Integrated molecular network and HPLC-UV-FLD analysis to explore antioxidant ingredients in camellia nitidissima Chi.

At present, screening of active ingredients from natural products for pharmacological and clinical research is mostly time-consuming and costly. In this study, a molecular network (MN) guided high performance liquid chromatography-ultraviolet-fluorescence detector (HPLC-UV-FLD) method was carried out to profile the global antioxidant activity compounds, including the trace amount ingredients in Camellia nitidissima Chi (CNC). Firstly, HPLC-UV-FLD postcolumn derivatization system was utilized to screen the antioxidants. Then the MN of CNC was established via mass spectrometry (MS) data for getting the connection between ingredient structures. As a result, HPLC-UV-FLD indicated three antioxidant ingredients: gallic acid (126.3 mg/g), catechin (564.8 mg/g), and salicylic acid (24.3 mg/g). Combined with the MN, the actives' precise location and connection relationship were clarified based on the structural similarities. A new antioxidant ingredient, okicamelliaside, was suggested and evaluated at free radical scavenging and enzymatic protection. The novel method of activity and structural correlation analysis based on MN could provide a useful guide for screening trace active ingredients in natural products. PRACTICAL APPLICATION: Three main ingredients were screened out from Camellia nitidissima Chi by HPLC-UV-FLD postcolumn derivatization system. Integrated molecular network and HPLC-UV-FLD analysis, a new type of antioxidant okicamelliaside was selected. The novel method of activity and structural correlation analysis based on molecular network could provide a useful guide for screening trace active ingredients in natural products.

Exogenous application of salicylic acid to control coffee rust

The exogenous application of salicylic acid can induce plant resistance against pathogens. However, little is known about the potential uses of this bioregulator for controlling coffee diseases. In this study, we assessed the effect of applying salicylic acid (SA – 150 mg L-1) on the management of coffee rust (Hemileia vastatrix) in a 7-year-old coffee plantation with low crop load (651.6 kg ha-1 in 2017). For comparison, plants were sprayed with protectant fungicide (copper hydroxide – CH) and standard fungicides (SF) used by local farmers (boscalid, pyraclostrobin + epoxiconazole, and copper hydroxide). Non-treated plants were included as a negative control. Five monthly applications were performed from November 2016 to March 2017. Rust incidence and severity, defoliation, and growth of plagiotropic branches were evaluated monthly. The activity of catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and total proteins was assessed one day after the first, third, and fifth product applications. Compared to untreated plants, SA reduced the severity and incidence of rust from 36.3 to 54.7%, while CH and SF reduced disease from 31.8 to 54.6% and from 83.8 to 88%, respectively. SA reduced defoliation by 54.1%. SA increased the concentration of CAT, APX, and SOD after the first application. However, this effect was not observed after subsequent applications. Foliar application of SA reduces the severity and incidence of coffee rust and defoliation in plants with a low crop load.

Comparative study of salicylic acid contents in young wheat and rice plants and their anticancer activities in HepG2 and Caco-2 cells.

Some recent results reported that aspirin (acetylsalicylic acid) had a positive effect on the treatment of certain types of cancer. However, the results cannot be generalized and it is not always clear whether it is a direct anticancer effect or a general health effect. Since plants produce different amounts of salicylic acid, we have sought a relationship between the salicylic acid content of some plant extracts and their anticancer activity. Growing of wheat and rice plants were carried out under controlled conditions. The salicylic acid content was determined by high-performance liquid chromatography. The viability and cell cycle assays were performed on HepG2 and Caco-2 cell lines. Despite the high content of salicylic acid, the extracts from rice plants did not show significant anticancer activity. In spite of the low salicylic acid content, the positive effect of wheat germ was confirmed in both tests. There is no direct relationship between the salicylic acid content of the plant extracts and their anticancer activity. However, it has been proven that young wheat germ is more effective than mature leaf.

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.

Development of a structure-switching aptamer-based nanosensor for salicylic acid detection.

