Database-aided ultrahigh-performance liquid chromatography Q-Exactive-Orbitrap tandem mass spectrometry putatively identifies 16 unexpected compounds and three anticounterfeiting pharmacopoeia quality markers for Perillae Fructus.

RATIONALE: Perillae Fructus (PF) is a common traditional Chinese medicine (TCM) for the treatment of asthma. It has not been effectively characterized by rosmarinic acid (RosA), which is currently designed as the sole quality indicator in the Chinese Pharmacopoeia. METHODS: This study introduced a database-aided ultrahigh-performance liquid chromatography equipped with quadrupole-Exactive-Orbitrap mass spectrometry (UHPLC/Q-Exactive-Orbitrap MS/MS) technology to putatively identify the compounds in PF, followed by literature research, quantum chemical calculation, and molecular docking to screen potential quality markers (Q-markers) of PF. RESULTS: A total of 27 compounds were putatively identified, 16 of which had not been previously found from PF. In particular, matrine, scopolamine, and RosA showed relatively high levels of content, stability, and drug-likeness. They exhibited interactions with the asthma-related target and demonstrated the TCM properties of PF. CONCLUSIONS: The database-aided UHPLC/Q-Exactive-Orbitrap MS/MS can identify at least 27 compounds in PF. Of these, 16 compounds are unexpected, and three compounds (matrine, scopolamine, and RosA) should be considered anticounterfeiting pharmacopoeia Q-markers of PF.

Protective Effect of Perilla Seed Meal and Perilla Seed Extract against Dextran Sulfate Sodium-Induced Ulcerative Colitis through Suppressing Inflammatory Cytokines in Mice.

An excessive inflammatory response of the gastrointestinal tract is recognized as one of the major contributors to ulcerative colitis (UC). Despite this, effective preventive approaches for UC remain limited. Rosmarinic acid (RA), an enriched fraction from Perilla frutescens, has been shown to exert beneficial effects on disease-related inflammatory disorders. However, RA-enriched perilla seed meal (RAPSM) and perilla seed (RAPS) extracts have not been investigated in dextran sulfate sodium (DSS)-induced UC in mice. RAPSM and RAPS were extracted using the solvent-partitioning method and analyzed with high-pressure liquid chromatography (HPLC). Mice with UC induced using 2.5% DSS for 7 days were pretreated with RAPSM and RAPS (50, 250, 500 mg/kg). Then, the clinical manifestation, colonic histopathology, and serum proinflammatory cytokines were determined. Indeed, DSS-induced UC mice exhibited colonic pathological defects including an impaired colon structure, colon length shortening, and increased serum proinflammatory cytokines. However, RAPSM and RAPS had a protective effect at all doses by attenuating colonic pathology in DSS-induced UC mice, potentially through the suppression of proinflammatory cytokines. Concentrations of 50 mg/kg of RAPSM and RAPS were sufficient to achieve a beneficial effect in UC mice. This suggests that RAPSM and RAPS have a preventive effect against DSS-induced UC, potentially through alleviating inflammatory responses and relieving severe inflammation in the colon.

Effects of the dose of administration, co-antioxidants, food matrix, and digestion-related factors on the in vitro bioaccessibility of rosmarinic acid - A model study.

This study aimed to determine the effect of the administration dose, combinations with co-antioxidants (vitamin C, caffeic acid, chlorogenic acid, catechin, rutin), and different food matrices (cooked and lyophilized hen eggs, chicken breast, soybean seeds, potatoes) on the potential bioaccessibility of rosmarinic acid (RA) in simulated digestion conditions, depending on the digestion stage (gastric and intestinal) and the contribution of physicochemical and biochemical digestion factors. The in vitro bioaccessibility of RA depended on the digestion stage and conditions. The physicochemical factors were mainly responsible for the bioaccessibility of RA applied alone. The higher RA doses improved its bioaccessibility, especially at the intestinal stage of digestion. Furthermore, the addition of vitamin C and protein-rich food matrices resulted in enhanced intestinal bioaccessibility of RA. In the future, the knowledge of factors influencing the bioaccessibility of RA can help enhance its favorable biological effects and therapeutic potential.

