dehydroandrographolide and neoandrographolide

Andrographis paniculata is an important traditional Chinese herbal material widely used for treatment of fever and diarrhea. The three diterpenoids [andrographolide (AP), dehydroandrographolide (DP) and neoandrographolide (NP)] in A. paniculata were characterized as the key active components for its extractive and related medicinal preparations. A rapid and accurate quantitative analysis of these three diterpenoids in A. paniculata capsules was accomplished by means of a valid and reliable high-performance liquid chromatography with tandem mass spectrometric (HPLC-MS/MS) analytical method. An optimized gradient elution method, which under the mobile phase consisting of methanol and water, was established successfully. An XDB C18 column (3.5 μm, 2.1 mm × 50 mm) was successfully applied to separate these three diterpenoids at the flow rate of 0.55 mL/min. A triple quadrupole mass spectrometer with multiple reaction monitoring (MRM) mode using an electrospray ionization source was served for analytical detection. The linearity, specificity, accuracy, recovery, precision, stability and repeatability were demonstrated to fully confirm the validity of this study. Good linear regression relationships for the three analytes were obtained over the range of 0.50-1000 ng/mL. Intra-day and inter-day precisions were evaluated with relative standard deviation < 7.62%, and the extraction recovery varied from 93.8% to 102.0%. The rapid and accurate HPLC-MS/MS analytical method was developed for the quality evaluation of A. paniculata capsules successfully. The content of AP, DP and NP obtained were 3.90-4.08, 4.77-5.04, 4.32-4.48 mg/g in A. paniculata capsules, respectively. Moreover, it is expected that this analytical technique can be applied to detect three effective components rapidly and accurately in other different medicinal preparations of A. paniculata as a method for their quality control.

Topics: Andrographis; Capsules; Chromatography, High Pressure Liquid; Diterpenes; Drugs, Chinese Herbal; Glucosides; Quality Control; Tandem Mass Spectrometry; Tetrahydronaphthalenes

Topics: Andrographis; Diagnostic Equipment; Diterpenes; Glucosides; Principal Component Analysis; Taste; Tetrahydronaphthalenes

In this study, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determinate andrographolide (AP), dehydroandrographolide (DP), and neoandrographolide (NP) in plasma of beagle dogs after oral administration of Andrographis paniculata tablet (A. paniculata). The analytes and bilobalide (internal standard) were separated on an Agilent ZORBAX XDB-C18 column (50mm×2.1mm, 3.5μm) by using gradient elution consisting of methanol and water at a flow rate of 0.50mL/min in 7min. Multiple reaction monitoring (MRM) mode was performed to quantify data under monitoring precursor-product ion transitions of m/z 348.8→286.9, 330.9→107.9, 479.1→160.8 and 325.0→163.0 for AP, DP, NP and internal standard (IS) at negative ion mode, respectively. This method was developed at linearity ranging from 0.50 to 250ng/mL for AP, 1.00 to 500ng/mL for DP and 0.20 to 100ng/mL for NP. The accuracy of each analyte ranged between 94.8% and 107.1% and the precision was within 14.6%. No significant matrix effect was observed. AP, DP and NP were stable during sample storage, preparation and analytic procedures. Furthermore, this method was successfully applied in the investigation of the pharmacokinetic profile of AP, DP and NP in beagle dogs after oral administration of A. paniculata tablet (49.5mg for AP, 7.0mg for DP, 22.0mg for NP). Biological half-life (t1/2) was 2.08±0.99, 3.13±1.19 and 1.07±0.38h for AP, DP and NP, respectively. The areas under curves (AUC0-t) of AP, DP and NP was 494.50±150.64, 26.01±8.72 and 78.78±18.29ngh/mL, respectively.

