Pimenta dioica (L.) Merr. Bioactive Constituents Exert Anti-SARS-CoV-2 and Anti-Inflammatory Activities: Molecular Docking and Dynamics, In Vitro, and In Vivo Studies.

In response to the urgent need to control Coronavirus disease 19 (COVID-19), this study aims to explore potential anti-SARS-CoV-2 agents from natural sources. Moreover, cytokine immunological responses to the viral infection could lead to acute respiratory distress which is considered a critical and life-threatening complication associated with the infection. Therefore, the anti-viral and anti-inflammatory agents can be key to the management of patients with COVID-19. Four bioactive compounds, namely ferulic acid 1, rutin 2, gallic acid 3, and chlorogenic acid 4 were isolated from the leaves of Pimenta dioica (L.) Merr (ethyl acetate extract) and identified using spectroscopic evidence. Furthermore, molecular docking and dynamics simulations were performed for the isolated and identified compounds (1-4) against SARS-CoV-2 main protease (Mpro) as a proposed mechanism of action. Furthermore, all compounds were tested for their half-maximal cytotoxicity (CC50) and SARS-CoV-2 inhibitory concentrations (IC50). Additionally, lung toxicity was induced in rats by mercuric chloride and the effects of treatment with P. dioca aqueous extract, ferulic acid 1, rutin 2, gallic acid 3, and chlorogenic acid 4 were recorded through measuring TNF-α, IL-1ß, IL-2, IL-10, G-CSF, and genetic expression of miRNA 21-3P and miRNA-155 levels to assess their anti-inflammatory effects essential for COVID-19 patients. Interestingly, rutin 2, gallic acid 3, and chlorogenic acid 4 showed remarkable anti-SARS-CoV-2 activities with IC50 values of 31 µg/mL, 108 µg/mL, and 360 µg/mL, respectively. Moreover, the anti-inflammatory effects were found to be better in ferulic acid 1 and rutin 2 treatments. Our results could be promising for more advanced preclinical and clinical studies especially on rutin 2 either alone or in combination with other isolates for COVID-19 management.

Phenolic Profiles of Ten Australian Faba Bean Varieties.

Although Australia is the largest exporter of faba bean globally, there is limited information available on the levels of bioactive compounds found in current commercial faba bean varieties grown in this country. This study profiled the phenolic acid and flavonoid composition of 10 Australian faba bean varieties, grown at two different locations. Phenolic profiling by HPLC-DAD revealed the most abundant flavonoid to be catechin, followed by rutin. For the phenolic acids, syringic acid was found in high concentrations (72.4-122.5 mg/kg), while protocatechuic, vanillic, p-hydroxybenzoic, chlorogenic, p-coumaric, and trans-ferulic acid were all found in low concentrations. The content of most individual phenolics varied significantly with the variety, while some effect of the growing location was also observed. This information could be used by food processors and plant breeders to maximise the potential health benefits of Australian-grown faba bean.

Neochlorogenic acid: an anti-HIV active compound identified by screening of Cortex Mori [Morus Alba L. (Moraceae)].

CONTEXT: Chinese herbs such as Cortex Mori [Morus alba L. (Moraceae)] may inhibit human immunodeficiency virus (HIV), but active compounds are unknown. OBJECTIVE: Screening of Cortex Mori and other herbs for anti-HIV active compounds. MATERIALS AND METHODS: HIV-1 virus (multiplicity of infection: 20), and herbs (dissolved in dimethyl sulfoxide, working concentrations: 10, 1, and 0.1 mg/mL) such as Cortex Mori, etc., were added to 786-O cells (105 cell/well). Zidovudine was used as a positive control. Cell survival and viral inhibition rates were measured. The herb that was the closest inactivity to zidovudine was screened. Mass spectrometry identified the active compounds in herbs (mobile phase: 0.05% formic acid aqueous solution and acetonitrile, gradient elution, detection wavelength: 210 nm). The effect of the compounds on reverse transcriptase (RT) products were evaluated by real-time PCR. Gene enrichment was used to analyse underlying mechanisms. RESULTS: With a dose of 1 mg/mL of Cortex Mori, the cell survival rate (57.94%) and viral inhibition rate (74.95%) were closest to the effect of zidovudine (87.87%, 79.81%, respectively). Neochlorogenic acid, one of the active ingredients, was identified by mass spectrometry in Cortex Mori. PCR discovery total RT products of neochlorogenic acid group (mean relative gene expression: 6.01) significantly inhibited (control: 35.42, p < 0.0001). Enrichment analysis showed that neochlorogenic acid may act on haemopoietic cell kinase, epidermal growth factor receptor, sarcoma, etc., thus inhibiting HIV-1 infection. CONCLUSIONS: For people of low socioeconomic status affected by HIV, Chinese medicine (such as Cortex Mori) has many advantages: it is inexpensive and does not easily produce resistance. Drugs based on active ingredients may be developed and could have important value.