Salicylic acid (SA) is a phytohormone regulating immune responses against pathogens. SA and its derivatives can be found in diverse food products, medicines, cosmetics and preservatives. While salicylates have potential disease-preventative activity, they can also cause health problems to people who are hypersensitive. The current SA detection methods are costly, labor-intensive and require bulky instruments. In this study, a structure-switching aptamer-based nanopore thin film sensor was developed for cost-effective, rapid, sensitive and simple detection of SA in both buffer and plant extracts. SA is a challenging target for aptamer selection using conventional systemic evolution of ligands by exponential enrichment (SELEX) due to its small size and scarcity of reactive groups for immobilization. By immobilizing the SELEX library instead of SA and screening the library using a structure-switching SELEX approach, a high affinity SA aptamer was identified. The nanopore thin film sensor platform can detect as low as 0.1 µM SA. This is much better than the sensitivity of antibody-based detection method. This nanosensor also exhibited good selectivity among SA and its common metabolites and can detect SA in Arabidopsis and rice using only about 1 µl plant extracts within less than 30 min. The integration of SA aptamer and nanopore thin film sensor provides a promising solution for low-cost, rapid, sensitive on-site detection of SA.

Effect of Major Tea Insect Attack on Formation of Quality-Related Nonvolatile Specialized Metabolites in Tea ( Camellia sinensis) Leaves.

Insect attack is known to induce a high accumulation of volatile metabolites in tea ( Camellia sinensis). However, little information is available concerning the effect of insect attack on tea quality-related nonvolatile specialized metabolites. This study aimed to investigate the formation of characteristic nonvolatile specialized metabolites in tea leaves in response to attack by major tea insects, namely, tea green leafhoppers and tea geometrids, and determine the possible involvement of phytohormones in metabolite formation resulting from insect attack. Both tea green leafhopper and tea geometrid attacks increased the jasmonic acid and salicylic acid contents. The abscisic acid content was only increased under tea green leafhopper attack, perhaps due to special continuous piercing-sucking wounding. Tea green leafhopper attack induced the formation of theaflavins from catechins under the action of polyphenol oxidase, while tea geometrid attack increased the l-theanine content. Exogenous phytohormone treatments can affect the caffeine and catechin contents. These results will help to determine the influence of major tea pest insects on important tea quality-related metabolites and enhance understanding of the relationship of phytohormones and quality-related nonvolatile metabolite formation in tea exposed to tea pest insect attacks.

Transcriptome and metabolome analysis reveal candidate genes and biochemicals involved in tea geometrid defense in Camellia sinensis.

Tea plant (Camellia sinensis (L) O. Kuntze) respond to herbivore attack through large changes in defense related metabolism and gene expression. Ectropis oblique (Prout) is one of the most devastating insects that feed on tea leaves and tender buds, which can cause severe production loss and deteriorate the quality of tea. To elucidate the biochemicals and molecular mechanism of defense against tea geometrid (TG), transcriptome and metabolome of TG interaction with susceptible (SG) and resistance (RG) tea genotypes were analyzed by using UPLC-Q-TOF-MS, GC-MS, and RNA-seq technologies. This revealed that jasmonic acid was highly induced in RG, following a plethora of secondary metabolites involved in defense against TG could be induced by jasmonic acid signaling pathway. However, the constitutively present of salicylic acid in SG might be a suppressor of jasmonate signaling and thus misdirect tea plants against TG. Furthermore, flavonoids and terpenoids biosynthesis pathways were highly activated in RG to constitute the chemical barrier on TG feeding behavior. In contrast, fructose and theanine, which can act as feeding stimulants were observed to highly accumulate in SG. Being present in the major hub, 39 transcription factors or protein kinases among putative candidates were identified as master regulators from protein-protein interaction network analysis. Together, the current study provides a comprehensive gene expression and metabolite profiles, which can shed new insights into the molecular mechanism of tea defense against TG. The candidate genes and specific metabolites identified in the present study can serve as a valuable resource for unraveling the possible defense mechanism of plants against various biotic stresses.

Potato tubers contamination with nitrate under the influence of nitrogen fertilizers and spray with molybdenum and salicylic acid.

