A simple and reproducible High-Performance Thin-Layer Chromatography (HPTLC) densitometric method was developed for the simultaneous quali-quantitative determination of rosavin and salidroside in commercial products based on Rhodiola rosea L. Their content was compared with Vitango (R), one of the first Traditional Herbal Medicine released on the market. Fingerprints of commercial product extracts and their rosavin and salidroside quantifications were performed, on the same layer, by densitometric evaluation in absorption mode (272 nm). Their antioxidant activity was also evaluated in fluorescence mode (517 nm). The Retention Factors (Rf) were 0.39 +/- 0.02 for rosavin and 0.58 +/- 0.02 for salidroside. Linear regression analysis data in the concentration range of 1.0-5.0 mu g/spot showed a strong linearity relationship with R2 = 0.9888 for rosavin and R2 = 0.9982 for salidroside. The amount of rosavin and salidroside in formulations ranged from 0.7 to 6.0% and 0.9 to 10.7% respectively. Simultaneous HPTLC rosavin and salidroside quantification and their antioxidant evaluation, can be considered quick, cost-effective and green method, for plant extracts/formulations analysis.

Ephedrine is one component of a popular traditional Chinese multi-plant formulation used to treat asthma, fever, coughs and lately COVID-19. The aim of this study is to develop and validate a simple and rapid quali-quantitative High Performance Thin Layer Chromatography (HPTLC) densitometry method to detect ephedrine, the most abundant alkaloid of Ephedra plant, and to analyze free sale ephedra-based capsules. This quick, simple and low requirement method is cost-effective and allows screening up to 19 samples all together in a single layer. During the development of this method, an extraction solvent mixture composed of methanol/water (2:1, v/v) and a mobile phase constituted of ammonia/methanol/dichloromethane (1:10:40, v/v/v) gave the best results. The identity and purity of ephedrine peak were confirmed by matching Rf value and UV-spectrum with its reference standard. Lastly, samples were quantitated by densitometry at 500 nm after ninhydrin derivatization. Linearity was obtained in the concentration range of 0.062–0.146 μg/band. Limit of detection and limit of quantification (LOQ) were found to be 0.0020 μg/band and 0.0067 μg/band, respectively. This technique is useful to screen multiple samples at the same time with a limited use of solvents and reagents so that it can be considered a fast and simple procedure.

Carica papaya Linn. is one of the most common plants in almost all tropical countries. Its fruit is widely used as food or an ointment in traditional medicine. It is one of the few tropical fruits to contain glucotropaeolin (benzyl glucosinolate) detected in great quantity, mainly in the seeds of papaya. When cells in plant tissue are damaged, glucotropaeolin is hydrolysed by the enzyme myrosinase to benzyl isothiocyanate. The anticancer activity of this latter compound has been widely evidenced, but the metabolic profile of the papaya seed extracts is not reported in the literature. The objective of this study is to investigate the seed extracts of C. papaya L. by UHPLC-PDA ESI/MS, with and without the inactivation of myrosinase, and compare the recovery of some main components using two different inactivation procedures. The extracts (methanol/water, 60:40, v/v) were studied in negative and positive ionisation modes. Separations were carried out on an Acquity BEH C18 (50 × 2.1 mm i.d.) 1.7 μm analytical column, and 0.02% formic acid in water and acetonitrile was used as the mobile phase at a flow rate of 0.6 mL min−1. Beyond the amino acids and glucotropaeolin already detected in papaya seeds, 4-hydroxybenzoic acid, which has never been detected in papaya seeds with this technique before, was identified. Moreover, mono-, di- and tri-glycosides of 4-hydroxybenzoic acid were putatively assigned. Glucotropaeolin, 4-hydroxybenzoic and tryptophan were quantified in seeds extracted after myrosinase deactivation. These three components were more effectively recovered using an oven than microwave myrosinase inactivation.

Most women with ovarian cancer are treated with chemotherapy before or after surgery. Unfortunately, chemotherapy treatment can cause negative side effects and the onset of multidrug resistance (MDR). The aim of this study is to evaluate the chemosensitizing effect of a natural compound, voacamine (VOA), in ovarian (A2780 DX) and colon (LoVo DX) cancer drug-resistant cell lines which overexpress P-glycoprotein (P-gp), in combination with paclitaxel (PTX), or doxorubicin (DOX) or 5-fluorouracil (5-FU). VOA, a bisindole alkaloid extracted from Peschiera fuchsiaefolia, has already been shown to be effective in enhancing the effect of doxorubicin, because it interferes with the P-gp function. Ovarian cancer cytotoxicity test shows that single treatments with VOA, DOX and PTX do not modify cell viability, while pretreatment with VOA, and then PTX or DOX for 72 h, induces a decrease. In colon cancer, since 5-FU is not a-substrate for P-gp, VOA has no sensitizing effect while in VOA + DOX there is a decrease in viability. Annexin V/PI test, cell cycle analysis, activation of cleaved PARP1 confirm that VOA plus PTX induce apoptotic cell death. Confocal microscopy observations show the different localization of NF-kB after treatment with VOA + PTX, confirming the inhibition of nuclear translocation induced by VOA pretreatment. Our data show the specific effect of VOA which only works on drugs known to be substrates of P-gp.