Effect of Different Durations of Solid-Phase Fermentation for Fireweed (Chamerion angustifolium (L.) Holub) Leaves on the Content of Polyphenols and Antioxidant Activity In Vitro.

Fireweed has recently been recognized as a plant with high antioxidant potential and phenolic content. Its leaves can be fermented to prepare an infusion with ideal antioxidant activity. The aim of this study was to investigate and to determine the influence of solid-phase fermentation of different durations on the variation of polyphenols in the leaves of fireweed. Laboratory experiments were conducted in 2017-2018. The leaves of fireweed, naturally growing, were fermented for different periods of time: not fermented (control) and fermented for 24 and 48 h. The evaluation of polyphenols and antioxidant activity in leaves was performed using high- performance liquid chromatography (HPLC). Additionally, principal component analysis was used to characterize differences in bioactive compounds between fireweed samples fermented at different durations. Solid-phase fermented leaves were characterized by higher contents of oenothein B, quercetin and benzoic acid but had lower contents of quercetin-3-O-rutinoside, luteolin and chlorogenic and gallic acids. Antioxidant activity in short- (24 h) and long-term (48 h) fermentation (compared to control) gave the highest level of regression in 2017, but in 2018 the effect was observed only with short-term fermentation and control. In conclusion, solid-phase fermentation can be used to modulate biologically active compounds in fireweed leaves.

Identification of Transient Receptor Potential Vanilloid 3 Antagonists from Achillea alpina L. and Separation by Liquid-Liquid-Refining Extraction and High-Speed Counter-Current Chromatography.

Bioassay-guided fractionation of the ethanol extract of whole herbs of Achillea alpina led to the isolation of isochlorogenic acids A and B as transient receptor potential vanilloid 3 (TRPV3) channel antagonists by using a calcium fluorescent assay. The structures were identified by spectroscopic analysis and the inhibitory activities of isochlorogenic acids A and B were confirmed by whole-cell patch clamp recordings of human embryonic kidney 293 (HEK293) cells expressing human TRPV3. Molecular docking results revealed that these two compounds reside in the same active pocket of human TRPV3 channel protein with lower binding energy than the agonist 2-aminoethoxydiphenyl borate (2-APB). High-speed counter-current chromatography (HSCCC) coupled with a liquid-liquid extraction approach was successfully established for the separation of isochlorogenic acids A and B from the whole herbs of A. alpina. Ethyl acetate and n-hexane-ethyl acetate-water (3:3:4 and 1:5:4, v/v/v) were selected as liquid-liquid extraction solvent systems to remove high- and low-polarity impurities in the mixture. Sixty g of ethanol extract was refined by solvent partition to yield 1.7 g of the enriched fraction, of which 480 mg in turn obtained 52.5 mg of isochlorogenic acid B (purity 98.3%) and 37.6 mg isochlorogenic acid A (purity 96.2%) after HSCCC with n-hexane-ethyl acetate-water containing 1% acetic acid (1:4:8, v/v/v).

Chemical Diversity of Bastard Balm (Melittis melisophyllum L.) as Affected by Plant Development.

