Optimization of extraction with salicylic acid, rheological behavior and antiproliferative activity of pectin from Citrus sinensis peels.

A Box-Behnken design was used to optimize extraction temperature, extraction time and concentration of the salicylic acid to obtain a maximum polysaccharide yield from Citrus sinensis peels. The optimal settings were: extraction time 3 h, extraction temperature 80 °C and concentration of the salicylic acid 1.5%. Under these conditions, the experimental yield and uronic acid content were 11.74% and 66.9% respectively. Preliminary characterization was performed via FT-IR, SEC/MALS/VD/DRI and GC-MS after hydrolysis. SEC analysis showed that the extracted polysaccharide had a weight average molar mass of 350 kDa and an intrinsic viscosity of 640 mL/g. The GC-MS results revealed that the extracted polysaccharide was composed of arabinose 56.7%, galactose 17.8%, xylose 13.8%, rhamnose 5.1%, mannose 2.5% and glucose 1.5% suggested a rhamnogalacturonan pectin type I with a degree of esterification of 50.9% (IRTF). The flow curve and the dynamic frequency sweep were obtained at 10, 20, 30 and 40 g/L in water and at 30 g/L in presence of CaCl2 or NaCl at 1 mol/L. The solutions showed shear-thinning behavior fitted with Ostwald-De Waele model, except 10 g/L with a Newtonian behavior. The apparent viscosity and, the G' and G" moduli increase with PACO concentration in agreement with a slow-down of the dynamic chain. In the presence of CaCl2 or NaCl the reduction of electrostatic repulsions between pectin chains decreases the rheological parameters. The effect is less sensitive with CaCl2 due to intermolecular interactions. The antiproliferative activity of the extracted pectin on human Caco-2 and Hep-2 cells was very interesting with an IC50 1.4 and 1.8 µg/mL respectively.

A new aliphatic ester of hydroxysalicylic acid from fermented Carica papaya L. preparation with a potential hair growth stimulating activity.

An aliphatic ester of hydroxysalicylic acid (6), reported for the first time from a natural source in addition to five known compounds were isolated from the fermented Carica papaya L. preparation, a commercialized functional food. The known compounds were identified as 5-hydroxymethylfurfuraldehyde (1), trans-caffeic acid (2), butyl 4-hydroxybenzoate (butylparaben) (3), lycopene (4), benzyl isothiocyanate (5). Compounds 1 and 3 were reported for the first time from Papaya fruits through this study. The new compound showed a moderate antioxidant activity and a potent hair growth stimulating activity in vitro.

Systematic evaluation of sample preparation for fractionation of phytohormone salicylic acid in fresh leaves.

The determination of salicylic acid (SA), an important phytohormone responsible for stress signaling in plants, is of great importance in agricultural studies. However, a critical evaluation of the procedures for the extraction of the analytes and hydrolysis of the conjugated forms of SA is lacking in the literature and the available alternatives are complex, time-consuming, and laborious. In this study, the sample preparation methods for SA fractionation were critically evaluated to develop a simpler and faster alternative procedure. Microwave-assisted extractions were carried out with 2.0 g of fresh leaves and 8.0 mL of a 75% v/v ethanol:water solution at 40 °C for 10 min, followed by alkaline hydrolysis using 100 µL of 0.1 mol L-1 NaOH at 80 °C for 60 min. Free and total SA were determined in crude and hydrolyzed extracts, respectively, by fluorimetry after chromatographic separation of the sample matrix under isocratic elution (25% v/v acetonitrile/phosphate buffer) using a C18 column. Recovery experiments using methyl salicylate and acetylsalicylic acid model compounds demonstrated that the soft microwave-assisted extraction did not decompose the SA derivatives and that alkaline hydrolysis was quantitative. The proposed procedure was successfully applied for fractionation of SA in sugarcane, corn, and soybean leaves with extraction and hydrolysis yields up to 70 and 20% higher than those achieved in previously proposed approaches, respectively. The developed procedure is a simple, fast, and reliable alternative for SA fractionation in crude extracts without sample clean-up, and utilizes dilute reagents and green solvents.

Methyl Salicylate Glucosylation Regulates Plant Defense Signaling and Systemic Acquired Resistance.