α-Glucosidase Inhibitory Components from Garcinia pedunculata Fruits.

From Garcinia pedunculata Roxb. fruits, two undescribed aromatic compounds including a benzofuran and a depsidone derivative, and a new natural product, together with four known compounds were isolated. Through the analysis of spectroscopic data, high resolution mass spectrum and calculated nuclear magnetic resonance, their structures were determined. The α-glucosidase inhibitory activity of the isolates was evaluated. And compound 3 exhibited a moderate inhibitory effect on α-glucosidase. The molecular docking of compound 3 was performed to elucidate the interaction with α-glucosidase.

Mechanism Behind the Programmed Biosynthesis of Heterotrimeric Fungal Depside Thielavin A.

Thielavin A (1) is a fungal depside composed of one 3-methylorsellinic acid and two 3,5-dimethylorsellinic acid units. It displays diverse biological activities. However, the mechanism underlying the assembly of the heterotrimeric structure of 1 remains to be clarified. In this study, we identified the polyketide synthase (PKS) involved in the biosynthesis of 1. This PKS, designated as ThiA, possesses an unusual domain organization with the C-methyltransferase (MT) domain situated at the C-terminus following the thioesterase (TE) domain. Our findings indicated that the TE domain is solely responsible for two rounds of ester bond formation, along with subsequent chain hydrolysis. We identified a plausible mechanism for TE-catalyzed reactions and obtained insights into how a single PKS can selectively yield a specific heterotrimeric product. In particular, the tandem acyl carrier protein domains of ThiA are critical for programmed methylation by the MT domain. Overall, this study highlighted the occurrence of highly optimized domain-domain communication within ThiA for the selective synthesis of 1, which can advance our understanding of the programming rules of fungal PKSs.

Anti-Inflammatory and Antioxidant Effects of Rosmarinic Acid Trimetallic (Cu0.5Zn0.5Fe2O4) Nanoparticles.

Newly, green metallic-nanoparticles (NPs) have received scientists' interest due to their wide variable medicinal applications owned to their economical synthesis and biologically compatible nature. In this study, we used rosmarinic acid (RosA) to prepare Cu0.5Zn0.5FeO4 NPs and later encapsulated them using PEG polymer. Characterization of NPs was done using the XRD method and SEM imaging. Further, we explored the encapsulated NPs for anti-inflammatory properties by downregulating the expression of pro-inflammatory cytokines mRNA in LPS-stimulated Raw 264.7 cells. Besides, employing DPPH, NO and ABTS radical scavenging assays to examine the antioxidant activity of the synthesized Cu0.5Zn0.5FeO4 NPs. Cu0.5Zn0.5FeO4 NPs revealed moderate antioxidant activity by scavenging DPPH and nitric oxide. We demonstrated that the NPs showed high potential anti-inflammatory activity by suppressing the mRNA and protein levels of pro-inflammatory cytokines in a dose-dependent manner, in LPS-induced Raw 264.7 cells. To our best knowledge, this is the first report where RosA was found to be a suitable phyto source for the green synthesis of Cu0.5Zn0.5FeO4 NPs and their in vitro anti-inflammatory and antioxidant effects. Taken together, our findings suggest that the RosA is a green resource for the eco-friendly synthesis of Cu0.5Zn0.5FeO4/PEG NPs, which further can be employed as a novel anti-inflammatory therapeutic agent.

Botryorhodines K and L, two new cytotoxic depsidones from a fungus of the genus Arcopilus.

Botryorhodines K (1) and L (2), two new depsidone derivatives, along with one known metabolite, 4-O-demethylbarbatic acid (3), were isolated from the culture extract of a fungus of the genus Arcopilus. The structures of 1‒3 were determined by the analysis of NMR and MS spectral data and the absolute configuration of 1 was established by single-crystal X-ray diffraction analysis. Compounds 1 and 2 showed antimicrobial activity against Gram-positive bacteria and cytotoxicity against murine leukemia P388 cells.