The phytochemical diversity of Melittis melissophyllum was investigated in terms of seasonal changes and age of plants including plant organs diversity. The content of phenolics, namely: coumarin; 3,4-dihydroxycoumarin; o-coumaric acid 2-O-glucoside; verbascoside; apiin; luteolin-7-O-glucoside; and o-coumaric; p-coumaric; chlorogenic; caffeic; ferulic; cichoric acids, was determined using HPLC-DAD. Among these, luteolin-7-O-glucoside, verbascoside, chlorogenic acid, and coumarin were the dominants. The highest content of flavonoids and phenolic acids was observed in 2-year-old plants, while coumarin in 4-year-old plants (272.06 mg 100 g-1 DW). When considering seasonal changes, the highest content of luteolin-7-O-glucoside was observed at the full flowering, whereas verbascoside and chlorogenic acid were observed at the seed-setting stage. Among plant organs, the content of coumarin and phenolic acids was the highest in leaves, whereas verbascoside and luteolin-7-O-glucoside were observed in flowers. The composition of essential oil was determined using GC-MS/GC-FID. In the essential oil from leaves, the dominant was 1-octen-3-ol, whilst from flowers, the dominant was α-pinene.

The chemistry of chlorogenic acid from green coffee and its role in attenuation of obesity and diabetes.

An overview of green coffee, the unroasted bean enriched with antioxidants, is presented in the following article. Green coffee beans are known to have a higher content of chlorogenic acid (CGA) with potential health benefits like activity against hypertension, diabetes, obesity, etc. There are three major classes of chlorogenic acids present in green coffee beans, namely: caffeoylquinic acid (CQA), di-caffeoylquinic acid (diCQA) and feruloylquinic acid (FQA). Another pivotal component of the green beans is caffeic acid. A compilation of the different research studies and reviews pertaining to the diverse biomolecules present in the green coffee, their structure and the different sources of CGA is presented. The traditional and modern methods of the extraction of CGA are also studied. Green coffee upon roasting develops its aromatic characteristics but the flavor development comes with a reciprocation of reduced chlorogenic acid content. Thus, the effect of processing is also addressed. There are numerous studies conducted to show the health benefits associated with the consumption of green coffee out of which, anti-diabetic and anti-obesity effects are particularly concentrated in this article.

Optimization of an Extraction Solvent for Angiotensin-Converting Enzyme Inhibitors from Hibiscus sabdariffa L. Based on Its UPLC-MS/MS Metabolic Profiling.

Hibiscus species (Malvaceae) have been long used as an antihypertensive folk remedy. The aim of our study was to specify the optimum solvent for extraction of the angiotensin-converting enzyme inhibiting (ACEI) constituents from Hibiscus sabdariffa L. The 80% methanol extract (H2) showed the highest ACEI activity, which exceeds that of the standard captopril (IC50 0.01255 ± 0.00343 and 0.210 ± 0.005 µg/mL, respectively). Additionally, in a comprehensive metabolomics approach, an ultra-performance liquid chromatography (UPLC) coupled to the high resolution tandem mass spectrometry (HRMS) method was used to trace the metabolites from each extraction method. Interestingly, our comprehensive analysis showed that the 80% methanol extract was predominated with secondary metabolites from all classes including flavonoids, anthocyanins, phenolic and organic acids. Among the detected metabolites, phenolic acids such as ferulic and chlorogenic acids, organic acids such as citrate derivatives and flavonoids such as kaempferol have been positively correlated to the antihypertensive potential. These results indicates that these compounds may significantly contribute synergistically to the ACE inhibitory activity of the 80% methanol extract.

Cost-effective imprinting to minimize consumption of template in room-temperature ionic liquid for fast purification of chlorogenic acid from the extract of E. ulmoides leaves.

One of the main challenges in large-scale applications of molecularly imprinted polymers (MIPs) is the significant amount of template needed in polymer preparation. A new strategy based on room-temperature ionic liquids (RTILs) was suggested to solve this problem by reducing the amount of template in the polymerization recipe. The MIP was synthesized with a mixture of dimethyl sulfoxide and RTIL (1-butyl-3-methylimidazolium tetrafluoroborate) as porogen, in which chlorogenic acid (CGA) was used as template, 4-vinylpyridine (4-VP) as functional monomer, and ethylene glycol dimethacrylate (EDMA) as cross-linker. The influence of polymerization variables, including CGA concentrations, and the ratio of 4-VP to EDMA on imprinting effect were investigated comprehensively. Moreover, the properties involving the column permeability, the number of binding sites, and the polymer morphology of the CGA-MIP monoliths were studied thoroughly. The MIP monolith had an excellent column permeability (1.53 × 10-13 m2) and allowed an ultra-fast on-line SPE, which dramatically shortens the separation time (< 10 min) and improves the separation efficiency. At high flow velocity (5.0 mL min-1), 50 µL of the extract from Eucommia ulmoides leaves can be loaded directly on the CGA-MIP monoliths and CGA with high purity can be obtained with a recovery of 89.01 ± 0.05%. As a conclusion, the resulting RTIL-induced approach of preparing MIP may be an effective tool in fabricating MIP in a low-cost way. Graphical abstract ᅟ.