Plant systemic acquired resistance (SAR) provides an efficient broad-spectrum immune response to pathogens. SAR involves mobile signal molecules that are generated by infected tissues and transported to systemic tissues. Methyl salicylate (MeSA), a molecule that can be converted to salicylic acid (SA), is an essential signal for establishing SAR, particularly under a short period of exposure to light after pathogen infection. Thus, the control of MeSA homeostasis is important for an optimal SAR response. Here, we characterized a uridine diphosphate-glycosyltransferase, UGT71C3, in Arabidopsis (Arabidopsis thaliana), which was induced mainly in leaf tissue by pathogens including Pst DC3000/avrRpt2 (Pseudomonas syringae pv tomato strain DC3000 expressing avrRpt2). Biochemical analysis indicated that UGT71C3 exhibited strong enzymatic activity toward MeSA to form MeSA glucosides in vitro and in vivo. After primary pathogen infection by Pst DC3000/avrRpt2, ugt71c3 knockout mutants exhibited more powerful systemic resistance to secondary pathogen infection than that of wild-type plants, whereas systemic resistance in UGT71C3 overexpression lines was compromised. In agreement, after primary infection of local leaves, ugt71c3 knockout mutants accumulated significantly more systemic MeSA and SA than that in wild-type plants. whereas UGT71C3 overexpression lines accumulated less. Our results suggest that MeSA glucosylation by UGT71C3 facilitates negative regulation of the SAR response by modulating homeostasis of MeSA and SA. This study unveils further SAR regulation mechanisms and highlights the role of glucosylation of MeSA and potentially other systemic signals in negatively modulating plant systemic defense.

Chitosan tailor-made membranes as biopolymeric support for electromembrane extraction.

A chitosan membrane composed by 60% (w/w) chitosan and 40% (w/w) Aliquat®336 has been proposed as a new biopolymeric support for electromembrane extraction. The new support has been characterized by Scanning Electron Microscopy, resulting a 30-35 µm thickness. Amoxicillin, nicotinic acid, hippuric acid, salicylic acid, anthranilic acid, ketoprofen, naproxen and ibuprofen have been successfully extracted using the proposed support. Better enrichment factors were obtained for the acidic polar analytes than for the non-steroidal anti-inflammatory compounds (ranging from 118 for hippuric acid and 20 for ibuprofen). Electromembrane extraction was developed applying a DC voltage of 100 V, 1-octanol as supported liquid membrane and 20 min of extraction. The target analytes have also been satisfactorily extracted from human urine samples, providing high extraction efficiencies. The chitosan membrane is presented as a promising alternative for supporting liquid membrane compared to commonly used materials for this purpose.

Preparation of inverse opal adsorbent by water-soluble colloidal crystal template to obtain ultrahigh adsorption capacity for salicylic acid removal from aqueous solution.

Diethylenetriamine (DETA) modified inverse opal (IO) poly (glycidyl methacrylate-co-divinylbenzene) (IO PGMD-DETA) was fabricated through the water-soluble colloidal crystal template and further applied as salicylic acid (SA) adsorbent with the ultrahigh adsorption capacity in aqueous solution. Compared that with the reported adsorbents, IO PGMD-DETA possessed the performance of ultrahigh adsorption capacity, comparatively short equilibration time, and relatively good reusability. The influences of the specific surface area and group content on adsorption capacity showed that the high content of group was favorable to improve the hydrophilicity and polarity of the materials, resulting in high adsorption capacity. IO PGMD-DETA-95 (the number of 95 is the mass ratio of GMA monomer) exhibited the largest adsorption capacity towards SA. Langmuir and Liu models could well fit the data of isothermal adsorption. The maximum adsorption capacity was estimated by the Langmuir and Liu models, which could reach out to an excellent value of 905.0 mg/g and 908.4 mg/g at 40 ℃ respectively. Furthermore, the pseudo-first-order rate equation could describe the kinetic data. The adsorption equilibrium of SA on IO PGMD-DETA-95 could be reached within 30-40 min. After five adsorption-desorption cycles, the excellent performance of IO PGMD-DETA-95 and the morphology were well retained.

Preparation of particle electrodes from manganese slag and its degradation performance for salicylic acid in the three-dimensional electrode reactor (TDE).