A field trial was conducted through 2015 and 2016 growing seasons to study the effect of nitrogen fertilizer sources and foliar spray with molybdenum (Mo), salicylic acid (SA) and their combination on tubers yield, some chemical constituents, nutrients uptake, nitrate accumulation and nitrate reductase activity in potato tubers. N source was added at a rate of 350 kg N ha-1in five equal doses as two different forms, the first is urea and the second is ammonium sulfate plus calcium nitrate equally. SA was sprayed with three rates of 0, 75 and 150 mg l-1. Also, Mo as ammonium molybdate was sprayed using three rates 0, 50 and 100 mg l-1Mo. Both treatments of SA and Mo were applied separately as well as with each other, at three successive times 30, 50 and 70 days after planting of potato plants. Results indicated that the addition of 350 kg N ha-1 as ammonium sulfate and calcium nitrate equally caused a significant elevation (P > 0.05) in fresh weight, chlorophyll b, carotenoids, chlorophyll a, nitrate reductase activity, dry weight and NPK uptake by potato tubers compared with the same amount of nitrogen in the form of urea only. All the aforementioned characteristics were improved with increasing concentration of Mo and/or SA. The highest accumulation of nitrate was recorded under the addition of 350 kg N ha-1 as urea alone. The highest average of all the aforementioned characteristics was observed at the treatment of 350 kg N ha-1 as ammonium sulfate and calcium nitrate equally plus spraying with 100 mg l-1Mo and 150 mg l-1 SA. In contrast, this treatment gave the lowest accumulation of nitrates in potato tubers.

Overall Content of Salicylic Acid and Salicylates in Food Available on the European Market.

The study aimed to determine the salicylates content in 112 products available on the European market. Quantitative determination of free and conjugated forms of salicylic acid in food was performed using reversed-phase high-performance liquid chromatography with fluorescence detection. The salicylates contents ranged from 0 to 1675.79 (µg/100 g). The results of this study confirm the presence of salicylates in food products, as well as a broad content diversity of these compounds depending on the species, variety, and method of processing the food items. The results can be very helpful for nutritionists and dieticians in planning low-salicylates or high-salicylates diets.

Development of an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of salicylic acid, jasmonic acid, and abscisic acid in rose leaves.

This paper describes a method to detect and quantitate the endogenous plant hormones (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in hybrid rose leaf matrices. Deuterium-labeled [(2)H6] (+)-2-cis-4-trans-abscisic acid, [(2)H6] (±)-jasmonic acid, and [(2)H4]-salicylic acid were used as internal standards. Rose samples (10 mg) were extracted with methanol/water/acetic acid (10:89:1) and subsequently purified on an Oasis MCX 1 cm(3) Vac SPE cartridge. Performance characteristics were validated according to Commission Decision 2002/657/EC. Recovery, repeatability, and within-laboratory reproducibility were acceptable for all phytohormones tested at three different concentrations. The decision limit and detection capability for (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid were 0.0075 and 0.015 µg/g, 0.00015 and 0.00030 µg/g, and 0.0089 and 0.018 µg/g, respectively. Matrix effects (signal suppression or enhancement) appeared to be high for all substances considered, implying the need for quantitation based on matrix-matched calibration curves.

Quality and antioxidant properties on sweet cherries as affected by preharvest salicylic and acetylsalicylic acids treatments.

The effects of salicylic acid (SA) or acetylsalicylic acid (ASA) treatments during on-tree cherry growth and ripening on fruit quality attributes, especially those related with the content on bioactive compounds and antioxidant activity were analysed in this research. For this purpose, two sweet cherry cultivars, 'Sweet Heart' and 'Sweet Late', were used and SA or ASA treatments, at 0.5, 1.0 and 2.0mM concentrations, were applied at three key points of fruit development (pit hardening, initial colour changes and onset of ripening). These treatments increased fruit weight and ameliorated quality attributes at commercial harvest, and led to cherries with higher concentration in total phenolics and in total anthocyanins, as well as higher antioxidant activity, in both hydrophilic and lipophilic fractions. Thus, preharvest treatments with SA or ASA could be promising tools to improve sweet cherry quality and health beneficial effects for consumers.

Jasmonic acid is involved in the signaling pathway for fungal endophyte-induced volatile oil accumulation of Atractylodes lancea plantlets.

