Antihyperlipidemic effect of Vaccinium dunalianum buds based on biological activity screening and LC-MS.

ETHNOPHARMACOLOGICAL RELEVANCE: The buds of Vaccinium dunalianum Wight are used as folk medicine in the Yi settlement of the Yunnan Province, China. It has long been used as herbal tea in the local area owing to its effects of lowering blood lipids and body weight. However, there are only a few studies on its antihyperlipidemic effects, effective substances and mechanisms, especially its effectiveness in diet-induced hyperlipidemia. AIM OF THE STUDY: This study aimed to elucidate the therapeutic effects, pharmacodynamic material bases, and mechanisms of V. dunalianum buds on diet-induced hyperlipidemia. MATERIALS AND METHODS: A high-fat diet-induced hyperlipidemic Sprague-Dawley (SD) rat model was established. Rats were gavaged with different doses of aqueous extract of V. dunalianum(VDW) for 8 weeks and their sera and organ samples were collected. The antihyperlipidemic effect of VDW on SD rats was evaluated based on the biochemical indices and histopathological outcomes. Liquid chromatography-mass spectrometry(LC-MS) was used to determine the main components in VDW, which were separated and purified using sequential chromatographic methods. Their chemical structures were determined using high-resolution electrospray ionization mass spectroscopy and nuclear magnetic resonance spectroscopy. 6'-O-caffeoyl-arbutin, as the principal component of VDW, was also evaluated for its antihyperlipidemic activity using an approach similar to that used for VDW. Lastly, the potential targets of VDW and 6'-O-caffeoyl-arbutin in lowering blood lipids were screened out using network pharmacology, and the selected targets were docked with arbutin derivatives. The expression of target proteins was determined using western blotting to illustrate the antihyperlipidemic mechanisms of VDW and 6'-O-caffeoyl-arbutin. RESULTS: VDW reduced triglyceride, total cholesterol, low-density lipoprotein, alanine transaminase, and aspartate transaminase levels in the serum of modeled rats, and increased high-density lipoprotein levels. There was an improvement in steatoses, and lipid droplet accumulation decreased in vivo after VDW intervention. LC-MS revealed that VDW mainly contained arbutin and chlorogenic acid derivatives. Sixteen compounds were isolated and identified. 6'-O-caffeoyl-arbutin was the main compound of VDW (>21.67%) that showed obvious antihyperlipidemic effect with low hepatic damage at different doses. PTGS2, ADH1C, and MAOB were screened out using network pharmacology and they showed strong correlations with arbutin derivative through molecular docking. Results from WB showed that VDW and 6'-O-caffeoyl-arbutin could reduce blood lipid levels by reducing the protein expression of PTGS2, ADH1C, and MAOB. CONCLUSIONS: 6'-O-caffeoyl-arbutin was the main component of V. dunalianum buds. VDW and 6'-O-caffeoyl-arbutin could regulate blood lipid levels in the high-fat diet-induced rat model of hyperlipidemia without damaging their vital organs. Furthermore, they could regulate the expression of PTGS2, ADH1C, and MAOB proteins and play a role in lowering blood lipids. The findings of this study lay a foundation for the further development of V. dunalianum and 6'-O-caffeoyl-arbutin as health supplements or drugs for the management of hyperlipidemia.

Quantification of Arbutin in Cosmetics, Drugs and Food Supplements by Hydrophilic-Interaction Chromatography.

