A new diacylated labdane diterpenoid from Andrographis wightiana

A new acylated labdane diterpenoid, 14-deoxy-3,19-diacetyl-11,12-didehydroandrographolide (1), together with three known labdane diterpenoids, wightionolide (2), andrographolide (3) and neoandrographolide (4), and three known flavones, echioidinin (5), skullcapflavone I 2'-methyl ether (6) and echioidin (7), were isolated from the whole plant of Andrographis wightiana. The structure of compound 1 was elucidated by 1D and extensive 2D-NMR spectral studies.     

Simultaneous determination of four andrographolides in Andrographis paniculata Nees by silver ion reversed-phase high-performance liquid chromatography

A rapid and sensitive silver ion high-performance liquid chromatographic (Ag[I]-HPLC) method is developed for the simultaneous determination of the biologically active diterpenoids andrographolide, 14-deoxy-11,12-didehydroandrographolide, 14-deoxyandrographolide, and neoandrographolide in Andrographis paniculata Nees. HPLC is carried out for determining andrographolide and its derivatives with methanol-water (55:45, v/v) as the mobile phase on a C18 column (5 microm, 150 mm x 4.6 mm i.d.) with UV detection at 205 nm. Four andrographolides are baseline separated in a novel way: by adding silver ions (0.005 mol L(-1)) to the previously mentioned mobile phase. Validation of the method challenges specificity, linearity, limit of detection, limit of quantitation, accuracy, and repeatability, and the results met the acceptance criteria for all analytes. The molecular mechanism of retention is demonstrated by comparing partition coefficients (logP) of different andrographolides and andrographolide-Ag(I) complexes. Thus, the method is successfully applied to characterize and determine the four andrographolides in Andrographis paniculata Nees extract and its commercial product.     

Rapid Simultaneous Determination of Andrographolides in Andrographis paniculata by Near-Infrared Spectroscopy

The potential of near-infrared spectroscopy (NIRS) for the quality control of traditional Chinese medicine has been evaluated. Seven quantitative parameters, andrographolide, deoxyandrographolide, dehydroandrographolide, neoandrographolide, moisture, ash content, and alcohol-soluble extract of Andrographis paniculata, were evaluated by NIRS. The reference values of andrographolides were determined by high-performance liquid chromatography, and the others were obtained using the standard methods of the 2015 Chinese Pharmacopoeia. The predicted values were determined by a quantitative model using NIRS based on partial least square regression. Different spectral preprocessing methods, spectral ranges, and optimum number of factors were selected to optimize the models. All models were estimated by the combination of various parameters, including the correlation coefficient of calibration for andrographolide, deoxyandrographolide, dehydroandrographolide, neoandrographolide, moisture, ash content, alcohol-soluble extract (values of 0.980, 0.984, 0.989, 0.983, 0.987, 0.988, 0.979, respectively), root mean square error of calibration (values of 0.156, 0.038, 0.050, 0.029, 0.604, 0.431, 0.135, respectively), root mean square error of prediction (values of 0.169, 0.041, 0.050, 0.033, 0.280, 0.493, 0.140, respectively), root mean square error of cross-validation (values of 0.626, 0.114, 0.158, 0.046, 1.145, 0.774, 0.508, respectively), and ratio of standard deviation to standard error of prediction (values of 4.583, 4.690, 4.796, 4.899, 4.899, 4.690, 5.099, respectively). The results show that the calibration models by NIRS are reliable and can be applied for the quantification for seven parameters from A. paniculata for quality control in traditional Chinese medicine production and processing.     

A new diterpene from the leaves of Andrographis paniculata Nees

Phytochemical investigation of the methanolic extract of the leaves of Andrographis paniculata Nees yielded a minor, new diterpene, 21-nor-3,19-isopropylidine-14-deoxy-ent-labda-8(17),13-dien-16,15-olide (1), together with five known labdane type diterpenes, andrographolide 3, 14-deoxyandrographolide 4, 14-deoxy-12-hydroxyandrographolide 5, 14-deoxy-11,12-dihydroandrographolide 6, neoandrographolide 7, and two acids, cinnamic acid 8, and ferulic acid 9. The chemical structures were established by spectroscopic analyses, including 1D- and 2D-NMR spectroscopic experiments, and on the basis of HR-ESI MS analyses. The methanolic extract of the leaves exhibited moderate antibacterial and antifungal activities at a dose of 200 microg/mL in comparison with the reference standards.     