Salicylic acid (SA) is a class of trace pollutants widely presented in the environment belonged to pharmaceuticals and personal care products. It is difficult to remove SA by the traditional treatment processes because of its toxicity. In this paper, the degradation of SA by Mn-loaded Cu/Fe particle electrodes was studied. Firstly, the particle electrodes were prepared by impregnation-roasting method and then characterized by SEM, XRF and XRD. The diffraction peaks of Fe2O3 and CuO in the XRD patterns of the particle electrodes which had the dense spherical particles were significantly increased and the content of CuO and Fe2O3 increased by 1.9% and 3.6% respectively. Secondly, single factor experiments were carried out under conditions of cell voltage, electrolyte concentration, pH, HRT, inter-electrode distance and initial pollutant concentration. Under the optimum conditions of all the factors, the degradation rate of SA reached 76.9%. Then, HPLC and GCMS were employed to deduce the degradation pathways of SA by the TDE with Mn-particle electrodes (Mn-PETDE). Under the action of •OH, SA underwent decarboxylation and substitution reactions and then mineralized after the ring-opening reaction. All results demonstrated that this Mn-PETDE was effective for degradation of SA.

Use of Polymer Inclusion Membranes (PIMs) as support for electromembrane extraction of non-steroidal anti-inflammatory drugs and highly polar acidic drugs.

The use of polymer inclusion membranes (PIMs) as support of 1-octanol liquid membrane in electromembrane extraction (EME) procedure is proposed. Synthesis of PIMs were optimized to a composition of 29% (w/w) of cellulose triacetate as base polymer and 71% (w/w) of Aliquat®336 as cationic carrier. Flat PIMs of 25µm thickness and 6mm diameter were used. EME protocol was implemented for the simultaneous extraction of four non-steroidal anti-inflammatory drugs (NSAIDs) (salicylic acid, ketoprofen, naproxen and ibuprofen) and four highly polar acidic drugs (anthranilic acid, nicotinic acid, amoxicillin and hippuric acid). Posterior HPLC separation of the extracted analytes was developed with diode array detection. Recoveries in the 81-34% range were obtained. EME procedure was applied to human urine samples.

Ion cum molecularly dual imprinted polymer for simultaneous removal of cadmium and salicylic acid.

Ion cum molecularly dual imprinted polymer (DMIP) was synthesized for the simultaneous removal of salicylic acid (SA) and cadmium (Cd) by suspension polymerization method using chitosan (CTS) as functional polymer, epichlorohydrin as cross-linker, and 4-hydroxy benzoic acid (4HBA) as well as Cd as organic and inorganic templates, respectively. Use of the dummy template 4HBA during the synthesis of DMIP had the advantage of creating imprinted cavities in DMIP, which depicted good uptake for SA. Scanning electron microscopy and Fourier transform infrared spectroscopy indicated successful preparation of DMIP. Particle size analysis confirmed polydispersity, and thermal and swelling studies indicated the mechanical stability in DMIP. The rebinding capacities of the DMIP for Cd and SA were found to be 38.46 and 23.81 mgg-1 , respectively, under the optimize condition of the time, dose, and concentration. Adsorption isotherm results fitted into Langmuir adsorption isotherm model with the R2 values of 0.994 and 0.995 for Cd and SA, respectively. The presence of intramolecular hydrogen bonding in SA, stability of the template-monomer complexes (CTS-SA and CTS-4HBA), and the involvement of the hydroxyl groups on DMIP for the uptake of SA has been supported by molecular modeling studies using Gaussian 03 software. The electron doublet of the amino groups of DMIP was involved for the uptake of Cd. Lower binding efficiency of DMIP for SA as compared to Cd may be due to the partial participation of hydroxyl group in cross-linking with epichlorohydrin during the synthesis of DMIP.

Adsorption performance of salicylic acid on a novel resin with distinctive double pore structure.

Two approaches were used to synthesize two resins with different pore structures. In one way, the CH2Cl groups in macroporous chloromethylated polystyrene resin were transformed to methylene bridges, and achieved a hypercrosslinked resin with plentiful micropores (denoted GQ-06). In the other way, 50% of the CH2Cl groups in chloromethylated polystyrene resin was used to produce micropores, while the residual 50% of the CH2Cl groups was reacted with 2-aminopyridine, and prepared another resin with double pore structure of hypercrosslinked resin and macroporous resin (denoted GQ-11). The adsorption of salicylic acid (SA) on GQ-11 was investigated using GQ-06 as the reference adsorbent. The effect of pH on the adsorption of SA on GQ-06 was consistent with the dissociation curve of SA. The maximum adsorption capacity of SA on GQ-11 was observed at the solution pH of 2.64. The greater adsorption rate of SA on GQ-11 than that of GQ-06 was attributed to its double pore structure. The multifunctional adsorption mechanism of anion exchange and hydrophobic interaction resulted in the larger equilibrium capacity of SA on GQ-11 than that of GQ-06. GQ-06 and GQ-11 could be regenerated by absolute alcohol and 80% of alcohol -0.5mol/L of sodium hydroxide aqueous solution, respectively.

