Antioxidant and Antimicrobial Effects of Baby Leaves of Amaranthus tricolor L. Harvested as Vegetable in Correlation with Their Phytochemical Composition.

Amaranth is used as a spinach replacement; therefore, it is sometimes called Chinese Spinach. So far, the activity of the plant has not been associated with the presence of specific compounds. Three cultivars of Amaranthus tricolor L. were investigated for their antioxidant and antimicrobial activities. The correlation between the bioactivity and metabolite profiles was investigated in order to indicate active compounds in A. tricolor. The phytochemical profile of a total of nine extracts was studied by HPLC-DAD-ESI/HRMS, revealing the presence of 52 compounds. The highest antioxidant activity was noticed in the Red cultivar (0.06 mmol TE/g DE (Trolox Equivalent/Dry Extract Weight) and was related to the presence of amino acids, flavonoids and phenolic acids, as well as individual compounds such as tuberonic acid hexoside. All studied extracts revealed antimicrobial activity. Gram-positive bacteria were more susceptible to N-(carboxyacetyl) phenylalanine, phenylalanine, tuberonic acid and succinic acid and Gram-negative bacteria to dopa, tryptophan, norleucine, tuberonic acid hexoside, quercetin-O-hexoside, luteolin-O-rhamnosylhexoside, luteolin-6-C-hexoside succinic acid, gallic acid-O-hexoside, dihydroxybenzoic acid and hydroxybenzoic acid. Maleic acid showed promising antifungal activity. In summary, A. tricolor is a good source of antioxidant and antimicrobial compounds.

Enhanced marine monitoring and toxicity study of oil spill dispersants including Corexit EC9500A in the presence of diluted bitumen.

Observations made for the analysis of the oil spill dispersant tracer dioctyl sulfosuccinate (DOSS) during LC50 toxicity testing, highlighted a stability issue for this tracer compound in seawater. A liquid chromatography high-resolution quadrupole time-of-flight mass spectrometry (LC/QToF) was used to confirm monooctyl sulfosuccinate (MOSS) as the only significant DOSS breakdown product, and not the related isomer, 4-(2-ethylhexyl) 2-sulfobutanedioate. Combined analysis of DOSS and MOSS was shown to be applicable to monitoring of spill dispersants Corexit® EC9500A, Finasol OSR52, Slickgone NS, and Slickgone EW. The unassisted conversion of DOSS to MOSS occurred in all four oil spill dispersants solubilized in seawater, although differences were noted in the rate of MOSS formation. A marine microcosm study of Corexit EC9500A, the formulation most rapid to form MOSS, provided further evidence of the stoichiometric conversion of DOSS to MOSS under conditions relevant to real world dilbit spill. Results supported combined DOSS and MOSS analysis for the monitoring of spill dispersant in a marine environment, with a significant extension of sample collection time by 10 days or longer in cooler conditions. Implications of the unassisted formation of MOSS and combined DOSS:MOSS analysis are discussed in relation to improving dispersant LC50 toxicity studies.

Callus growth kinetics and accumulation of secondary metabolites of Bletilla striata Rchb.f. using a callus suspension culture.

Bletilla striata is an endangered traditional Chinese medicinal plant with multiple uses and a slow regeneration rate of its germplasm resources. To evaluate the callus growth kinetics and accumulation of secondary metabolites (SMs), a callus suspension culture was proven to be a valuable approach for acquiring high yields of medicinal compounds. An effective callus suspension culture for obtaining B. striata callus growth and its SMs was achieved with the in vitro induction of calluses from B. striata seeds. The callus growth kinetics and accumulation of SMs were analyzed using a mathematical model. The resulting callus growth kinetic model revealed that the growth curves of B. striata suspension-cultured calluses were sigmoidal, indicating changes in the growth of the suspension-cultured calluses. Improved Murashige and Skoog callus growth medium was the most favorable medium for B. striata callus formation, with the highest callus growth occurring during the stationary phase of the cultivation period. Callus growth acceleration started after 7 days and thereafter gradually decreased until day 24 of the cultivation period and reached its highest at day 36 period in both the dry weight and fresh weight analyses. The coelonin concentration peaked during the exponential growth stage and decreased afterward during the stationary stage of the callus suspension culture. The maximum content of coelonin (approximately 0.3323 mg/g callus dry weight) was observed on the 18th day of the cultivation cycle, while dactylorhin A and militarine reached the highest concentrations at day 24, and p-hydroxybenzyl alcohol at day 39. This investigation also laid a foundation for a multimathematical model to better describe the accumulation variation of SMs. The production of SMs showed great specificity during callus growth and development. This research provided a well-organized way to increase the accumulation and production of SMs during the scaled-up biosynthesis of calluses in B. striata callus suspension cultures.

