A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

Introduction – Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of β‐glucosidase inhibitors but not for α‐glucosidase. Nonetheless, α‐glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti‐viral infections. Objective – To develop a TLC bioautographic method to detect α‐ and β‐glucosidase inhibitors in plant extracts. Methodology – The enzymes α‐ and β‐d ‐glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of α‐d ‐glucosidase, and 37°C for 20 min in the case of β‐d ‐glucosidase. For detection of the active enzyme, solutions of 2‐naphthyl‐α‐D‐glucopyranoside or 2‐naphthyl‐β‐D‐glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for α‐d ‐glucosidase) or 1 : 4 (for β‐d ‐glucosidase) and sprayed onto the plate to give a purple background colouration after 2–5 min. Results – Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited α‐d ‐glucosidase and β‐d ‐glucosidase down to 0.1 µg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 µg for α‐glucosidase and 50 µg for β‐glucosidase. Conclusion – The screening test was able to detect inhibition of α‐ and β‐glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

Validated TLC method for simultaneous quantitation of kutkoside and picroside‐I from Kutki extract

Introduction – The two iridoid glycosides kutkoside and picroside‐I are the active hepatoprotective principles of Picrorhiza kurroa Royle ex Benth (Scrophulariaceae), commonly known as Kutki. Quantitation of these phytoconstituents is important for the routine quality control of Kutki extract. Objective – To develop and validate a simple, precise and rapid thin‐layer chromatography (TLC) method for the simultaneous quantitation of kutkoside and picroside‐I in Kutki extract. Methodology – The analysis was performed on a TLC precoated silica gel 60 F₂₅₄ plate with ethyl acetate:methanol:glacial acetic acid:formic acid (25:5:1:1, v/v/v/v) as mobile phase. Densitometric evaluation of kutkoside and picroside‐I was carried out at 265 nm and the mobile phase showed good resolution with R_f values 0.42 ± 0.03 and 0.61 ± 0.03 for kutkoside and picroside‐I, respectively. The method was validated in terms of specificity, linearity, accuracy and precision. Results – The content of kutkoside and picroside‐I was found to be 2.18 and 1.90%, respectively, and was comparable with those obtained by HPLC. The linearity was found to be in the range of 80–480 ng/spot for both kutkoside and picroside‐I. The average recovery values were found to be 96.5 and 96.0% for kutkoside and picroside‐I, respectively. Conclusion – The developed method was found to be relatively simple, precise and reproducible for the simultaneous quantitation of kutkoside and picroside‐I. The method does not employ any derivatisation procedure and can be used as a quality control tool for the routine analysis of commercial Kutki extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of polyphenolics in extracts of Potentilla species by high‐performance thin‐layer chromatography photodensitometry method

Introduction – Separation of polyphenolics in different plant materials using high‐performance thin‐layer chromatography (HPTLC) represents an effective method for their detection and quantification. Objective – To develop a simple, specific, precise, sensitive and accurate method for the simultaneous quantification of tiliroside (TRS), methyl brevifolincarboxylate (MBR) and ellagic acid (EA) in a plant extract using the HPTLC‐photodensitometry method. Methodology – Aerial parts of the selected Potentilla species, P. anserina, P. erecta, P. grandiflora and P. nepalensis var. ‘Miss Willmott’, were extracted with methanol. After solvent evaporation, the methanolic extracts were diluted with water and successively partitioned between chloroform and then diethyl ether. The diethyl ether extracts from each sample were used for quantification. The analyses were performed on HPTLC precoated silica gel 60F₂₅₄ plates with toluene–ethyl formate–formic acid (6 : 4 : 1 v/v/v) as the mobile phase (distance of 7.5 cm). Densitometric detections of TRS, MBR and EA were performed at 320, 287 and 280 nm, respectively. The amounts of these compounds were calculated using the regression equations of the calibration curves, which were linear within a range of 0.05–0.5 μg/spot (R² = 0.9957) for TRS, 0.05–0.525 μg/spot (R² = 0.9965) for MBR and 0.0525–0.5 μg/spot (R² = 0.9998) for EA. Results – The amounts of marker compounds measured by the method developed are expressed in mg/g of dry extracts. TRS ranged from 20.3 ± 0.3 mg/g for P. erecta herbs to 197.7 ± 2.9 mg/g for P. grandiflora herbs; MBR ranged from 5.0 ± 0.6 mg/g for P. erecta herbs to 68.5 ± 3.4 mg/g for P. nepalensis flowers; and EA ranged from 24.0 ± 0.6 mg/g for P. erecta herbs to 216.2 ± 3.2 mg/g for P. anserina leaves. Conclusion – The proposed method was found to be relatively simple, specific, precise, sensitive and accurate and may be used for the routine assay of simultaneous determination of TRS, MBR and EA in other extracts and phytomedicines containing Potentilla species. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid validated HPTLC method for estimation of betulinic acid in Nelumbo nucifera (Nymphaeaceae) rhizome extract

