Spectrophotometric and Chromatographic Assessment of Total Polyphenol and Flavonoid Content in Rhododendron tomentosum Extracts and Their Antioxidant and Antimicrobial Activity.

In the literature, the chemical composition of Rhododendron tomentosum is mainly represented by the study of isoprenoid compounds of essential oil. In contrast, the study of the content of flavonoids will contribute to the expansion of pharmacological action and the use of the medicinal plant for medical purposes. The paper deals with the technology of extracts from Rh. tomentosum shoots using ethanol of various concentrations and purified water as an extractant. Extracts from Rh. tomentosum were obtained by a modified method that combined the effects of ultrasound and temperature to maximize the extraction of biologically active substances from the raw material. Using the method of high-performance thin-layer chromatography in a system with solvents ethyl acetate/formic acid/water (15:1:1), the following substances have been separated and identified in all the extracts obtained: rutin, hyperoside, quercetin, and chlorogenic acid. The total polyphenol content (TPC) and total flavonoid content (TFC) were estimated using spectrophotometric methods involving the Folin-Ciocalteu (F-C) reagent and the complexation reaction with aluminum chloride, respectively. A correlation analysis was conducted between antioxidant activity and the polyphenolic substance content. Following the DPPH assay, regression analysis shows that phenolic compounds contribute to about 80% (r2 = 0.8028, p < 0.05) of radical scavenging properties in the extract of Rh. tomentosum. The extract of Rh. tomentosum obtained by ethanol 30% inhibits the growth of test cultures of microorganisms in 1:1 and 1:2 dilutions of the clinical strains #211 Staphylococcus aureus and #222 Enterococcus spp. and the reference strain Pseudomonas aeruginosa ATCC 10145.

Phytochemical and Pharmacological Evaluation of the Residue By-Product Developed from the Ocimum americanum (Lamiaceae) Postdistillation Waste.

The yield of essential oils in plants is not high and postdistillation wastes rich in phenolic compounds could be used to enhance the profitability of essential oil-bearing plants. The aim of the study was to evaluate polyphenols in a dry extract obtained from the postdistillation waste of the American basil (Ocimum americanum L.) herb, and to conduct the screening of its pharmacological activities. Rosmarinic acid, caffeic acid and rutin were identified in the extract using high-performance thin-layer chromatography. The high-performance liquid chromatography analysis found the presence of a plethora of polyphenols in the extract. Rosmarinic acid, luteolin-7-O-glucoside and rutin were as the main compounds. The total phenolic content in the extract was 106.31 mg GAE/g and free radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl evaluated as IC50 was 0.298 mg/mL. The tested extract dose-dependently decreased the paw edema in rats, suggesting its potent anti-inflammatory property. The acute toxicity study indicates its safety. Thus, the O. americanum hydrodistilled residue by-product is the promising source of biologically active compounds with significant antioxidant and anti-inflammatory effects.

Polyphenols and Pharmacological Screening of a Monarda fistulosa L. dry Extract Based on a Hydrodistilled Residue By-Product.

This study aimed to determine the composition and content of polyphenols in the dry extract obtained from the hydrodistilled residue by-product of the wild bergamot (Monarda fistulosa L., Lamiaceae Martinov family) herb (MFDE) and to evaluate its safety and pharmacological properties. The total phenolic content (TPC) in the MFDE was 120.64 mg GAE/g. The high-performance liquid chromatography (HPLC) analysis showed the presence of a plethora of phenolic compounds, including hydroxycinnamic acids and flavone derivatives in the MFDE, with rosmarinic acid and luteolin-7-O-glucoside being the main components. With an IC50 value of 0.285 mg/mL, it was found to be a strong DPPH radical scavenger. The acute toxicity study results indicate that the oral administration of MFDE to rats at the doses of 500-5,000 mg/kg did not produce any side effects or death in animals which indicates its safety. The results of the in vivo assay showed that the MFDE dose-dependently inhibited paw oedema and significantly reduced the number of writings in mice induced by the acetic acid injection suggesting its potent anti-inflammatory and analgesic activities, respectively. The conducted studies revealed that M. fistulosa hydrodistilled residue by-product could be regarded as a new natural source of polyphenols with valuable pharmacological properties.

Phytochemical Evaluation of Tinctures and Essential Oil Obtained from Satureja montana Herb.