BACKGROUND: Jasmonic acid (JA) is a well-characterized signaling molecule in plant defense responses. However, its relationships with other signal molecules in secondary metabolite production induced by endophytic fungus are largely unknown. Atractylodes lancea (Asteraceae) is a traditional Chinese medicinal plant that produces antimicrobial volatiles oils. We incubated plantlets of A. lancea with the fungus Gilmaniella sp. AL12. to research how JA interacted with other signal molecules in volatile oil production. RESULTS: Fungal inoculation increased JA generation and volatile oil accumulation. To investigate whether JA is required for volatile oil production, plantlets were treated with JA inhibitors ibuprofen (IBU) and nordihydroguaiaretic acid. The inhibitors suppressed both JA and volatile oil production, but fungal inoculation could still induce volatile oils. Plantlets were further treated with the nitric oxide (NO)-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the H2O2 inhibitors diphenylene iodonium (DPI) and catalase (CAT), and the salicylic acid (SA) biosynthesis inhibitors paclobutrazol and 2-aminoindan-2-phosphonic acid. With fungal inoculation, IBU did not inhibit NO production, and JA generation was significantly suppressed by cPTIO, showing that JA may act as a downstream signal of the NO pathway. Exogenous H2O2 could reverse the inhibitory effects of cPTIO on JA generation, indicating that NO mediates JA induction by the fungus through H2O2-dependent pathways. With fungal inoculation, the H2O2 scavenger DPI/CAT could inhibit JA generation, but IBU could not inhibit H2O2 production, implying that H2O2 directly mediated JA generation. Finally, JA generation was enhanced when SA production was suppressed, and vice versa. CONCLUSIONS: Jasmonic acid acts as a downstream signaling molecule in NO- and H2O2-mediated volatile oil accumulation induced by endophytic fungus and has a complementary interaction with the SA signaling pathway.

Thaxtomin A-deficient endophytic Streptomyces sp. enhances plant disease resistance to pathogenic Streptomyces scabies.

Each plant species in nature harbors endophytes, a community of microbes living within host plants without causing any disease symptom. However, the exploitation of endophyte-based phytoprotectants is hampered by the paucity of mechanistic understandings of endophyte-plant interaction. We here reported two endophytic Streptomyces isolates IFB-A02 and IFB-A03 recovered from a stress-tolerant dicotyledonous plant Artemisia annua L. After the determination of their non-pathogenicity at the genomic level and from the toxin (thaxtomin A, TXT) level, the endophytism of both isolates was supported by their successful colonization in planta. Of the two endophytes, IFB-A03 was further studied for the mechanism of endophyte-conferred phytoprotection owing to its plant growth promotion in model eudicot Arabidopsis thaliana. Using the endophyte-Arabidopsis co-cultivation system into which pathogenic Streptomyces scabies was introduced, we demonstrated that IFB-A03 pre-inoculation could activate the salicylic acid (SA)-mediated plant defense responses upon pathogen challenge. Moreover, IFB-A03 was shown to partially rescue the defense deficiency in eds5 (enhanced disease susceptibility 5) Arabidopsis mutants, putatively acting at the upstream of SA accumulation in the defense signaling pathway associated with the systemic acquired resistance (SAR). These data suggest that endophytic Streptomyces sp. IFB-A03 could be a promising candidate for biocontrol agents against S. scabies--a causative pathogen of common scab diseases prevailing in agronomic systems.

Simultaneous determination and pharmacokinetics of protein unbound aspirin and salicylic acid in rat blood and brain by microdialysis: an application to herbal-drug interaction.

Aspirin is commonly used for the prevention of myocardial infarction and ischemic stroke; whereas the Chinese people employ the bu-yang-huan-wu-tang (BYHWT) as a routine herbal formulation for the treatment and prevention of transient ischemic stroke. The current study develops a microdialysis technique coupled to a validated liquid chromatography system to measure free-form aspirin and salicylic acid for herbal-drug interaction in rat blood and brain. The intra- and inter-day precisions in biological dialysates were within 0.1-9.4% in the concentration ranges of 0.1-50 µg/mL and the accuracies ranged from -4.7 to 6.1%. The pharmacokinetic data demonstrate that the area under the concentration time curve (AUC) of the aspirin was 2031 ± 266 min µg/mL after aspirin administration (100mg/kg, i.v.). The AUC of salicylic acid was 12660 ± 1799 min µg/mL, which suggests that aspirin is quickly hydrolyzed to salicylic acid in blood and the metabolite can also be detected within 15 min in brain dialysate. The herbal-drug pharmacokinetic interaction showed no significant effect in blood and brain. The results of pharmacodynamics for the bleeding time suggested that there were no significant differences between the aspirin alone group and the BYHWT pretreated group. However, the bleeding time has been prolonged when compared aspirin alone or the group pretreated with BYHWT to the blank control. The conclusion provides practical information for clinical practice for the herbal formulation BYHWT and aspirin used concurrently.