Introduction – Betulinic acid (pentacyclic triterpenoid) is an important marker component present in Nelumbo nucifera Gaertn. rhizome. N. nucifera rhizome has several medicinal uses including hypoglycaemic, antidiarrhoeal, antimicrobial, diuretic, antipyretic, psychopharmacological activities. Objective – To establish a simple, sensitive, reliable, rapid and validated high‐performance thin‐layer chromatography method for estimation of betulinic acid in hydro‐alcoholic extract of N. nucifera Gaertn. rhizome. Materials and methods – The separation was carried out on a thin‐layer chromatography aluminium plate pre‐coated with silica gel 60F₂₅₄, eluted with chloroform, methanol and formic acid (49 : 1 : 1 v/v). Post chromatographic derivatisation was done with anisaldehyde–sulphuric acid reagent and densitometric scanning was performed using a Camag TLC scanner III, at 420 nm. Results – The system was found to produce a compact spot for betulinic acid (R_f = 0.30). A good linear precision relationship between the concentrations (2–10 µg) and peak areas were obtained with the correlation coefficient (r) of 0.99698. The limit of detection and limit of quantification of betulinic acid were detected to be 0.4 and 2.30 µg per spot. The percentage of recovery was found to be 98.36%. The percentage relative standard deviations of intra‐day and inter‐day precisions were 0.82–0.394 and 0.85–0.341, respectively. Conclusion – This validated HPTLC method provides a new and powerful approach to estimate betulinic acid as phytomarker in the extract. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous analysis of diosgenin and sarsasapogenin in Asparagus officinalis byproduct by thin‐layer chromatography

Introduction – Asparagus officinalis L. has several biological activities including antifungal, antiviral and antitumoral activities due to the steroidal saponins. Normally diosgenin and sarsasapogenin are analysed separately by thin‐layer chromatography or high‐performance liquid chromatography (HPLC‐UV or HPLC‐ELSD), which is time‐consuming and expensive, so we need to find a rapid solution to this problem. Objective – To develop a sensitive, rapid and validated TLC method for simultaneous detection and quantification of diosgenin and sarsasapogenin. Methodology – Samples were prepared by extraction of A. officinalis with 70% aqueous ethanol to get steroidal saponins, and then hydrolysed using 36 mL 2 m hydrochloric acid for 3 h. The hydrolysis product was extracted with chloroform, and then analysed by TLC, the results of which were verified by HPLC and HPLC‐MS. Results – The retention factor (R_f) of diosgenin and sarsasapogenin on TLC plate were 0.49 and 0.6, respectively. After calculation from the regression equation of the standard curve, the contents of diosgenin and sarsasapogenin in the A. officinalis extract were 0.27–0.46 and 0.11–0.32%, respectively. Conclusion – The study showed that thin‐layer chromatography can be applied for the determination of diosgenin and sarsasapogenin in the oldest tissue of A. officinalis, and also can be conducted for screening of sapogenin in other plant or extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantitative analysis of safranal in saffron extract and nanoparticle formulation by a validated high‐performance thin‐layer chromatographic method†