Arbutin, the glucoside of hydroquinone, exists in two isomers, α-arbutin and ß-arbutin. The synthetic α isomer is mainly used as a skin brightening agent, while ß-arbutin occurs naturally, for instance in bearberry, and is used in drugs for treatment of lower urinary tract infections and as a food supplement. Since both isomers can be harmful at high concentrations, methods for their quantification are required. Classically they have been determined by reversed-phase chromatography, but separation of both isomers is often unsatisfactory. Here we present a simple and reliable method for quantification of α- and ß-arbutin based on hydrophilic-interaction chromatography. Prior to analysis, interfering compounds that would frequently be present in cosmetics and drugs, particularly biopolymers, were efficiently removed by precipitation with acetonitrile. In this paper, for separation, a Cyclobond I 2000 5 µm 250 × 4.6 mm column was employed as stationary phase and acetonitrile/water 92/8 (v/v) was used as an eluent at a flow rate of 0.8 mL min−1. For quantification, a UV detector operating at 284 nm was applied. Although analysis took less than 10 min, baseline separation of α- and ß-arbutin was achieved. The response was highly linear (r > 0.999) and the method had, for both α- and ß-arbutin, a LOD of 0.003% (w/w) and a LOQ of 0.009% (w/w). Moreover, the method showed excellent intra-day and inter-day repeatability with relative standard deviations in the range of 0.5% to 2.3% and 1.0% to 2.2%, respectively, with cosmetics, drugs and food supplements as samples.

Proteome changes in human bladder T24 cells induced by hydroquinone derived from Arctostaphylos uva-ursi herbal preparation.

ETHNOPHARMACOLOGICAL RELEVANCE: Arctostaphylos uva-ursi (L.) Spreng. (bearberry) is a well-known traditional herbal plant used as a urinary tract disinfectant. Its antiseptic and diuretic properties can be attributed to hydroquinone, obtained by hydrolysis of arbutin. AIM OF THE STUDY: This study aimed to determine the toxic profile of free hydroquinone on urinary bladder cells (T24) as a target of therapeutic action. MATERIALS AND METHODS: Quantitative and qualitative analysis of the extract and the digestive stability and bioavailability of arbutin and hydroquinone were performed by HPLC assay and simulated in vitro digestion, respectively. Cytotoxic effect, reactive oxygen species induction and proteome changes in T24 cells after hydroquinone treatment were determined using Neutral red assay, 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay and mass spectrometry, respectively. RESULTS: Through in vitro digestion, arbutin was stable, but hydroquinone increased after pepsin treatment (109.6%) and then decreased after the small intestine phase (65.38%). The recommended doses of Uva-ursi had a cytotoxic effect on T24 cells only when all hydroquinone conjugates were converted to free hydroquinone (320 and 900 µg/mL) and the toxic effect was enhanced by recovery. One cup of the therapeutic dose had a prooxidative effect after 4 h of incubation. Shorter time of cell exposure (2 h) to hydroquinone did not have any impact on reactive oxygen species induction. Proteomic analysis found 17 significantly up-regulated proteins compared to control. Hydroquinone activated proteins related to oxidative stress response, stress-adaptive signalling, heat shock response and initiation of translation. CONCLUSIONS: Despite the therapeutic properties of bearberry, up-regulated T24 cell proteins are evidence that plant compounds, although from a natural source, may exhibit negative properties.

Identification of Sitogluside as a Potential Skin-Pigmentation-Reducing Agent through Network Pharmacology.

Many traditional Chinese medicines (TCMs) with skin-whitening properties have been recorded in the Ben-Cao-Gang-Mu and in folk prescriptions, and some literature confirms that their extracts do have the potential to inhibit pigmentation. However, no systematic studies have identified the specific regulatory mechanisms of the potential active ingredients. The aim of this study was to screen the ingredients in TCMs that inhibit skin pigmentation through a network pharmacology system and to explore underlying mechanisms. We identified 148 potential active ingredients from 14 TCMs, and based on the average "degree" of the topological parameters, the top five TCMs (Fructus Ligustri Lucidi, Hedysarum multijugum Maxim., Ampelopsis japonica, Pseudobulbus Cremastrae Seu Pleiones, and Paeoniae Radix Alba) that were most likely to cause skin-whitening through anti-inflammatory processes were selected. Sitogluside, the most common ingredient in the top five TCMs, inhibits melanogenesis in human melanoma cells (MNT1) and murine melanoma cells (B16F0) and decreases skin pigmentation in zebrafish. Furthermore, mechanistic research revealed that sitogluside is capable of downregulating tyrosinase (TYR) expression by inhibiting the ERK and p38 pathways and inhibiting TYR activity. These results demonstrate that network pharmacology is an effective tool for the discovery of natural compounds with skin-whitening properties and determination of their possible mechanisms. Sitogluside is a novel skin-whitening active ingredient with dual regulatory effects that inhibit TYR expression and activity.

