Identification of Andrographis Herba and its common products using mini-barcode / 中国天然药物

Andrographis Herba, the aerial part of Andrographis paniculata (Burm. f.) Wall. ex Nees (Acanthaceae), has a wide geographic distribution and has been used for the treatment of fever, cold, inflammation, and other infectious diseases. In markets, sellers and buyers commonly inadvertently confuse with related species. In addition, most Chinese medicinal herbs are subjected to traditional processing procedures, such as steaming and boiling, before they are sold at dispensaries; therefore, it is very difficult to identify Andrographis Herba when it is processed into Chinese medicines. The identification of species and processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA barcoding has received considerable attention as a new potential means to identify species and processed medicinal materials. In this study, 17 standard reference materials of A. paniculata, 2 standard decoctions, 27 commercial products and two adulterants were collected. Based on the ITS2 sequence, it could successfully identify A. paniculata and adulterants. Moreover, a nucleotide signature consisting of 71 bp was designed, this sequence is highly conserved and specific within A. paniculata while divergent among other species. Then, we used these new primers to amplify the nucleotide signature region from processed materials. In conclusion, the DNA barcoding method developed in the present study for authenticating A. paniculata is rapid and cost-effective. It can be used in the future to guarantee the quality of Andrographis Herba of each regulatory link for clinical use.

Current quality standards of Andrographis Herba of different countries and regions / 中国中药杂志

Andrographis Herba is a commonly used plant medicine, and has been recorded in pharmacopeias of different countries. However, there are some differences in the quality standards. Based on this, this paper compare the quality standards of Andrographis Herba between Chinese Pharmacopoeia, Hong Kong Chinese Materia Medica Standards, United States Pharmacopoeia, European Pharmacopoeia and Indian Pharmacopoeia, including origin, botanical characteristics, identification(microscopic identification and chromatographic identification), content determination, specific test(such as impurities, loss on drying, extractives, pesticides, heavy metals, mycotoxins, and other items) and storage requirements, so as to provide a reference for studying international quality standards of Andrographis.

Mechanism of salicylic acid ameliorates salt-induced changes in Andrographis paniculata / 中国中药杂志

In this study, Andrographis paniculata seedlings were used as experimental materials to study the effects of salicylic acid(SA) on the growth and effective component accumulation of A. paniculata under NaCl stress. The results showed that with the increase of NaCl concentration, the growth of A. paniculata seedlings was significantly inhibited, and the content of carotene and carotenoid decreased. The activity of antioxidant enzyme was enhanced. At the same time, the contents of proline, proline and soluble protein were on the rise. The contents of andrographolide, new andrographolide and deoxyandrographolide showed an upward trend, while deoxyandrographolide showed a downward trend. Treatment with 100 mmol·L~(-1) NaCl+5 mg·L~(-1) SA showed a significant increase in antioxidant enzyme activity in A. paniculata leaves. Treatment with 100 mmol·L~(-1) NaCl+10 mg·L~(-1) SA showed significant changes in soluble protein and proline content in A. paniculata leaves, while MDA content in A. paniculata leaves significantly decreased. 10 mg·L~(-1) SA had the best effect on the growth of A. paniculata seedlings under salt stress. Under the treatment of 50 mmol·L~(-1) NaCl+10 mg·L~(-1) SA, fresh weight, dry weight and leaf dry weight of A. paniculata seedlings reached the highest level, which were 1.02, 1.09 and 1.11 times of those in the control group, respectively. The concentrations of NaCl and 10 mg·L~(-1) SA were significantly higher than those of the control group. Four key enzyme genes of A. paniculata diterpene lactone synthesis pathway were selected to explore the molecular mechanism of salicylic acid to alleviate salt stress. With the increase of salt stress, the relative expressions of HMGR, GGPS and ApCPS were up-regulated, indicating that salt stress may enhance the synthesis of A. paniculata diterpene lactone through MVA pathway. SA can effectively promote the growth and development of A. paniculata under salt stress, improve its osmotic regulation and antioxidant capacity, improve its salt tolerance, and alleviate the effects of salt stress on A. paniculata.