Introduction – Safranal is an effective anticonvulsant shown to act as an agonist at GABA_A receptors. Nose to brain delivery via nanoparticle formulation might improve its brain delivery. A selective and sensitive analytical method is required for evaluation of safranal‐based novel drug delivery systems. Objective – To develop and validate a high‐performance thin‐layer chromatographic (HPTLC) method for the quantitative analysis of safranal as bulk, in saffron extract and in developed safranal‐loaded nanoparticle formulation. Methodology – Chromatographic separation was achieved on silica gel pre‐coated TLC aluminium plates 60F‐254, using n‐hexane:ethyl acetate (9 : 1, v/v) as the mobile phase. Quantitative analysis was carried out by densitometry at a wavelength of 310 nm. The method was validated and applied to detect related impurities, to analyse safranal in saffron extract and to evaluate safranal‐loaded nanoparticles. Results – Compact spots of safranal were observed at R_f value 0.51 ± 0.02. The method was linear (r = 0.9991) between 0.5 and 5.0 μg/spot. The intra‐ and inter‐day precisions were 1.08–2.17 and 1. 86–3.47%, respectively. The limit of detection was 50 ng/spot and the limit of quantification was 150 ng/spot. The method proved to be accurate (recovery 97.4–102.0%) and was selective for safranal. Evaluation of safranal‐loaded nanoparticle formulation demonstrated drug loading of 23.0%, encapsulation efficiency of 42.0% and sustained drug release following biphasic pattern. Conclusion – The present method is useful for the quantitative and qualitative analysis of safranal and safranal‐loaded nanoparticle formulation. It provides significant advantages in terms of greater specificity and rapid analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Essential Oils and Diethyl Ether Extracts of Serbian Xeranthemum cylindraceum and X. annum: Chemical Composition,Antimicrobial Activity,and Chemotaxonomic Implications

Detailed GC and GC‐MS analyses of the essential oils and Et₂O extracts of two Xeranthemum species – X. cylindraceum and X. annum – resulted in the identification of 254 components, in total. Terpenoids constituted the major part of both X. cylindraceum and X. annum essential oils and extracts (51.8–65.7%, and 50.7%, resp.). Among the sesquiterpenoids, the extracts of both investigated taxa contained the guaianolide xerantholide, its 11,13‐dihydro derivatives, and two additional sesquiterpene lactones: an eudesmanolide, 11,13‐dihydroisoalantolactone, and a pseudoguaianolide, confertin. The last two lactones and both isomers of 11,13‐dihydroxerantholide have not been previously detected in Xeranthemum species. The isolated extracts of X. cylindraceum and X. annum were tested in a broth microdilution assay against a panel of microorganisms. The tested extracts demonstrated significant antimicrobial inhibitory activity, ranging from 30 to 260 μg/ml, being most active against Bacillus cereus and Staphylococcus aureus, an important human pathogen, with MIC close in value to those of chloramphenicol. Chemotaxonomic significance of the sesquiterpene lactones' distribution in the taxa investigated in this study and those detected earlier in phylogenetically close species (up to the level of the tribe Cardueae) was also discussed.     

A qualitative and quantitative HPTLC densitometry method for the analysis of cannabinoids in Cannabis sativa L.

Introduction – Cannabis and cannabinoid based medicines are currently under serious investigation for legitimate development as medicinal agents, necessitating new low‐cost, high‐throughput analytical methods for quality control. Objective – The goal of this study was to develop and validate, according to ICH guidelines, a simple rapid HPTLC method for the quantification of Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC) and qualitative analysis of other main neutral cannabinoids found in cannabis. Methodology – The method was developed and validated with the use of pure cannabinoid reference standards and two medicinal cannabis cultivars. Accuracy was determined by comparing results obtained from the HTPLC method with those obtained from a validated HPLC method. Results – Δ⁹‐THC gives linear calibration curves in the range of 50–500 ng at 206 nm with a linear regression of y = 11.858x + 125.99 and r² = 0.9968. Conclusion – Results have shown that the HPTLC method is reproducible and accurate for the quantification of Δ⁹‐THC in cannabis. The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of carbohydrates in medicinal plants--comparison between TLC, mf-MELDI-MS and GC-MS