A Natural Alternative Treatment for Urinary Tract Infections: Itxasol©, the Importance of the Formulation.

Genito-urinary tract infections have a high incidence in the general population, being more prevalent among women than men. These diseases are usually treated with antibiotics, but very frequently, they are recurrent and lead to the creation of resistance and are associated with increased morbidity and mortality. For this reason, it is necessary to develop new compounds for their treatment. In this work, our objective is to review the characteristics of the compounds of a new formulation called Itxasol© that is prescribed as an adjuvant for the treatment of UTIs and composed of ß-arbutin, umbelliferon and n-acetyl cysteine. This formulation, based on biomimetic principles, makes Itxasol© a broad-spectrum antibiotic with bactericidal, bacteriostatic and antifungal properties that is capable of destroying the biofilm and stopping its formation. It also acts as an anti-inflammatory agent, without the adverse effects associated with the recurrent use of antibiotics that leads to renal nephrotoxicity and other side effects. All these characteristics make Itxasol© an ideal candidate for the treatment of UTIs since it behaves like an antibiotic and with better characteristics than other adjuvants, such as D-mannose and cranberry extracts.

Pharmacoinformatics and UPLC-QTOF/ESI-MS-Based Phytochemical Screening of Combretum indicum against Oxidative Stress and Alloxan-Induced Diabetes in Long-Evans Rats.

This research investigated a UPLC-QTOF/ESI-MS-based phytochemical profiling of Combretum indicum leaf extract (CILEx), and explored its in vitro antioxidant and in vivo antidiabetic effects in a Long-Evans rat model. After a one-week intervention, the animals' blood glucose, lipid profile, and pancreatic architectures were evaluated. UPLC-QTOF/ESI-MS fragmentation of CILEx and its eight docking-guided compounds were further dissected to evaluate their roles using bioinformatics-based network pharmacological tools. Results showed a very promising antioxidative effect of CILEx. Both doses of CILEx were found to significantly (p < 0.05) reduce blood glucose, low-density lipoprotein (LDL), and total cholesterol (TC), and increase high-density lipoprotein (HDL). Pancreatic tissue architectures were much improved compared to the diabetic control group. A computational approach revealed that schizonepetoside E, melianol, leucodelphinidin, and arbutin were highly suitable for further therapeutic assessment. Arbutin, in a Gene Ontology and PPI network study, evolved as the most prospective constituent for 203 target proteins of 48 KEGG pathways regulating immune modulation and insulin secretion to control diabetes. The fragmentation mechanisms of the compounds are consistent with the obtained effects for CILEx. Results show that the natural compounds from CILEx could exert potential antidiabetic effects through in vivo and computational study.

Bioactivity-Guided Isolation of Phytochemicals from Vaccinium dunalianum Wight and Their Antioxidant and Enzyme Inhibitory Activities.

Vaccinium dunalianum Wight, usually processed as a traditional folk tea beverage, is widely distributed in the southwest of China. The present study aimed to investigate the antioxidant, α-glucosidase and pancreatic lipase inhibitory activities of V.dunalianum extract and isolate the bioactive components. In this study, the crude extract (CE) from the buds of V. dunalianum was prepared by the ultrasound-assisted extraction method in 70% methanol and then purified with macroporous resin D101 to obtain the purified extract (PM). Five fractions (Fr. A-E) were further obtained by MPLC column (RP-C18). Bioactivity assays revealed that Fr. B with 40% methanol and Fr. D with 80% methanol had better antioxidant with 0.48 ± 0.03 and 0.62 ± 0.01 nM Trolox equivalent (TE)/mg extract for DPPH, 0.87 ± 0.02 and 1.58 ± 0.02 nM TE/mg extract for FRAP, 14.42 ± 0.41 and 19.25 ± 0.23 nM TE/mg extract for ABTS, and enzyme inhibitory effects with IC50 values of 95.21 ± 2.21 and 74.55 ± 3.85 for α-glucosidase, and 142.53 ± 11.45 and 128.76 ± 13.85 µg/mL for pancreatic lipase. Multivariate analysis indicated that the TPC and TFC were positively related to the antioxidant activities. Further phytochemical purification led to the isolation of ten compounds (1-10). 6-O-Caffeoylarbutin (7) showed significant inhibitory effects on α-glucosidase and pancreatic lipase enzymes with values of 38.38 ± 1.84 and 97.56 ± 7.53 µg/mL, and had the highest antioxidant capacity compared to the other compounds.

