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Development of methods for determination of the residues of 15 pesticides in medicinal herbs Isatis indigotica Fort. by capillary gas chromatography with electron capture or flame photometric detection.

Two multiresidue methods were developed for the determination of 15 pesticides (organochlorines, organphosphorus compounds, pyrethroids, and fungicides) in medicinal herbs Isatis indigotica Fort. and its formulations. The analytical procedure is based on ultrasonic assisted extraction and liquid-liquid extraction (LLE). After solvents were added, the raw material or granule sample was sonicated in an ultrasonic water bath and then centrifuged, filtered, and cleaned up by LLE. The infusion sample was extracted with petroleum ether by LLE. The pesticide residues were determined by capillary gas chromatography with electron-capture or flame photometric detection. Recoveries with the method at concentrations between 0.4 microg/kg and 10 mg/kg ranged from 70.2 to 119.5% for raw material, 73.2 to 105.1% for granule formulation, and 72.8 to 113.3% for infusion formulation. The relative standard deviation values were <20% for all of the pesticides studied. The pesticide detection limits were within the ranges 0.3-0.5 microg/L for endosulfan, 3-7.5 microg/L for pyrethroids, 0.7-32.5 microg/L for organophosphorus pesticides, and 0.1-0.6 microg/L for the other pesticides. The proposed methods are simple and rapid and provide simultaneous determination of pesticide residues in Isatis indigotica Fort. with acceptable recoveries and repeatability and an adequate limit of determination.

Chromatography, Gas↗

[Study on the extraction of indirubin from Isatis indigotica Fort].

OBJECTIVE: To study the optimal process of the extraction of indirubin from Isatis indigotica Fort. METHODS: The process was studied by supersonic extraction, refluxing and orthogonal design with the content of indirubin as the detective marker. Then the extraction of indirubin with supersonic extraction and other methods were compared basing on the yield of extracts. RESULTS: Among them, the supersonic extraction was the simplest and the most rapid and the most complete in extraction. And the optimal conditions were A1 B1 C2 D3: supersonic extraction with 60% ethanol, 1 hour, 10-fold solvent and 3 times. CONCLUSION: The supersonic extraction can extract more indirubin from Isatis indigotica Fort in shorter time with less energy. It also shows a promising prospect for leaching the effective constituents from Chinese herbal medicine by supersonic extraction.

Analysis of Variance↗

Quantitative determination of the dual COX-2/5-LOX inhibitor tryptanthrin in Isatis tinctoria by ESI-LC-MS.

Isatis tinctoria L. is an old European and Chinese dye plant and anti-inflammatory herb from which the potent cyclooxygenase-2 and 5-lipoxygenase inhibitor tryptanthrin (1) (indolo-[2,1-b]-quinazoline-6,12-dione) was recently isolated as one of the active principles. An HPLC method for the quantitative analysis of the compound in plant material was developed. Reproducible extraction was achieved by accelerated solvent extraction (ASE). Detection by UV at 254 and 387 nm and by electrospray-MS were compared. The low tryptanthrin content in the herb and possible interferences required isocratic high-performance liquid chromatography coupled with electrospray-MS in single ion mode. More than 70 Isatis samples of different origin were analyzed. The tryptanthrin content in leaf samples varied from 0.56 to 16.74 x 10(-3) %.

Arachidonate 5-Lipoxygenase↗

The elusive indigo precursors in woad (Isatis tinctoria L.)--identification of the major indigo precursor, isatan A, and a structure revision of isatan B.

A metabolite-profiling study of shock-frozen leaves of Isatis tinctoria L., an old indigo dye plant and medicinal herb, revealed a complex pattern of indigo-forming compounds with higher polarities than the known indigo precursors isatan B and indican. These highly unstable compounds underwent rapid post-harvest transformation and were not detected in air-dried leaves. The major indigo precursor, named isatan A (4), was isolated by rapid normal-phase and gel chromatography, along with isatan B (3). A full spectral data set of 3 showed that the previous structure assignment as 'indoxyl-5-ketogluconate' has to be revised to 1H-indol-3-yl beta-D-ribohex-3-ulopyranoside. Isatan A (4) was identified as 1H-indol-3-yl 6'-O-(carboxyacetyl)-beta-D-ribohex-3'-ulopyranoside. In aqueous solution, glycosides 3 and 4 occur as hydrates and undergo rapid hydrolysis under very mild acidic or basic conditions.

