Bisbibenzyls from Serbian Primula veris subsp. Columnae (Ten.) Lȕdi and P. acaulis (L.) L.

Bisbibenzyls are specialized metabolites found exclusively in liverworts, until recently; they represent chemical markers of liverworts. Their occurrence in vascular plants was noticed in 2007, when they were found in Primula veris subsp. macrocalyx from Russia. This report prompted us to chemically analyze the two most common Serbian Primula species, P. veris subsp. columnae and P. acaulis, in order to determine the presence of bisbibenzyls in them. Our study revealed nine structurally distinct bisbibenzyls (1-9), identified based on 1D and 2D NMR, IR, UV and HRESIMS data. Among them were five previously undescribed compounds (2-6). The remaining compounds found and previously described in the literature were: the bisbibenzyls riccardin C (1), isoperrottetin A (7), isoplagiochin E (8) and 11-O-demethylmarchantin I (9), as well as 4-hydroxyphenylmethylketone (10) and 4-hydroxy-3-methoxyphenylmethylketone (11). Riccardin C was the most dominant bisbibenzyl in both species studied. Previously, it was the first bisbibenzyl found in vascular plants (P. veris subsp. macrocalyx). An assessment of the cytotoxic activity of the isolated compounds against A549 lung cancer and healthy MRC5 cell lines was also the subject of our study. Compounds 6 and 9 exhibited significant cytotoxic activity expressed by IC50 values of 12 µM, but the selectivity was not satisfactory.

Very-Long-Chain Wax Constituents from Primula veris and P. acaulis: Does the Paradigm of Non-Branched vs. Branched Chain Dominance Universally Hold in all Plant Taxa?

Herein n-, iso- and anteiso-series of very-long-chained (VLC) alkanes (C21 -C35 ), fatty acid benzyl esters (FABEs; C20 -C32 ), and 2-alkanones (C23 -C35 ) were identified in the wax of Primula veris L. and P. acaulis (L.) L. (Primulaceae). For the very first time in a sample of natural origin, the presence of iso- and anteiso-VLC FABEs and 2-alkanones was unequivocally confirmed by synthetic work, derivatization, and NMR. It should be noted that the studied species produced unusually high amounts of branched wax constituents (e. g., >50 % of 2-alkanones were branched isomers). The domination of iso-isomers, probably biosynthesized from leucine-derived starters, is a unique feature in the Plant Kingdom. The plant organ distribution of these VLC compounds in P. acaulis samples (different habitats and phenological phases) pointed to their possible ecological value. This was supported by a eutectic behavior of binary blends of FABEs and alkanes, as well as by high UV-C absorption by FABEs.

Bioactive Phenolic Compounds from Primula veris L.: Influence of the Extraction Conditions and Purification.

Our experiments may help to answer the question of whether cowslip (Primula veris L.) is a rich source of bioactive substances that can be obtained by efficient extraction with potential use as a food additive. A hypothesis assumed that the type of solvent used for plant extraction and the individual morphological parts of Primula veris L. used for the preparation of herbal extracts will have key impacts on the efficiency of the extraction of bioactive compounds, and thus, the health-promoting quality of plant concentrates produced. Most analysis of such polyphenolic compound contents in extracts from Primula veris L. has been performed by using chromatography methods such as ultra-performance reverse-phase liquid chromatography (UPLC-PDA-MS/MS). Experiments demonstrated that the most effective extraction agent for fresh study material was water at 100 °C, whereas for dried material it was 70% ethanol. The richest sources of polyphenolic compounds were found in cowslip primrose flowers and leaves. The aqueous and ethanol extracts from Primula veris L. were characterized by a quantitatively rich profile of polyphenolic substances, and a high antioxidative potential. Selective extraction with the use of mild conditions and neutral solvents is the first step to obtaining preparations from cowslip primrose with a high content of bioactive substances.

Arabian Primrose leaf extract mediated synthesis of silver nanoparticles: their industrial and biomedical applications.