The Influence of Methyl Jasmonate on Expression Patterns of Rosmarinic Acid Biosynthesis Genes, and Phenolic Compounds in Different Species of Salvia subg. Perovskia Kar L.

Salvia yangii B.T. Drew and Salvia abrotanoides Kar are two important fragrant and medicinal plants that belong to the subgenus Perovskia. These plants have therapeutic benefits due to their high rosmarinic acid (RA) content. However, the molecular mechanisms behind RA generation in two species of Salvia plants are still poorly understood. As a first report, the objectives of the present research were to determine the effects of methyl jasmonate (MeJA) on the rosmarinic acid (RA), total flavonoid and phenolic contents (TFC and TPC), and changes in the expression of key genes involved in their biosynthesis (phenylalanine ammonia lyase (PAL), 4-coumarate-CoA ligase (4CL), and rosmarinic acid synthase (RAS)). The results of High-performance liquid chromatography (HPLC) analysis indicated that MeJA significantly increased RA content in S. yungii and S. abrotanoides species (to 82 and 67 mg/g DW, respectively) by 1.66- and 1.54-fold compared with untreated plants. After 24 h, leaves of Salvia yangii and Salvia abrotanoides species treated with 150 M MeJA had the greatest TPC and TFC (80 and 42 mg TAE/g DW, and 28.11 and 15.14 mg QUE/g DW, respectively), which was in line with the patterns of gene expression investigated. Our findings showed that MeJA dosages considerably enhanced the RA, TPC, and TFC contents in both species compared with the control treatment. Since increased numbers of transcripts for PAL, 4CL, and RAS were also detected, the effects of MeJA are probably caused by the activation of genes involved in the phenylpropanoid pathway.

Deciphering the therapeutical potentials of rosmarinic acid.

Lemon balm is herbal tea used for soothing stomach cramps, indigestion, and nausea. Rosmarinic acid (RA) is one of its chemical constituents known for its therapeutic potentials against cancer, inflammatory and neuronal diseases such as the treatment of neurofibromatosis or prevention from Alzheimer's diseases (AD). Despite efforts, recovery and purification of RA in high yields has not been entirely successful. Here, we report its aqueous extraction with optimal conditions and decipher the structure by nuclear magnetic resonance (NMR) spectroscopy. Using various physical-chemical and biological assays, we highlight its anti-aggregation inhibition potentials against the formation of Tau filaments, one of the hallmarks of AD. We then examine its anti-cancer potentials through reduction of the mitochondrial reductase activity in tumor cells and investigate its electrochemical properties by cyclic voltammetry. Our data demonstrates that RA is a prominent biologically active natural product with therapeutic potentials for drug discovery in AD, cancer therapy and inflammatory diseases.

A Comprehensive Review of Rosmarinic Acid: From Phytochemistry to Pharmacology and Its New Insight.

Polyphenolic acids are the widely occurring natural products in almost each herbal plant, among which rosmarinic acid (RA, C18H16O8) is well-known, and is present in over 160 species belonging to many families, especially the Lamiaceae. Aside from this herbal ingredient, dozens of its natural derivatives have also been isolated and characterized from many natural plants. In recent years, with the increasing focus on the natural products as alternative treatments, a large number of pharmacological studies have been carried out to demonstrate the various biological activities of RA such as anti-inflammation, anti-oxidation, anti-diabetes, anti-virus, anti-tumor, neuroprotection, hepatoprotection, etc. In addition, investigations concerning its biosynthesis, extraction, analysis, clinical applications, and pharmacokinetics have also been performed. Although many achievements have been made in various research aspects, there still exist some problems or issues to be answered, especially its toxicity and bioavailability. Thus, we hope that in the case of natural products, the present review can not only provide a comprehensive understanding on RA covering its miscellaneous research fields, but also highlight some of the present issues and future perspectives worth investigating later, in order to help us utilize this polyphenolic acid more efficiently, widely, and safely.

Biomedical features and therapeutic potential of rosmarinic acid.