Effect of Polygonum multiflorum-Andrographis paniculata intercropping system on rhizosphere soil actinomycetes community structure and diversity of P. multiflorum / 中国中药杂志

To investigate the effect of Polygonum multiflorum-Andrographis paniculata intercropping system on rhizosphere soil actinomycetes of P. multiflorum, the community structure and diversity of soil actinomycetes were studied by using the original soil as the control group and the rhizosphere soil actinomycetes communities of P. multiflorum under monoculture and intercropping systems as the experimental group. In this study 655 221 effective sequences were obtained with an average length of 408 bp. OTU coverage and rarefaction curve showed that the sequencing could represent the real situation of soil actinomycetes. According to the results of alpha diversity analysis, the diversity soil actinomycetes varied as follows: original soil>intercropping soil>monoculture soil. The soil actinomycetes community structure and the relative abundance of dominant genera were significantly changed by both monoculture and intercropping, especially monoculture. OTU clustering and PCA analysis of soil samples showed that all the soil samples were divided into three distinct groups and the original soil was more similar to intercropping soil. In addition, intercropping increased the relative abundance of some beneficial actinomyces, such as Kitasatospora and Mycobacterium, which was beneficial to maintain soil health and reduce the occurrence of soil-borne diseases. The results show that, P. multiflorum-A. paniculata intercropping reduced the change of community structure and the decrease of diversity of soil actinomycetes caused by P. multiflorum monoculture, and made the actinomycete community in rhizosphere soil of P. multiflorum close to the original soil.

Establishment of QAMS method with dehydroandrographolide as internal reference substance for quality control of Andrographis Herba / 中国中药杂志

To solve the problems of the poor resolution of chromatographic separation,the weak durability of the relative correction factors,and the low accuracy of content determination results in the quantitative analysis of multi-components by single-marker( QAMS) method with andrographolide as the internal reference substance in the existing research of Andrographis Herba,a new QAMS method using dehydroandrographolide as the internal reference substance was established for the first time in this study. This new method can be used to simultaneously determine four diterpene lactones,including andrographolide( A),neoandrographolide( B),14-deoxyandrographolide( C),and dehydroandrographolide( S) through the optimization of chromatographic conditions and systematic investigation of methodology. At the present HPLC chromatographic conditions,four components could be well separated( R > 1. 5),and the methodology validations could satisfy the requirement of quantitative analysis. The relative correction factors( RCFs) of fA/S,fB/S,fC/S were determined as 0. 65,0. 54,0. 78,respectively. The relative standard deviations( RSDs) of their RCFs ranged between 1. 3%-5. 1%,0. 25%-0. 33%,0. 070%-0. 15%,0. 070%-0. 22%,respectively with three brands of HPLC instruments,five brands of C18 column,different flow rates( 0. 9,1. 0,1. 1 m L·min~(-1)),and different column temperatures( 25,30,35 ℃),indicating good durability of the RCFs. The relative retention value( RRV) method was used to locate the chromatographic peak of the components to be determined.The RRVs of rA/S,rB/S,and rC/Swere 0. 44,0. 86,0. 97,respectively. The RSDs of the RRVs ranged between 0. 030%-1. 6% with different HPLC instruments and columns,showing accurate peak location. The present QAMS method and the external standard method( ESM)were both used to determine the contents of four diterpene lactones from Andrographis Herba( 6 batches of medicinal materials and 18 batches of cut crude drugs). The relative errors of the determined content results between two methods were less than 2. 0%. It demonstrated that there was no significant difference in content results between these two methods,indicating good accuracy of the present QAMS method. Therefore,in this study,an accurate and highly durable QAMS method using dehydroandrographolide as the internal reference substance was established for simultaneous determination of four diterpene lactones. This method could be used to effectively control the quality of Andrographis Herba and provide technical basis for the formulation of traditional Chinese medicine industry standard and improvement of the Chinese Pharmacopoeia standard of Andrographis Herba.

Correlation analysis of main agronomic traits and contents of diterpene lactones of Andrographis paniculata / 中国中药杂志

In order to study the correlation between the traits of Andrographis paniculata. The main agronomic traits and the content of four kinds of diterpene lactons were measured by the seedlings and the unmutagenized seeds carried by the spacecraft,and multiple comparisons,correlations and principal component analysis were carried out. The results showed that the agronomic traits of A. paniculata have different degrees of difference after being carried by space. The contents of diterpene lactones were quite different. The variation coefficients of deoxyandrographolide content,fresh weight,leaf dry weight,dehydrated andrographolide content,dry weight,neoandrographolide content and andrographolide content were all over 35%. There was a significant correlation between multiple traits,and the leaf weight ratio was significantly positively correlated with the number of primary tillers,leaf dry weight and dry weight,and was significantly negatively correlated with the content of deoxyandrographolide. Andrographolide content was a significantly negatively correlated with the number of leaves and the number of primary tillers,and positively correlated with the other three lactones. Five principal components were extracted from principal component analysis,and the cumulative contribution rate was 83. 127%,which were yield factor,plant type factor,leaf type factor,component factor and seed weight factor. Among the traits affecting the quality of A. paniculata,the yield factor for reliability of the selection of A. paniculata is higher than other factors. There are abundant variations among the traits of A. paniculata,carried in space which increase the genetic diversity. The principal component analysis method can be used to select the principal component factors according to the breeding requirements,which provides a theoretical basis for the breeding of high-yield and high-quality A. paniculata and the high-yield and stable cultivation of A. paniculata.