Glycosides↗

Anti-SARS coronavirus 3C-like protease effects of Isatis indigotica root and plant-derived phenolic compounds.

The 3C-like protease (3CLpro) of SARS-coronavirus mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, becoming an important target for the drug development. In this study, Isatis indigotica root extract, five major compounds of I. indigotica root, and seven plant-derived phenolic compounds were tested for anti-SARS-CoV 3CLpro effects using cell-free and cell-based cleavage assays. Cleavage assays with the 3CLpro demonstrated that IC50 values were in micromolar ranges for I. indigotica root extract, indigo, sinigrin, aloe emodin and hesperetin. Sinigrin (IC50: 217 microM) was more efficient in blocking the cleavage processing of the 3CLpro than indigo (IC50: 752 microM) and beta-sitosterol (IC50: 1210 microM) in the cell-based assay. Only two phenolic compounds aloe emodin and hesperetin dose-dependently inhibited cleavage activity of the 3CLpro, in which the IC50 was 366 microM for aloe emodin and 8.3 microM for hesperetin in the cell-based assay.

Animals↗

HPLC based activity profiling for 5-lipoxygenase inhibitory activity in Isatis tinctoria leaf extracts.

In the pursuit of the anti-inflammatory constituents in lipophilic woad extracts, the 5-lipoxygenase (5-LOX) inhibitory activity was investigated by HPLC-based activity profiling. In a low-resolution profiling, two time windows with peaks of activity were found. The first coincided with tryptanthrin, a known dual inhibitor of cyclooxygenase-2 (COX-2) and 5-LOX, whereas the major inhibitory fraction was towards the end of the HPLC run. The active fractions were profiled in a peak-resolved manner, and the compounds analyzed by LC-MS, GC and TLC. The activity in the lipophilic fractions of the Isatis extract could be linked to an unsaturated fatty acid, alpha-linolenic acid.

Chromatography, High Pressure Liquid↗

Chemical fingerprinting of Isatis indigotica root by RP-HPLC and hierarchical clustering analysis.

The aim was to establish a method for extraction and chemical fingerprinting of extracts of Isatis indigotica roots ("Ban-Lan-Gen") and to apply the method developed to 18 Ban-Lan-Gen samples. RP-HPLC with gradient elution was performed on authentic reference standards of powdered I. indigotica roots, indigotin and indirubin purchased from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP) of China. Eighteen "Ban-Lan-Gen" samples (including the reference powdered herb) were bought from Singapore and different regions in China. Comparisons of the chromatograms showed that the samples can be divided into three groups. The chromatograms of the extracts of five samples were found to be similar to that of the extract of the authentic sample. Eight other samples had similar peaks as the authentic sample but the intensities of the peaks were generally lower, except for the peaks between retention times of 10-40 min. Peaks in these regions were more intense than those found in the extract of the authentic sample. Forty-five characteristic peaks could be found in the extracts of all the above samples. Peaks at retention times 52 and 53 min were determined to be indigotin and indirubin, respectively. The remaining four samples had similar chemical fingerprints to each other but were different from that of the authentic sample. Hierarchical clustering analysis gave similar results as the visual comparison. The RP-HPLC method developed allows simple identification and comparisons of I. indigotica roots. This is the first report of hierarchical clustering analysis of I. indigotica root.

Chromatography, High Pressure Liquid↗

The content of indigo precursors in Isatis tinctoria leaves--a comparative study of selected accessions and post-harvest treatments.

