phase and stationary phase were exchanged when the rotation direction was reversed. The two purified alkaloids (purity over 98% as determined by HPLC) were identified by ESI-MS, (1)H-NMR and (13)C-NMR.

Capillary electrophoresis of tropane alkaloids and glycoalkaloids occurring in Solanaceae plants.

This chapter examines the role of capillary electrophoresis (CE) in the separation of tropane alkaloids, glycoalkaloids, and closely related compounds that have either pharmaceutical value or toxicological effects on humans. The latest significant developments in CE analysis have been selected and critically discussed. When the conventional CE mode was found unable to provide an acceptable selectivity towards the analytes, the addition of either an organic solvent, a chiral selector, or a surfactant to the running buffers was exploited. Likewise, nonaqueous CE (NACE) was also employed to increase solute solubilities and for a better compatibility of this media with mass spectrometry. It turns out that, upon selecting the most appropriate experimental conditions, the CE separation of tropane alkaloids and steroidal glycoalkaloids of Solanaceae plants was successfully accomplished. All major steps involved in the separation and detection of these secondary metabolites in complex samples are described and the relevant aspects of each application are examined with emphasis on the main aspects entailed a typical assay. More applications have yet to be developed in order to encourage more labs to exploit the tremendous potential of capillary electrophoresis.

Studies on tropane alkaloid extraction by volatile organic solvents: dichloromethane vs. chloroform.

In order to investigate the production of tropane alkaloids by hairy roots of Atropa baetica, transgenic for the gene h6h encoding the enzyme hyoscyamine 6beta-hydroxylase, solvent extraction with chloroform and with dichloromethane of the metabolites present in the liquid medium and in the root tissue was compared. The extraction of scopolamine from the liquid medium was equally effective with either solvent, giving maximum values of around 850 microg/flask. For the roots, three different extraction methods were employed: A, employing chloroform:methanol: (25%) ammonia (15:5:1) for initial extraction, followed by treatment with sulfuric acid and ammonia, and using chloroform for the final extraction and washes; B, as A but using dichloromethane for extraction and washes; and C, as B but substituting chloroform for dichloromethane in the extraction cocktail. Scopolamine was the most abundant metabolite (present in amounts of 3250-3525 microg/g dry weight) and presented similar extraction efficiencies with all of the extraction methods employed. The highest amounts of hyoscyamine and the intermediate 6beta-hydxoxyhyoscyamine were present on day 31 (800 and 975 microg/g dry weight, respectively) and no statistical differences between the three extraction methods employed were detected. This study confirms that, for the extraction of tropane alkaloids, dichloromethane can replace the commonly employed chloroform, the use of which incurs major health, security and regulation problems.

Uniformly sized molecularly imprinted polymer for atropine and its application to the determination of atropine and scopolamine in pharmaceutical preparations containing Scopolia extract.

A uniformly sized molecularly imprinted polymer (MIP) for atropine has been prepared. The MIP was prepared using 2-(trifluoromethyl) acrylic acid and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and thermal polymerization method. The selectivity factor, which is defined as the ratio of the retention factors (k) on the molecularly imprinted and non-imprinted polymers, k(imprinted)/k(non-imprinted), was 2.2 for atropine on the MIP. The obtained MIP was applied for the determination of tropane alkaloids (atropine and scopolamine) in a commercial gastrointestinal drug by a column-switching HPLC system, consisting of an MIP material as a pre-column, and a conventional cation-exchange analytical column. An interference peak was observed at the retention time of atropine derived from pre-column. However, since the peak area was less than 0.5% the peak area of atropine of a standard solution under the analytical conditions of this study (0.2 microg of atropine was loaded), this interference was negligible in the determination of atropine. On the other hand, no interference peak was observed at the retention time of scopolamine. Calibration curves of atropine and scopolamine showed good linearity in the range of 0.02-0.9 microg/ml (r=0.9999) and 0.003-0.09 microg/ml (r=0.9998), respectively. The mean recoveries of atropine and scopolamine from a placebo pharmaceutical preparation sample were 98.9 and 99.9%, respectively. The intra-day precision (measured by relative standard deviation, R.S.D. (%)) of both ingredients was less than 2.0%. The optimized column-switching system was applied successfully to the determination of atropine and scopolamine in a commercial gastrointestinal drug.