Morphological and chemical studies of artificial Andrographis paniculata polyploids / 中国天然药物

Andrographis paniculata (Burm. f.) Nees (AP) is commonly used for the treatment of many infectious diseases and has been cultivated widely in Asian countries, and has been included in United States Pharmacopoeia as a dietary supplement, but the cultivars of A. paniculata are not abundant due to its self-pollinated. With the aims to enrich AP resources and provide materials for after breeding we explored the polyploidy induction. Different explants, colchicine concentration, and treatment time were tested. After identification by flow cytometry, eleven polyploid plants with different morphologic traits were obtained. The agronomic traits and andrographolide concentration of the polyploids were improved greatly. One of the polyploids (serial 3-7) was chosen for further study. The traits of the second and third generation polyploids (serial 3-7) were stable. Compared with the normal plants, the seeds (2nd generation) weight increased by 31%, and the andrographolide concentration of the leaves increased by 14% (2nd) and 28% (3rd). In conclusion, AP autopolyploids with different morphologic traits were established successfully for the first time, and the polyploids induction might be effective for crop improvement of AP.

Morphological and chemical studies of artificial Andrographis paniculata polyploids / 中国天然药物

Andrographis paniculata (Burm. f.) Nees (AP) is commonly used for the treatment of many infectious diseases and has been cultivated widely in Asian countries, and has been included in United States Pharmacopoeia as a dietary supplement, but the cultivars of A. paniculata are not abundant due to its self-pollinated. With the aims to enrich AP resources and provide materials for after breeding we explored the polyploidy induction. Different explants, colchicine concentration, and treatment time were tested. After identification by flow cytometry, eleven polyploid plants with different morphologic traits were obtained. The agronomic traits and andrographolide concentration of the polyploids were improved greatly. One of the polyploids (serial 3-7) was chosen for further study. The traits of the second and third generation polyploids (serial 3-7) were stable. Compared with the normal plants, the seeds (2nd generation) weight increased by 31%, and the andrographolide concentration of the leaves increased by 14% (2nd) and 28% (3rd). In conclusion, AP autopolyploids with different morphologic traits were established successfully for the first time, and the polyploids induction might be effective for crop improvement of AP.

Cloning and functional identification of a new NADPH-cytochrome P450 reductase in Andrographis paniculata / 中国中药杂志

Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata，with a variety of pharmacological activity，widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study，the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment，named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification，the enzyme activity was identified . The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its function，ApCPR4 and CYP76AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate (MeJA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by MeJA，suggesting that ApCPR4 was associated with biosynthesis of andrographolide.

Two new diterpenoid lactones isolated from Andrographis paniculata / 中国天然药物

In the present study, two new diterpenoid lactones, 3-deoxy-andrographoside (1) and 14-deoxy-15-methoxy-andrographolide (2), were isolated from the aerial parts of Andrographis paniculata. Their structures were elucidated by combination of NMR, MS, and chemical methods. The configurations of 1 and 2 were established based on the analysis of ROESY data and single crystal X-ray diffraction experiment.

A new biocompatible microemulsion increases extraction yield and bioavailability of Andrographis paniculata / 中国天然药物

The purpose of this study was to design and prepare a biocompatible microemulsion of Andrographis paniculata (BMAP) containing both fat-soluble and water-soluble constituents. We determined the contents of active constituents of BMAP and evaluated its bioavailability. The biocompatible microemulsion (BM), containing lecithin and bile salts, was optimized in the present study, showing a good physical stability. The mean droplet size was 19.12 nm, and the average polydispersity index (PDI) was 0.153. The contents of andrographolide and dehydroandrographolide in BMAP, as determined by high performance liquid chromatography (HPLC), were higher than that in ethanol extraction. The pharmacokinetic results of BMAP showed that the AUC0-7 and AUC0→∞ values of BMAP were 2.267 and 27.156 μg·mL(-1)·h(-1), respectively, and were about 1.41-fold and 6.30-fold greater than that of ethanol extraction, respectively. These results demonstrated that the bioavailability of and rographolide extracted by BMAP was significantly higher than that extracted by ethanol. In conclusion, the BMAP preparation displayed ann improved dose form for future clinical applications.