The Bioactive Effects of Chicoric Acid As a Functional Food Ingredient.

Chicoric acid, a hydroxycinnamic acid, has been reported to possess a variety of health benefits, including antivirus, antioxidant, anti-inflammation, obesity prevention, and neuroprotection effects. The purpose of this article is to summarize current knowledge of pharmacological and biological effects of chicoric acid. Since most studies to date on chicoric acid have limited their focus to cell cultures and animals, more human and mechanistic studies are therefore needed to further determine the beneficial effects of chicoric acid as a potential functional food ingredient.

Which Plant Part of Purple Coneflower (Echinacea purpurea (L.) Moench) Should be Used for Tea and Which for Tincture?

Medicinal plants are widely used for the relief of disease symptoms or as dietary supplements. In recent decades, purple coneflower has become extremely well known. An infusion or tincture of purple coneflower can be prepared by anyone simply, inexpensively, and ecologically safely. Three plant parts of purple coneflower were used in the study: extracts from roots, flowers, and leaves were obtained using three different solvents (100% and 40% ethanol and water). High-performance liquid chromatography-mass spectrophotometer identified and quantified 23 individual phenolics. Pure (100%) ethanol gave the lowest yield of all the investigated phenolic compounds in all herb parts. Chicoric and caftaric acids were the major phenolic compounds in coneflower. Caftaric acid, with health promoting properties, was extracted best in a water solution from purple coneflower leaves (2673.31 mg/100 g dry weight [DW]) and chicoric acid, also with a beneficial effect on human health, yielded the highest levels in 40% ethanol solution from flowers (1571.79 mg/100 g DW) and roots (1396.27 mg/100 g DW).

Qualitative and quantitative analysis of Yifei Tongluo granules to identify main bioactive components using LC-DAD/MS and pharmacokinetic studies.

A standard fingerprint containing twelve common peaks was constructed from ten batches of Yifei Tongluo granules to evaluate batch-to-batch consistency by using HPLC-DAD. Additionally, the corresponding medicinal material attributes of these chemical constituents were analyzed according to the data acquired from the HPLC method and the identification was further carried out using the LC-MS/MS method. Comparing the retention time or accurate mass with previous studies or standards, the common components were tentatively identified in 50 min for ten batches of samples. At the same time, a reliable LC-MS/MS method was established to quantify marker substances simultaneously in 25 min, and the linear relationship of the standard curves was good in the experimental range. The validations of the method were successfully applied to the quality control and pharmacokinetic study. The results obtained from this study suggest that militarine was most abundant and the components in the granules caused pharmacokinetic herb-drug interactions in rats. This study provides a meaningful basis for evaluating the viability of Yifei Tongluo granules for clinical applications.

In-vivo metabolite profiling of chicoric acid in rat plasma, urine and feces after oral administration using liquid chromatography quadrupole time of flight mass spectrometry.

Chicoric acid (CA) is an active derivative of caffeic acid, which is naturally present in many medicinal plants and vegetables. In the present study, the metabolic profile of CA was determined in rat plasma, urine and feces and was subsequently used to propose the metabolic pathways of CA. CA (100 mg/kg) was orally administered to rats by gastric intubation. Then, the plasma, urine and feces samples were collected and treated with methanol and acetonitrile (1:1, V/V) to precipitate the proteins. The pretreated samples were separated by ultra performance liquid chromatography (UPLC) equipped with an HSS T3 column (2.1 mm × 100 mm I.D., 1.7 µm) and with quadrupole time-of-flight mass spectrometry (Q-TOF-MS) as the detection method. A total of nineteen metabolites were detected and identified based on the characteristics of their deprotonated ions in the plasma, urine and feces samples. The results revealed that the metabolism of CA followed a number of known in-vivo mammalian biotransformation pathways including hydrolysis, reduction, methylation, sulfation, glucuronidation, acetylation, isomerization and deoxygenation.