The present study encompasses green synthesis of silver nanoparticles (AgNPs) using aqueous leaf extract of Arabian Primrose within 6 min of reaction at 60 °C, pH 7 and their characterisation using physico-chemical analytical techniques. UV-Visible spectroscopy elucidated the surface plasmon resonance around 420 nm. FESEM and TEM images revealed that AgNPs were spherical with average diameter 10-60 nm. XRD pattern confirmed their crystalline nature. The leaf extract rich in phenolics and flavonoids was subjected to GC-MS analysis that identified bioactive compounds helping in reduction and stabilisation of AgNPs. The synthesised AgNPs possessed high anti-oxidant potential against DPPH and H2O2 radicals. Incidentally, the AgNPs acted as excellent nanocatalyst towards borohydride reduction and degradation of structurally different organic dyes. The AgNPs also exhibited selective colorimetric sensing of hazardous mercuric, ferric ions and ammonia. Such AgNPs were cytotoxic against HeLa cells (IC50 7.18 µg/mL) and compatible towards normal L20B cells. These AgNPs showed effective anti-microbial activity against different human pathogens tested (bacterial and fungal). This is probably the first report of AgNPs synthesis using Arabian Primrose leaf extract showing strong anti-oxidant, catalytic, biosensing, anti-cancer and anti-microbial activities and find remarkable applications in medical, industrial and ecological sectors.

Effects of co-administration of rapamycin and evening primrose/hemp seed oil supplement on immunologic factors and cell membrane fatty acids in experimental autoimmune encephalomyelitis.

BACKGROUND: The immune response is influenced by the administration of omega-3 polyunsaturated fatty acids (PUFA). Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE) are affected by PUFA. The combination of evening primrose/hemp seed oil (EPO/HSO) has essential fatty acids (EFAs) for human optimal health due to the favorable ratio of omega-6/omega-3 and antioxidantal properties. The study was conducted to evaluate the effects of EPO/HSO on improving the membrane fatty acids composition of spleen and blood cells and immunologic factors in compared to rapamycin (RAPA) in the EAE model. METHODS AND MATERIALS: Chronic-EAE was induced by induction of MOG in C57BL/6J mice (female, age: 6-8 weeks, weight 18-21). Mice were assigned to 5 groups (6/group) to evaluate the therapeutic effects of EPO/HSO supplement in comparison with rapamycin: A group; EPO/HSO + RAPA, B group; RAPA, C group; EPO/HSO. Results were compared to two control groups (EAE and naive). The fatty acid profile of the spleen and blood cell membrane was evaluated. Real-time-polymerase chain reaction was used for the evaluate the genes expression levels of interleukin (IL) -4, IL-5, and IL-13 in lymphocytes. Also, IL-4 of serum was evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Our findings indicated that EPO/HSO therapy significantly increased the percentage of essential fatty acids in cell membrane of the spleen and blood. The relative expression of IL-4, IL-5, and IL-13 genes in lymphocytes and serum level of IL-4 was significantly increased in the HSO/EPO treated group versus other groups. CONCLUSION: These results point to potential therapeutic effects on the repair of the structure of cell membranes and suppression of inflammation by EPO/HSO in EAE.

Assessment of the Bioactive Compounds in White and Red Wines Enriched with a Primula veris L.

The aim of this paper was to analyze selected physicochemical properties and the pro-health potential of wines produced in southeastern Poland, in the Subcarpathian region, and commercial Carlo Rossi wines enhanced with cowslip (Primula veris L.). This study used ultra-performance reverse-phase liquid chromatography (UPLC)-PDA-MS/MS to perform most of the analysis, including the polyphenolic compounds and saponin content in wines enriched by Primula veris L. The initial anthocyanin content in Subcarpathian (Regional) red wine samples increased four times to the level of 1956.85 mg/L after a 10% addition of Primula veris L. flowers. For white wines, a five-fold increase in flavonol content was found in Subcarpathian (Regional) and wine samples, and an almost 25-fold increase in flavonol content was found in Carlo Rossi (Commercial) wine samples at the lowest (2.5%) Primula veris L. flower addition. Qualitative analysis of the regional white wines with a 10% Primula veris L. flower enhancement demonstrated the highest kaempferol content (197.75 mg/L) and a high quercetin content (31.35 mg/L). Thanks to wine enrichment in triterpenoid saponins and in polyphenolic compounds from Primula veris L. flowers, which are effectively extracted to wine under mild conditions, both white and red wines can constitute a highly pro-health component of diets, which is valuable in preventing chronic heart failure.