Fatal Datura poisoning: identification of atropine and scopolamine by high performance liquid chromatography/photodiode array/mass spectrometry.

A forensic method comprising solid phase extraction and HPLC analysis was developed for the detection and confirmation of atropine and scopolamine, the main toxic alkaloids of Datura stramonium and Datura ferox. This method allowed the direct coupling of an electrospray (ZMD) mass selective detector to the HPLC system. Under these conditions, atropine and scopolamine were well separated from other components and detected on the PDA (LOD = 1 microg/ml) and ZMD (LOD(atropine) = 10 pg/ml; LOD(scopolamine) = 100 pg/ml) detectors. Four geographically isolated populations of each of D. stramonium and D. ferox were analysed for seed alkaloids and it was found that the two species were diagnostically different in their atropine-scopolamine ratios. The optimised HPLC method was used to analyse three viscera samples of an adult Caucasian male whose death was ascribed to a fatal heart attack. Atropine and scopolamine were detected in the stomach and its contents, which contained Datura seeds. The chemical profile of the seeds found in the stomach contents was similar to those from four geographically different D. ferox plants.

Tropane alkaloids from Latua pubiflora.

Four known tropane alkaloids were isolated from the leaves of the endemic Chilean plant Latua pubiflora (Solanaceae). For the first time; 3alpha-cinnamoyloxitropane and apoatropine are reported in this plant. Scopolamine and hyoscyamine were previously reported.

Preparation of a molecularly imprinted polymer for the solid-phase extraction of scopolamine with hyoscyamine as a dummy template molecule.

Molecularly imprinted polymers (MIPs) selective for scopolamine were produced using hyoscyamine (a close structural analogue) as template molecule. The produced polymers were used as media for solid-phase extraction, exhibiting selective binding properties for the analyte from biological samples. Human and calf urine and serum were processed on the MIP under various extraction protocols. The best performance was observed after loading the analyte in aqueous environment facilitating retention on the MIP by non-selective hydrophobic interactions. The MIPs were subsequently washed using an optimised solvent system to enable selective desorption of the analyte. Other related and non-related compounds were accessed to evaluate molecular recognition properties. Recoveries of up to 79% were achieved for the analyte of interest from biological samples.

[Chirality of natural products: hyoscyamine and scopolamine].

Monthly changes of the content-ratio between S-(-)- and R-(+)-hyoscyamine as well as those between S-(-)- and R-(+)-scopolamine in the leaves of Datura metel L. cultivated in the field, were quantitatively analyzed by the use of HPLC with a chiral adsorbent. It was found that S-(-)-isomer was predominant for hyoscyamine and the ratio of R-(+)-isomer gradually increased during the growth, whereas in the case of scopolamine, S-(-)-isomer was the sole one found throughout the cultivation period. The 1H-NMR study in the CD3OD solution has suggested that S-(-)-hyoscyamine (1) and S-(-)-scopolamine (2) take a "face-to-face" conformation between their tropane skeletons and the benzene rings of the tropic acid moieties. In the presence of an equimolar NaOD in the CD3OD solution, the racemization at C-2' of 1 and 2 proceeded more rapidly than the hydrolysis at the tropic acid ester bond, presumably due to the steric hindrance caused by their "face-to-face" conformations. In the D2O and H2O solutions, on the other hand, the racemization and the hydrolysis of 1 proceeded smoothly, while those of 2 did not occur. It has been supposed that these individual reaction manners are ascribable in considerable extent to the different basicity of N atom in each tropane skeleton of 1 and 2 and to stronger intramolecular hydrogen bond occurring between the carbonyl oxygen at C-1' and the hydroxyl group at C-3' in the tropic acid moiety of 1.

Determination of the main tropane alkaloids from transformed Hyoscyamus muticus plants by capillary zone electrophoresis.