Eudesmane-type sesquiterpene glycosides: sonneratiosides A-E and eudesmol ß-D-glucopyranoside from the leaves of Sonneratia alba.

Five eudesmane-type sesquiterpene glycosides, named sonneratiosides A-E (1-5), were isolated from the leaves of Sonneratia alba (Lythraceae). The aglycone of sonneratioside A was identified as cryptomeridiol also known as proximadiol. X-ray crystallographic analysis of sonneratioside A confirmed its structure and its absolute stereochemistry. Eudesmol ß-D-glucopyranoside (6) was also isolated from nature for the first time. The tyrosinase inhibitory activity was assayed for the new compounds together with seven known compounds. Among them, arbutin (12) showed the expected activity and luteolin 7-O-rutinoside (10) showed comparable activity to arbutin.

Chemical and Biocatalytic Routes to Arbutin †.

Arbutin (also called ß-arbutin) is a natural product occurring in the leaves of a variety of different plants, the bearberries of the Ericaceae and Saxifragaceae families being prominent examples. It is a ß-glucoside derived from hydroquinone (HQ; 1,4-dihydroxybenzene). Arbutin has been identified in traditional Chinese folk medicines as having, inter alia, anti-microbial, anti-oxidant, and anti-inflammatory properties that useful in the treatment of different ailments including urinary diseases. Today, it is also used worldwide for the treatment of skin ailments by way of depigmenting, which means that arbutin is a component of many products in the cosmetics and healthcare industries. It is also relevant in the food industry. Hundreds of publications have appeared describing the isolation, structure determination, toxicology, synthesis, and biological properties of arbutin as well as the molecular mechanism of melanogenesis (tyrosinase inhibition). This review covers the most important aspects with special emphasis on the chemical and biocatalytic methods for the production of arbutin.

Protein-polysaccharide complex coated W/O/W emulsion as secondary microcapsule for hydrophilic arbutin and hydrophobic coumaric acid.

This study aimed to examine the modification effect of whey protein concentrate (WPC), WPC-gum arabic (WPC-GA) or WPC-high methoxyl pectin (WPC-PEC) complex to tailor-modify W/O/W emulsion for secondary microencapsulation of hydrophilic arbutin and hydrophobic coumaric acid. The stability and rheological properties of coated emulsions, encapsulation yield, release and degradation kinetics of arbutin and coumaric acid were investigated. Results revealed that WPC-PEC complex (at the ratio of 1:3) coating W/O/W emulsion exhibited the highest viscosity and stability, with the highest encapsulation yield of 91.08% for arbutin and 80.92% for coumaric acid, respectively. Tighter coating structure of the WPC-PEC complex (1:3) forming a stronger gel network structure was confirmed, accounting for the larger mean particle size of 569.67 nm. Moreover, the WPC-PEC (1:3) coating W/O/W emulsion also showed controlled release of arbutin and coumaric acid in simulated conditions. The k value of degradation kinetics for arbutin (7.99 × 10-4 at pH = 1.2, 4.19 × 10-4 at 90 °C and 7.52 × 10-4 at UV-C treatment) and coumaric acid (5.18 × 10-4 at pH = 1.2, 3.24 × 10-4 at 90 °C and 6.90 × 10-4 at UV-C treatment) indicated low degradation rate. The present study revealed that the WPC-PEC (1:3) coating W/O/W emulsion could provide a better synergistic effect on higher encapsulation yield and stability of arbutin and coumaric acid.

New Alkaloid and Aromatic Glucoside from the Flowers of Cymbidium Lunagrad Eternal Green.