Winter Savory (Satureja montana L.) has been used in traditional medicine and as a spice or natural food preservative in the Mediterranean region for centuries. In this paper, some technological and analytical aspects of the S. montana tinctures development and an evaluation of the essential oil composition are provided. The total phenolic and flavonoid contents and phenolic compounds profile analyzed spectrophotometrically and by high-performance thin-layer chromatography (HPTLC), respectively, were evaluated in the developed tinctures. The results showed that the tinctures prepared from the S. montana herb by maceration or remaceration are rich in polyphenols, and there is an influence of the technological factors (particle size and extraction mode) on the total phenolic and flavonoid contents. Caffeic, rosmarinic, and chlorogenic acids, (-)-catechin and rutin were identified in the tinctures using the HPTLC method. p-Thymol (81.79%) revealed by gas chromatography-mass spectrometry (GC-MS) was the predominant compound of the essential oil of this plant. Thus, the high contents of polyphenols and flavonoids in the developed tinctures and p-thymol among the volatile components of the S. montana essential oil could indicate the promising antioxidant and antimicrobial properties of these herbal preparations. The obtained results are a ground for the organization of the manufacture of the S. montana tincture and essential oil with the purpose of performing preclinical studies.

Development of high-performance thin layer chromatography method for identification of phenolic compounds and quantification of rosmarinic acid content in some species of the Lamiaceae family.

AIM: Representatives of Nepetoideae Burnett subfamily are promising species of the Lamiaceae Martinov family because of accumulating such valuable groups of secondary metabolites as terpenoids and polyphenols. A high-performance thin layer chromatography (HPTLC) fingerprint method for the qualitative determination of phenolic compounds and for the quantification of rosmarinic acid (RA) content in methanol extracts of five species of this subfamily was developed for the first time. MATERIALS AND METHODS: Dried aerial parts of Dracocephalum moldavica L., Ocimum americanum L., Lophanthus anisatus (Nutt.) Benth., Monarda fistulosa L., and Satureja hortensis L. collected in flowering period were macerated with methanol. The HPTLC analysis was conducted using the CAMAG analytical system (Muttenz, Switzerland). The comparative analysis of RA contents was performed by HPTLC densitometric detection at λ = 366nm. RESULTS: Identification of polyphenols in the investigated herbs was performed by comparison of a color and R f of the chromatographic zones with six reference standards: rutin, apigenin, luteolin, caffeic acid, chlorogenic acid, and RA. HPTLC method was also validated for the quantification of RA in the extracts of investigated herbs. RA contents decreased in such a sequence: D. moldavica (24.83 ± 0.78mg/g) > M. fistulosa (20.32 ± 0.64mg/g) > O. americanum (19.59 ± 0.61mg/g) > S. hortensis (18.77 ± 0.52mg/g) > L. anisatus (12.61 ± 0.43mg/g). CONCLUSION: Obtained data can facilitate the differentiation of investigated species using the chromatographic fingerprints of their phenolic compounds. Developed and validated HPTLC method provides an approach to estimate RA content as a common marker of investigated herbs.

Development of a high performance thin layer chromatography method for the rapid qualification and quantification of phenolic compounds and abscisic acid in honeys.

Honey is a natural product with a complex chemical composition consisting of sugars and other bioactive compounds. It is important in many traditional systems of medicine, exhibiting interesting bioactivities, in particular antimicrobial, anti-inflammatory and antioxidant effects. Authentication of botanical origin of honeys is particularly important in this context. Therefore, methods for quality control of honey and detection of its adulteration are very important. A HPTLC method for the quantitative determination of phenolic compounds in honey was developed for the first time. Seven phenolic compounds were detected and determined quantitatively in lime and acacia honey samples. The obtained results show that the HPTLC profiles of phenolic compounds and abscisic acid can be useful for determining the floral origin of honeys. Chromatographic tests were supplemented by statistical analysis (PCA and HCA) led to the successful separation of acacia and lime honey samples.

Secondary metabolites from the aerial parts of Cytisus villosus Pourr.

Phytochemical investigation of the aerial parts of Cytisus villosus Pourr. resulted in the isolation and characterization of a new isoflavan, (3S, 4S)-2',4'-dihydroxy-3'-methoxy-6,7-methylenedioxyisoflavan- 4-ol (1), and a new monoterpene, (4R,6S)-4-hydroxy-2,2,6-trimethyl-9-oxabicyclo [4.2.1] non-1(8)-en-7-one (2), together with four known flavonoids: geinstein (3), chrysin (4), chrysin -7-O-ß-D-glucopyranoside (5) and 2″-O-α-L-rhamnosylorientin (6). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analysis, including 1D, 2D NMR (1H, 13C, COSY, TOCSY, HMBC and HSQC) and HRESIMS. The absolute configurations of 1 and 2 were established by the comparison of experimental and calculated electronic circular dichroism (ECD) spectra.

