Development and cross-validation of simple HPLC-fluorescence and UPLC-MS-UV methods for rapid determination of oleuropein in olive leaves.

INTRODUCTION: Olive leaves, abundant by-products of the olive oil industry, are a rich source of oleuropein, an important polyphenol in olive leaves. So far, no published methods have been validated using matrix standards for oleuropein quantification in olive leaves. OBJECTIVES: The study aimed to develop an HPLC method for oleuropein determination in olive leaves using spiked matrix standards prepared from a blank olive leaf matrix, to validate the method with respect to aqueous standards, and cross-validate the HPLC method with UPLC-MS and UPLC-UV techniques. METHODOLOGY: Oleuropein was extracted into methanol and analysed by HPLC with fluorescence detection (FLD; excitation and emission wavelengths 281 and 316 nm, respectively) and by UPLC-MS-UV. For validation, calibration curves of spiked matrix standards (0.4 to 4.8 mg/g) were analysed by the three methods over several days. Oleuropein was then analysed in French olive varieties. RESULTS: For the HPLC-FLD method, repeatability and intermediate precision were less than 5% RSD and linearity was demonstrated by the Fischer test. Differences in results of the spiked placebos by the three methods were non-significant, as confirmed by ANOVA. Extraction recovery was >90%, and there was a strong linear relationship between authentic and spiked matrix standards. The determination of oleuropein in French olive varieties is reported, including analysis in "Olivière" cultivar for the first time, leaves of which contained twice the amount of oleuropein compared with "Picholine". CONCLUSION: Accurate quantification of oleuropein is possible using aqueous standards. Cross-validation indicates that selective analysis can equally be carried out by HPLC or by UPLC-MS techniques.

Preparative separation of iridoid glucosides and crocins from Gardeniae Fructus using sequential macroporous resin column chromatography and evaluation of their anti-inflammatory and antioxidant activities.

Iridoid glycosides (geniposide (GP), genipin-1-gentiobioside (GB), etc.) and crocins (crocin Ⅰ (CR1), crocin Ⅱ(CR2), etc.) are two main bioactive components in Gardeniae Fructus (GF), which is a famous traditional Chinese medicine. Iridoid glycosides exhibit many activities and are used to manufacture gardenia blue pigment for the food industry. Crocins are rare natural water-soluble carotenoids that are often used as food colorants. A sequential macroporous resin column chromatography technology composed of HC-500B and HC-900B resins was developed to selectively separate iridoid glucosides and crocins from GF. The adsorption of GP on HC-900B resin was an exothermic process. The adsorption of CR1 on HC-500B resin was an endothermic process. The two kinds of components were completely separated by a sequential resin column. GB and GP were mainly found in product 1 (P1) with purities of 11.38% and 46.83%, respectively, while CR1 and CR2 were mainly found in product 2 (P2) with purities of 12.32% and 1.40%, respectively. The recovery yields of all the compounds were more than 80%. The above results showed that sequential resin column chromatography technology achieved high selectivity and recovery yields. GF extract, P1 and P2 could significantly inhibit the secretion of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-induced RAW264.7 cells, indicating that iridoid glycosides and crocins provide a greater contribution to the anti-inflammatory activity of GF. At the same time, compared to the GF extract and P1, P2 exhibited stronger scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, indicating that crocins may provide a significant contribution to the antioxidant activity of GF.

A new iridoid glucoside from the roots of Morinda officinalis.

A new iridoid glucoside, moridoside (1), and nine known compounds, asperulosidic acid (2), 6-O-epi-acetylscandoside (3), geniposidic acid (4), 2-hydroxymethylanthraquinone (5), 2-hydroxymethyl-3-hydroxyanthraquinone (6), damnacanthol (7), lucidine-ω-methyl ether (8), 2-hydroxy-1-methoxyanthraquinone (9), and 3,8-dihydroxy-1,2-dimethoxyanthraquinone (10) were isolated from the methanol extract of Morinda officinalis How. roots. Their structural identification was carried out based on the spectroscopic evidence. All compounds were evaluated for their nitric oxide (NO) production inhibitory activities in LPS-stimulated RAW264.7 macrophages. Compounds 5-7 significantly inhibited the production of NO with IC50 values of 28.4, 33.6, and 30.5 µM, respectively.