Introduction – Quality control in the pharmaceutical and phytopharmaceutical industries requires fast and reliable methods for the analysis of raw materials and final products. Objective – This study evaluates different analytical approaches in order to recognise the most suitable technique for the analysis of carbohydrates in herbal drug preparations. Methodology – The specific focus of the study is on thin‐layer chromatography (TLC), gas chromatography (GC), and a newly developed mass spectrometric method, i.e. matrix free material enhanced laser desorption/ionisation time of flight mass spectrometry (mf‐MELDI‐MS). Samples employed in the study were standards and microwave‐assisted water extracts from Quercus. Results – TLC analysis proved the presence of mono‐, di‐ and trisaccharides within the biological sample and hinted at the existence of an unknown carbohydrate of higher oligomerisation degree. After evaluation of different derivatisation techniques, GC‐MS confirmed data obtained via TLC for mono‐ to trisaccharides, delivering additionally quantified values under a considerable amount of time. A carbohydrate of higher oligomerisation degree could not be found. The application of mf‐MELDI‐MS further confirmed the presence of carbohydrates up to trisaccharides, also hinting at the presence of a form of tetrasaccharide. Besides this information, mf‐MELDI‐MS delivered further data about other substances present in the extract. Quantitative determination resulted in 1.750, 1.736 and 0.336 mg/mL for glucose, sucrose and raffinose respectively. Conclusion – Evaluation of all three techniques employed, clearly proved the heightened performance of mf‐MELDI‐MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requires only a few minutes. In addition, GC‐MS is suitable for quantitative analysis. Copyright © 2011 John Wiley & Sons, Ltd.     

Validation of a quantitative assay of arbutin using gas chromatography in Origanum majorana and Arctostaphylos uva‐ursi extracts

Introduction – Arbutin is a skin‐whitening agent that occurs naturally in the bark and leaves of various plants. It is commonly quantified in plant extracts and skin‐whitening products by HPLC. Objective – To develop an alternative gas chromatographic method for the separation and quantification of arbutin in Origanum majorana and Arctostaphylos uva‐ursi extracts. Methodology – N,O‐Bis(trimethylsilyl)acetamide and trimethylchlorosilane were used as silylation reagents, and the gas chromatographic separation of silylated extracts and standards was performed using a DB‐5 narrow bore column. GC‐MS was used for the compound identification, and the quantification was carried out by GC‐FID. The quantitative results were compared with those of HPLC analysis. Results – The developed method gave a good sensitivity with linearity in the range 0.33–500 mg/mL and recovery >98%, allowing the quantification of arbutin in O. majorana and A. uva‐ursi extracts. The relative standard deviations (RSD) relating to intra‐day and inter‐day precision were <0.002% and <4.8%, respectively. The GC results correlated well with those obtained by HPLC analysis. Conclusion – The analysis of marjoram and bearberry samples showed that the established GC method was rapid, selective, and demonstrated that arbutin could be screened alternatively by gas chromatography. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative analysis of major dibenzocyclooctane lignans in Schisandrae fructus by online TLC-DART-MS

Introduction – Direct analysis in real time (DART) ion source is a powerful ionising technique for the quick and easy detection of various organic molecules without any sample preparation steps, but the lack of quantitation capacity limits its extensive use in the field of phytochemical analysis. Objective – To improvise a new system which utilize DART‐MS as a hyphenated detector for quantitation. Methodology – A total extract of Schisandra chinensis fruit was analyzed on a TLC plate and three major lignan compounds were quantitated by three different methods of UV densitometry, TLC‐DART‐MS and HPLC‐UV to compare the efficiency of each method. To introduce the TLC plate into the DART ion source at a constant velocity, a syringe pump was employed. The DART‐MS total ion current chromatogram was recorded for the entire TLC plate. The concentration of each lignan compound was calculated from the calibration curve established with standard compound. Results – Gomisin A, gomisin N and schisandrin were well separated on a silica‐coated TLC plate and the specific ion current chromatograms were successfully acquired from the TLC‐DART‐MS system. The TLC‐DART‐MS system for the quantitation of natural products showed better linearity and specificity than TLC densitometry, and consumed less time and solvent than conventional HPLC method. Conclusion – A hyphenated system for the quantitation of phytochemicals from crude herbal drugs was successfully established. This system was shown to have a powerful analytical capacity for the prompt and efficient quantitation of natural products from crude drugs. Copyright © 2010 John Wiley & Sons, Ltd.     