Phytochemical composition of wormwood (Artemisia gmelinii) extracts in respect of their antimicrobial activity.

BACKGROUND: Extracts from medicinal plants with phytochemicals with known antimicrobial properties can be an effective adjunct in the complex treatment of infectious diseases. This study aimed to evaluate the antimicrobial activity of wormwood extracts collected in Kazakhstan (Artemisia gmelinii Weber ex Stechm.), along with their phytochemical analysis. METHODS: The ethanolic and chloroform extracts were subjected to HPLC combined with quadrupole time-of-flight mass spectrometry method. For quantitative assessment of antimicrobial activity, minimal inhibitory concentration (MIC) of the tested extracts was determined by micro-dilution broth method for the panel of the reference microorganisms. Minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) were also determined. RESULTS: LC/MS analysis showed the presence of 13 compounds in the tested extracts, including flavonoids: apigenin, luteolin, rutin, two O-methylated flavonols (isorhamnetin, rhamnazine), coumarin compounds (umbelliferone, scopoletin and scopolin (scopoletin 7-glucoside), 3-hydroxycoumarin and 4-hydroxycoumarin), chlorogenic acid and two dicaffeoylquinic acid isomers. Quantitative HPLC analysis showed that umbelliferone was dominant in the chloroform extract while chlorogenic acid was identified as a main compound in the ethanolic extract. The antibacterial and antifungal activity of chloroform and ethanolic extracts was comparable. The most sensitive were the Gram-positive bacteria represented by staphylococci, Micrococcus luteus and Bacillus spp. (MIC = 1.25-5 mg/ml) and yeasts represented by Candida spp. (MIC = 2.5-5 mg/ml), irrespective of the assayed extract. CONCLUSIONS: Extracts of wormwood Artemisia gmelinii have shown a wide spectrum of antibacterial and antifungal activity. Luteolin, rutin, isorhamnetin and scopolin were identified in A. gmelinii species for the first time. The determining of the most potential compounds of Artemisia gmelinii can be used to develop effective antibacterial and antifungal agents.

Comparison of Artificial Neural Networks and Response Surface Methodology towards an Efficient Ultrasound-Assisted Extraction of Chlorogenic Acid from Lonicera japonica.

Chlorogenic acid (CGA), a bioactive compound commonly found in plants, has been demonstrated possessing nutraceutical potential in recent years. However, the more critical issue concerning how to improve production efficacy of CGA is still limited. It is a challenge to harvest a large amount of CGA without prolonging extraction time. In this study, the feasibility of using ultrasound for CGA extraction from Lonicera japonica was investigated. A central composite design (CCD) was employed to evaluate the effects of the operation parameters, including temperature, ethanol concentration, liquid to solid ratio, and ultrasound power on CGA yields. Meanwhile, the process of ultrasound-assisted extraction was optimized through modeling response surface methodology (RSM) and artificial neural network (ANN). The data indicated that CGA was efficiently extracted from the flower of Lonicera japonica by ultrasound assistance. The optimal conditions for the maximum extraction of CGA were as follows: The temperature at 33.56 °C, ethanol concentration at 65.88%, L/S ratio at 46:1 mL/g and ultrasound power at 150 W. ANN possessed greater optimization capacity than RSM for fitting experimental data and predicting the extraction process to obtain a maximum CGA yield. In conclusion, the process of ultrasound-assisted extraction can be well established by a methodological approach using either RSM or ANN, but it is worth mentioning that the ANN model used here showed the superiority over RSM for predicting and optimizing.

The Impact of Temperature and Ethanol Concentration on the Global Recovery of Specific Polyphenols in an Integrated HPLE/RP Process on Carménère Pomace Extracts.