Dimethyl 3, 3', 4, 4'-tetrahydroxy-delta-truxinate isolated from the leaves of Andrographis lineata.Wall. ex. nees suppress adipogenesis in 3T3-L1 preadipocytes for type 2 diabetes

The present investigation elucidates the isolation and characterization of bioactive compound from the ethanolic leaf extract of Andrographis lineata [EtALL] which suppress the differentiation of 3T3-L1 adipocytes. The ethanolic leaf extract was subjected to bioassay guided fractionation in 3T3-L1 cell lines. Five fractions were isolated from the EtALL extract by column chromatography. All the Fractions [I-V] along with EtALL were screened for adipogenesis activity [Oil-Red-O staining].The fraction which showed maximum adipogenesis activity was purified by thin layer chromatography. The bioactive Fraction IV was found to have maximum adipogenic [96.83%] activity and the activity was comparable to Rosiglitazone. The spectroscopic data analysis reveals that, the isolated bioactive compound was Dimethyl 3, 3', 4, 4'-tetrahydroxy-delta-truxinate [DT[delta]T], a combination of truxillic and truxinic acid derivative. DT[delta]T showed insulin mimicking [131.2%], sensitizing [810.02%] and adipogenic activity [80.23%]. Hence our present study concluded that, Dimethyl 3, 3', 4, 4'-tetrahydroxy-delta-truxinate isolated from the ethanolic leaf extract of Andrographis lineata stimulates glucose uptake, potentiates insulin-stimulated glucose in 3T3-L1 adipocytes without increasing adiposity

Cloning and functional characterization of phytoene desaturase in Andrographis paniculata / 中国中药杂志

A full-length cDNA of phytoene desaturase (PDS) gene from Andrographis paniculata was obtained through RACE-PCR. The cDNA sequence consists of 2 224 bp with an intact ORF of 1 752 bp (GeneBank: KP982892), encoding a ploypeptide of 584 amino acids. Homology analysis showed that the deduced protein has extensive sequence similarities to PDS from other plants, and contains a conserved NAD ( H) -binding domain of plant dehydrase cofactor binding-domain in N-terminal. Phylogenetic analysis demonstrated that ApPDS was more related to PDS of Sesamum indicum and Pogostemon cablin. The semi-quantitative RT-PCR analysis revealed that ApPDS expressed in whole aboveground tissues with the highest expression in leaves. Virus induced gene silencing (VIGS) was performed to characterize the functional of ApPDS in planta. Significant photobleaching was not observed in infiltrated leaves, while the PDS gene has been down-regulated significantly at the yellowish area. To the best of our knowledge, this represents the first report of PDS gene cloning and functional characterization from A. paniculata, which lays the foundation for further investigation of new genes, especially that correlative to andrographolide biosynthetic pathway.

Preparation of hydrophilic matrix sustained release tablets of total lactones from Andrographis paniculata and study on its in vitro release mechanism / 中国中药杂志

In this study, hydrophilic matrix sustained release tablets of total lactones from Andrographis paniculata were prepared and the in vitro release behavior were also evaluated. The optimal prescription was achieved by studying the main factor of the type and amount of hydroxypropyl methylcellulose (HPMC) using single factor test and evaluating through cumulative release of three lactones. No burst drug release from the obtained matrix tablets was observed. Drug release sustained to 14 h. The release mechanism of three lactones from A. paniculata was accessed by zero-order, first-order, Higuchi and Peppas equation. The release behavior of total lactones from A. paniculata was better agreed with Higuchi model and the drug release from the tablets was controlled by degradation of the matrix. The preparation of hydrophilic matrix sustained release tablets of total lactones from A. paniculata with good performance of drug release was simple.

Research on UPLC-PDA fingerprint of andrographis paniculata and quantitative determination of 4 major constituents / 中国中药杂志

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.

Genetic diversity analysis of Andrographis paniculata in China based on SRAP and SNP / 中国中药杂志

In order to reveal genetic diversity of domestic Andrographis paniculata and its impact on quality, genetic backgrounds of 103 samples from 7 provinces in China were analyzed using SRAP marker and SNP marker. Genetic structures of the A. paniculata populations were estimated with Powermarker V 3.25 and Mega 6.0 software, and polymorphic SNPs were identified with CodonCode Aligner software. The results showed that the genetic distances of domestic A. paniculata germplasm ranged from 0. 01 to 0.09, and no polymorphic SNPs were discovered in coding sequence fragments of ent-copalyl diphosphate synthase. A. paniculata germplasm from various regions in China had poor genetic diversity. This phenomenon was closely related to strict self-fertilization and earlier introduction from the same origin. Therefore, genetic background had little impact on variable qualities of A. paniculata in domestic market. Mutation breeding, polyploid breeding and molecular breeding were proposed as promising strategies in germplasm innovation.