In this paper, we investigated the chemical components of the flowers of Cymbidium Lunagrad Eternal Green for the first time. In the whole post-fertilization, a new alkaloid, named Lunagrad A (1), and a new aromatic glucoside, named Lunagrad B (2), were isolated from the MeOH extract of the flowers of Cymbidium Lunagrad Eternal Green, along with other six known aromatic compounds (3-8) and three flavone glucosides (9-11). These structures were determined on the basis of NMR experiments, as well as chemical evidence.

The effects of strawberry tree water leaf extract, arbutin and hydroquinone on haematological parameters and levels of primary DNA damage in white blood cells of rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Strawberry tree (Arbutus unedo L., Ericaceae) leaves represent a potent source of biologically active compounds and have been used for a long to relieve symptoms of various health impairments and diseases. Two major compounds related to their beneficial activities in animals and humans are arbutin and hydroquinone. AIM OF THE STUDY: To establish potential benefit/risk ratio associated with daily oral administration of strawberry tree water leaf extract, arbutin and hydroquinone in doses expected to be non-toxic. MATERIALS AND METHODS: We performed a 14-day and a 28-day study on male and female Lewis rats and evaluated main haematological parameters and the effects of treatments on the levels of primary DNA damage in white blood cells (WBC) using the alkaline comet assay. RESULTS: Our findings suggest no significant changes in the haematological parameters following prolonged exposure to strawberry tree water leaf extract, arbutin, and hydroquinone. However, hydroquinone causes increased, and extract as well as arbutin decreased WBC count in male rats compared to control after 14 days of treatment. DNA damage measured in WBC of rats treated with all compounds was below 10% of the DNA in the comet tail, which indicates low genotoxicity. The genotoxic potential of strawberry water leaf extract was within acceptable limits and reflected effects of a complex chemical composition upon DNA. We also observed slight gender- and exposure time- related differences in primary DNA damage in the leucocytes of control and treated rats. CONCLUSIONS: Future studies should investigate which doses of strawberry tree water leaf extract would be most promising for the potential use as a substitute for bearberry leaves for treatment of urinary infection.

Iridoid glycosides and polyphenolic compounds from Teucrium chamaedrys L.

In this work, the phytochemical analysis of Teucrium chamaedrys L. collected in Italy was reported. Eight compounds were isolated and identified by means of classical column chromatography and spectroscopic techniques, such as NMR and MS. In detail, these compounds were: verbascoside (1), forsythoside b (2), samioside (3), alyssonoside (4), harpagide (5), 8-O-acetyl-harpagide (6), cirsiliol (7) and ß-arbutin (8). The presence of these compounds, in particular iridoids and phenyl-ethanoid glycosides, has a chemotaxonomic relevance and results to be in perfect accordance with the current botanical classification of the species. In addition, it provides a phytochemical rationale for the use of this particular plant in the ethno-pharmacological field. Conversely, it is worth of mention the absence of potentially toxic components, unlike to what observed in other species of the genus which can no longer be used for ethno-medicinal purposes.

Labdane-Type Diterpenes, Galangalditerpenes A-C, with Melanogenesis Inhibitory Activity from the Fruit of Alpinia galanga.

In our continuing study of biologically active natural products from the fruit of Alpinia galanga (Zingiberaceae), we newly isolated three new labdane-type diterpenes, termed galangalditerpenes A-C (1-3), along with four known sesquiterpenes (4-7) and two diterpenes (8 and 9). The stereostructures of 1-3 were elucidated on the basis of their spectroscopic properties. The melanogenesis inhibitory activities in theophylline-stimulated murine B16 melanoma 4A5 cells of these isolates, including the new diterpenes (1-3, IC50 = 4.4, 8.6, and 4.6 µM, respectively), were found to be more than 6-87-fold higher than that of arbutin (174 µM), a commercially available positive control.

Antimicrobial and Antiradical Activity of Extracts Obtained from Leaves of Five Species of the Genus Bergenia: Identification of Antimicrobial Compounds.