Sequential extraction and purification stages are required to obtain extracts rich in specific polyphenols. However, both separation processes are often optimized independently and the effect of the integrated process on the global recovery of polyphenols has not been fully elucidated yet. We assessed the impact of hot-pressurized liquid extraction (HPLE) conditions (temperature: 90-150 °C; ethanol concentration: 15%-50%) on the global recovery of specific phenolic acids, flavanols, flavonols and stilbenes from Carménère grape pomace in an integrated HPLE/resin purification (RP) process. HPLE of phenolic acids, flavanols and stilbenes were favored when temperature and ethanol concentration increased, except for chlorogenic acid which showed an increment of its Gibbs free energy of solvation at higher ethanol contents. Ethanol concentration significantly impacted the global yield of the integrated HPLE/RP process. The lower the ethanol content of the HPLE extracts, the higher the recovery of phenolic acids, flavanols and stilbenes after RP, except for flavonols which present more polar functional groups. The best specific recovery conditions were 150 °C and ethanol concentrations of 15%, 32.5% and 50% for phenolic acids, flavanols and stilbenes, and flavonols, respectively. At 150 °C and 32.5% of ethanol, the extracts presented the highest total polyphenol content and antioxidant capacity. The integrated HPLE/RP process allows a selective separation of specific polyphenols and eliminates the interfering compounds, ensuring the safety of the extracts at all evaluated conditions.

Carpesium divaricatum Sieb. & Zucc. Revisited: Newly Identified Constituents from Aerial Parts of the Plant and Their Possible Contribution to the Biological Activity of the Plant.

Carpesium divaricatum Sieb. & Zucc. has a long history of use as both a medicinal and a food plant. However, except for terpenoids, its chemical constituents have remained poorly investigated. The composition of hydroalcoholic extract from aerial parts of C. divaricatum was analyzed by HPLC-DAD-MSn, revealing the presence of numerous caffeic acid derivatives that were formerly unknown constituents of the plant. In all, 17 compounds, including commonly found chlorogenic acids and rarely occurring butyryl and methylbutyryl tricaffeoylhexaric acids, were tentatively identified. Fractionation of lipophilic extract from cultivated shoots led to the isolation of 12-oxo-phytodienoic acid (12-OPDA), which is a newly identified constituent of the plant. The compound, at concentrations of 0.5, 1.0, and 2.5 µM, significantly reduced IL-8, IL-1ß, TNFα, and CCL2 excretion by lipopolysaccharide (LPS)-stimulated human neutrophils. Reactive oxygen species (ROS) production induced by f-MLP was also significantly diminished in the neutrophils pretreated by 12-OPDA. The newly identified constituents of the plant seem to be partly responsible for its pharmacological activity and elevate the value of C. divaricatum as a potential functional food.

Solidago graminifolia L. Salisb. (Asteraceae) as a Valuable Source of Bioactive Polyphenols: HPLC Profile, In Vitro Antioxidant and Antimicrobial Potential.

Solidago species are often used in traditional medicine as anti-inflammatory, diuretic, wound-healing and antimicrobial agents. Still, the bioactive compounds and biological activities of some species have not been studied. The present work aimed to investigate the polyphenolic profile and the biological properties of Solidago graminifolia L. Salisb., a poorly explored medicinal plant. The hydroalcoholic extracts from aerial parts were evaluated for total phenolic content (TPC), total flavonoid content (TFC) and the polyphenolic compounds were investigated by HPLC-MS. The antioxidant potential in vitro was determined using DPPH and FRAP assays. Antibacterial and antifungal effects were evaluated by dilution assays and MIC, MBC and MFC were calculated. The results showed that Solidago graminifolia aerial parts contain an important amount of total phenolics (192.69 mg GAE/g) and flavonoids (151.41 mg RE/g), with chlorogenic acid and quercitrin as major constituents. The hydroalcoholic extracts showed promising antioxidant and antimicrobial potential, with potent antibacterial activity against Staphylococcus aureus and important antifungal effect against Candida albicans and C. parapsilosis. The obtained results indicated that the aerial parts of Solidago graminifolia could be used as novel resource of phytochemicals in herbal preparations with antioxidant and antimicrobial activities.