Iridoids Isolation from a Phytochemical Study of the Medicinal Plant Teucrium parviflorum Collected in Iraqi Kurdistan.

Herbal medicines are still widely practiced in Kurdistan Region-Iraq, especially by people living in villages on mountainous regions. Among plants belonging to the genus Teucrium (family Lamiaceae), which are commonly employed in the Kurdish traditional medicine, we have analyzed, for the first time, the methanol and aqueous methanol extracts of T. parviflorum aerial parts. The plant is mainly used by Kurds to treat jaundice, liver disorders and stomachache. We aimed to determine the phytochemical profile of the extracts and the structures of the main components, so to provide a scientific rationale for the ancient use of the plant in the ethno-pharmacological field. TLC analysis of the two extracts on silica gel and reversed phase TLC plates, using different visualization systems, indicated similar contents and the presence of phenolics, flavonoids, terpenoids and sugars. The chlorophyll-free extracts exhibited weak/no antimicrobial activities against a panel of bacteria (MICs = 800-1600 µg/mL) and fungal strains (MICs ≥ 5 mg/mL). At the concentration of 600 µg/mL, the methanol extract showed moderate antiproliferative effects against A549 and MCF-7 cancer cell lines in the MTS assay. Moreover, both extracts exhibited a significant dose-dependent free radical scavenging action against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (EC50 = 62.11 and 44.25 µg/mL, respectively). In a phytochemical study, a high phenolic content (77.08 and 81.47 mg GAE/g dry extract, respectively) was found in both extracts by the Folin-Ciocalteu assay. Medium pressure liquid chromatographic (MPLC) separation of the methanol extract on a reversed phase cartridge eluted with a gradient of MeOH in H2O, afforded two bioactive iridoid glucosides, harpagide (1) and 8-O-acetylharpagide (2). The structures of 1 and 2 were established by spectral data, chemical reactions, and comparison with the literature. Interestingly, significant amounts of hepatotoxic furano neo-clerodane diterpenoids, commonly occurring in Teucrium species, were not detected in the extract. The wide range of biological activities reported in the literature for compounds 1 and 2 and the significant antiradical effects of the extracts give scientific support to the traditional use in Iraqi Kurdistan of T. parviflorum aerial parts for the preparation of herbal remedies.

Olive Leaves (Olea europaea L) Extract Loaded Lipid Nanoparticles: Optimization of Processing Parameters by Box-Behnken Statistical Design, in-vitro Characterization, and Evaluation of Anti-oxidant and Anti-microbial Activity.

The present study was aimed to prepare and evaluated solid lipid nanoparticles (SLNs) of olive leaves extract powder (OLP) which contained many anti-oxidant and antimicrobial agents like oleuropein, a natural polyphenol. The major issue concern OLP was the instability due to environmental conditions and hence compromised bioactivity. To overcome this problem, SLNs were designed by hot homogenous followed by sonication technique to protect the drug and improve its antioxidant and antimicrobial activity. Lipids like compritol 888ATO and surfactant like tween 80 were used for the development and stabilization of SLNS and optimization was done by Box-Behnken statistical design (3x3). The optimized batch (F9) showed particle size, entrapment efficiency, PDI, and zeta potential 277.46 nm, 80.48%, 0.275, and －23.18 mV respectively. Optimized formulation (F9) exhibited a sustained release pattern up to 24 h with first-order release kinetic (R2 = 0.9984) and the mechanism of drug release was found to be Fickian diffusion type (n = 0.441). Upon the stability study, it could be found that SLNs formulation was stable. Anti-oxidation and anti-microbial studies were conducted on optimized formulation and findings suggested that SLNs showed an improved radical scavenging activity and anti-microbial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria. Finally, it was concluded that developed SLNs were able to protect and suitable for the delivery of OLP.