Topics: Andrographis; Animals; Chromatography, Liquid; Diterpenes; Dogs; Drug Stability; Glucosides; Linear Models; Male; Plant Extracts; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry; Tetrahydronaphthalenes

To establish the specific fingerprint of alcohol extract of Andrographis paniculata by HPLC.. The analysis was performed on Cosmosil 5C18 -MS-II (250 mm x 4. 6 mm, 5 µm) column, with gradient phase consisting of acetonitrile and water at the flow rate of 1. 0 mL/min. The UV detection wavelength was set at 225 nm, and column temperature was 30 °C.. The specific fingerprint chromatogram was established and seven common peaks were identified by comparison with the reference standards and LC-MS. The relative retention times were 1. 00 (No. 1, andrographolide), 1. 04 (No. 2, deoxyandrographoside), 1. 07 (No. 3, isoandrographolide), 1. 10 (No. 4, 14-deoxy-11, 12-didehydroandrographoside), 1. 50 (No. 5, neoandrographolide), 1. 75 ( No. 6, deoxyandrographolide) and 1. 79 (No. 7, dehydroandrographolide).. The method is simple and reproducible with high precision, which can provide the basis for the quality control and evaluation of alcohol extract of Andrographis paniculata and its preparations.

Topics: Andrographis; Chromatography, High Pressure Liquid; Diterpenes; Ethanol; Glucosides; Plant Extracts; Plants, Medicinal; Tetrahydronaphthalenes

To establish the high-performance thin layer chromatography (HPTLC) fingerprint of andrographolides from Andrographis paniculata, and to valuate the fingerprint similarity of samples from different habitats, markets, used parts and so on.. Chromatographic conditions were as follows: stationary phase: precoated HPTLC GF254 silica-gel plate (20 cm x 10 cm); developing solvent system: chloroform-toluene-methanol (80:10:15); Relative humidity: 42%; Color development reagent: 5% H2SO4 ethanolic solution, heating at 105 degrees C and observing the fluorescent chromatogram in a UV cabinet at 366 nm. The common patterns of HPTLC fingerprint were obtained through CHROMAP 1.5 solution software.. The HPTLC fingerprint of andrographolides was consisted of 9 characteristic peaks (fluorescent bands) including andrographolide, neoandrographolide and dehydroandrographolide which were chemical reference substances. The investigation and analysis of 51 batches of Andrographis paniculata showed that there were remarkable differences among different samples, so was the content of andrographolide and total lactones.. This method is simple and rapid, which can serve as an effective identification and quality assessment method for Andrographis paniculata.

Topics: Andrographis; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Desiccation; Diterpenes; Drug Stability; Glucosides; Plant Components, Aerial; Plants, Medicinal; Tetrahydronaphthalenes

Andrographis paniculata from different parts and origins were analyzed by UPLC-PDA fingerprint to provide refererice for related preparation technology. Using the peak of andrographolide as reference, 27 common peaks were identified, and digitized UPLC-PDA fingerprints for 23 batches of andrographis paniculata were established in this research. Principal component analysis (PCA) was carried out after feature extraction. The contents of andrographolide, neoandrographolide, deoxyandrographolide, dehydroandrographolide were determined by external standard method. The Plackett-Burman design combined with pareto chart was used to analyze the factors influencing the robustness of the method. It was found that the medicinal part has a more remarkable influence on the quality of andrographis paniculata than the origin. The contents of the 4 lactones the differ greatly in the different parts of andrographis paniculata, and the pH of the mobile phase is an important factor that influenced the robustness of the method.

Topics: Andrographis; Chromatography, Liquid; Diterpenes; Drug Stability; Glucosides; Principal Component Analysis; Tetrahydronaphthalenes

A rapid resolution liquid chromatography/time-of-flight tandem mass spectrometry (RRLC-TOF/MS) method was developed for qualitative and quantitative analysis of the major chemical constituents in Andrographis paniculata. Fifteen compounds, including flavonoids and diterpenoid lactones, were unambiguously or tentatively identified in 10 min by comparing their retention times and accurate masses with standards or literature data. The characteristic fragmentation patterns of flavonoids and diterpenoid lactones were summarized, and the structures of the unknown compounds were predicted. Andrographolide, dehydroandrographolide and neoandrographolide were further quantified as marker substances. It was found that the calibration curves for all analytes showed good linearity (R² > 0.9995) within the test ranges. The overall limits of detection (LODs) and limits of quantification (LOQs) were 0.02 μg/mL to 0.06 μg/mL and 0.06 μg/mL to 0.2 μg/mL, respectively. The relative standard deviations (RSDs) for intra- and inter-day precisions were below 3.3% and 4.2%, respectively. The mean recovery rates ranged from 96.7% to 104.5% with the relative standard deviations (RSDs) less than 2.72%. It is concluded that RRLC-TOF/MS is powerful and practical in qualitative and quantitative analysis of complex plant samples due to time savings, sensitivity, precision, accuracy and lowering solvent consumption.