Effects of ultrasonication, agitation and stirring extraction techniques on the physicochemical properties, health-promoting phytochemicals and structure of cold-brewed coffee.

BACKGROUND: Ultrasonication, agitation and stirring or a combination of ultrasonication, agitation and stirring extraction techniques were applied to observe their effects on the physicochemical properties, health-promoting phytochemicals, and structure of cold brewed coffee. RESULTS: All the extraction techniques led to significant (P < 0.05) increases in the color values, total soluble solids, antioxidant activities and most organic acids, while a combination of extraction techniques increased the chlorogenic acid and caffeine content significantly (P < 0.05) compared with that by conventional methods. Fourier transform infrared spectroscopy allowed us to identify the different compounds in the cold-brewed coffee extract rapidly. The partial least square regression model presented good predictability because experimental and predicted values were close to each other. Principal component analysis revealed that levels of all phytochemicals correlated with the use of non-conventional methods. CONCLUSION: The combination of ultrasonication and agitation might be the best option to enhance the various phytochemicals in cold-brewed coffee. © 2018 Society of Chemical Industry.

Evaluation of Antioxidant Capacity, Protective Effect on Human Erythrocytes and Phenolic Compound Identification in Two Varieties of Plum Fruit (Spondias spp.) by UPLC-MS.

Plum edible part was used to obtained extracts by during a 4 h maceration process using three different solvents (ethanol, methanol and water) for the determination of total phenols and flavonoids, antioxidant capacity by (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hemolysis inhibition in human blood assays. Subsequently, phenolic compounds were identified using ultra-performance liquid chromatography (UPLC-MS). The results indicated that the ethanolic extract of plum fruit being a good source of phenolic (12⁻18 mg GAE/g FW) and flavonoids (2.3⁻2.5 mg QE/g FW) content in both varieties of plum. Also, the fruits proved a good source of antioxidants as measured by DPPH and ABTS; likewise, plum aqueous extracts showed the highest protective effect on human erythrocytes with 74.34 and 64.62% for yellow and red plum, respectively. A total of 23 bioactive compounds were identified by UPLC-MS, including gallic acid, rutin, resorcinol, chlorogenic acid, catechin, and ellagic acid, and the antioxidant capacity can be attributed to these species. The edible part of plum contains compounds of biological interest, suggesting that this fruit has antioxidant potential that can be exploited for various technologies.

Development and Validation of a HPLC-UV Method for Extraction Optimization and Biological Evaluation of Hot-Water and Ethanolic Extracts of Dendropanax morbifera Leaves.

Dendropanax morbifera Leveille (Araliaceae) has been used in traditional oriental remedies for cancer, inflammation, diabetes, and thrombosis. However, a validated analytical method, standardization, and optimization of extraction conditions with respect to biological activity have not been reported. In this study, a simple and validated HPLC method for identifying and quantifying active substances in D. morbifera was developed. Hot water and ethanolic D. morbifera leaf extracts from different production regions were prepared and evaluated with regard to their chemical compositions and biological activities. The contents of active compounds such as rutin and chlorogenic acid were determined in four samples collected from different regions. The 80% ethanolic extract showed the best antioxidant activity, phenolic content, reducing power, and xanthine oxidase (XO) inhibitory activity. The validated HPLC method confirmed the presence of chlorogenic acid and rutin in D. morbifera leaf extracts. The antioxidant and XO inhibitory activity of D. morbifera extract could be attributed to the marker compounds. Collectively, these results suggest that D. morbifera leaves could be beneficial for the treatment or prevention of hyperuricemia-related disease, and the validated HPLC method could be a useful tool for the quality control of food or drug formulations containing D. morbifera.

Direct Observation of Hydrangea Blue-Complex Composed of 3-O-Glucosyldelphinidin, Al3+ and 5-O-Acylquinic Acid by ESI-Mass Spectrometry.