HPTLC Fingerprinting-Rapid Method for the Differentiation of Honeys of Different Botanical Origin Based on the Composition of the Lipophilic Fractions.

Bee honey possess various nutritional and medicinal functions, which are the result of its diverse chemical composition. The numerous bioactive compounds in honey come from flower nectar; thus, the identification of the specific chemical profiles of honey samples is of great importance. The lipophilic compounds from eight monofloral honeys (rape, buckwheat, clover, willow, milk thistle, dandelion, raspberry and sweet yellow clover) were investigated. Analyses of the lipophilic fractions obtained by UAE (ultrasound assisted extraction) and SPE (solid phase extraction) extractions were performed using high-performance thin layer chromatography (HPTLC). Chromatographic and cluster analyses allowed the identification of a unique, colorful pattern of separated compounds with specific Rf values on the HPTLC plate for each type of monofloral honey. HPTLC is a simple and effective visual method of analysis, and it can serve as a basis for authenticating different types of honey.

Polish Yellow Sweet Clover (Melilotus officinalis L.) Honey, Chromatographic Fingerprints, and Chemical Markers.

A case study of Polish Melilotus officinalis honey was presented for the first time. Gas chromatography-mass spectrometry (GC-MS) (after steam distillation, Soxhlet extraction, ultrasonic solvent extraction, and solid phase extraction (SPE)) and targeted high performance liquid chromatography with a photodiode array detector (HPLC-PAD) were applied to determine the characteristic components of honey. While ubiquitous in most honeys, carbohydrates, terpene derivatives, and phenylacetic acid dominated in the Soxhlet extracts (25.54%) and in the application of SPE (13.04%). In addition, lumichrome (1.85%) was found, and may be considered as a marker of this honey. Due to the presence of these compounds, Polish yellow sweet clover honey is similar to French lavender honeys. The major compounds determined in the methanolic extract were (+)-catechine (39.7%) and gallic acid (up to 30%), which can be regarded as specific chemical markers of the botanical origin of melilot honey. With respect to total phenolic and flavonoid contents, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were determined spectrophotometrically. The honey exhibited a moderate antioxidant activity, typical for light honeys, which correlates well with its phenolic and flavonoid composition.

Research on acute toxicity and the behavioral effects of methanolic extract from psilocybin mushrooms and psilocin in mice.

The pharmacological activities and acute toxicity of the psilocin (PC) and dried residues of the crude extracts of psychotropic mushrooms were investigated in mice. The hallucinogenic substances were effectively isolated, by using methanol, from the species of Psilocybe semilanceata and Pholiotina cyanopus, that were collected in the north-east region of Poland. The chemical analysis of these extracts, which was performed by liquid chromatography with mass spectrometry detection (LC-MS), indicated the presence of psilocin and other hallucinogenic substances, including indolealkylamines and their phosphorylated analogues. When the pure psilocin or fungal extracts were used, slight differences in determined LD50 values were observed. However, the application of PC evoked the highest level of toxicity (293.07 mg/kg) compared to the activity of extracts from Ph. cyanopus and P. semilanceata, where the level of LD50 was 316.87 mg/kg and 324.37 mg/kg, respectively. Furthermore, the behavioral test, which considered the head-twitching response (HTR), was used to assess the effects of the studied psychotropic factors on the serotonergic system. Both, the fungal extracts and psilocin evoked characteristic serotoninergic effects depending on the dose administered to mice, acting as an agonist/partial agonist on the serotonergic system. A dose of 200 mg/kg 5-hydroxytryptophan (5-HTP) induced spontaneous head-twitching in mice (100% effect), as a result of the formation of 5-hydroxytryptamine (5-HT) in the brain. Compared to the activity of 5-HTP, the intraperitoneal administration of 1mg/kg of psilocin or hallucinogenic extracts of studied mushrooms (Ph. cyanopus and P. semilanceata) reduced the number of head-twitch responses of about 46% and 30%, respectively. In contrast, the administration of PC exhibited a reduction of about 60% in HTR numbers.

Do differences in chemical composition of stem and cap of Amanita muscaria fruiting bodies correlate with topsoil type?