Chromatographic analysis of simple phenols in some species from the genus Salix

Introduction – Salicis Cortex, made from willow bark is a herbal remedy, which is standardised based on the content of salicin, a compound with analgesic and antiphlogistic properties. However, clinical trials suggest that other compounds also present in Salicis Cortex can contribute to the pharmacological effects. Objective – To characterise the composition of phenolic acids in the barks of different species and clones from the genus Salix by use of chromatographic methods—HPTLC and HPLC. Methodology – The phenolic acid composition was analysed by MGD (multiple gradient development)–HPTLC technique. The separation was performed on HPTLC Diol plates with gradient elution using a mixture of chloroform:hexane:ethyl acetate with increasing concentration of ethyl acetate from 10 to 25%. Derivatisation with thymol reagent was employed for the first time for specific detection of phenolic acids containing methoxyl groups. Results – The presence of all phenolic acids previously reported in the genus Salix was confirmed, namely p‐hydroxybenzoic, vanillic, cinnamic, p‐coumaric, ferulic and caffeic acids. Furthermore, pyrocatechol as a constituent of willow bark was revealed. The highest concentration of this compound was observed in the S. purpurea bark (2.25 mg/g). Conclusion – The presence of a relatively high content of pyrocatechol in Salix species may raise doubts about the safe application of this herbal medicine. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of a naturally‐occurring 8‐[α‐D‐glucopyranosyl‐(1→6)‐β‐D‐glucopyranosyl]daidzein from cultivated kudzu root

Introduction – Kudzu root (Radix puerariae) is a rich source of isoflavones that are effective in preventing osteoporosis, heart disease and symptoms associated with menopause. The major isoflavonoids in kudzu root extracts were reported as puerarin, daidzin and daidzein. Recently, an unknown isoflavonoid (compound 1) was detected from one‐year‐old kudzu root cultivated in Vietnam. Objective – To identify a novel compound 1 in kudzu root extract and determine the structure of the compound by ESI⁺ TOF MS‐MS, ¹H‐, ¹³C‐NMR and enzymatic hydrolysis. Methodology – Samples were prepared by extraction of one‐year‐old kudzu root with 50% ethanol and the isoflavonoids were purified using recycling preparative HPLC. Unknown compound 1 was detected using UV‐light at 254 nm in TLC and HPLC analyses. The molecular weight of 1 was determined using a TOF mass spectrometer equipped with an electrospray ion source. The structure of 1 was determined from the ¹³C and ¹H NMR spectra recorded at 100.40 and 400.0 MHz, respectively. Results – ESI⁺ TOF MS‐MS analysis shows that 1 is a puerarin diglycoside. The interglycosidic linkage of diglycoside determined by ¹H‐, ¹³C‐NMR, and enzymatic hydrolysis suggests that 1 has a glucosyl residue linked to puerarin by an α‐1,6‐glycosidic bond. This compound is the first naturally‐occurring 8‐[α‐D ‐glucopyranosyl‐(1→6)‐β‐D ‐glucopyranosyl]daidzein in kudzu root. The concentration of glucosyl‐α‐1,6‐puerarin in kudzu root was 2.3 mg/g as determined by HPLC. Conclusion – The results indicate that puerarin diglycoside is one of the major isoflavonoids in kudzu root and has a significant impact on the preparation of highly water‐soluble glycosylated puerarin. Copyright © 2009 John Wiley & Sons, Ltd.     

High-performance thin-layer chromatographic method for the detection and determination of lansoprazole in human plasma and its use in pharmacokinetic studies

A rapid and sensitive high-performance thin-layer chromatography (HPTLC) method has been developed for the measurement of lansoprazole in human plasma and its use for pharmacokinetic study has been evaluated. Detection and quantitation were performed without using an internal standard. A single stage extraction procedure was followed for extracting lansoprazole from plasma and a known amount of the extract was spotted on precoated silica gel 60 F₂₅₄ plates using a Camag Linomat IV autosampler. Lansoprazole was quantified using a Camag TLC Scanner 3. The recovery study of authentic analytes added to plasma at 0.05 to 0.25 μg/ml was 95.37±2.15% and the lowest amount of lansoprazole that could be detected was 20 ng/ml plasma. The method provides a direct estimate of the amount of lansoprazole present in plasma. The method was used for the determination of plasma levels as well as pharmacokinetic parameters of lansoprazole after oral administration of two marketed preparations to healthy volunteers.     