Removal of salicylic acid as an emerging contaminant by a polar nano-dendritic adsorbent from aqueous media.

A polar nano-dendritic adsorbent containing amine groups (SAPAMAA) was synthesized onto the nanoparticles of SiO2Al2O3 and its uptake of salicylic acid (SA) from the synthetic and real water was investigated. The synthesized nanomaterials were fully studied by nuclear magnetic resonance spectrum (1H NMR and 13C NMR), Fourier transform infrared spectroscopy (FT-IR), zeta potential (ζ), inductively coupled plasma atomic emission spectroscopy (ICP-AES), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Brunauer-Emmett-Teller (BET) and elemental analysis. Various parameters such as the effect of the contact time, adsorbent dosage, initial SA concentrations, effect of solution's temperature, interfering ions, the hydrophobicity of the nanoadsorbent and initial pH were assessed. The contact time to approach equilibrium for higher adsorption was 15min (252.8mgg-1). The isotherms could be fitted by Sips model (with the average relative error of 6.6) and the kinetic data could be characterized by pseudo-second-order rate equation (with the average relative error of 13.0), implying chemical adsorption as the ratelimiting step of uptake process which was supported by the experimental data from the effect of interfering ions, zeta potential, and altering of the adsorbent's hydrophobicity. The uptake capacities decreased with temperature increasing, and showed that the uptake of SA was chemically exothermic in nature between 15 and 80°C. In addition, the spent SAPAMAA could be regenerated by the removal of adsorbed SA with NaOH and ethanol to regain the original SAPAMAA, the regenerated SAPAMAA also exhibited the high adsorption capacity after 10 runs. Moreover, SAPAMAA could also be applied to uptake SA from a real water (Anzali lagoon water). We envisage that the prepared nano-dendritic with remarkable characteristics such as environmentally friendly, low-cost, easy preparation in large quantity, high mechanical and chemical stability will play a significant role in developing a new generation of emerging contaminants adsorbent.

New nanostructured support for carrier-mediated electromembrane extraction of high polar compounds.

A new support has been proposed to be used for carrier-mediated electromembrane extraction purposes. The new support (Tiss®-OH) is a 100µm thickness sheet nanofiber membrane manufactured by electrospinning and composed by acrylic nanofibers. It has been used in an electromembrane extraction (EME) combined with a HPLC procedure using diode array detection. The proposed method has been used for the extraction of four high polarity acidic compounds: nicotinic acid, amoxicillin, hippuric acid and salicylic acid. Analytes were extracted from an aqueous sample solution (pH 4) (donor phase) using a Tiss®-OH sheet that supports a 5% (w/v) Aliquat®336 in 1-octanol liquid membrane. Aqueous solution (pH 6) was used as acceptor phase. The electrical field was generated from a d.c. electrical current of 100V through two spiral shaped platinum wires placed into donor and acceptor phases. Analytes were extracted in 10min with recoveries in the 60-85% range. The proposed EME procedure has been successfully applied to the determination of the target analytes in human urine samples.

High-capacity hollow porous dummy molecular imprinted polymers using ionic liquid as functional monomer for selective recognition of salicylic acid.

The existence of strong intramolecular hydrogen bond in salicylic acid (SA) weakens its intermolecular hydrogen bonding with functional monomer, then it is a challenge work to fabricate molecularly imprinted polymers (MIPs) for SA recognition with high capacity and good selectivity. Here, hollow porous dummy MIPs (HPDMIPs) were prepared using benzoic acid (BA) as dummy template, ionic liquid (i.e. 1-vinyl-3-methylimidazolium chloride) as functional monomer, and MCM-48 as sacrificial support. Factors that affected adsorption, such as type of template and porogen, mole ratio of template-functional monomer-cross-linker and type of binding solvent, were optimized in detail. Multiple strong interactions between SA and ionic liquid in HPDMIPs deduced higher binding capacity (29.75mg/g), imprinting factor (5.61) and selectivity than any previously reported MIPs by traditional or surface imprinting technology. The large surface area (543.9m2/g) with hollow porous structure resulted in faster kinetic binding (25min). The equilibrium data fitted well to Freundlich equation and the adsorption process could be described by pseudo-second order model. Finally, HPDMIPs were successfully applied to selectively extract and enrich SA from Actinidia chinensis with a relatively high recovery (84.6-94.5%).