[Simultaneous determination of seven components in Pudilan Xiaoyan oral liquid by HPLC].

OBJECTIVE: To establish an HPLC method for the content determination of baicalin, wogonin, chlorogenic acid, caffeic acid, cichoric acid, corynoline and adenosine in Pudilan Xiaoyan oral liquid. METHOD: The analysis was performed on a Phenomenex Luna C18 column (4.6 mm x 250 mm, 5 µm) with a gradient mobile phase of methanol-0.1% trifluoroacetic acid solution system at flow rate of 1.0 mL · min(-1). The detective wavelength was at 280 nm. The column temperature was 30 °C. RESULT: The standard curves of seven studied components show good linearity in their concentration ranges with r ≥ 0.999 6. The average recovery was 98.73%-102.1% with RSD less than 2.6%. CONCLUSION: The method is rapid, simple and accurate, and can be applied for the quality control of Pudilan Xiaoyan oral liquid.

Synthesis of anaerobic degradation biomarkers alkyl-, aryl- and cycloalkylsuccinic acids and their mass spectral characteristics.

Anaerobic biodegradation of petroleum hydrocarbons has been reported to proceed predominantly via fumarate addition to yield substituted succinate metabolites. These metabolites, commonly regarded as signature biomarkers, are specific indicators of anaero- bic hydrocarbon degradation by microbial activity. To the best of our knowledge, mass spectrometry information for 2-(1-methylalkylj succinic acids, 2-arylsuccinic acids, 2-cycloalkylsuccinic acids and/or their derivatives is still incomplete, especially for the analysis of environmental samples. Here, a novel approach is proposed for the successful synthesis of five hydrocarbon-derived succinic acids. The characteristic fragments of 2-[1-methylalkyllsuccinic acid diesters were investigated by four derivatization processes (methyl, ethyl, n-butyl and trimethylsilyl esterification], some of which are not available in official Libraries. Under electron ionization mass spec- trometry conditions, informative fragments of various molecular masses have been obtained. Results confirmed characteristic differ- ences among the derivatization processes of the chemically synthesized compounds. In the case of 2-[cyclo)alkylsuccinate esters, four intermediate fragments were observed at m/z 114 + 14n, 118 + 28n, [M - [17 + 14n1]]+ and [M - (59 + 14n)]+ (n = 1, 2 and 4 for methyl, ethyl and n-butyl ester]. However, for silylation the abundant fragment ions are at m/z 262, 217, 172, 147, 73 and [M - 15]+. These data provide information for the identification of hydrocarbon-derived succinic acids as anaerobic biodegradation intermediates in hydrocarbons- rich environments.

Influence of postharvest processing and storage conditions on key antioxidants in puha (Sonchus oleraceus L.).

OBJECTIVES: To investigate effects of different postharvest drying processes and storage conditions on key antioxidants in Sonchus oleraceus L. leaves. METHODS: Fresh leaves were oven-dried (60°C), freeze-dried or air-dried (∼25°C) for 6 h, 24 h and 3 days, respectively. Design of experiments (DOE) was applied to study the stability of antioxidants (caftaric, chlorogenic and chicoric acids) in S. oleraceus leaves and leaf extracts stored at different temperatures (4, 25 and 50°C) and relative humidities (15%, 43% and 75%) for 180 days. The concentration of antioxidants was quantified by a HPLC-2,2'-diphenylpicrylhydrazyl post-column derivatisation method. Antioxidant activity was assessed by a cellular antioxidant activity assay. KEY FINDINGS: The three antioxidants degraded to unquantifiable levels after oven-drying. More than 90% of the antioxidants were retained by freeze-drying and air-drying. Both leaf and extract samples retained >90% of antioxidants, except those stored at 75% relative humidity. Leaf material had higher antioxidant concentrations and greater cellular antioxidant activity than corresponding extract samples. CONCLUSION: Freeze-drying and air-drying preserved more antioxidants in S. oleraceus than oven-drying. From DOE analysis, humidity plays an important role in degradation of antioxidants during storage. To preserve antioxidant activity, it is preferable to store S. oleraceus as dried leaf material.

Hydroxycinnamic acids in Crepidiastrum denticulatum protect oxidative stress-induced retinal damage.