Aucuba japonica Extract and Aucubin Prevent Desiccating Stress-Induced Corneal Epithelial Cell Injury and Improve Tear Secretion in a Mouse Model of Dry Eye Disease.

Dry eye disease is affected by a broad range of causes such as age, lifestyle, environment, medication and autoimmune diseases. These causes induce tear instability that activates immune cells and promotes expression of inflammatory molecules. In this study, we investigated the therapeutic effects of an ethanolic extract of Aucuba japonica (AJE) and its bioactive compound, aucubin, on dry eye disease. The human corneal cells were exposed to desiccation stress induced by exposing cells to air, so that viability was decreased. On the other hand, pre-treatment of AJE and aucubin restored cell survival rate depending on the dose under the dry condition. This result was confirmed again by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The mRNA expression of inflammatory molecules was reduced by the pretreatment of AJE and aucubin under the dry state. The therapeutic effects of AJE and aucubin were examined in the animal model for dry eye induced by unilateral excision of the exorbital lacrimal gland. Declined tear volumes and corneal irregularity in the dry eye group were fully recovered by the administration of AJE and aucubin. The apoptotic cells on the cornea were also decreased by AJE and aucubin. Therefore, this study suggests that administration of AJE can be a novel therapeutic for dry eye disease and that the pharmacological activities of AJE may be in part due to its bioactive compound, aucubin.

Simultaneous Quantification of Eight Marker Compounds in Yongdamsagan-Tang Using a High-Performance Liquid Chromatography Equipped with Photodiode Array Detector.

Yongdamsagan-tang (YDSGT) has been used clinically for the treatment of acute- and chronic-urethritis, cystitis, orchitis and hypertension in Korea. In this study, a powerful method based on high-performance liquid chromatography (HPLC) with photodiode array (PDA) detection was established and validated for the quantitative analysis of eight components: chlorogenic acid, gentiopicroside, liquiritin apioside, liquiritin, nodakenin, baicalin, wogonoside and glycyrrhizin in YDSGT extract. The compounds were separated with a Gemini C18 analytical column (column temperature: 40°C; mobile phase: 0.1% (v/v) aqueous trifluoroacetic acid (A) and acetonitrile (B); flow rate: 1.0 mL/min; injection volume: 10 µL). The PDA detector scanned the range 190-800 nm and the marker compounds were monitored at 254, 275, 325 and 335 nm. The correlation coefficients of all compounds were 1.000 and the results showed excellent linearity. The lower limits of detection and quantification of the analytes were 0.01-0.09 µg/mL and 0.03-0.28 µg/mL, respectively. The extraction recoveries of the marker compounds were 98.13-103.86%, with relative standard deviation values not exceeding 2.10%. The precision of intra- and inter-day measurements were 0.09-1.78% and 0.12-2.09%, respectively. The content of the eight marker compounds in the freeze-dried YDSGT extract were 1.41-23.71 mg/g.

Unusual molecular pattern in Ajugoideae subfamily: the case of Ajuga genevensis L. from Dolomites.

We analysed the ethanolic extract from Ajuga genevensis L. (Lamiaceae) growing in Dolomites, part of Italian Alps. Three new compounds for this species were identified: rosmarinic acid (1), oleanolic acid (2) and maslinic acid (3), representative of two different classes of chemical compounds (phenylpropanoids and pentacyclic triterpenes). A. genevensis resulted to be a valuable source of these compounds endowed with interesting biological activities (i.e. antioxidant, neuroprotective, anti-inflammatory, antiproliferative). The recognition of compounds (1), (2) and (3) may also confirm the ethnomedicinal uses of this plant. From a chemotaxonomical point of view, it is worth noting that iridoids were not evidenced in this accession. Iridoids are considered chemotaxonomic marker in Lamiales, and, in contrast with a previous study on this species, the presence of aucubin was not confirmed. In addition, the presence of large amounts of rosmarinic acid (1) was unexpected for a species that does not belong to subfamily Nepetoideae.