Topics: Andrographis; Calibration; Chromatography, High Pressure Liquid; Diterpenes; Drugs, Chinese Herbal; Ethanol; Flavones; Glucosides; Limit of Detection; Solid Phase Extraction; Solvents; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Tetrahydronaphthalenes

A quantitative proton nuclear magnetic resonance technique (qHNMR) has been successfully introduced to quantify andrographolide, dehydroandrographolide, deoxyandrographolide and neoandrographolide in Andrographis paniculata, a commonly used important traditional Chinese medicine. Creative use of trifluoroacetic acid-d, which satisfactorily resolved the overlapping signals of these compounds in crowded regions of δ 4.5-5.6 ppm in (1)H NMR spectrum, made their quantification possible. Optimization of other experimental conditions, including internal standard, NMR pulse sequence, and NMR relaxation delay time, finally established the (1)H NMR based quantification approach, which was validated with satisfactory accuracy, precision, repeatability, and recovery. Except for deoxyandrographolide and neoandrographolide in two compound recipes, this method was successfully applied to quantify the four major components in fourteen raw herb materials and five commercial preparations, providing quantification results in good agreement with those determined by HPLC. The inherent advantages of qHNMR, such as its rapidity and simplicity, make itself a feasible alternative to HPLC for the quality control of A. paniculata raw material and herbal preparations.

Topics: Andrographis; Calibration; Diterpenes; Feasibility Studies; Glucosides; Magnetic Resonance Spectroscopy; Plant Preparations; Quality Control; Reference Standards; Reproducibility of Results; Tetrahydronaphthalenes; Trifluoroacetic Acid

In inflammation, the responses to noxious stimuli are controlled by the highly modulated interactions between various immune cells and chemical mediators. The purpose of this study is to evaluate and compare the anti-inflammatory effect of diterpenoids isolated from Andrographis paniculata, including dehydroandrographolide (AP1), andrographolide (AP2) and neoandrographolide (AP3), on the production of inflammatory cytokines and COX activities. Furthermore, the alteration of gene expression involved in this activity was investigated in the most potent compound to elucidate the other possible molecular mechanisms. AP1 (30.1 μM; 10 μg/ml) and AP2 (28.5 μM; 10 μg/ml) markedly inhibited COX-1 in ionophore A23187-induced human platelets. AP2 (28.5 μM) and AP3 (20.8 μM; 10 μg/ml) strongly suppressed the LPS-stimulated COX-2 activity in human blood. In addition, AP2 modulated the level of LPS-induced TNF-α, IL-6, IL-1β and IL-10 secretion in human blood in a concentration-dependent manner. The results revealed that AP2 exhibited the highest efficacy. Therefore, changes in the levels of mRNA transcripts by AP2 were further measured using human cDNA microarrays. The molecular response to AP2 was complex and mediated by various processes. Among the altered gene expressions, the genes involved in immune and inflammation processes were selectively down-regulated, such as cytokines and cytokine receptors (TNFSF14, TNF, TNFRSF6, and IL1A), chemokines (CCL8 and CXCL11), JAK/STAT signaling (JAK3 and STAT5A), TLRs family (TLR4 and TLR8) and NF-κB (NFKB1). Expression of some genes was validated using RT-PCR. The results demonstrated that AP1, AP2 and AP3 exhibited the anti-inflammatory effect by interfering COX and inflammatory cytokines and the underlying mechanisms of AP2 may be related to down-expression of genes involved in inflammatory cascade.

Topics: Adult; Andrographis; Anti-Inflammatory Agents, Non-Steroidal; Blood Platelets; Calcimycin; Cyclooxygenase 1; Cyclooxygenase 2; Cytokines; Diterpenes; Down-Regulation; Gene Expression; Glucosides; Humans; Male; Receptors, Cytokine; Tetrahydronaphthalenes; Toll-Like Receptors; Young Adult