Unusual Flavones from Primula macrocalyx as Inhibitors of OAT1 and OAT3 and as Antifungal Agents against Candida rugosa.

A bioactivity guided program exploring the interaction of phytochemicals in the entire plant Primula macrocalyx with the organic anion transporters (OAT1 and OAT3) and microorganisms led to the elucidation of ten known flavones (1-4, 6-10, 12) and two previously undescribed flavones (5, 11). The structures of the compounds were determined by extensive analysis of spectroscopic data, as well as by comparison with data from previous reports. Two known flavones (9, 12) are reported for the first time from the family Primulaceae. All compounds were evaluated for inhibition of OAT1 and OAT3. Six flavones (2, 3, 6-8, 12) showed potent inhibitory activity on OAT1, while seven flavones (2, 3, 6-9, 12) showed marked inhibitory activity on OAT3, with IC50 ≤ 10.0 µM. Antimicrobial activities of crude fractions against sixteen microorganisms were tested to give a target yeast strain Candida rugosa for further evaluation of MICs on the isolates. Three flavones (7, 8, 12) showed marked antifungal activity with MIC < 2.0 µM. To our knowledge, this study is the first to evaluate these flavones as inhibitors of the OAT1 and OAT3, and as antifungal agents.

Composition and cardioprotective effects of Primula veris L. solid herbal extract in experimental chronic heart failure.

BACKGROUND: High interest in chronic heart failure (CHF) is accounted for by its high incidence, poor prognosis, growing number of hospital admissions due to the heart failure relapse, and inadequate treatment. These facts necessitate a search for new pharmacological agents for the CHF correction. Herbal medicinal products appear to be very promising as they have a noticeable therapeutic effect and tend to be more harmless in comparison to the most of synthesized medications. PURPOSE: Our aim was to study the composition of the Primula veris L. solid herbal extract (PVSHE) and its effects on the myocardial contractile function in animals with experimental CHF. STUDY DESIGN: The study design involved the identification of the raw material composition of the P. veris L. extract. For the experimental part of our research, we used the model of CHF to elucidate the cardioprotective properties of PVSHE. METHODS: The active extract constituents were isolated by thin-layer chromatography and column chromatography; the extract components were identified by high-performance liquid chromatography, ultraviolet spectroscopy (UVS), and nuclear magnetic resonance spectroscopy (NMRS). To model CHF, L-isoproterenol at a dose of 2.5 mg/kg was intraperitoneally injected to the experimental rats twice a day for 21 days. Cardiac output was assessed with the loading test, adrenoreactivity test, and maximum isometric loading test; CHF markers adrenomedullin and copeptin were detected in blood plasma with ELISA kit for adrenomedullin and copeptin (Coud-Clone Corp., USA). RESULTS: P. veris L. solid herbal extract contains flavonoid aglycons (apigenin, quercetine, kaemferol), flavonoid glycosides (cinarozid, rutin, hyperozid), as well as polymethoxylated flavonoids acting as chemotaxonomic markers for the genus Primula (8-methoxy-flavone; 3',4'methylenedioxy-5'-methoxyflavone). The substance 3',4'methylenedioxy-5'-methoxyflavone has been isolated from the primrose herb for the first time. We showed that the PVSHE has a cardioprotective effect when it was administered at a dose of 30 mg/kg in the experimental CHF, as evidenced by a lower number of animal death, lower level of CHF markers in the blood plasma of the experimental animals, the higher increase in rate of myocardial contraction and relaxation, the higher level of left ventricular pressure (LVP) and of maximum intensity of structural performance (MISP), as compared to the control group. CONCLUSION: P. veris L. solid herbal extract contains flavonoid aglycons, flavonoid glycosides, and polymethoxylated flavonoids. The herbal agent increases the myocardial contractility in experimental CHF.

[Influence of the dense extract from herb of Primula veris L. On the oxidative stress development and the functional state of the cardiomyocytes mitochondria of rats with experimental chronic heart failure]. / Vliianie gustogo ékstrakta iz travy pervotsveta vesennego na razvitie oksidativnogo stressa i funktsional'noe sostoianie mitokhondrii kardiomiotsitov krys s éksperimental'noi khronicheskoi serdechnoi nedostatochnost'iu.