Phytochemical investigation of crude methanol extracts of different species of Swertia from Nepal.

BACKGROUND: The genus Swertia is reported to contain potent bitter compounds like iridoids, xanthones and c-glucoflavones that are known to heal many human disorders. In contrast to high ethnomedicinally valued Swertia chirayita, its other species have not been studied extensively, in spite of their common use in traditional medicinal system in Nepalese communities. So, the present study attempts to investigate the content of total polyphenols, flavonoids, antioxidant activity and estimate the rough content of amarogentin, swertiamarin and mangiferin from different species of Swertia from Nepalese Himalayas. METHODS: Whole plant parts of S. chirayita (SCH), S. angustifolia (SAN), S. paniculata (SPA), S. racemosa (SRA), S. nervosa (SNE), S. ciliata (SCI) and S. dilatata (SDI) were collected; total phenolic and flavonoid contents were quantified spectrophotometrically and in vitro DPPH free radical scavenging assay was measured. Thin layer chromatography was performed on TLC aluminium plates pre-coated with silica gel for identification of swertiamarin, amarogentin and mangiferin from those species and semi quantitative estimation was done using GelQuant.NET software using their standard compounds. RESULTS: The phenolic content was highest in the methanol extract of SCH (67.49 ± 0.5 mg GAE/g) followed by SDI, SRA, SNE, SCI, SPA and SAN. The contents of flavonoids were found in the order of SCH, SPA, SRA, SNE, SDI, SCI and SAN. Promising concentration of phenolics and flavonoids produced promising DPPH free radical scavenging values. The IC50 values for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test was lowest in SCH (23.35 ± 0.6 µg/ml), even lower than the standard ascorbic acid among the seven studied species. A significant correlation of 0.977 was observed between the polyphenol content and antioxidant values. The TLC profile showed the presence of all three major phytochemicals; amarogentin, swertiamarin and mangiferin in all of the plant samples. CONCLUSION: Among the seven studied species, SCH showed anticipating results in total phenol content, flavonoid content and DPPH radical scavenging test. The less considered species of Swertia can be a potential source of bioactive amarogentin, and other useful therapeutic compounds in the alarming status of Swertia chirayita as shown by the phytochemical analysis.

Comparative studies for screening of bioactive constituents from various parts of Incarvillea emodi.

A comparative study was performed on various parts (shoots, roots and flowers) of Incarvillea emodi. The alcoholic extracts of different parts were fractionated with solvents of different polarity and studied for the determination of total polyphenol content and total antioxidant potential. Furthermore, we have isolated major iridoid glucosides from the dried flowers of I. emodi followed by the comparative cytotoxicity studies of these iridoids against five different human cancer cell lines. The results have demonstrated that ethyl acetate fraction of all parts have higher phenolic content (167.87-294.31 mg/g as gallic acid equivalent) and higher total antioxidant potential (252.95-384.64 mg/g as trolox equivalent). The results of in vitro cytotoxicity studies have indicated that boschnaloside (2) possesses promising anticancer potential against three human cancer cell lines, THP-1, A-549 and PC-3, which belong to leukaemia, lung and prostate cancers, respectively, while plantarenaloside (1) expressed relevant cytotoxic activity against THP-1 cell lines of leukaemia.