An important focus of modern medicine is the search for new substances and strategies to combat infectious diseases, which present an increasing threat due to the growth of bacterial resistance to antibiotics. Another problem concerns free radicals, which in excess can cause several serious diseases. An alternative to chemical synthesis of antimicrobial and antiradical compounds is to find active substances in plant raw materials. We prepared extracts from leaves of five species of the genus Bergenia: B. purpurascens, B. cordifolia, B. ligulata, B. crassifolia, and B. ciliata. Antimicrobial and antiradical features of extracts and raw materials were assessed, and the quantities of phenolic compounds were determined. We also evaluated, using high-performance liquid chromatography, the amounts of arbutin and hydroquinone, compounds related to antimicrobial activity of these raw materials. The strongest antiradical properties were shown by leaves of B. crassifolia and B. cordifolia, the lowest by leaves of B. ciliata. The antiradical activity of extracts showed a strong positive correlation with the amount of phenols. All raw materials have significant antimicrobial properties. Among them, the ethyl acetate extracts were the most active. Antimicrobial activity very weakly correlated with the amount of arbutin, but correlated very strongly with the contents of both hydroquinone and phenolic compounds. Additional experiments using artificially prepared mixtures of phenolic compounds and hydroquinone allowed us to conclude that the most active antimicrobial substance is hydroquinone.

New arbutin derivatives from the leaves of Heliciopsis lobata with cytotoxicity.

Heliciopsis lobata is a medicinal plant, which is exclusively used to treat tumor in Li folk region. Two new arbutin derivatives, 6'-((E)2-methoxy-5-hydroxycinnamoyl) arbutin (1) and 2'-((E)2, 5-dihydroxycinnamoyl) arbutin (2) along with five known compounds (3-7), were isolated from the leaves of Heliciopsis lobata. Their structures were elucidated on the basis of extensive spectroscopic interpretations. They were evaluated for their potential anticancer activity. Compounds 6 and 7 exhibited cytotoxicity against MGC-803 cells with IC50 values being 44.1 and 11.3 µg·mL-1, respectively. Additionally, compounds 1, 2 and 5-7 exhibited a moderate inhibition of MGC-803 cells invasion; compound 2 at 20 µg·mL-1 inhibited the invasion of MGC-803 cells by 43.0%, compared with the controls.

Arbutin, an intracellular hydroxyl radical scavenger, protects radiation-induced apoptosis in human lymphoma U937 cells.

Ionizing radiation (IR) can generate reactive oxygen species (ROS). Excessive ROS have the potential to damage cellular macromolecules including DNA, proteins, and lipids and eventually lead to cell death. In this study, we evaluated the potential of arbutin, a drug chosen from a series of traditional herbal medicine by measuring intracellular hydroxyl radical scavenging ability in X-irradiated U937 cells. Arbutin (hydroquinone-ß-D-glucopyranoside), a naturally occurring glucoside of hydroquinone, has been traditionally used to treat pigmentary disorders. However, there are no reports describing the effect of arbutin on IR-induced apoptosis. We confirmed that arbutin can protect cells from apoptosis induced by X-irradiation. The combination of arbutin and X-irradiation could reduce intracellular hydroxyl radical production and prevent mitochondrial membrane potential loss. It also could down-regulate the expression of phospho-JNK, phospho-p38 in whole cell lysate and activate Bax in mitochondria. Arbutin also inhibits cytochrome C release from mitochondria to cytosol. To verify the role of JNK in X-irradiation-induced apoptosis, the cells were pretreated with a JNK inhibitor, and found that JNK inhibitor could reduce apoptosis induced by X-irradiation. Taken together, our data indicate that arbutin plays an anti-apoptotic role via decreasing intracellular hydroxyl radical production, inhibition of Bax-mitochondria pathway and activation of the JNK/p38 MAPK pathway.

Grevillosides J-Q, arbutin derivatives from the leaves of Grevillea robusta and their melanogenesis inhibitory activity.