Experimental chronic heart failure (CHF), caused by administration of L-isoproterenol (2.5 mg/kg twice a day intraperitoneally for 21 days), promotes uncoupling of respiration and oxidative phosphorylation. The rate of mitochondrial oxygen consumption in the metabolic state V3 by Chance in animals with CHF decreased by 53.3% (p<0.05) with malate using (as an oxidation substrate feeding сomplex I of the electron transport chain (ETC)), by 70.6% (p<0.05) with succinate using (сomplex II substrate) and by 63.6% (p<0.05) when malate and succinate were added simultaneously. The respiratory control ratio significantly decreased 2.3 times for сomplex I, 2.5 for сomplex II, and 2.6 times for the simultaneous operation of two respiratory chain complexes in mitochondria of CHF rats compared to intact animals. Mitochondrial dysfunction in experimental CHF is evidently due to the development of oxidative stress. It was revealed that the content of malonic dialdehyde (MDA) in the group of rats with experimental CHF was higher by 54.7% (p<0.05), as compared with intact animals. The activity of superoxide dismutase (SOD) and catalase was lower by 17.5% (p<0.05), and by 18.4%, respectively than in the intact group. The dense extract from herba of Primula veris L. (DEHPV) 30 mg/kg limits the development of mitochondrial dysfunction in rats with experimental CHF, as evidenced by an increase in the role of V3 respiration for the first and second respiratory chain complexes in 1.7 (p<0.05) and 2.0 times (p<0.05), respectively, the ratio of respiratory control (RCR) - 1.7 times (p<0.05) for сomplex I and 2 times (p<0.05) for сomplex II compared with the negative control. The concentration of MDA was by 15.7% (p<0.05), lower and the activity of SOD was by 56.3% (p<0.05) higher.

Phytochemistry of European Primula species.

The genus Primula is the largest among the Primulaceae and is widespread mainly in the cold and temperate regions of the Northern Hemisphere. Since the beginning of the Twentieth century, several studies on the phytochemical composition of different species of Primula have been carried out. The main constituents examined were tissue and epicuticular flavonoids and saponins, which are of therapeutic significance. Only in recent years studies of the volatiles emitted by leaves and flowers have been carried out as well, but they are restricted to a small number of species. Only a few authors have documented the morphology and function of glandular trichomes in relation to the production of flavonoids and volatile organic compounds (VOCs). The use of Primula in folk medicine is described in the literature. Investigation of the biological and pharmacological activities of Primula are reported. This study aims at providing a collection of publications on the genus Primula along with a critical revision of literature data. It focuses on the possible taxonomic significance of the secondary metabolites and on their ecological role as attractors for pollinators and deterrents against herbivores and parasites, in order to build the base for further studies.

Striking Diversification of Exudate Profiles in Selected Primula Lineages.

In continuation of previous studies on glandular exudates of Primula, we analyzed eleven so far unstudied species and several populations for exudate composition. Unsubstituted flavone and unusually substituted flavones, normally predominant in Primula exudates, were not detected in all of the analyzed samples. Instead, some species exhibited regular substituted flavonoids, and in some cases, no flavonoids could be detected at all. The detection of a diterpene (1) in P. minima exudates is new to Primula. On basis of MS and NMR, 1 was structurally characterized as ent-kaur-16-en-19-oic acid. Comparative profiling of exudates as performed by HPLC and TLC against authentic markers indicated further the presence of the benzoquinone primin and derivatives in some exudates. Thus, exudates of newly studied species contrast markedly with those analyzed so far. The significance of observed exudate diversification is discussed in view of the phylogeny of derived lineages in European alpine regions.

Essential Fatty Acids as Transdermal Penetration Enhancers.

The aim of this study was to investigate the effect of different penetration enhancers, containing essential fatty acids (EFAs), on the transdermal delivery of flurbiprofen. Evening primrose oil (EPO), vitamin F, and Pheroid technology all contain fatty acids and were compared using a cream-based formulation. This selection was to ascertain whether EFAs solely, or EFAs in a Pheroid delivery system, would have a significant increase in the transdermal delivery of a compound. Membrane release studies were performed, and the results indicated the following rank order for flurbiprofen release from the different formulations: vitamin F >> control > EPO >> Pheroid. Topical skin delivery results indicated that flurbiprofen was present in the stratum corneum-epidermis and the epidermis-dermis. The average percentage flurbiprofen diffused to the receptor phase (representing human blood) indicated that the EPO formulation showed the highest average percentage diffused. The Pheroid formulation delivered the lowest concentration with a statistical significant difference (p < 0.05) compared with the control formulation (containing 1% flurbiprofen and no penetration enhancers). The control formulation presented the highest average flux, with the EPO formulation following the closest. It could, thus, be concluded that EPO is the most favorable chemical penetration enhancer when used in this formulation.