[Content of salicylic and jasmonic acids in pea roots (Pisum sativum L.) at the initial stage of symbiotic or pathogenic interaction with bacteria of the family Rhizobiaceae].

A change in the contents of endogenous salicylic and jasmonic acids in the roots of the host plant at the preinfectious stage of interaction with symbiotic (Rhizobium leguminosarum) and pathogenic (Agrobacterium rizogenes) bacteria belonging for to the family Rhizobiaceae was studied. It was found that the jasmonic acid content increased 1.5­2 times 5 min after inoculation with these bacterial species. It was shown that dynamics of the change in the JA and SA contents depends on the type of infection. Thus, the JA content decreased in the case of pathogenesis, while the SA content increased. At the same time, an increased JA content was observed during symbiosis. The observed regularities could indicate the presence of different strategies of hormonal regulation for interaction with symbiotic and pathogenic bacteria belonging to the family Rhizobiaceae in peas plants.

E. coli metabolic engineering for gram scale production of a plant-based anti-inflammatory agent.

In this report, the heterologous production of salicylate (SA) is the basis for metabolic extension to salicylate 2-O-ß-d-glucoside (SAG), a natural product implicated in plant-based defense mechanisms. Production was optimized through a combination of metabolic engineering, gene expression variation, and co-culture design. When combined, SA and SAG production titers reached ~0.9g/L and ~2.5g/L, respectively. The SAG compound was then tested for anti-inflammatory properties relative to SA and acetylsalicylate (aspirin). Results indicate comparable activity between SAG and aspirin in reducing nitric oxide (NO) and reactive oxygen species (ROS) from macrophage cells while no discernable negative effects on cellular viability were observed.

Simultaneous Determination of Aspirin, Dipyridamole and Two of Their Related Impurities in Capsules by Validated TLC-Densitometric and HPLC Methods.

Aspirin (ASP) and dipyridamole (DIP) are widely used as a combination in pharmaceutical formulations for treatment of strokes. Many of these formulations are containing tartaric acid as an excipient (in DIP pellets formulation for sustained release), which increases the probability of formation of dipyridamole tartaric acid ester impurity (DIP-I). On the other hand, salicylic acid (SAL) is considered to be one of the synthesis impurities and a degradation product of ASP. In this work, two chromatographic methods, namely, TLC-densitometry and HPLC, have been established and validated for simultaneous determination of ASP, DIP, SAL and DIP-I. Good separation was achieved by using silica gel as stationary phase and toluene-methanol-ethyl acetate (2:3:5, by volume) as mobile phase in the case of TLC-densitometry and Zorbax ODS column with mobile phase consisting of phosphate buffer (pH 3.3)-acetonitrile-triethylamine (40:60:0.03, by volume) for HPLC. Influence of different organic solvents in mobile phase composition has been studied to optimize the separation efficiency in TLC densitometry. Moreover, factors affecting the efficiency of HPLC, like pH of the buffer used, organic solvent ratio in the mobile phase and flow rate, have been carefully studied using one variable at a time approach. Finally, the proposed methods were validated as per ICH guidelines.

Benzoylsalicylic acid isolated from seed coats of Givotia rottleriformis induces systemic acquired resistance in tobacco and Arabidopsis.