The blue sepal color of hydrangea is due to a metal complex anthocyanin composed of 3-O-glucosyldelphinidin (1) and an aluminum ion with the co-pigments 5-O-caffeoylquinic acid (2) and/or 5-O-p-coumaroylquinic acid (3). The three components, namely anthocyanin, Al3+ and 5-O-acylquinic acids, are essential for blue color development, but the complex is unstable and only exists in an aqueous solution. Furthermore, the complex did not give analyzable NMR spectra or crystals. Therefore, many trials to determine the detailed chemical structure of the hydrangea-blue complex have not been successful to date. Instead, via experiments mixing 1, Al3+ and 2 or 3 in a buffered solution at pH 4.0, we obtained the same blue solution derived from the sepals. However, the ratio was not stoichiometric but fluctuated. To determine the composition of the complex, we tried direct observation of the molecular ion of the complex using electrospray-ionization mass spectrometry. In a very low-concentration buffer solution (2.0 mM) at pH 4.0, we reproduced the hydrangea-blue color by mixing 1, 2 and Al3+ in ratios of 1:1:1, 1:2:1 and 1:3:1. All solution gave the same molecular ion peak at m/z = 843, indicating that the blue solution has a ratio of 1:1:1 for the complex. By using 3, the observed mass number was m/z = 827 and the ratio of 1, 3 and Al3+ was also 1:1:1. A mixture of 1, 3-O-caffeoylquinic acid (4) and Al3+ did not give any blue color but instead was purple, and the intensity of the molecular ion peak at m/z = 843 was very low. These results strongly indicate that the hydrangea blue-complex is composed of a ratio of 1:1:1 for 1, Al3+ and 2 or 3.

Phytochemical Analysis, Antioxidant and Antimicrobial Activities of Helichrysum arenarium (L.) Moench. and Antennaria dioica (L.) Gaertn. Flowers.

Antennaria dioica (L.) Gaertn. and Helichrysum arenarium (L.) Moench. are two species of the Asteraceae family, known in Romanian traditional medicine for their diuretic, choleretic, and anti-inflammatory properties. The aim of the present study was to evaluate the phenolic and sterolic composition of flowers from the two species and to assess their antioxidant, antibacterial and antifungal properties. LC-MS analyses were performed on methanolic, ethanolic and 70% v/v ethanolic extracts, before and after acid hydrolysis, and revealed high amounts of polyphenols. Chlorogenic acid was found as the main compound for the flowers of A. dioica (502.70 ± 25.11 mg/100 g d.w.), while quercitrin was dominant in H. arenarium (424.28 ± 21.21 mg/100 g d.w.) in 70% v/v ethanolic extracts before hydrolysis. Antioxidant capacity assays showed an important antioxidant potential, which can be correlated with the determined polyphenolic compounds, showing the 70% v/v ethanolic extracts of the two species as being the most effective antioxidant samples for the DPPH assay. Antibacterial and antifungal assays confirm a modest biological potential for the same extract of both species. Results obtained in the present study bring important data and offer scientific evidence on the chemical composition and on the biological activities of the flowers belonging to the two species.

Metabolite Profiles, Bioactivity, and HPLC Fingerprint of Different Varieties of Eucommia ulmoides Oliv.: Towards the Utilization of Medicinal and Commercial Chinese Endemic Tree.

Eucommia ulmoides Oliv. is widely regarded in China as a precious medicinal and commercial endemic tree. Due to cross-breeding or natural variation of E. ulmoides, the metabolite composition may vary significantly, making control of the medical quality difficult. In order to improve the rational development and utilization, the quality of seven varieties of E. ulmoides were evaluated based on metabolite profiles (total phenolic, total flavonoid, gutta-percha, aucubin, geniposidic acid, chlorogenic acid, geniposide, pinoresinol diglucoside, rutin, hyperoside, and astragalin), bioactivities (in vitro, in vivo antioxidant activities, and antibacterial activities) and HPLC fingerprint combined with chemometrics analysis. On this basis, the differences of medicinal parts (leaf and bark) were further carried out. For the traditional use of bark, Purple-leaf E. ulmoides was the most suitable. For the use of leaf, Qinzhong 1 and Purple-leaf E. ulmoides were appropriate. HPLC fingerprint analysis showed that significant differences in metabolite profiles exist among seven varieties of E. ulmoides. Combined with chemometrics analysis, seven varieties of E. ulmoides were divided into three groups from the use of leaf and bark. The analysis not only evaluated quality of seven varieties of E. ulmoides, but also could distinguish different varieties and different regions of origin. The results can provide theoretical basis for E. ulmoides resources utilization and cultivation of fine varieties.