Iberin (IBR) is an isothiocyanate (ITC) with chemopreventive properties on different types of cancer. The need of quantifying IBR either in natural products or in food supplements makes the availability of suitable and stable analytical standards necessary. The choice of proper solvents for ITCs storage and experiment setup is a critical point due to ITCs reactivity. As IBR reference standard is sometimes supplied in ethanol, methanol is also frequently utilised as a solvent in ITCs analysis. A study on IBR stability in the most common solvents used in liquid chromatography and at different pH is presented. A fast UHPLC-PDA-ESI/MS analysis was used to separate IBR from degradation products, to monitor IBR stability at three temperatures and at different pH. The fastest degradation was observed in methanol/water mixture. IBR degraded always faster in methanol than in ethanol. At 20 degrees C, the degradation in ethanol and in water was comparable. At 30 degrees C and 40 degrees C the degradation in water was faster than either in ethanol or methanol. Ethyl thiocarbamate, methyl thiocarbamate and disubstituted thiourea degradation products, in ethanol, methanol and water respectively, were tentatively assigned. IBR was stable in acetonitrile and at acidic pH but it was unstable at alkaline pH.

Search for natural substances in association with conventional chemotherapeutic drugs with a chemiosensitizing action easily accessible to the tumor mass has encouraged our studies on voacamine (VOA) and its monomeric units, voacangine and vobasine. Our previous results showed that VOA sensitized multidrug resistant (MDR) osteosarcoma cells (U-2 OS/DX) to doxorubicin (DOX) cytotoxicity. VOA, extracted by Peschiera fuchsiaefolia plant, is a bisindole alkaloid consisting of an Iboga skeleton (voacangine) directly linked to a 2-acyl indole unit (vobasine). High-performance thin-layer chromatography densitometry demonstrated the purity of VOA, voacangine and vobasine samples. Flow cytometry analysis showed that VOA, voacangine and vobasine enhanced DOX accumulation of U-2 OS/DX cells, in equally way, whereas VOA reduced more efficiently DOX efflux. Optical microscopy and clonogenic assay confirmed that VOA was more effective than voacangine and vobasine in enhancing DOX cytotoxic effect. These results showed that monomers linked together are necessary to modulate resistant phenotype of osteosarcoma cells. To complete the study, we evaluated the effect of three compounds on microtubules by confocal microscopy, suggesting that only the whole molecule depolymerizes the microtubules blocking so DOX efflux-mediated by vesicles.

Introduction. Turmeric is the common name for the rhizome of Curcuma longa L. In the recent years, food supplements containing turmeric have been marketed and widely used by an increasing number of consumers. Spontaneous reports of suspected adverse reactions to food supplements are collected within the Phytovigilance system. Methods. An ad hoc multidisciplinary group investigated the suspected cases of hepatotoxicity reported to the Italian Phytovigilance system associated with the assumption of turmeric food supplements with the methodology specific to pharmacovigilance as well as for the evaluation of the quality and safety of food supplements. Results. A cluster of 28 spontaneous reports of acute hepatitis, mostly with cholestasis, associated with turmeric products were sent to the Italian Phytovigilance system in the first six months of 2019. In all cases, except one, the causality assessment was at least possible. The suspected products were collected and analysed for the presence of drugs, heavy metals, aflatoxins, pesticides, synthetic dyes and pyrrolizidine alkaloids. Conclusion. On the basis of the results of all the activities performed by multidisciplinary group, regulatory intervention was taken. This study highlights the importance of developing an integrated evaluation approach for the evaluation of the adverse effects associated with the use of food supplements.

A comparison between High-Performance Thin-Layer Chromatography (HPTLC) analysis and Liquid Chromatography High Resolution Mass Spectrometry (LC–HRMS), coupled with Principal Component Analysis (PCA) was carried out by performing a combined metabolomics study to discriminate Arbutus unedo (A. unedo) plants. For a rapid digital record of A. unedo extracts (leaves, yellow fruit, and red fruit collected in La Maddalena and Sassari, Sardinia), HPTLC was used. Data were then analysed by PCA with the results of the ability of this technique to discriminate samples. Similarly, extracts were acquired by non-targeted LC–HRMS followed by unsupervised PCA, and then by LC–HRMS (MS) to identify secondary metabolites involved in the differentiation of the samples. As a result, we demonstrated that HPTLC may be applied as a simple and reliable untargeted approach to rapidly discriminate extracts based on tissues and/or geographical origins, while LC–HRMS could be used to identify which metabolites are able to discriminate samples.