We investigated the effects of an ethanol extract of C. denticulatum (EECD) in a mouse model of glaucoma established by optic nerve crush (ONC), and found that EECD significantly protected against retinal ganglion cell (RGC) death caused by ONC. Furthermore, EECD effectively protected against N-methyl-d-aspartate-induced damage to the rat retinas. In vitro, EECD attenuated transformed retinal ganglion cell (RGC-5) death and significantly blunted the up-regulation of apoptotic proteins and mRNA level induced by 1-buthionine-(S,R)-sulfoximine combined with glutamate, reduced reactive oxygen species production by radical species, and inhibited lipid peroxidation. The major EECD components were found to be chicoric acid and 3,5-dicaffeoylquinic acid (3,5-DCQA) that have shown beneficial effects on retinal degeneration both in vitro and in vivo studies. Thus, EECD could be used as a natural neuroprotective agent for glaucoma, and chicoric acid and 3,5-DCQA as mark compounds for the development of functional food.

Echinacea purpurea (L.) Moench modulates human T-cell cytokine response.

The study objective was to evaluate the composition of a neutral and weakly acidic water-soluble extract from Echinacea purpurea (L.) Moench (EchNWA) previously shown to modify murine influenza infection, and to assess immunomodulatory effects on human T-cells. EchNWA extract from fresh aerial parts was extracted with water, ethanolic precipitation, and size-exclusion chromatography. The chemical profile of EchNWA was characterized by chromatography (size-exclusion, HPLC, GC-MS), and small molecule fingerprint analysis performed by HPLC-PDA. Jurkat T-cells at high and low cell density were pretreated or not with doses of EchNWA, followed by activation with phorbol 12-myristate 13-acetate plus ionomycin (PMA+I). Interleukin-2 (IL-2) and interferon gamma (IFNg) cytokine secretions were measured by multi-cytokine luminex technology. Results showed that EchNWA contains 80% polysaccharides, predominantly a 10kDa entity; phenolic compounds, cynarin, cichoric and caftaric acids, but no detectable alkylamides. Cytokine production required stimulation and was lower after PMA+I activation in high-density compared to low-density conditions. EchNWA mediated a strong dose-dependent enhancement of high-density T-cell production of IL-2 and IFNg response to PMA+I. EchNWA alone did not stimulate T-cells. EchNWA enhanced mean fluorescence intensity of IL-2 in Jurkat T-cells activated by PMA+1 or ionomycin alone. Conversely EchNWA mediated modest but significant suppression of IFNg response and reduced the percentage of CD25+ T-cells under low-density conditions. Conclusions are that EchNWA polysaccharides, but not phenolic compounds have dose-related adjuvant effects on human T-cell cytokine responses characterized by enhancing and suppressive effects that are regulated by T-cell density.

[Determination of dactylorhin A and militarine in three varieties of Cremastrae Pseudobulbus/Pleiones Pseudobulbus by HPLC].

To establish an HPLC method for determination of dactylorhin A and militarine in Cremastrae Pseudobulbus/Pleiones Pseudobulbus. The analysis was achieved on an Alltech Prevail C18 column (4. 6 mm x 250 mm, 5 microm) using a mobile phase of acetonitrile (A), water (B) gradient elution in a total run time of 35 min (0 min, 20:80; 30 min, 55:45; 35 min, 55:45) and a diode array detector was set at 224 nm. The flow rate was 0.8 mL x min(-1). The assay displayed good linearity over the concentration range of 0.257-9.95 microg (r = 0.999 8), and 0.128-10.27 microg (r = 0.999 9), respectively. The average recoveries (n = 9) were 94.70% and 102.8% for dactylorhin A and militarine, respectively. The method is accurate, quick, simple and reproducibility. It can be used for the quality control of Pleione bulbocodioides and Pleione yunnanensis.

[Simultaneous determination of five organic acids in Kudiezi injection by HPLC].

The aim was to develop a high performance liquid chromatography method for simultaneous determination of five organic acids in Kudiezi injection. The Diamonsil C18 column (4.6 mm x 200 mm, 5 microm) was adopted with acetonitrile and water as the mobile phase at a gradient mode program. The flow rate was 1.0 mL min-1 , detection wavelength was 325 nm, and column temperature was 35 degree C. The linear range of monocaffeyltartaric acid, chlorogenic acid, caffeic acid, ferulic acid, and chicoric acid were 0. 64-81.60 (r =0. 999 9),0.09-11. 10 (r =0.999 8) ,0.09-11.30 (r =0. 999 8),0.10-12.80 (r =0.999 9),0.43-55. 50 mg L-1 (r = 0.999 8) , respectively. The average recoveries were 101.8% ,100. 9% ,99. 24% ,99. 83% ,101.9%, respectively, with RSD of less than 2.0%. The developed HPLC method was simple, sensitive and accurate with good repeatability. This work provided helpful information for comprehensive quality control of Kudiezi injection. [Key words] Kudiezi injection; organic acids; content determination; HPLC

Quantification of phenylpropanoids in commercial Echinacea products using TLC with video densitometry as detection technique and ANN for data modelling.