Traditionally used Veronica officinalis inhibits proinflammatory mediators via the NF-κB signalling pathway in a human lung cell line.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts from Veronica officinalis L. are traditionally used for the treatment of lung diseases; however, the effective compounds and the mode of action are still unknown. AIM OF THE STUDY: Here we analyzed the effects of a standardized Veronica extract on genes expression and signalling protein production associated with the development of inflammatory lung diseases. MATERIAL AND METHODS: The degranulation capacity of primary mast cells, as well as gene expression and release of inflammatory mediators from human lung epithelial cells (A549 cells) were analyzed in relation to the synthetic drugs azelastine and dexamethasone. Gene and protein expression of cyclooxygenase-2 were investigated by semi-quantitative RT-PCR and western blotting, respectively. The involvement of phosphorylated mitogen-activated protein kinases and NF-κB signaling in regulation of these molecules were characterized by western blotting and electrophoretic mobility shift assays. Characteristic extract components were identified by LC-MS and verminoside was quantified by HPLC analysis. RESULTS: We demonstrated that Veronica officinalis has a small influence on the degranulation capacity of mast cells but rather inhibits gene and protein expression of the chemokine eotaxin in A549 lung epithelial cells, which is essential for recruitment of inflammatory-associated cells in lung diseases. Furthermore, release of the inflammatory mediator PGE(2) was diminished through inhibition of COX-2 expression via the NF-κB signaling pathway in TNF-α-activated A549 cells. Phytochemical analysis identified verproside and verminoside as the most abundant iridoid glycosides. CONCLUSION: Our results are a contribution to explaining the observed anti-inflammatory effects of Veronica offcinalis extract on a molecular level. However, its clinical potency has at first to be proven in animals and subsequently in clinical trials.

Hepatoprotective effects of Gentiana asclepiadea L. extracts against carbon tetrachloride induced liver injury in rats.

This study is an attempt to evaluate the hepatoprotective activity of Gentiana asclepiadea L. against carbon tetrachloride-induced liver injury in rats. Methanol extracts of aerial parts (GAA) and roots (GAR) of G. asclepiadea at doses of 100, 200, and 400mg/ kg b.w. were orally administered to Wistar rats once daily for 7 days before they were treated with CCl(4). The hepatoprotective activity of the extracts in this study was compared with the reference drug silymarin. In CCl(4) treated animals, GAA and GAR significantly decreased levels of serum transaminases, alkaline phosphatase and total bilirubin, and increased the level of total protein. Treatment with the extracts resulted in a significant increase in the levels of catalase, superoxide dismutase and reduced glutathione, accompanied with a marked reduction in the levels of malondialdehyde, as compared to CCl(4) treated group. The histopathological studies confirmed protective effects of extracts against CCl(4)-induced liver injuries. No genotoxicity was observed in liver cells after GAA treatment, while GAR showed only slight genotoxic effects by comet assay. Phytochemical analysis revealed the presence of sweroside, swertiamarin and gentiopicrin in high concentrations in both extracts. It could be concluded that the use of G. asclepiadea extracts in the treatment of chemical-induced hepatotoxicity.

Effect of Feining on bleomycin-induced pulmonary injuries in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The flowers of Gentiana veitchiorum has been widely used in decoction form in the traditional medicine of Tibet against tussis, tracheitis, angina for their anti-inflammatory, antimicrobial and alexipharmic properties. AIM OF THE STUDY: The aim of current study was to evaluate the therapeutic effects of Feining, a Chinese herbal formula (national invention patent: ZL200510042636.3) against pulmonary injuries and to clarify the mechanisms involved. MATERIALS AND METHODS: Experimental pulmonary injuries were induced by bleomycin (BLM) in rats with or without subsequent treatment of Feining or prednisone as positive control. The pulmonary injuries were evaluated by histological analysis. Also, the levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH) and hydroxyproline (Hyp) in the lung tissue were determined. To clarify one of the possible active principles responsible for Feining, high performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MS) method was applied to identify the components of Gentiana veitchiorum, one of major ingredients of Feining. RESULTS: Feining significantly improved lung alveolitis scores and reduced the Hyp content of lungs, which is an index of collagen accumulation. Moreover, Feining played a role against the oxidative damages by decreasing the MDA level, whereas increasing SOD and GSH activity, which correlated with oxidation resistance and scavenging of free radicals. In addition, Feining alleviated inflammatory lung injury by decreasing tumor necrosis factor-α (TNF-α) expression. HPLC-DAD-MS analysis revealed that there was 1.97% gentiopicroside in Gentiana veitchiorum. CONCLUSION: Feining has certain therapeutic effects against pulmonary injuries.