We recently clarified the nature of indigo precursors in woad (Isatis tinctoria L.), by identifying the major indoxyl glycoside as isatan A (indoxyl-3-O-(6'-O-malonyl-beta-D-ribohexo-3-ulopyranoside)), and by correcting the structure of the related isatan B (indoxyl-3-O-beta-D-ribohexo-3-ulopyranoside). A quantitative densitometric assay for isatans A and B, and indican, was established and validated. HPTLC separation on silica gel was followed by densitometric analysis of indigoid pigments formed after treatment with dilute acid or base. The seasonal variation of indoxyl glycosides in woad leaves was investigated with first-year plants (rosette stage) of five defined I. tinctoria L. and one I. indigotica L. accessions. Isatan A content reached up to 7.6% of dry weight in I. tinctoria, and up to 21.8% in I. indigotica. The influence of various post-harvest treatments was studied. High concentrations of isatans A and B were found in freeze-dried leaf samples, whereas the content of indican was lowest. Conventional drying at ambient or 40 degrees C led to complete disappearance of isatans A and B. The concentration of indican, in contrast, was 3- to 5-fold higher in leaf samples submitted to drying at ambient and 40 degrees C, respectively.

Chromatography, High Pressure Liquid↗

Formation of natural indigo derived from woad (Isatis tinctoria L.) in relation to product purity.

There is an increasing commercial demand for naturally sourced indigo that meets the purity standards set by the synthetic product. This study concerns the indigo made from leaves of woad (Isatis tinctoria L.), and in particular its interaction with particulate impurities arising from soil and plant materials. Also, a more reliable method using N-methyl-2-pyrrolidone has been developed for the spectrophotometric determination of indigo. In a novel application of fluorescence spectroscopy, indoxyl intermediates in indigo formation are shown to be stable for minutes. The main indigo precursor from woad can be adsorbed onto Amberlite XAD16 in conformity with a Langmuir isotherm, but indigo precursors break down on this and other resin beads to yield indigo and red compounds. Indigo made from indoxyl acetate aggregates into particles, the size distribution of which can be modified by the inclusion of a fine dispersion of calcium hydroxide. Bright field microscopy of indigo products made under defined conditions and scanning electron microscopy combined with energy-dispersive X-ray analysis reveal the relationship of indigo with particulate materials. A model illustrating the interaction of indigo with particulate contaminants is developed on the basis of the results obtained, and recommendations are made for improving the purity of natural indigo.

Indigo Carmine↗

Bisindigotin, a TCDD antagonist from the Chinese medicinal herb Isatis indigotica.

A new indigoid derivative, bisindigotin (1), with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-antagonistic activity was isolated from the ethanol extract of the Chinese medicinal herb Isatis indigotica. Its structure was determined by spectroscopic methods. In the human HepG2 hepatoma cell model, 1 (50 nM to 2 microM) was found to dose-dependently inhibit TCDD-induced ethoxyresorufin O-deethylase (EROD) activity.

Carcinoma, Hepatocellular↗

Production and analysis of organic acids in hairy-root cultures of Isatis indigotica Fort. (indigo woad).

Hairy roots were induced from both cotyledon and hypocotyl explants of Isatis indigotica Fort. (indigo woad) through transformation with Agrobaterium rhizogenes strain A4, R1601 and ATCC15834. The results showed that the cotyledons were the preferred explants to hypocotyls and A4 was the most suitable A. rhizogenes strain for the transformation and induction of hairy roots of I. indigotica. High-voltage paper electrophoresis (HVPE) analysis demonstrated the production of mannopine in hairy roots and confirmed the successful transfer of Ri T-DNA (root-inducing transferred DNA) of A. rhizogenes into the I. indigotica genome. Five organic acids, namely CPQ [3-(2-carboxyphenol)-4(3 H )-quinazolinone], syringic acid, salicylic acid, benzoic acid and 2-aminobenzoic acid, which were considered as main antiviral components of I. indigotica, were detected in natural roots, hairy roots and liquid media with high-performance capillary electrophoresis. The results showed CPQ production in hairy roots was significantly higher than that in natural roots. Our results also revealed that all the five organic acids could be excreted from hairy roots into liquid media, and the concentrations of organic acids in the liquid media paralleled those in hairy roots. The hairy roots of I. indigotica grew fast and showed an S-shaped growth curve that reached its apex on the day 24 of culture with a 20-fold increase in fresh weight compared with the starting inoculums. The accumulation of the two organic acids CPQ and syringic acid in liquid media paralleled the growth of hairy roots. MS [Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15, 473-497] medium or half-strength MS medium supplemented with 30 g/l maltose was found to be best for hairy-root culture and accumulation of CPQ.