INTRODUCTION: Echinacea preparations are among the most popular herbal remedies worldwide. Although it is generally assigned immune enhancement activities, the effectiveness of Echinacea is highly dependent on the Echinacea species, part of the plant used, the age of the plant, its location and the method of extraction. OBJECTIVE: The aim of this study was to investigate the capacity of an artificial neural network (ANN) to analyse thin-layer chromatography (TLC) chromatograms as fingerprint patterns for quantitative estimation of three phenylpropanoid markers (chicoric acid, chlorogenic acid and echinacoside) in commercial Echinacea products. MATERIAL AND METHODS: By applying samples with different weight ratios of marker compounds to the system, a database of chromatograms was constructed. One hundred and one signal intensities in each of the TLC chromatograms were correlated to the amounts of applied echinacoside, chlorogenic acid and chicoric acid using an ANN. RESULTS: The developed ANN correlation was used to quantify the amounts of three marker compounds in Echinacea commercial formulations. The minimum quantifiable level of 63, 154 and 98 ng and the limit of detection of 19, 46 and 29 ng were established for echinacoside, chlorogenic acid and chicoric acid respectively. CONCLUSION: A novel method for quality control of herbal products, based on TLC separation, high-resolution digital plate imaging and ANN data analysis has been developed. The method proposed can be adopted for routine evaluation of the phytochemical variability in Echinacea formulations available in the market.

Seasonal variations in the concentrations of lipophilic compounds and phenolic acids in the roots of Echinacea purpurea and Echinacea pallida.

Roots of Echinacea purpurea and Echinacea pallida cultivated for 4 years in a North European climate were analyzed for seasonal variations in the concentrations of lipophilic constituents (alkamides, ketoalkenes, and ketoalkynes) and phenolic acids by harvesting five times during 1 year to establish the optimal time for harvest. A total of 16 alkamides, three ketoalkenes, two ketoalkynes, and four phenolic acids (echinacoside, cichoric acid, caftaric acid, and chlorogenic acid) were identified in aqueous ethanolic (70%) extracts by liquid chromatography-mass spectrometry and quantified by reverse-phase high-performance liquid chromatography. The major alkamides in the roots of E. purpurea were at their lowest concentration in the middle of autumn and early winter, and the total concentration of lipophilic compounds in E. pallida showed the same pattern. Moreover, all of the major phenolic acids in E. purpurea were at their highest concentrations in spring. The optimal harvest time in spring is in contrast to normal growing guidelines; hence, this specific information of seasonal variations in the concentrations of lipophilic and phenolic compounds in E. purpurea and E. pallida is valuable for research, farmers, and producers of medicinal preparations.

Analysis of eleven phenolic compounds including novel p-coumaroyl derivatives in lettuce (Lactuca sativa L.) by ultra-high-performance liquid chromatography with photodiode array and mass spectrometry detection.

INTRODUCTION: Lettuce is a widely consumed vegetable and a good source of phenolic compounds. Several factors (genetic, agronomical and environmental) can influence the lettuce composition; their effects are not completely defined and more studies are needed on this topic. OBJECTIVE: To develop an improved ultra-high-performance liquid chromatography (UHPLC) method to quantify the main target intact phenolic compounds in lettuce. METHODOLOGY: UHPLC identification of the compounds was supported by PAD spectra and MS(n) analyses. Quantification was carried out by PAD, by creating matrix-matched calibration curves at the specific wavelength for each compound. RESULTS: Sample pretreatment was simplified, with neither purification nor hydrolysis steps. Chromatographic conditions were chosen to minimise matrix interferences and to give a suitable separation of the major phenolic compounds within 27 min. The method allowed the quantification of 11 intact phenolic compounds in Romaine lettuces, including phenolic acids (caffeoyl and p-coumaroyl esters) and flavonoids (quercetin glycosides). Four p-coumaroyl esters were tentatively identified and quantified for the first time in lettuce. CONCLUSION: The main intact phenolic compounds, including four novel p-coumaroyl esters, were simultaneously quantified in lettuce with optimal performances and a reduced total time of analysis. These findings make headway in the understanding of the lettuce phytochemicals with potential nutritional relevance.