Fly agaric (Amanita muscaria) was investigated using a 1H NMR-based metabolomics approach. The caps and stems were studied separately, revealing different metabolic compositions. Additionally, multivariate data analyses of the fungal basidiomata and the type of soil were performed. Compared to the stems, A. muscaria caps exhibited higher concentrations of isoleucine, leucine, valine, alanine, aspartate, asparagine, threonine, lipids (mainly free fatty acids), choline, glycerophosphocholine (GPC), acetate, adenosine, uridine, 4-aminobutyrate, 6-hydroxynicotinate, quinolinate, UDP-carbohydrate and glycerol. Conversely, they exhibited lower concentrations of formate, fumarate, trehalose, α- and ß-glucose. Six metabolites, malate, succinate, gluconate, N-acetylated compounds (NAC), tyrosine and phenylalanine, were detected in whole A. muscaria fruiting bodies but did not show significant differences in their levels between caps and stems (P value>0.05 and/or OPLS-DA loading correlation coefficient <0.4). This methodology allowed for the differentiation between the fruiting bodies of A. muscaria from mineral and mineral-organic topsoil. Moreover, the metabolomic approach and multivariate tools enabled to ascribe the basidiomata of fly agaric to the type of topsoil. Obtained results revealed that stems metabolome is more dependent on the topsoil type than caps. The correlation between metabolites and topsoil contents together with its properties exhibited mutual dependences.

Chemometrics as a tool of origin determination of Polish monofloral and multifloral honeys.

The aim of this study was to evaluate the application of chemometrics studies to determine the botanical origin of Polish monofloral honeys using NMR spectroscopy. Aqueous extracts of six kinds of honeys, namely, heather (Calluna vulgaris L.), buckwheat (Fagopyrum esculentum L), lime (Tilia L), rape (Brassica napus L. var. napus), acacia (Acacia Mill.), and multifloral ones, were analyzed. Multivariate chemometric data analysis was performed using principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). Chemometric analysis supported by pollen analysis revealed the incorrect classification of acacia honeys by the producers. Characteristic motives for each honey were identified, which allowed chemical profiles of tested honeys to be built. Thus, phenylacetic acid and dehydrovomifoliol (4-hydroxy-4-[3-oxo-1-butenyl]-3,5,5-trimethylcyclohex-2-en-1-one) were proposed to be markers of Polish heather honey. Formic acid and tyrosine were found to be the most characteristic compounds of buckwheat honey, whereas 4-(1-hydroxy-1-methylethyl)cyclohexane-1,3-dienecarboxylic acid was confirmed as a marker of lime honey.

Possible fungistatic implications of betulin presence in betulaceae plants and their hymenochaetaceae parasitic fungi.

Betulin and its derivatives (especially betulinic acid) are known to possess very interesting prospects for their application in medicine, cosmetics and as bioactive agents in pharmaceutical industry. Usually betulin is obtained by extraction from the outer layer of a birch bark. In this work we describe a simple method of betulin isolation from bark of various species of Betulaceae trees and parasitic Hymenochaetaceae fungi associated with these trees. The composition of the extracts was studied by GC-MS, whereas the structures of the isolated compounds were confirmed by FTIR and 1H NMR. Additionally, the significant fungistatic activity of betulin towards some filamentous fungi was determined. This activity was found to be strongly dependent on the formulation of this triterpene. A betulin-trimyristin emulsion, in which nutmeg fat acts as emulsifier and lipophilic carrier, inhibited the fungal growth even in micromolar concentrations--its EC50 values were established in the range of 15 up to 50 microM depending on the sensitivity of the fungal strain. Considering the lack of fungistatic effect of betulin applied alone, the application of ultrasonic emulsification with the natural plant fat trimyristin appeared to be a new method of antifungal bioassay of water-insoluble substances, such as betulin.

Crotonic acid as a bioactive factor in carrot seeds (Daucus carota L.).

Water extracts from the carrot seed (Daucus carota L.) var. Perfekcja exhibit plant growth inhibitory properties against cress, cucumber, onion and carrot in a dose-dependant manner. This property results from the action of low-and high-molecular components of the extract. The low-molecular component was identified as crotonic acid ((E)-2-butenoic acid). Its presence was also confirmed in other late varieties of carrot. The determined strong herbicidal properties of crotonic acid and its availability after release to soil combined with its high level in seeds suggest that it might be considered as an allelopathic and autotoxic factor in the seeds.

Antifungal activity of the carrot seed oil and its major sesquiterpene compounds.

Carrot seed oil is the source of the carotane sesquiterpenes carotol, daucol and beta-caryophyllene. These sesquiterpenic allelochemicals were evaluated against Alternaria alternata isolated from the surface of carrot seeds cultivar Perfekcja, a variety widely distributed in horticultural practise in Poland. Alternaria alternata is one of the most popular phytotoxic fungi infesting the carrot plant. The strongest antifungal activity was observed for the main constituent of carrot seed oil, carotol, which inhibited the radial growth of fungi by 65% at the following concentration.