For decades, the use of secondary metabolites of various herbs has been an attractive strategy in combating human diseases. Rosmarinic acid (RA) is a bioactive phenolic compound commonly found in plants of Lamiaceae and Boraginaceae families. RA is biosynthesized using amino acids tyrosine and phenylalanine via enzyme-catalyzed reactions. However, the chemical synthesis of RA involves an esterification reaction between caffeic acid and 3,4-dihydroxy phenyl lactic acid contributing two phenolic rings to the structure of RA. Several studies have ascertained multiple therapeutic benefits of RA in various diseases, including cancer, diabetes, inflammatory disorders, neurodegenerative disorders, and liver diseases. Many previous scientific papers indicate that RA can be used as an anti-plasmodic, anti-viral and anti-bacterial drug. In addition, due to its high anti-oxidant capacity, this natural polyphenol has recently gained attention for its possible application as a nutraceutical compound in the food industry. Here we provide state-of-the-art, flexible therapeutic potential and biomedical features of RA, its implications and multiple uses. Along with various valuable applications in safeguarding human health, this review further summarizes the therapeutic advantages of RA in various human diseases, including cancer, diabetes, neurodegenerative diseases. Furthermore, the challenges associated with the clinical applicability of RA have also been discussed.

Intramolecular transesterification of depsides yields fluorescent 1H-isochromen-1-ones: Application as a chemical probe for lichen determination.

The reactivity of eight purified depsides obtained from six european lichens and that display as 2-oxoalkyl chain in ortho-position of the ester bond was explored. These depsides were found to lead to 1H-Isochromen-1-ones, which exhibit a distinctive blue fluorescence at 365 nm, in the presence of a 10% aqueous solution of KOH. A mechanistic explanation, involving the formation of an enolate intermediate and intramolecular transesterification, was proposed and validated by DFT. By exploiting this fluorescent phenomenon, we conceived a chemical probe (the KUV probe) that is useful for lichen determination, as exemplified on a selection of European Porpidia species.

The Role of Rosmarinic Acid on the Bioproduction of Gold Nanoparticles as Part of a Photothermal Approach for Breast Cancer Treatment.

Breast cancer is a high-burden malignancy for society, whose impact boosts a continuous search for novel diagnostic and therapeutic tools. Among the recent therapeutic approaches, photothermal therapy (PTT), which causes tumor cell death by hyperthermia after being irradiated with a light source, represents a high-potential strategy. Furthermore, the effectiveness of PTT can be improved by combining near infrared (NIR) irradiation with gold nanoparticles (AuNPs) as photothermal enhancers. Herein, an alternative synthetic method using rosmarinic acid (RA) for synthesizing AuNPs is reported. The RA concentration was varied and its impact on the AuNPs physicochemical and optical features was assessed. Results showed that RA concentration plays an active role on AuNPs features, allowing the optimization of mean size and maximum absorbance peak. Moreover, the synthetic method explored here allowed us to obtain negatively charged AuNPs with sizes favoring the local particle accumulation at tumor site and maximum absorbance peaks within the NIR region. In addition, AuNPs were safe both in vitro and in vivo. In conclusion, the synthesized AuNPs present favorable properties to be applied as part of a PTT system combining AuNPs with a NIR laser for the treatment of breast cancer.

Extraction, preparative monomer separation and antibacterial activity of total polyphenols from Perilla frutescens.