The marketing of new argan-based products is greatly increased in the last few years and consequently, it has enhanced the number of control analysis aimed at detecting counterfeit products claiming argan oil as a major ingredient. Argan oil is produced in Morocco and it is quite expensive. Two simple methods for the rapid screening of pure oil and argan-oil based products, focused on the analysis of the triacylglycerol profile, have been developed. A three-minute-run by UHPLC-PDA allows the identification of a pure argan oil, while the same run with the MS detector allows also the analysis of products containing the oil down to 0.03%. On the other hand, by HPTLC the simultaneous analysis of twenty samples, containing argan oil down to 0.5%, can be carried out in a forty-five-minute run. The triglyceride profile of the most common vegetable fats such as almond, coconut, linseed, wheat germ, sunflower, peanut, olive, soybean, rapeseed, hemp oils as well as shea butter used either in cosmetics or commonly added for the counterfeiting of argan oil, has been also investigated. Over sixty products with different formulations and use have been successfully analyzed and argan oil in the 2.4-0.06% concentration range has been quantified. The methods are suitable either for a rapid screening or for quantifying argan oil in different formulations. (C) 2017 Elsevier B.V. All rights reserved.

The aim of this study was to get a rapid metabolic fingerprinting and to gain insight into the metabolic profiling of Arctostaphylos pungens H. B. K., a plant morphologically similar to Arctostaphylos uva-ursi (L.) Spreng. (bearberry) but with a lower arbutin (Arb) content. According to the European Pharmacopoeia the Arb content in the dried leaf of A. uva-ursi (L.) Spreng. must be at least 7% (wt/wt) but other species, like A. pungens, are unintentionally or fraudulently marketed instead of it. Therefore, methanolic leaf extracts of nine A. uva-ursi and six A. pungens samples labeled and marketed as "bearberry leaf" have been analyzed. A five-minute gradient with a UHPLC-PDA-ESI-TOF/MS on an Acquity BEH C18 (50 × 2.1 mm i.d.) 1.7 μm analytical column has been used for the purpose. A comprehensive assignment of secondary metabolites has been carried out in a comparative study of the two species. Among twenty-nine standards of natural compounds analyzed, fourteen have been identified, while other fifty-five metabolites have been tentatively assigned. Moreover, differences in both metabolic fingerprinting and profiling have been evidenced by statistical multivariate analysis. Specifically, main variations have been observed in the relative content for Arb, as expected, and for some galloyl derivative like tetra- and pentagalloylglucose more abundant in A. uva-ursi than in A. pungens. Furthermore, differences in flavonols profile, especially in myricetin and quercetin glycosilated derivatives, were observed. Based on principal component analysis myricetrin, together with a galloyl arbutin isomer and a disaccharide are herein proposed as distinctive metabolites for A. pungens.

Natural substances present in herbal preparations should be carefully used because they can give toxic or therapeutic effects despite of their amount or the way of administration. The safety of products of vegetable origin must be assessed before commercialisation by monitoring the active ingredients and their metabolites. This study was therefore designed to identify and quantify arbutin and its metabolite hydroquinone, naturally present in Arctostaphylos uva-ursi (L.) Spreng plant in rat plasma, after an acute and subacute administration of aqueous arbutin solution in Wistar rats. For this purpose a reversed-phase high-performance liquid chromatography coupled with photodiode array detection was developed to assess the pharmacokinetic of arbutin and hydroquinone in plasma of female rats treated with aqueous arbutin solutions. The detection (arbutin: 0.0617 µg/ml and hydroquinone 0.0120 µg/ml) and quantification (arbutin: 0.2060 µg/ml and hydroquinone: 0.0400 µg/ml) limits were determined. At the arbutin concentration level of 10.7 µg/ml repeatability was 13.33% and its recovery 93.4±6.93%, while at the hydroquinone concentration level of 10.6 µg/ml repeatability was 11.66% and its recovery 92.9±7.75%. Furthermore the method was fully validated and the obtained data indicate that the new method provides good performances.

Previous investigations demonstrated that pretreatment with non-cytotoxic concentrations of voacamine had a chemosensitizing effect on cultured multidrug resistant osteosarcoma cells exposed to doxorubicin; whereas when used alone at high concentrations voacamine induced apoptosis-independent cell death on both sensitive and resistant cells. To gain insight into the mechanism of action of voacamine at the subcellular level, we developed an analytical high-performance thin-layer chromatography technique to assess the intracellular content of voacamine that could be correlated with the induction of cell death and consequent morphological and ultrastructural changes. The results of the quantitative analysis not only did allow us to measure both the amount of unmodified voacamine molecules (determined by the method) and the amount of molecules which reacted with cellular components (undetectable), but also to confirm the findings of our previous studies and support the validity of this method.