Systemic acquired resistance (SAR), a whole plant defense response to a broad spectrum of pathogens, is characterized by a coordinated expression of a large number of defense genes. Plants synthesize a variety of secondary metabolites to protect themselves from the invading microbial pathogens. Several studies have shown that salicylic acid (SA) is a key endogenous component of local and systemic disease resistance in plants. Although SA is a critical signal for SAR, accumulation of endogenous SA levels alone is insufficient to establish SAR. Here, we have identified a new acyl derivative of SA, the benzoylsalicylic acid (BzSA) also known as 2-(benzoyloxy) benzoic acid from the seed coats of Givotia rottleriformis and investigated its role in inducing SAR in tobacco and Arabidopsis. Interestingly, exogenous BzSA treatment induced the expression of NPR1 (Non-expressor of pathogenesis-related gene-1) and pathogenesis related (PR) genes. BzSA enhanced the expression of hypersensitivity related (HSR), mitogen activated protein kinase (MAPK) and WRKY genes in tobacco. Moreover, Arabidopsis NahG plants that were treated with BzSA showed enhanced resistance to tobacco mosaic virus (TMV) as evidenced by reduced leaf necrosis and TMV-coat protein levels in systemic leaves. We, therefore, conclude that BzSA, hitherto unknown natural plant product, is a new SAR inducer in plants.

Toxicity of titanium dioxide nanoparticles on Pseudomonas putida.

The increasing use of engineered nanoparticles (NPs) in industrial and household applications will very likely lead to the release of such materials into the environment. As wastewater treatment plants (WWTPs) are usually the last barrier before the water is discharged into the environment, it is important to understand the effects of these materials in the biotreatment processes, since the results in the literature are usually contradictory. We proposed the use of flow cytometry (FC) technology to obtain conclusive results. Aqueous solutions of TiO2 nanoparticles (0-2 mg mL(-1)) were used to check its toxicity effect using Pseudomonas putida as simplified model of real sludge over room light. Physiological changes in P. putida from viable to viable but non-culturable cells were observed by flow cytometry in presence of TiO2. The damaged and dead cell concentrations were below 5% in all cases under study. Both FSC and SSC parameter increased with TiO2 dose dependent manner, indicating nanoparticles uptake by the bacteria. The biological removal of salicylic acid (SA) was also significantly impacted by the presence of TiO2 in the medium reducing the efficiency. The use of FC allows also to develop and fit segregated kinetic models, giving the impact of TiO2 nanoparticles in the physiological subpopulations growth and implications for SA removal.

Simultaneous determination of plant hormones in peach based on dispersive liquid-liquid microextraction coupled with liquid chromatography-ion trap mass spectrometry.

Fruit development is influenced greatly by endogenous hormones including salicylic acid (SA) and abscisic acid (ABA). Mass spectrometry with high sensitivity has become a routine technology to analyze hormones. However, pretreatment of plant samples remains a difficult problem. Thus, dispersive liquid-liquid microextraction (DLLME) was used to concentrate trace plant hormones before liquid chromatography-ion trap mass spectrometry (LC-ITMS) analysis. Standard curves were linear within the ranges of 0.5-50, 0.2-20ng/mL for SA and ABA, respectively. The correlation coefficients were greater than 0.9995 with recoveries above 87.5%. The limits of detection were 0.2ng/mL for SA and 0.1ng/mL for ABA in spiked water solution, respectively (injection 20µL). The successful analysis of SA and ABA in fruit samples indicated our DLLME-LC-ITMS approach was efficient, allowing reliable quantification of both two compounds from very small amounts of plant material. Moreover, this research revealed the relationship between SA and ABA content and development of peach fruit at different growth stages.

Identification of didecyldimethylammonium salts and salicylic acid as antimicrobial compounds in commercial fermented radish kimchi.

Daikon radish (Raphanus sativus) fermented with lactic acid bacteria, especially Leuconostoc or Lactobacillus spp., can be used to make kimchi, a traditional Korean fermented vegetable. Commercial Leuconostoc/radish root ferment filtrates are claimed to have broad spectrum antimicrobial activity. Leuconostoc kimchii fermentation products are patented as preservatives for cosmetics, and certain strains of this organism are reported to produce antimicrobial peptides (bacteriocins). We examined the antimicrobial agents in commercial Leuconostoc/radish root ferment filtrates. Both activity-guided fractionation with Amberlite XAD-16 and direct extraction with ethyl acetate gave salicylic acid as the primary agent with activity against Gram-negative bacteria. Further analysis of the ethyl acetate extract revealed that a didecyldimethylammonium salt was responsible for the Gram-positive activity. The structures of these compounds were confirmed by a combination of (1)H- and (13)C NMR, high-performance liquid chromatography, high-resolution mass spectrometry, and tandem mass spectrometry analyses. Radiocarbon dating indicates that neither compound is a fermentation product. No antimicrobial peptides were detected.