Altitude Variation in the Composition of Essential Oils, Fatty Acid Methyl Esters, and Antimicrobial Activities of Two Subspecies of Primula vulgaris Grown in Turkey.

In this study, the changes caused by variation of altitude to the essential oils (EOs), fatty acid methyl esters (FAMEs), and antimicrobial activities of Primula vulgaris Huds. subsp. vulgaris (Pvv) and P. vulgaris Huds. subsp. sibthorpii (Hoffmanns) W.W.Sm. & Forrest (Pvs)) grown in Turkey were investigated. Major fluctuations in the composition of Pvv and Pvs oils included methyl-4-methoxy salicylate (4.5-35.3%; Pvv and 3.2-37.2%; Pvs), (Z,Z,Z)-7,10,13- hexadecatrienal (5.1-21.8%; Pvv and 4.4-15.2%; Pvs ) and flavone (5.5-14.9%; Pvv and 1.6-18.0%; Pvs). Fatty acid profile (C60-CI6o) changes were noted in .Pvv and Pvs. Methyl hexadecanoate (2.4-9.3%) and methyl octadecanoate (1.0-4.7%) were present in all the FAME samples of the plants. The antimicrobial activity of the EOs of Pvv and Pvs were tested against nine bacterial species, which showed activity against Mycobacterium smegmatis with minimum inhibitory concentrations (MIC) varying from 8.5 to 59.2 pg/mL in all samples, respectively, depending on the altitude at which the oils were obtained.

[Treatment of cough in respiratory tract infections - the effect of combining the natural active compounds with thymol].

Cough is one of the characteristic symptoms of upper respiratory tract infections (URI). Herbal medicines are often used in the treatment of the cough associated with infection and to accelerate recovery or support the immune system. An example of such products are extracts of thyme and primrose, and also their combination with thymol. Thymus vulgaris is a spasmolytic, antimicrobial, anti-inflammatory, immunomodulatory and antioxidant agent. The most important component responsible for the activity of thyme is thymol contained in the volatile thyme oil. Primrose is, most of all, a saponine agent with expectorant and secretolytic activity, showing also spasmolytic, anti-inflammatory and antimicrobial effects. Because of the additive effects of the mentioned extracts and their wide activity profile they are often used as a combination drug. The use of this combination was assessed in the URI with the effects of alleviation of cough and dyspnea, and shortening the length of the disease. The effectiveness of the drug containing the extracts of thyme and primrose with the addition of thymol (Bronchosol®) was comparable to synthetic ambroxol and its safety has been proved. Moreover, the in-vitro antibacterial and antifungal effects of this drug have been evaluated.

Evaluation of Blood Alcohol Concentrations after Oral Administration of a Fixed Combination of Thyme Herb and Primrose Root Fluid Extract to Children with Acute Bronchitis.

BACKGROUND: The application of alcohol-containing medicinal products to children has been a subject of discussion for many years. A safety threshold of 0.125‰ blood alcohol concentration following the administration of a single dose has been recommended by the European Medicines Agency.The aim of this clinical study was to prove the safety of administering a fixed combination of thyme herb and primrose root fluid extract (Bronchicum(®) Elixir) containing 4.9% ethanol. METHODS: The herbal drug was administered for a period of 7-9 days to 16 children (ages 1-12 years) suffering from acute bronchitis for ≤ 48 h. After 3-5 days, a blood sample was taken 45 min (children ≥ 5 years: also 0 and 90 min) after application of the drug. The efficacy was assessed using the Bronchitis Severity Score. Global efficacy and tolerability were rated by the investigator and patients. RESULTS: All measured blood ethanol concentrations were below the threshold (mean value after 45 min: 0.0029 ± 0.0057‰ and after 90 min: 0.0051 ± 0.0078‰). The Bronchitis Severity Score decreased from 6.6 ± 1.0 to 0.9 ± 1.6 points. Global efficacy was assessed as "very good" and "good" in 60% (investigator) and 80% (patients) of cases. Global tolerability was rated as "very good" and "good" in more than 90% of cases. CONCLUSION: In conclusion, oral administration of the drug containing 4.9% ethanol to children (age 1-12 years) demonstrated a favourable risk/benefit ratio of the drug.