Benzoic Acid↗

Anti-inflammatory and antiallergic activity in vivo of lipophilic Isatis tinctoria extracts and tryptanthrin.

The effects of a supercritical CO2 (SFE) extract, a dichloromethane (DCM) extract from Isatis tinctoria leaf and the alkaloidal constituent tryptanthrin were studied in acute and subchronic experimental models of inflammation. The SFE and DCM extracts showed anti-inflammatory activity in the carrageenan-induced acute mouse paw oedema (ED50 values of 78 mg/kg and 165 mg/kg P. O., respectively) and in the acute tetradecanoylphorbol acetate (TPA)-induced mouse ear oedema in oral (62% and 32% oedema reduction at 100 and 125 mg/kg, respectively) and topical application (37% and 33% reduction of oedema at 0.5 mg/ear). In contrast, tryptanthrin showed no significant anti-inflammatory effect. The DCM extract inhibited oedema formation and neutrophil infiltration in subchronic inflammation in mice induced by repeated application of TPA. The extract showed activity after oral and topical administration by reducing the various parameters of the inflammatory response. The DCM extract (1 mg/ear) inhibited the delayed-type hypersensitivity (DTH) reaction induced by application of dinitrofluorobenzene (DNFB) after topical application. The response during the induction phase (24 h) was decreased by 48%, and the inflammatory phase (48 to 96 h) was reduced by 53 to 56%. The extract had no effect in this model when administered orally. The DCM extract (200 mg/kg P. O.) inhibited the acetic acid-induced writhing by 49%.

Administration, Cutaneous↗

Molecular cloning and characterization of a novel calcium-dependent protein kinase gene IiCPK2 Responsive to polyploidy from tetraploid Isatis indigotica.

A novel calcium-dependent protein kinase gene (designated as IiCPK2) was cloned from tetraploid Isatis indigotica. The full-length cDNA of IiCPK2 was 2585 bp long with an open reading frame (ORF) of 1878 bp encoding a polypeptide of 625 amino acid residues. The predicted IiCPK2 polypeptide included three domains: a kinase domain, a junction domain (or autoinhibitory region), and a C-terminal calmodulin-like domain (or calcium-binding domain), which presented a typical structure of plant CDPKs. Further analysis of IiCPK2 genomic DNA revealed that it contained 7 exons, 6 introns and the length of most exons was highly conserved. Semi-quantitative RTPCR revealed that the expression of IiCPK2 in root, stem and leaf were much higher in tetraploid sample than that in diploid progenitor. Further expression analysis revealed that gibberellin (GA3), NaCl and cold treatments could upregulate the IiCPK2 transcription. All our findings suggest that IiCPK2 might participate in the cold, high salinity and GA3 responsive pathways.

Amino Acid Sequence↗

[Evaluation on antiendotoxic action and antiviral action in vitro of tetraploid Isatis indigotica].

OBJECTIVE: To evaluate the pharmacological effect of tetraploid Isatis indigotica Fort. METHODS: Comparing these actions of tetraploid I. indigotica with those of the diploid parent. RESULTS: These actions of tetraploid I. indigotica were obviously stronger than those of the diploid parent. The pharmacological effect of leaves was greater than that of roots with identical ploidy. CONCLUSION: Tetraploid I. indigotica is a new line of better quality, deserving to be exploited and utilized on a large scale.