Polyphenols exhibit potential functional activities, especially rosmarinic acid (RosA) and caffeic acid (CafA). In this study, two different methods, ultrasonic-assisted ethanol extraction (60%) and ultrasound-assisted cellulase (≥15 000 Ug-1, 2%) hydrolysis, were used for the extraction of the total phenolics from 44 species of Perilla frutescens. The Folin-Ciocalteu method of detection showed that the content of the total phenolics extracted by cellulase hydrolysis was the highest and attained up to 28.00 mgGAE per gextracts for ZB1. Continuously, the extracts were purified using XDA-8 macroporous resin and medium-pressure liquid chromatography (MPLC), and the content of the total phenolics improved to 66.62 mgGAE per gextract. A high-performance liquid chromatography (HPLC) assay showed that the total polyphenols were mainly composed of gallic acid, caffeic acid, rosmarinic acid, luteolin and apigenin. Besides, a sequential XDA-8 macroporous resin combined with high-speed counter-current chromatography (HSCCC)/MPLC system was established for the simultaneous isolation and preparation of RosA (purity 98.29%) and CafA (purity 97.01%) from the extracts. Furthermore, the antibacterial activities of the total polyphenols were evaluated by the disc diffusion method and scanning electron microscopy (SEM) observation. The results verified that the total polyphenols had effective antibacterial activity on three kinds of bacteria including E. coli, S. aureus, and B. subtilis in a concentration-dependent manner. All of these results demonstrated that the ultrasound-assisted cellulase hydrolysis extraction of the total polyphenols and the proposed three-step separation of RosA and CafA gave high yields and good purity, and they exhibited effective antibacterial ability.

A new depsidone from the lichen Usnea ceratina Arch.

Chemical investigation of the lichen Usnea ceratina Arch led to the isolation of five depsidones, including one new compound ceratinalone (1) along with four known compounds bailesidone (2), stictic acid (3), 8'-O-methylstictic acid (4) and 8'-O-ethylstictic acid (5). The structures were determined by analysis of their MS and NMR data as well as by comparison with literature values. Compounds 1 and 4 were evaluated the cytotoxic activity against HeLa (human epithelial carcinoma), NCI-H460 (human lung cancer), HepG2 (liver hepatocellular carcinoma), and MCF-7 (human breast cancer) cell lines, showing the moderate activity.

Sedative-hypnotic, anxiolytic and possible side effects of Salvia limbata C. A. Mey. Extracts and the effects of phenological stage and altitude on the rosmarinic acid content.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia limbata C. A. Mey. (Persian name: Maryam Goli-e-labeh dar) has been used for treating central nervous disorders such as insomnia, anxiety and depression in Persian traditional medicine. S. limbata is known for its pharmacological activities which could be at least in a part, upon the presence of rosmarinic acid (RA). However, the sedative-hypnotic effect, anxiolytic activity, possible side effects, and the mechanism of action of S. limbata extract has not yet been examined. AIM OF THE STUDY: In the current study the sedative-hypnotic effect, anxiolytic activity, possible side effects, and the mechanism of action of S. limbata extracts were evaluated. Besides, the effects of altitude and phenological stage on the RA content of S. limbata were investigated. MATERIALS AND METHODS: Sedative-hypnotic and anxiolytic effects were evaluated through the pentobarbital induced loss of righting reflex test and open field test, respectively. Flumazenil was used to reveal the mechanism of action. Possible side effects were investigated in the passive avoidance and grip strength tests. Besides, the effects of altitude and phenological stage (vegetative, flowering, and seed setting) on the RA content of S. limbata were evaluated using reversed-phase high-performance liquid chromatography (RP-HPLC). RESULTS: Following behavioral tests, sedative-hypnotic and anxiolytic effects were observed. Since the observed effects were reversed by flumazenil and no side effect on the memory and muscle strength was reported, modulation of the α1-containing GABA-A receptors could be proposed as one of the involved mechanisms. According to the RP-HPLC analysis, harvesting S. limbata in the vegetative stage at the altitude of 2500 m led to the highest content of RA (8.67 ± 0.13 mg/g dry matter). Among different extract of the plant samples collected in the vegetative stage at the altitude of 2500 m, the hydroalcoholic extract showed the highest rosmarinic acid content. CONCLUSION: The obtained results help to find the optimum situation to gain the highest content of RA as well as the pharmacological activity that could be economically important for the pharmaceutical industries.

Parmosidone K, a new meta-depsidone from the lichen Parmotrema tsavoense.

Further phytochemical investigation on P. tsavoense led to one new meta-depsidone, parmosidone K together with one known compound, barbatic acid. Their structures were determined by 1D and 2D NMR analysis, high resolution mass spectroscopy, and comparison their NMR data with those reported in literatures. Parmosidone K was evaluated for α-glucosidase inhibition and revealed the powerful activity with IC50 value of 3.12 µM.