New flavonol glycosides from the leaves and flowers of Primula sieboidii.

Three flavonol glycosides were isolated from the leaves of Primula sieboldii. They were identified as quercetin 3-O-ß-[xylopyranosyl-(1-->2)-ß- glucopyranosyl-(1-->6)-ß-glucopyranoside] (1), kaempferol 3-O-ß-[glucopyranosyl-(1-->2)-ß-glucopyranosyl-(1-->6)-ß-glucopyranoside] (2) and kaempferol 3- O-ß-[xylopyranosyl-(1-->2)-ß-glucopyranosyl-(1-->6)-ß-glucopyranoside] (3). Their chemical structures were determined by UV, 1H and 13C NMR spectroscopy, LC-MS and acid hydrolysis. Compounds 1 and 3 are found in nature for the first time. They were also detected in the flowers, together with two anthocyanins, malvidin 3,5-di-O-glucoside and a minor petunidin dihexoside.

Primula latifolia Lapeyr. and Primula vulgaris Hudson flavonoids.

Three flavonoids were isolated from the leaf MeOH extracts of Primula latifolia Lapeyr. and Primula vulgaris Hudson collected from Italian Alps: rutin (1) and kaempferol 3-neohesperidoside (2) from P. latifolia, and kaempferol 3-ß-O-glucopyranosyl-(1 → 2) gentiobioside (3) from P. vulgaris. The structures were assigned on the basis of their (1)H and (13)C NMR data, including those derived from 2D NMR, as well as on HPLC-MS results. This article is the first to report on P. vulgaris tissue flavonoids after Harborne's study in 1968 and the first work ever on these compounds from P. latifolia.

Farinose alpine Primula species: phytochemical and morphological investigations.

This work investigated the epicuticular and tissue flavonoids, the volatiles and the glandular trichome structure of the leaves of four species of Primula L. that grow in the Italian Eastern Alps. Primula albenensis Banfi and Ferlinghetti, P. auricula L., P. farinosa L., P. halleri Gmelin produce farinose exudates that are deposited on the leaf surface as filamentous crystalloids. In addition to compounds already known, a new flavone, the 3,5-dihydroxyflavone, was isolated from the acetone extract of leaf farinas and three new flavonol glycosides, 3'-O-(ß-galactopyranosyl)-2'-hydroxyflavone, isorhamnetin 3-O-α-rhamnopyranosyl-(1→3)-O-[α-rhamnopyranosyl-(1→6)]-O-ß-galactopyranoside, quercetin 3-O-α-rhamnopyranosyl-(1→3)-O-[α-rhamnopyranosyl-(1→6)]-O-ß-galactopyranoside, were isolated from the MeOH extract of the leaves. All the structures were elucidated on the basis of their (1)H and (13)C NMR data and 2D NMR techniques, as well as on HPLC-MS. The leaf-volatiles emitted by these Primula species were mainly sesquiterpene hydrocarbons, with the exception of P. albenensis, which produced almost exclusively a non-terpene derivative; P. halleri flowers were also examined and the volatiles emitted by the flower parts (corolla and calyx) were compared with the corresponding leaves.

Orphan flavonoids and dihydrochalcones from Primula exudates.

Two orphan flavonoids containing an oxepin structure in ring A and named as Primcortusin (1) and 3'-OH-Primcortusin (2) were isolated from leaf exudates of Primula cortusoides, while P. glutinosa exudates yielded two dihydrochalcone derivatives (3,4). These novel structures have not been detected in other species of Primula so far. Chemical structures were elucidated by 2D NMR and mas spectrometry. The nature of compounds 1 and 2 is discussed, and ideas about their possible origin and that of unsubstituted flavone and other irregular substituted Primula flavones are presented.