Henna leaves are the raw material of commercial body and hair dyes.According to historical and ethnobotanical information, henna was one of the first plants used for such purpose.However, differences can be observed between henna products by the origin of the raw material, the presence of other plants, or the addition of various contaminants that may cause allergies and permanent scarring.Nowadays henna is used everywhere but it lacks the necessary controls.We report a pharmacognostic study focused on quality control of henna's raw materials from different countries or based on other plants.The analytic approach based on High Performance Thin Layer Chromatography (HPTLC) was proposed as a reliable technique to evaluate natural products complex mixtures, as it is also the case of derived botanical marketed products.

In previous studies it has been demonstrated that the plant alkaloid voacamine (1), used at noncytotoxic concentrations, enhanced the cytotoxicity of doxorubicin and exerted a chemosensitizing effect on cultured multidrug-resistant (MDR) U-2 OS-DX osteosarcoma cells. The in vitro investigations reported herein gave the following results: (i) the chemosensitizing effect of 1, in terms of drug accumulation and cell survival, was confirmed using SAOS-2-DX cells, another MDR osteosarcoma cell line; (ii) compound 1 enhanced the cytotoxic effect of doxorubicin also on the melanoma cell line Me30966, intrinsically drug resistant and P-glycoprotein-negative; (iii) at the concentrations used to sensitize tumor cells, 1 was not cytotoxic to normal cells (human fibroblasts). These findings suggest possible applications of voacamine (1) in integrative oncologic therapies against resistant tumors.

Herbal species different from the official bearberry, Arctostaphylos uva-ursi, are sold through conventional markets and also through non-controlled Internet websites, putting consumer safety at risk owing to the lack of quality control. Recently, Arctostaphylos pungens has become one of the most used species as a raw material for herbal medicines and dietary supplements in the place of official bearberry, a plant used for the treatment of various urinary disorders. A fingerprint identification based on an integrated application of different analytical techniques (HPTLC, NMR, HPLC-DAD and LC-ESI-MS) is here described to distinguish A. uva-ursi from A. pungens. The HPTLC and HPLC-DAD fingerprints resulted the simplest methods to differentiate the two species, whereas LC-ESI-MS was more useful to quantify arbutin, the main component of bearberry, and to evaluate its different content in the two species. This multidisciplinary study showed for the first time a specific phytochemical fingerprint of the new species A. pungens.

A high-performance thin-layer chromatography (HPTLC) method was developed for simple and rapid chemical analyses of multiingredient botanicals. The method was based on comparison of the fingerprint of the marketed multi-ingredient botanical with the fingerprints of the extracts of its component plants and of a handmade mixture of the constituent extracts in a ratio as similar as possible to that of the market product, when labeled. Planar chromatography analysis was completed by a densitometric scansion. The analyses revealed a good correspondence between the market products and the handmade mixture of standard extracts.

Efficient, useful and sensitive analytical devices for the quality control of complex mixtures of natural products are urgently needed. Controls should be based on simple, viable, comprehensible and low cost methods. High Performance Thin Layer Chromatography (HPTLC) the last evolution of planar chromatography, is a reliable candidate for analysis of botanicals. Although HPLC remains the best choice for organic substances, HPTLC can solve several problems in a quicker, simpler and easier method. Furthermore, the visualization and the analysis of similar products performed exactly in the same conditions, allow an evident, good and safe result of comparison. The paper is dedicated to HPTLC application on selected market products, in order to evidence benefits and limits of the HPTLC fingerprint approach.

Equisetum arvense L. is a herbaceous medicinal plant, commonly known as horsetail, whose extracts have been reported to possess diuretic and haemostatic properties. The aim of this study was to evaluate the use of fingerprint chromatographic methods on commercially available raw materials or preparations of E. arvense L. in order to ascertain their quality and identify possible adulterants using HPLC and HPTLC densitometry. Two chromatographic methods were used to determine the chemical fingerprints of E. arvense and other allied species. The first was based on HPTLC identification followed by densitometric measurement at 350 nm. The second was based on HPLC separation. The ease of sample preparation and the possibility of simultaneous analysis of several samples in a short time make HPTLC a method of choice for the comprehensive quality evaluation of herbal products.

Glaucium flavum collected in Sardinia was studied using a phytochemical approach in order to evaluate its alkaloid composition and obtain a comparison with the alkaloid contents of the same species in populations of other geographic proveniences. In fact, different chemoecotypes of G. flavum have been identified, on the basis of their particular content and composition in alkaloids, in accordance with the different distribution areas. The analysis showed that Sardinian G. flavum contains a homogeneous alkaloid pattern of aporphyne type, significantly different from those reported for populations from other parts of Europe.