Studies on the stability of salvianolic acid B as potential drug material

Introduction – Salvianolic acid B (Sal B) is one of the major water‐soluble compounds isolated from the roots of Salvia miltiorrhiza, which is widely used as a traditional Chinese medicine. Although much research on the general stability of Sal B has been undertaken and reported, there is still a need for further study of the stability required as a potential drug material. Objective – To study the stability of Sal B in the solid state and in normal saline (NS) solution during storage, as required in the ICH guidelines (2003) and Chinese Pharmacopoeia (2005). Methodology – Sal B stability was analysed using the high‐performance liquid chromatography (HPLC) method described in the Chinese Pharmacopoeia. HPLC coupled with time‐of‐flight mass spectrometry (HPLC‐TOFMS) was applied for the separation and identification of the degradation products of Sal B. Results – In the solid state, Sal B packaged in aluminium foil bags was stable for 6 months under ‘accelerated conditions’ (40°C, 75% relative humidity, RH). However, solid Sal B degradation was observed under open exposure to stress conditions of high temperature (60°C) or high humidity (92.5 or 75% RH). In NS solution, Sal B underwent severe degradation under accelerated conditions. Through HPLC‐TOFMS, nine degradation products were identified and the possible degradation pathway was deduced. Conclusion – The results demonstrate that the potential drug material Sal B could be used in a solid formulation, but is not suitable for use as a liquid formulation. Copyright © 2011 John Wiley & Sons, Ltd.     

Conformational Disorder Enhances Solubility and Photovoltaic Performance of a Thiophene–Quinoxaline Copolymer

The side‐chain architecture of alternating copolymers based on thiophene and quinoxaline (TQ) is found to strongly influence the solubility and photovoltaic performance. In particular, TQ polymers with different linear or branched alkyloxy‐phenyl side chains on the quinoxaline unit are compared. Attaching the linear alkyloxy side‐chain segment at the meta‐ instead of the para‐position of the phenyl ring reduces the planarity of the backbone as well as the ability to order. However, the delocalisation across the backbone is not affected, which permits the design of high‐performance TQ polymers that do not aggregate in solution. The use of branched meta‐(2‐ethylhexyl)oxy‐phenyl side‐chains results in a TQ polymer with an intermediate degree of order. The reduced tendency for aggregation of TQ polymers with linear meta‐alkyloxy‐phenyl persists in the solid state. As a result, it is possible to avoid the decrease in charge‐transfer state energy that is observed for bulk‐heterojunction blends of more ordered TQ polymers and fullerenes. The associated gain in open‐circuit voltage of disordered TQ:fullerene solar cells, accompanied by a higher short‐circuit current density, leads to a higher power conversion efficiency overall. Thus, in contrast to other donor polymers, for TQ polymers there is no need to compromise between solubility and photovoltaic performance.     

Optimization and development of antidiabetic phytosomes by the Box–Behnken design

Researchers have extensively reviewed on herbs and natural products for their marked clinical efficacy in some recent years, however, maximum of the newly discovered bioactive constituents offer poor bioavailability due to their large size molecules or to their poor miscibility with oils and lipids, thereby limiting their ability to pass across the lipid-rich outer membranes of the enterocytes of the small intestine. Phytosomes are more bioavailable as compared to herbal extracts owing to their enhanced capacity to cross the bio-membranes and thus reaching the systemic circulation. This study was aimed to investigate the development and optimization of antidiabetic phytosomes using a three-factor, three-level the Box–Behnken design (17 batches). The fruits of Citrullus colocynthis (L.) Momordica balsamina and Momordica dioica were extracted using Soxhlet’s apparatus. The phytochemical fingerprint profile of the combined methanolic extracts was done by using high-performance thin layer chromatography (HPTLC). The polynomial quadratic equation analysis was designed to study the response (entrapment efficiency (EE), % yield) of independent significant factors at different levels. Phytosomes were characterized in terms of drug content, particle size, EE, zeta potential and in vitro dissolution. TEM analysis revealed good stability and a spherical, self-closed structure of phytosomes in complex formulations. Average particle size was found to 450?nm. Total flavonoid content was found to be 10.0?±?0.002?μg/g. Optimized formulation was selected and was prepared using A (1:3), B (60?°C) and C (2.5?h) to give maximum yield and entrapment efficiencies (72% and 92.1?±?5.1%). Phytosomes were found to have antidiabetic activity comparable to metformin in low dose. HPTLC showed the presence of the phyto-constituent quercetin.     