Camphor sulphonic acid mediated quantitative 1,3–diol protection of major Labdane diterpenes isolated from Andrographis paniculata

Phytochemical survey of the methanol extract of the dried aerial parts of Andrographis paniculata led to the isolation of major labdane diterpenes, namely 14-deoxy-11,12-didehydroandrographolide, andrographolide and neoandrographolide. Andrographolide was found to be the major phytoconstituent of the plant which was biologically active. For better physiochemical characteristics and bioefficacy, andrographolide is subjected to semi-synthetic modifications. However, presence of several free hydroxyl groups associated with this molecule make it quite polar and poorly soluble in many organic solvents and hence unsuitable for synthetic modifications. One way of resolving its solubility issue is to protect 1,3-diol quantitatively under mild reaction condition without effecting other functional groups. Reaction conditions were optimised using different solvent systems and catalysts towards this direction. X-ray structure of 3,19-isopropylidene-14-deoxy-11,12-didehydroandrographolide is being reported here for the first time. Isolated compounds and derivatives were confirmed by spectral analysis or X-ray data analysis.     

Determination of bioactive diterpenoids from Andrographis paniculata by micellar electrokinetic chromatography.

The present paper describes the development of a micellar electrokinetic chromatographic (MEKC) method for simultaneous determination of andrographolide, deoxyandrographolide and neoandrographolide in ethanol extracts of Andrographis paniculata. Separations were carried out in a fused-silica capillary tube with UV detection at 214 nm. Good separation was achieved using a 20 mM borate buffer, containing 20 mM sodium dodecyl sulphate and 10 mM sodium cholate, adjusted to pH 8.3 at an operating voltage of 25 kV, temperature of 35°C and a hydrodynamic injection of 5 s. The method was validated with good correlation coefficients obtained (0.9986–0.9989) while relative standard deviation (RSD) of migration time was between 1.14 and 2.42. It is concluded that this method could be used for speedy and accurate qualitative and quantitative analysis of bioactive diterpenoids in andrographis herb and its derived products.     

On-line coupling of dynamic microwave-assisted extraction with high-performance liquid chromatography for determination of andrographolide and dehydroandrographolide in Andrographis paniculata Nees

A novel technique based on dynamic microwave-assisted extraction (DMAE) coupled on-line with high-performance liquid chromatography (HPLC) through a flow injection interface has been developed for determination of andrographolide and dehydroandrographolide in Andrographis paniculata Nees. A TM(010) microwave resonance cavity built in the laboratory was applied to concentrating the microwave energy. An extraction vessel was placed in microwave irradiation zone. The extraction was performed in a recirculating system. When a number of extraction cycles were completed, the fractional extract (20muL) was driven to the analytical column by 65% aqueous methanol and was measured by diode array detector (DAD) at 225nm. The optimized extraction conditions are follows: extraction solvent 60% aqueous methanol; microwave forward power 80W; extraction time 6min; extraction solvent flow-rate 1.0mLmin(-1). The detection and quantification limits obtained are 0.5 and 1.7microgmL(-1) for andrographolide and 0.6 and 1.9microgmL(-1) for dehydroandrographolide, respectively. The within-day and between-day precision (RSD) are 2.1% and 3.7% for andrographolide and 1.7% and 4.1% for dehydroandrographolide, respectively. Mean recoveries for andrographolide and dehydroandrographolide are 97.7% and 98.7%, respectively. Compared with ultrasonic extraction used in the Chinese pharmacopoeia, the proposed method was demonstrated to obtain higher extraction yield in a shorter time. In addition, only small quantities of solvent (5mL) and sample (10mg) were required.     

Determination of andrographolide and dehydroandrographolide in rabbit plasma by on-line solid phase extraction of high-performance liquid chromatography

The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining andrographolide and dehydroandrographolide in rabbit plasma. Plasma samples (100 μL) were injected directly into a C18 SPE column and the biological matrix was washed out for 6 min using 15% aqueous methanol. By rotation of the switching valve, andrographolide and dehydroandrographolide were eluted in the back-flush mode and transferred to the analytical column by the chromatographic mobile phase consisted of methanol:acetonitrile (ACN):water (50:10:40; v/v). The UV detection was performed at 225 nm. The calibration curves showed excellent linear relationship (R ≥ 0.9993) over the concentration range of 0.05-5.0 μg mL⁻¹. The within- and between-day precisions (R.S.D.) of two analytes were in the range of 1.2-6.5% and the accuracies were between 92.0% and 102.1%. Their recoveries were all greater than 94%. The limits of detection were 0.019 μg mL⁻¹ for andrographolide and 0.022 μg mL⁻¹ for dehydroandrographolide. This method was successfully applied to the plasma concentration-time curve study after oral administration of Andrographis paniculata Nees extract in rabbit.     