A capillary zone electrophoretic method (CZE) was developed using an uncoated fused silica capillary for the separation and determination of the main tropane alkaloids. The applicability of the developed method for analysis of plant samples was examined by analyzing samples of transgenic Egyptian henbane Hyoscyamus muticus (L.) plants. A simple 40 mM phosphate buffer at pH 7.8 using a voltage of 20 kV was found the best for this purpose. The main tropane alkaloids, atropine and scopolamine as well as nor-(-)-scopolamine, and tropic acid, the precursor of tropane alkaloids, could be separated in less than 13 min. The linear concentration range for atropine was 5.00-140 microg ml(-1), for scopolamine 7.50-210 microg ml(-1) and for tropic acid 2.50-70.0 microg ml(-1).

Microbore liquid chromatography of tertiary amine anticholinergic pharmaceuticals with tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection.

The post-column chemiluminescent reaction of six anticholinergic alkaloid compounds with tris(2,2'-bipyridine)ruthenium(III) (Ru(bpy)3(3+)) is applied to microbore high-performance liquid chromatography (HPLC). At flow rates less than 200 microL/min, the capillary mixing cell in which Ru(bpy)3(3+) and the analyte are mixed directly allows for good light detection. In contrast, a diminished signal occurs at these low flow rates with conventional post-column mixing in a tee. Optimal chemiluminescent pH conditions for atropine, scopolamine, dicyclomine, cyclopentolate, cyclobenzaprine, and procyclidine are determined at moderately basic conditions (pH 7 to 9). 2-Butanone is found to be compatible with the chemiluminescent reaction, whereas tetrahydrofuran and propionitrile cause an increase in background noise and a chemiluminescent signal loss. As 2-butanone is more nonpolar than acetonitrile, it assists in the elution of these hydrophobic anticholinergic compounds. Five anticholinergic compounds are resolved successfully with a PRP-1 polymeric column and a slightly basic mobile phase, but a C8 silica column is better suited for the more hydrophobic compounds (cyclobenzaprine, procyclidine, and dicyclomine).

Determination of the enantiomeric purity of scopolamine isolated from plant extract using achiral/chiral coupled column chromatography.

The optical purity of scopolamine derived from Datura sanguinea was determined using coupled column chromatography. A C18 column was used to separate scopolamine from the additional alkaloids and other biological material present in the vegetal extract. The C18 column was coupled through a six-port switching valve to two beta-cyclodextrin columns in series which were used to resolve the scopolamine enantiomers. A single acetylated beta-cyclodextrin column gives equivalent results to the native cyclodextrin columns because of slightly higher enantioselectivity for scopolamine. A multistep extraction procedure is used to isolate scopolamine from the vegetal material. 4-6% of the scopolamine in the final extract was found to be the d enantiomer. Sample extracts as well as commercial scopolamine hydrobromide were treated under various conditions commonly encountered during typical commercial extraction procedures and analyzed in order to determine if the d enantiomer was present in the original material or if it was produced during the extraction process and, if so, determine which step and conditions contribute to racemization. Both the salt and the extract were found to be susceptible to racemization under basic conditions (greater than or equal to pH 9) although the extract appeared to be more susceptible than the salt. Tropic acid formed from the hydrolysis of scopolamine seemed to be completely racemized even though the remaining scopolamine was only partially racemized. Within experimental error, no d enantiomer was found in the original fresh plant material.

[Study on separation and determination of four tropane alkaloids in crude drugs by micellar HPLC].

A new method for separation and determination of four important tropane alkaloids (hyoscyamine, scopolamine, anisodamine and anisodine) in crude drugs by micellar HPLC was developed. The mobile phase was optimized with the modified simplex method (MSM). The chromatographic response function (CRF) was used as the criterion of optimization and three dimension simplex was used in this course. After eight tests, the given accuracy was achieved. In the optimized system the four tropane alkaloids are separated not only from each other but also from the interfering components in the crude drugs. The CMC of SDS in mobile phase was determined to be 5 mmol/L by circle method. The experimental evidences show that the optimized system is micellar chromatography. This method is simple, sensitive and accurate. The extraction solution can be injected directly without the need of a general purification procedure. Several plant materials containing tropane alkaloids were determined. The results and chromatograms are reported in this paper.