Sucrose accelerates flower opening and delays senescence through a hormonal effect in cut lily flowers.

Sugars are generally used to extend the vase life of cut flowers. Such beneficial effects have been associated with an improvement of water relations and an increase in available energy for respiration by floral tissues. In this study we aimed at evaluating to what extent (i) endogenous levels of sugars in outer and inner tepals, androecium and gynoecium are altered during opening and senescence of lily flowers; (ii) sugar levels increase in various floral tissues after sucrose addition to the vase solution; and (iii) sucrose addition alters the hormonal balance of floral tissues. Results showed that endogenous glucose levels increased during flower opening and decreased during senescence in all floral organs, while sucrose levels increased in outer and inner tepals and the androecium during senescence. Sucrose treatment accelerated flower opening, and delayed senescence, but did not affect tepal abscission. Such effects appeared to be exerted through a specific increase in the endogenous levels of sucrose in the gynoecium and of glucose in all floral tissues. The hormonal balance was altered in the gynoecium as well as in other floral tissues. Aside from cytokinin and auxin increases in the gynoecium; cytokinins, gibberellins, abscisic acid and salicylic acid levels increased in the androecium, while abscisic acid decreased in outer tepals. It is concluded that sucrose addition to the vase solution exerts an effect on flower opening and senescence by, among other factors, altering the hormonal balance of several floral tissues.

Detection, characterization and quantification of salicylic acid conjugates in plant extracts by ESI tandem mass spectrometric techniques.

An approach for the detection and characterization of SA derivatives in plant samples is presented based on liquid chromatography coupled to electrospray ionization (ESI) tandem mass spectrometric techniques. Precursor ion scan methods using an ESI triple quadrupole spectrometer for samples from plants challenged with the virulent Pseudomonas syringae pv tomato DC3000 allowed us to detect two potential SA derivatives. The criterion used to consider a potential SA derivative is based on the detection of analytes in the precursor ion scan chromatogram upon selecting m/z 137 and m/z 93 that correspond to the salicylate and its main product ion, respectively. Product ion spectra of the newly-detected analytes as well as accurate m/z determinations using an ESI Q-time-of-flight instrument were registered as means of characterization and strongly suggest that glucosylated forms of SA at the carboxylic and at the phenol functional groups are present in plant samples. The specific synthesis and subsequent chromatography of salicylic glucosyl ester (SGE) and glucosyl salicylate (SAG) standards confirmed the chemical identity of both peaks that were obtained applying different tandem mass spectrometric techniques and accurate m/z determinations. A multiple reaction monitoring method has been developed and applied to plant samples. The advantages of this LC-ESI-MS/MS methods with respect to the traditional analysis of glucosyl conjugates are also discussed. Preliminary results revealed that SA and the glucosyl conjugates are accumulated in Arabidopsis thaliana in a time dependent manner, accordingly to the up-regulation of SA-dependent defenses following P. syringae infection. This technique applied to plant hormones or fragment ions may be useful to obtain chemical family members of plant metabolites and help identify their contribution in the signaling of plant defenses.

Simultaneous determination of different endogenetic plant growth regulators in common green seaweeds using dispersive liquid-liquid microextraction method.