Rapid Determination of Diterpenoids in Andrographis paniculata by Microemulsion Electrokinetic Capillary Chromatography with Short-End Injection

A rapid, simple, and reliable method has been developed for routine capillary electrophoretic analysis of two diterpenoids, andrographolide and dehydroandrographolide, in Andrographis paniculata. For this system, using ethyl acetate oil-based microemulsion electrokinetic chromatography (MEEKC) and the short-end injection technique, analysis was complete in less than 2.5 min. In method validation the relative standard deviations of migration time and peak area of the two constituents were, respectively, 0.54% and 1.70% for andrographolide and 0.45% and 2.11% for dehydroandrographolide. Regression equations revealed linear relationships (correlation coefficients 0.9994 for andrographolide and 0.9993 for dehydroandrographolide) between peak area and concentration. The effects of buffer pH, borate concentration, SDS concentration, co-surfactant type and concentration, injection time, and running potential were systematically investigated. The method can be successfully implemented in routine quality-control testing.     

反相高效液相法测定穿心莲药材及其制剂中的穿心莲内酯和脱水穿心莲内酯

 发展了穿心莲药材及其中成药中两种主要成分穿心莲内酯和脱水穿心莲内酯的反相高效液相测定方法。采用甲醇振荡提取法进行样品前处理 ,在以乙腈 水为流动相作梯度洗脱、ODS柱、检测波长为 2 2 5nm的条件下 ,穿心莲内酯和脱水穿心莲内酯在 1 5min内可达到基线分离。两种内酯在 1 0mg/L～ 1 0 0mg/L时其浓度与峰面积成良好的线性关系 ,加标回收率为 96 %～ 1 0 4 %。     

Determination of bioactive diterpenoids from Andrographis paniculata by micellar electrokinetic chromatography

The present paper describes the development of a micellar electrokinetic chromatographic (MEKC) method for simultaneous determination of andrographolide, deoxyandrographolide and neoandrographolide in ethanol extracts of Andrographis paniculata. Separations were carried out in a fused-silica capillary tube with UV detection at 214 nm. Good separation was achieved using a 20 mM borate buffer, containing 20 mM sodium dodecyl sulphate and 10 mM sodium cholate, adjusted to pH 8.3 at an operating voltage of 25 kV, temperature of 35°C and a hydrodynamic injection of 5 s. The method was validated with good correlation coefficients obtained (0.9986–0.9989) while relative standard deviation (RSD) of migration time was between 1.14 and 2.42. It is concluded that this method could be used for speedy and accurate qualitative and quantitative analysis of bioactive diterpenoids in andrographis herb and its derived products.     

Separation of enantiomers of butorphanol and cycloamine by capillary zone electrophoresis

Butorphanol tartrate is a synthetic opioid agonist-antagonist used as analgesic, possessing three chiral centres in the basic part of the molecule. Its chiral purity is routinely controlled only by optical rotation. A new capillary zone electrophoresis method, capable to separate the enantiomers of butorphanol and intermediate of its synthesis, cycloamine, was developed. Different electrolyte composition (type and concentration of carrier ion, pH, and organic solvent addition), and type and concentration of several chiral selectors (natural and modified cyclodextrins) were tested. Using the optimized conditions (acidic electrolyte with the addition of highly sulphated gamma-cyclodextrin) as low as 0.05% of undesirable enantiomers can be detected. Selected method characteristics, i.e., linearity (0-50 mg/l), precision (2.5% at 20 mg/l), and accuracy (101 +/- 2% at 20 mg/l) were evaluated. The optimized method was applied for the analysis of real batches of butorphanol and cycloamine. It was found that butorphanol tartrate manufactured by IVAX Pharmaceuticals contains less than 0.05% of undesirable enantiomer.     