Simultaneous quantification of 14 bioactive constituents in Forsythia Suspensa by liquid chromatography–electrospray ionisation–mass spectrometry

Introduction – Forsythia suspensa is a commonly used traditional Chinese medicine including phenylethanoid glycosides, lignans, flavonoids, terpenes and volatile oils. Quantification of multi‐components is important for the quality control of Forsythia suspensa. Objective – To establish a liquid chromatography–electrospray ionisation–mass spectrometry method for simultaneous quantification of 14 bioactive constituents of Forsythia suspensa in different places of China and different parts of this herb. Methodology – The optimal chromatographic conditions were achieved on a Kromasil C₁₈ column (150 ¥ 4.6 mm, 5 mm) with gradient elution of methanol, acetonitrile and 0.1% formic acid in 27 min. Detection was performed in negative ionisation mode by monitoring the precursor–product combination in multiple reaction monitoring (MRM) mode. The validation of the method included tests of linearity, sensitivity, precision, repeatability, stability and accuracy. Results – All calibration curves showed good linear regression (r > 0.9990) within test ranges. The established method showed good precision and accuracy with overall intra‐day and inter‐day variations of 0.7–4.3 and 1.1–3.9% respectively, and overall recoveries of 96.65–101.2% for the compounds analysed. Conclusion – The proposed method was successfully applied for the quantitative analysis of 14 constituents in 12 Forsythia suspensa samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of Nigella sativa and its Major Constituent, Thymoquinone on Sciatic Nerves in Experimental Diabetic Neuropathy

The aim of this study was designed to investigate the possible beneficial effects of Nigella sativa (NS) and thymoquinone (TQ) on histopathological changes of sciatic nerves in streptozotocin-induced diabetic rats. The rats were randomly allotted into one of four experimental groups: A (control), B (diabetic untreated), C (diabetic treated with NS) and D (diabetic treated with TQ); each group contain ten animals. B, C and D groups received streptozotocin (STZ) to induce diabetes. The rats in NS and TQ treated groups were given NS (in a dose of 400 mg/kg body weight) and TQ (50 mg/kg body weight) once a day orally by using intra-gastric intubation for 12 weeks starting 2 days after STZ injection, respectively. Blood and tissue samples were obtained for biochemical and histopathological investigation. The treatment of both NS and TQ caused a sharp decrease in the elevated serum glucose (P < 0.01, 0.05, respectively), and an increase in the lowered serum insulin concentrations (P < 0.01, 0.05, respectively), in STZ-induced diabetic rats. STZ induced a significant decrease in the area of insulin immunoreactive β-cells (P < 0.0001). NS (P < 0.001) and TQ (P < 0.01) treatment resulted in increased area of insulin immunoreactive β-cells significantly. To date, no histopathological changes of sciatic nerves in STZ induced diabetic rats by NS and TQ treatment have been reported. In this study, histologic evaluation of the tissues in diabetic animals treated with TQ and especially NS showed fewer morphologic alterations. Myelin breakdown decreased significantly after treatment with NS and TQ. The ultrastructural features of axons also showed remarkable improvement. We believe that further preclinical research into the utility of NS and TQ may indicate its usefulness as a potential treatment on peripheral neuropathy (PN) in STZ induced diabetic rats.     

Contribution of microwave accelerated distillation in the extraction of the essential oil of Zygophyllum album L.

Introduction – The aerial parts of Zygophyllum album L. are used in folk medicine as an antidiabetic agent and as a drug active against several pathologies. In this work we present the chemical composition of Algerian essential oils obtained by microwave accelerated distillation (MAD) extraction, a solventless method assisted by microwave. Objective – Under the same analytical conditions and using GC‐FID and GC‐MS, the chemical composition of the essential oil of Zygophyllum album L. extracted by MAD was compared with that achieved using hydrodistillation (HD). Methodology – The extracted compounds were hydrosoluble, and they were removed from the aqueous solution by a liquid extraction with an organic solvent. Results – Employing MAD (100°C, 30 min), the essential oil contained mainly oxygenated monoterpenes with major constituents: carvone and α‐terpineol. However, most of the compounds present in the hydrodistilled volatile fraction were not terpene species, with β‐damascenone as a major constituent. Conclusion – The MAD method appears to be more efficient than HD: after 30 min extraction time, the obtained yields (i.e. 0.002%) were comparable to those provided by HD after 3 h extraction. MAD seems to be more convenient since the volatile fraction is richer in oxygenated monoterpenes, species that are recognised for their olfactory value and their contribution to the fragrance of the essential oil. Copyright © 2010 John Wiley & Sons, Ltd.