Identification of main chemical constituents of diterpene lactone effective fraction of Andrographis panniculata by HPLC-DAD/ESI-MS and their preliminary pharmacodynamics research / 中国中药杂志

<p><b>OBJECTIVE</b>To establish an HPLC-DAD/ESI-MS method for quickly identifying chemical constituents in diterpene lactone effective fraction of Andrographis panniculata and to study its pharmacodynamics.</p><p><b>METHOD</b>The separation was performed on an Agilent SB-C18 column (2.1 mm x 150 mm, 5 μm) with a mobile phase of acetonitrile (A) and water (B). The flow rate was maintained at 0.4 mL x min(-1) and detection wavelength was set at 205 nm. The samples were analyzed in positive ion mode, and mass scan range was m/z 50-1 000. Using two kinds of tumor cell lines made living animal models, and studied preliminary pharmacodynamics on anti-tumor aspect.</p><p><b>RESULT</b>Five diterpene lactones in the diterpene lactone effective fraction of A. panniculata could be separated in one run. Pharmacodynamic experiments showed that the effectve fraction had an inhibitory effect on the growth of tumor.</p><p><b>CONCLUSION</b>A rapid and efficient HPLC-ESI-MS method to determine the chemical constituents in diterpene lactone effective fraction of A. panniculata has been established, and the preliminary pharmacodynamics research has been done, which could be used for the quality control and further studies of diterpene lactone effective fraction of A. panniculata in vivo.</p>

Study on key physical properties of granulated products of Andrographis mixed powder by high-speed mixing wet method / 中国中药杂志

The impact of key physical properties on granulated products by the high-speed mixing wet method was studied. Andrographis extracts were utilized as the model drug. Four processing methods were adopted to prepare mixed powder of microcrystalline cellulose (MCC) and starch with the mass ratio 1:0.5, 1:1 and 1:2 by the high-speed mixing wet method. The properties of the prepared granules were evaluated with such indexes as granule yield, the ratio of lumps and fine powder, granule-AOR and granule-HR. The impact of key physical properties on granulated products was analyzed through stepwise regression analysis. The results showed that angle of repose, moisture content, pore volume, density and contact angle with water were key physical properties of the powder. The key physical properties of Chinese medical extracts powder are the important factor impacting granulated products made by the high-speed mixing wet method. In this study, the impact of key physical properties on granulated products of Chinese medical extracts was analyzed from the physical angle.

Advance in studies on anti-infection of andrographolide and its derivatives in past 10 years / 中国中药杂志

Andrographolide (AG) is a main effective ingredient in leaves of Andrographis paniculata. AG and its derivatives have such effects in anti-infection, anti-tumor and immuno-regulation. Particularly, its antibacterial, antiviral and anti-protozoal activities play an increasingly important role in resisting opportunistic infections. On the basis of our studies on AG over the years, we made a summary for the basis and clinical studies on AG and its derivatives over the past 10 years.

Serological tests for the diagnosis of active tuberculosis: relevance for India.

Diagnostic tests for active tuberculosis (TB) based on the detection of antibodies (serological tests) have been commercially available for decades, although no international guidelines have recommended their use. An estimated 1.5 million serological TB tests, mainly enzyme-linked immunosorbent assays, are performed in India alone every year, mostly in the private sector. The cost of serological tests in India is conservatively estimated at US $15 million (825 million) per year. Findings from systematic reviews on the diagnostic accuracy of serological tests for both pulmonary and extra-pulmonary TB suggest that these tests are inaccurate and imprecise. A cost-effectiveness modelling study suggests that, if used as a replacement test for sputum microscopy, serology would increase costs to the Indian TB control sector approximately 4-fold and result in fewer disability-adjusted life years averted and more false-positive diagnoses. After considering all available evidence, the World Health Organization issued a strong recommendation against the use of currently available commercial serological tests for the diagnosis of TB disease. The expanding evidence base continues to demonstrate that the harms/risks of serological tests far outweigh the benefits. Greater engagement of the private sector is needed to discontinue the use of serological tests and to replace these tests with WHO-endorsed new diagnostics in India. The recent ban on import or sale of TB serological tests by the Indian health ministry is a welcome step in the right direction.