Inhibitor-Mediated Structural Transition in a Minimal Amyloid Model.

Despite the fundamental clinical importance of amyloid fibril formation, its mechanism is still enigmatic. Crystallography of minimal amyloid models was a milestone in the understanding of the architecture and biological activities of amyloid fibers. However, the crystal structure of ultimate dipeptide-based amyloids is not yet reported. Herein, we present the crystal structure of a typical amyloid-forming minimal dipeptide, Ac-Phe-Phe-NH2 (Ac-FF-NH2 ), showing a canonical ß-sheet structure at the atomic level. The simplicity of the structure helped in investigating amyloid-inhibition using crystallography, never previously reported for larger peptide models. Interestingly, in the presence of an inhibitor, the supramolecular packing of Ac-FF-NH2 molecules rearranged into a supramolecular 2-fold helix (21 helix). This study promotes our understanding of the mechanism of amyloid formation and of the structural transitions that occur during the inhibition process in a most fundamental model.

Phenethyl ester of rosmarinic acid attenuates autoimmune responses during type 1 diabetes development in mice.

AIMS: Rosmarinic acid (RA) is a polyphenol that occurs in plants of the Lamiaceae family. Phenethyl ester of RA (PERA), a novel RA derivative, has been developed and evaluated in vivo in an animal model of type 1 diabetes (T1D). METHODS: T1D was induced in male C57BL/6 mice using multiple low doses of streptozotocin (STZ) administered intraperitoneally for 5 consecutive days. Intraperitoneal administration of PERA (2.5 mg/kg bw) began from the first STZ injection and continued for 20 days. KEY FINDINGS: PERA-treated mice exhibited lower incidence of T1D (monitored up to 38 days from the disease induction), and fluorescent histochemical analysis showed that their pancreatic islets expressed more insulin. PERA treatment significantly down-regulated the proportions of CD11b+ and CD11c+ myeloid cells in the immune cell infiltrates in the pancreatic islets early during T1D pathogenesis (on day 9 after T1D induction), while on day 15, PERA significantly reduced the proportions of CD11c+, CD8+, Th1 and Th17 cells. Simultaneously, it was found that the cells from the pancreatic infiltrates of PERA-treated mice produced significantly less reactive oxygen species than cells from the control group. SIGNIFICANCE: These findings suggest that PERA efficiently prevented T1D development in mice. Interestingly, PERA attenuated the inflammatory process in the islets through temporally specific interference with the innate and adaptive immune response and therefore shows great promise for further clinical evaluation as a novel T1D therapeutic.

Rosmarinic Acid and Sodium Citrate Have a Synergistic Bacteriostatic Effect against Vibrio Species by Inhibiting Iron Uptake.

BACKGROUND: New strategies are needed to combat multidrug-resistant bacteria. The restriction of iron uptake by bacteria is a promising way to inhibit their growth. We aimed to suppress the growth of Vibrio bacterial species by inhibiting their ferric ion-binding protein (FbpA) using food components. METHODS: Twenty spices were selected for the screening of FbpA inhibitors. The candidate was applied to antibacterial tests, and the mechanism was further studied. RESULTS: An active compound, rosmarinic acid (RA), was screened out. RA binds competitively and more tightly than Fe3+ to VmFbpA, the FbpA from V. metschnikovii, with apparent KD values of 8 µM vs. 17 µM. Moreover, RA can inhibit the growth of V. metschnikovii to one-third of the control at 1000 µM. Interestingly, sodium citrate (SC) enhances the growth inhibition effect of RA, although SC only does not inhibit the growth. The combination of RA/SC completely inhibits the growth of not only V. metschnikovii at 100/100 µM but also the vibriosis-causative pathogens V. vulnificus and V. parahaemolyticus, at 100/100 and 1000/100 µM, respectively. However, RA/SC does not affect the growth of Escherichia coli. CONCLUSIONS: RA/SC is a potential bacteriostatic agent against Vibrio species while causing little damage to indigenous gastrointestinal bacteria.