Metabolites indicate hot spots of biodegradation and biogeochemical gradients in a high-resolution monitoring well.

Anaerobic degradation processes play an important role in contaminated aquifers. To indicate active biodegradation processes signature metabolites can be used. In this study field samples from a high-resolution multilevel well in a tar oil-contaminated, anoxic aquifer were analyzed for metabolites by liquid chromatography-tandem mass spectrometry and time-of-flight mass spectrometry. In addition to already known specific degradation products of toluene, xylenes, and naphthalenes, the seldom reported degradation products benzothiophenemethylsuccinic acid (BTMS), benzofuranmethylsuccinic acid (BFMS), methylnaphthyl-2-methylsuccinic acid (MNMS), and acenaphthene-5-carboxylic acid (AC) could be identified (BFMS, AC) and tentatively identified (BTMS, MNMS). The occurrence of BTMS and BFMS clearly show that the fumarate addition pathway, known for toluene and methylnaphthalene, is also important for the anaerobic degradation of heterocyclic contaminants in aquifers. The molar concentration ratios of metabolites and their related parent compounds differ over a wide range which shows that there is no simple and consistent quantitative relation. However, generally higher ratios were found for the more recalcitrant compounds, which are putatively cometabolically degraded (e.g., 2-carboxybenzothiophene and acenaphthene-5-carboxylic acid), indicating an accumulation of these metabolites. Vertical concentration profiles of benzylsuccinic acid (BS) and methyl-benzylsuccinic acid (MBS) showed distinct peaks at the fringes of the toluene and xylene plume indicating hot spots of biodegradation activity and supporting the plume fringe concept. However, there are some compounds which show a different vertical distribution with the most prominent concentrations where also the precursor compounds peaked.

Effect of high pressure pasteurization on bacterial load and bioactivity of Echinacea purpurea.

UNLABELLED: High hydrostatic pressure (HHP) technology was applied to organic Echinacea purpurea (E. purpurea) roots and flowers to determine the feasibility of using this technology for cold herb pasteurization, to produce microbiologically safe and shelf-stable products for the natural health products (NHPs) industry. HHP significantly (P < 0.01) reduced microbial contamination in both roots and flowers without affecting the phytochemical retention of chicoric and chlorogenic acids, and total alkamide contents. The antioxidant activity of E. purpurea methanol-derived extracts, evaluated in both chemical (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) [ABTS] and oxygen radical absorption capacity [ORAC] assay) and in cell culture models (RAW264, 7 macrophage, H(2)O(2)-induced intracellular oxidation, and lipopolysaccharide [LPS]-induced nitric oxide production), was not adversely affected by the application of HHP at both 2 and 5 min at 600 mPa. Furthermore, HHP did not affect the capacity of E. purpurea extracts to suppress nitric oxide production in LPS-activated macrophage cells. Therefore, our results show that HHP is an effective pasteurization process treatment to reduce microbial-contamination load while not adversely altering chemical and bioactive function of active constituents present in organic E. purpurea. PRACTICAL APPLICATION: Our study reports for the first time, the effectiveness of using high hydrostatic pressure (HHP) technology pressure to pasteurize E. purpurea root and flower, and the comparative retention of bioactive phytochemicals. Therefore, this technique can be used in food and natural health product industries to produce high-quality, microbiologically safe, and shelf-stable products.

[Determination of militarine in Rhizoma Bletillae by HPLC].

OBJECTIVE: To develop an HPLC method for determination of militarine in Rhizoma Bletillae. METHOD: Analysis was carried out on a Symmetry C18 analytical column, and a C18 guard column eluted with mobile phase consisted of acetonitrile and water (37:63) militarine the detection wavelength was set at 244 nm. RESULT: The contents of in 12 samples of Rhizoma Bletillae in market were in the ranges of 1.45%-3.32%. CONCLUSION: The method is simple, accurate and reproducible, and is suitable for the quality control of Rhizoma Bletillae.