A simple and rapid HPLC-based method was developed for simultaneous determination of major classes of plant growth regulators (PGRs) in Monostroma and different species of Ulva. The plant growth regulators determined included gibberellic acid (GA(3)), indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA), salicylic acid and kinetin riboside (KR) and their respective elution time was 2.75, 3.3, 3.91, 4.95, 5.39 and 6.59 min. The parameters optimized for distinct separation of PGRs were mobile phase (60:40 methanol and 0.6% acetic acid in water), column temperature (35°C) and flow rate (1ml/min). This method presented an excellent linearity (0.2-100µg/ml) with limit of detection (LOD) as 0.2µg/ml for ABA, 0.5µg/ml for KR and salicylic acid, and 1µg/ml for IAA, IBA and GA(3). The precision and accuracy of the method was evaluated after inter and intra day analysis in triplicates. The effect of plant matrix was compensated after spiking and the resultant recoveries estimated were in the range of 80-120%. Each PGR thereby detected were further characterized by ESI-MS analysis. The method optimized in this study determined IBA along with IAA for the first time in the seaweed species investigated except Ulva linza where the former was not detected. In all the species studied, ABA level was detected to be the highest while kinetin riboside was the lowest. In comparison to earlier methods of PGR analysis, sample preparation and analysis time were substantially reduced while allowing determination of more classes of PGRs simultaneously.

[Evaluation of anti-inflammatory activity of extracts from Siberian plants].

Experimental investigations have shown that water-alcohol extracts from plants containing alkaloids (Aconitum baikalense, Aconitum septentrionale, Delphinium elatum L., Conium maculatum) and salicylic acid (Filipendula ulmaria, Salix viminalis, Fragaria vesca, Rubus idaeus) inhibited the development of main symptoms of inflammation, viz. exudation, pain, fever, to the same extent as non-steroidal anti-inflammatory agents. The substances studied in this work may be used to develop new efficient pharmacological preparations for the treatment of different inflammatory conditions associated with severe pain syndrome.

[Determination of salylic acid, syringic acid, benzoic acid and anthranilic acid in Radix Isatidis by HPCE].

OBJECTIVE: To develop a simple and rapid capillary electrophoresis (CE) method for the separation and determination of four active organic acids including salicylic acid, syringic acid, benzoic acid, and anthranilic acid in Radix Isatidis. METHOD: The HPCE system consisted of a fused-silica capillary column of 47.3 cm (38.3 cm to the detector) x50 microm i.d. and a mixture ofacetonitrile-borate buffer (15% acetonitrile, 25 mmol L(-1) borate, 15 mmol L(-1) beta-CD, pH 9.10) solution as the operating buffer. The applied voltage was 11.5 kV and the UV detection was set at 220 nm. The effects of the applied voltage, detection wavelength, and the pH of buffer, the concentration of buffer, acetonitrile and beta-CD were investigated. RESULT: The linear calibration rang was 3.0-90 mg L(-1) (r=0.9994) for salylic acid, 4.0-120 mg L(-1) (r=0.9995) for syringic acid, 2.0-60 mg L(-1) (r=0.9998) for benzoic acid and 5.0-100 mg L(-1) (r=0.9992) for anthranilic acid. The recoveries of salylic acid, syringic acid, benzoic acid and anthranilic acid were 95.9%-102.6%, 98.6%-103.4%, 98.7%-104.1%, 96.1%-104.3% respectively. The detection limits of salylic acid, syringic acid, benzoic acid and anthranilic acid were 0.7, 1.1, 1.2 and 1.5 mg L(-1), respectively.

Simultaneous determination of active ingredients in ethnomedicine Gaultheria leucocarpa var. yunnanensis and its medicinal preparation by capillary electrophoresis with electrochemical detection.

A simple and rapid capillary electrophoresis (CE) with electrcochemical detection (ED) method has been established for the simultaneous determination of seven active ingredients in the stems and roots of Gaultheria leucocarpa var. yunnanensis and its medicinal preparation, including (+)-catechin, rutin, gentisic acid, vallinic acid, salicylic acid, quercetin, and protocatechuic acid. The effects of working potential, pH, and concentration of running buffer, separation voltage, and injection time on CE-ED are systematically investigated. Under the optimum conditions, the seven analytes could be completely separated within 23 min in a borax running buffer (pH 8.7). A good linear relationship is obtained over three orders of magnitude with detection limits (signal-to-noise ratio=3) ranging from 5x10(-8) g/mL to 3x10(-7) g/mL for the analytes. The proposed method is successfully used in the analysis of real samples after a relatively simple extraction procedure, and the assay results are satisfactory.