Animals↗

[Quantitative analysis of 4(3)-quinazolinedione in 4 "daqingye", leave of Isatis indigotica, Baphicacanthus cusia, Polygonum tinctorium and Clerodendron cyrtophyllum].

Using 4(3)-quinazolinedione, the active constituent of "Daqingye", as the quality control criteria, 4 samples of the herbal drug collected from Isatis indigotica, Baphicacanthus cusia, Polygonum tinctorium and Clerodendron cyrtophyllum were comparetively analyzed by RP-HPLC. The results showed that the average 4(3)-quinazolinedione contents (microgram/g) were 30.7, 7.2, 4.4 and 27.5 respectively. This method was proved to be quick, simple and reproducible, and provided a reliable basis for the quality control and evaluation of Chinese Traditional Drug "Daqingye".

Anti-Inflammatory Agents, Non-Steroidal↗

[Studies on chemical constituents in leaf of Isatis indigotica].

OBJECTIVE: To study the chemical constituents of the leaf of Isatis indigotica. METHOD: Chromatography and spectral analysis were respectively used to isolate and identify the constituents. RESULT: Three compounds were isolated from the ethanol extracts of theleaf of I. indigotica, and identified as indirubin, tryptanthrin and L-pyroglutamic acid. CONCLUSION: L-pyroglutamic acid was isolated from the genus for the first time, and tryptanthrin was isolated from the leaf of this plant for the first time.

Drugs, Chinese Herbal↗

[Establishment of a novel preliminary screening model for bioactive parts of folium of Isatis indigotica based on biothermodynamics].

OBJECTIVE: To apply a novel method for screening the bioactive parts and substances of traditional Chinese medicines (TCM) based on biothermodynamic theories. METHOD: The power-time curves of Escherchia coli proliferation at different concentrations of decoction and its five bioactive parts of folium of Isatis indigotica (FII) were determined by TAM Air Isothermal Calorimeter. The growth rate constants of promotive and inhibitory actions were calculated. Moreover, the potential pharmacological action of FII was analyzed from the point of view of TCM theory. RESULT: The growth rate constant k1 and Pm of the studied bacterium in the presence of the decoction of FII were concentration-dependant. The k1 and Pm increased with the mass increase of the decoction and its water-extract of FII. However, the value of k1 and Pm decreased with the increasing concentrations of its other four organic solvent parts. The experimental results revealed that the sequence of inhibitory activity of different extracts was n-BuOH extract > EtOAc extract > chloroform extract > PE extract. CONCLUSION: The decoction and its water-extract of FII stimulated the metabolism of E. coli. While the other four organic solvent extracts of the total decoction inhibited the growth of tested bacterium. The microcalorimetric method has many virtues such as extensive applicability, convenience, etc, and provides a new and useful means for primary screening the bioactive parts and substances of TCM.

Anti-Bacterial Agents↗

Identification of an indigo precursor from leaves of Isatis tinctoria (Woad).

Indole is presumably a product of indole-3-glycerol phosphate catabolism in Isatis tinctoria. It is oxidized into indoxyl and stored in young leaves as indigo precursor. Further oxidation and dimerization of indoxyl produces indigoid pigments. In this work, we describe an HPLC method dedicated to the identification and quantification of indigoid pigments (indigo, indirubin, isoindigo and isoindirubin) and indigo precursors produced in I. tinctoria (Woad). This work, carried out with two cultivars of I. tinctoria, has confirmed that the quantity of indigo precursors is dependent on the species and the harvest period. In addition we have shown for the first time that young leaves of I. tinctoria, harvested in June contained a new indigo precursor in addition to isatan B (indoxyl-5-ketogluconate) and indican (indoxyl-beta-D-glucoside). We suggest the name "isatan C" for this new indigo precursor in I. tinctoria. Its chemical characteristics point to an dioxindole ester with PM of 395. We have shown that isatan C reacts with isatan B increasing the red pigment production.

Brassicaceae↗