Andrographolide: Solving Chemical Instability and Poor Solubility by Means of Cocrystals

The bioactive agent andrographolide was screened with pharmaceutically acceptable coformers to discover a novel solid form that will solve the chemical instability and poor solubility problems of this herbal medicine. Liquid‐assisted grinding of andrographolide with GRAS (generally regarded as safe) coformers in a fixed stoichiometry resulted in cocrystals with vanillin (1:1), vanillic acid (1:1), salicylic acid (1:1), resorcinol (1:1), and guaiacol (1:1). All the crystalline products were characterized by thermal, spectroscopic, and diffraction methods. Interestingly, even though the cocrystals are isostructural, their physicochemical properties are quite different. The andrographolide–salicylic acid cocrystal completely inhibited the chemical transformation of andrographolide to its inactive sulfate metabolite, and moreover, the cocrystal exhibited a dissolution rate that was three times faster and a drug release that was two times higher than pure andrographolide.     

Application of a Multilayer Perceptron Artificial Neural Network for the Prediction and Optimization of the Andrographolide Content in Andrographis paniculata

Andrographolide, the principal secondary metabolite of Andrographis paniculata, displays a wide spectrum of medicinal activities. The content of andrographolide varies significantly in the species collected from different geographical regions. Therefore, this study aims at investigating the role of different abiotic factors and selecting suitable sites for the cultivation of A. paniculata with high andrographolide content using a multilayer perceptron artificial neural network (MLP-ANN) approach. A total of 150 accessions of A. paniculata collected from different regions of Odisha and West Bengal in eastern India showed a variation in andrographolide content in the range of 0.28–5.45% on a dry weight basis. The MLP-ANN was trained using climatic factors and soil nutrients as the input layer and the andrographolide content as the output layer. The best topological ANN architecture, consisting of 14 input neurons, 12 hidden neurons, and 1 output neuron, could predict the andrographolide content with 90% accuracy. The developed ANN model showed good predictive performance with a correlation coefficient (R²) of 0.9716 and a root-mean-square error (RMSE) of 0.18. The global sensitivity analysis revealed nitrogen followed by phosphorus and potassium as the predominant input variables influencing the andrographolide content. The andrographolide content could be increased from 3.38% to 4.90% by optimizing these sensitive factors. The result showed that the ANN approach is reliable for the prediction of suitable sites for the optimum andrographolide yield in A. paniculata.     

Elicitation of Andrographolide in the Suspension Cultures of Andrographis paniculata

Andrographis paniculata belonging to the family Acanthaceae produces a group of diterpene lactones, one of which is the pharmaceutically important??andrographolide. It is known to possess various important biological properties like anticancer, anti-HIV, anti-inflammatory, etc. This is the first report on the production of andrographolide in the cell suspension cultures of Andrographis paniculata by ??elicitation??. Elicitation was attempted to enhance the andrographolide content in the suspension cultures of Andrographis paniculata and also to ascertain its stimulation under stress conditions or in response to pathogen attack. The maximum andrographolide production was found to be 1.53?mg/g dry cell weight (DCW) at the end of stationary phase during the growth curve. The biotic elicitors (yeast, Escherichia coli, Bacillus subtilis, Agrobacterium rhizogenes 532 and Agrobacterium tumefaciens C 58) were more effective in eliciting the response when compared to the abiotic elicitors (CdCl₂, AgNO₃, CuCl₂ and HgCl₂). Yeast has shown to stimulate maximum accumulation of 13.5?mg/g DCW andrographolide, which was found to be 8.82-fold higher than the untreated cultures.     

Effect of andrographolide on cysteamine-induced duodenal ulcer in rats

The aim of this study was to evaluate the gastroprotective efficacy of andrographolide isolated from Andrographis paniculata in rats induced with duodenal ulcers. Duodenal ulcers were induced by cysteamine administration in rats pretreated with 3?mg?kg?1 BW?day?1 of andrographolide for 30 days. Ulcer score, myeloperoxidase activity, TBARS level, GSH/GSSG ratio and enzyme antioxidants were measured in the duodenal tissue. Brush border and basolateral membranes were isolated to assay sucrase, maltase, alkaline phosphatase and total ATPases. Ulcer score was significantly minimised in rats pretreated with andrographolide. Elevation in myeloperoxidase and TBARS levels were found to be minimised significantly due to andrographolide treatment. Membrane-bound enzyme activities and the thiol redox status of glutathione were significantly maintained in duodenal mucosa of rats that received andrographolide. This study reveals that the major component of A. paniculata, andrographolide, has potent antiulcer properties that are most likely caused by minimising inflammatory changes, counteracting free radical formation and maintaining the thiol redox status in the duodenum.     