From the 1-BuOH-soluble fraction of a MeOH extract of the leaves of Grevillea robusta, new arbutin derivatives and related compounds, named grevillosides J-Q (1-8), together with eight known compounds (9-18) were isolated. Various kind of acyl groups were attached to ß-D-glucose at the 6-position through an ester linkage. Their structures were mainly elucidated from one- and two-dimensional NMR spectroscopic data. For exploitation as skin-lightening and anti-chloasma agents, the inhibitory activities of the isolated compounds as well as ones isolated in previous experiments (19-31) toward tyrosinase and melanin-producing B16 cells were assayed. Several compounds showed promising activity.

Arbutin production via biotransformation of hydroquinone in in vitro cultures of Aronia melanocarpa (Michx.) Elliott.

Arbutin (hydroquinone ß-D-glucoside) is a compound of plant origin possessing valuable therapeutic (urinary tract disinfection) and cosmetic (skin whitening) properties, which can be obtained from in vitro cultures of plants belonging to different taxa via biotransformation of exogenously supplemented hydroquinone. Agitating cultures of Aronia melanocarpa were maintained on the Murashige and Skoog medium containing growth regulators: the cytokinin - BAP (6-benzylaminopurine), 2 mg/l and the auxin NAA (α-naphthaleneacetic acid), 2 mg/l. The biomass was cultured for 2 weeks and then hydroquinone was supplemented at the following doses: 96, 144, 192, 288 and 384 mg/l either undivided or divided into two or three portions added at 24-hour intervals. The content of the reaction product - arbutin, was determined using an HPLC method in methanolic extracts from biomass and lyophilized medium samples collected 24 hours after the addition of the last precursor dose. The total amounts of arbutin were very diverse, from 2.71 to 8.27 g/100g d.w. The production of arbutin rose with increasing hydroquinone concentration. The maximum content of the product was observed after hydroquinone addition at 384 mg/l divided into two portions. Biotransformation efficiency also varied widely, ranging from 37.04% do 73.80%. The identity of the product - arbutin, after its isolation and purification was confirmed by spectral analysis ((1)H-NMR spectrum). The maximum amount of arbutin obtained was higher than that required by the latest 9(th) Edition of the Polish Pharmacopoeia and by the newest 8th Edithion of European Pharmacopoeia for Uvae ursi folium (7.0 g/100g d.w.), and is interesting from practical point of view.

ID-CUBE direct analysis in real time high-resolution mass spectrometry and its capabilities in the identification of phenolic components from the green leaves of Bergenia crassifolia L.

RATIONALE: Bergenia crassifolia is a plant widely used in herbal medicine. Its chemical composition has been little studied, and no studies using high-resolution mass spectrometry (HRMS) have been performed. Its phenolic components are of particular interest, due to the interest in such compounds in medicine and cosmetics. The ID-CUBE, a simplified Direct Analysis in Real Time (DART) ion source, suitable for the fast MS analysis of liquids without complex sample preparation, offers a new method of studying extracts of such plant. Coupling the ID-CUBE with a high-resolution mass spectrometer can provide identification of extract components. METHODS: Mass spectral conditions were optimized for model solutions of the flavonoid naringenin and used for the identification of phenolic compounds in green leaves extracts of Bergenia crassifolia. OpenSpot sample cards with a metal grid surface were used for sample introduction into the ID-CUBE ion source on an Obitrap mass spectrometer. The samples were applied as 5-µL aliquots of the extract onto the metal grid of the card. Sample ionization was stimulated in the ion source within 20 s by applying an electric current to the metal grid to thermally desorb the analytes into the gas flow of metastable helium atoms from the ID-CUBE. RESULTS: Elemental compositions were assigned to abundant ions in the mass spectra of the extracts. The major phenolic components were confirmed by their [M-H](-) ions. Thirty-six other marker ions were found, and elemental compositions were suggested for 30% of them, based on a search for compounds found in herbal extracts. CONCLUSIONS: The ID-CUBE-Orbitrap MS coupling allowed the rapid accurate mass determination of the phenolic components (and other compounds) in herbal extracts. Higher confidence in component identification could be provided by using additional structural elucidation methods, including tandem mass spectrometry (MS/MS), and this will be the focus of future studies.