Development of andrographolide molecularly imprinted polymer for solid-phase extraction

A method employing molecularly imprinted polymer (MIP) as selective sorbent for solid-phase extraction (SPE) to pretreat samples was developed. The polymers were prepared by precipitation polymerization with andrographolide as template molecule. The structure of MIP was characterized and its static adsorption capacity was measured by the Scatchard equation. In comparison with C(18)-SPE and non-imprinted polymer (NIP) SPE column, MIP-SPE column displays high selectivity and good affinity for andrographolide and dehydroandrographolide for extract of herb Andrographis paniculata (Burm.f.) Nees (APN). MIP-SPE column capacity was 11.9±0.6 μmol/g and 12.1±0.5 μmol/g for andrographolide and dehydroandrographolide, respectively and was 2-3 times higher than that of other two columns. The precision and accuracy of the method developed were satisfactory with recoveries between 96.4% and 103.8% (RSD 3.1-4.3%, n=5) and 96.0% and 104.2% (RSD 2.9-3.7%, n=5) for andrographolide and dehydroandrographolide, respectively. Various real samples were employed to confirm the feasibility of method. This developed method demonstrates the potential of molecularly imprinted solid phase extraction for rapid, selective, and effective sample pretreatment.     

正交试验法优选穿心莲内酯硫酸酯化反应工艺

硫酸酯化反应以分子结构中的羟基为反应位点,向有机分子中引入硫酸酯基,是一种有效改善天然产物水溶性的结构修饰途径[1]。有些药物因水溶性差,致使其在临床应用中存在一些问题,如生物利用度不高,服用量大,制成的片剂或胶囊体内吸收缓慢等。这类化合物经硫酸酯化后不但可以增加     

Separation and purification of ergosterol and stigmasterol in Anoectochilus roxburghii (wall) Lindl by high-speed counter-current chromatography

Ergosterol and stigmasterol are the most common phytosterols in the traditional Chinese medicine. They are two major sterol compounds in Anoectochilus roxburghii (wall) Lindl (A. roxburghii) and have been proved to have many important biological activities. A method by using high-speed counter-current chromatography (HSCCC) has been successfully developed for separation and purification of ergosterol and stigmasterol in A. roxburghii simultaneously in this paper. The optimum conditions used in this method were as follows: The two-phase solvent system consisted of n-hexane-ethylacetate-butanol-methanol-water (3.5:0.3:0.5:2.5:0.3, v/v); the rotation speed was 900 rpm; the flow rate of the lower phase was 1.5 mL/min. About 36.5 mg of ergosterol and 43.6 mg of stigmasterol were obtained from 100 g of A. roxburghii. The purity of ergosterol and stigmasterol was examined to be 92.0 and 95.5%, respectively, by using HPLC. The chemical structures of these components were identified by UV spectra, FT-IR, MS, (1) H-NMR and (13) C-NMR. The results demonstrated that high-speed counter-current chromatography was a feasible method to separate and purify ergosterol and stigmasterol from the herb. This separation and purification method was more effective than many other conventional techniques.     

Optical fibre chemical sensor for trace vanadium(V) determination based on newly synthesized palm based fatty hydroxamic acid immobilized in polyvinyl chloride membrane

Fatty hydroxamic acid (FHA) immobilized in polyvinyl chloride (PVC) has been studied as a sensor element of an optical fibre chemical sensor for V(V). By using this instrument, V(V) in solution has been determined in the log concentration range of 0-2.5 (i.e. 1.0-300 mg/L). The detection limit was 1.0 mg/L. The relative standard deviation (R.S.D.) of the method for the reproducibility study at V(V) concentration of 200 mg/L and 300 mg/L were calculated to be 2.9% and 2.0%, respectively. Interference from foreign ions was also studied at 1:1 mole ratio of V(V):foreign ions. It was found that, Fe(III) ion interfered most in the determination of vanadium(V). Excellent agreement with ICP-AES method was achieved when the proposed method was applied towards determination of V(V).