Analysis on Isozyme and Diterpene Lactones Variability during Ontogenesis of Andrographis paniculata

Objective To understand the relationship between isozyme activities and diterpene lactone biosynthesis of Andrographispaniculata. Methods Plants were collected during ontogeny from seeding to seed maturity, and separated into leaves and stems for determination. Morphological and yield parameters were used to describe plant growing states. Isozyme changes were tested by polyacrylamide gel electrophoresis. HPLC was used to develop the fingerprints as well as to determine the diterpene lactone content. Results Significant increases were observed in the activities of isozymes, such as aspartate aminotransferase （AST）, malate dehydrogenase （MDH）, peroxidase （POD）, and catalase （CAT）, around the early stage of bud in leaves, and the activities of these four kinds of isozymes increased gradually as time progressed in stems. The content changes of diterpene lactones in leaves and stem were various. In the leaves, andrographolide （1） was recorded the highest [（23.63 ± 1.06） mg/g] at the early stage of bud, whereas deoxyandrographolide （2） was the lowest [（6.78 ± 0.27） mg/g] at this period and it reached the highest level at the seeding stage [（26.05 ± 1.04） mg/g]. Dehydroandrographolide （3） and neoandrographolide （4） fluctuated during growing stages. Meanwhile, the HPLC fingerprint showed that the content changes of two unknown compounds were related to that of dehydroandrographolide in leaves. In stems, andrographolide had increased gradually until the bud stage [（8.26 ± 0.33） mg/g], and other three diterpene lactones showed a trend of fluctuation. The yield of total diterpene lactones in aerial part reached the highest at the first flowering stage （806.71 mg/plant）. Conclusion These results lay the foundation for the future research on the relationship of isozymes and diterpene lactones, and for determining the most favorable time for harvesting A. paniculata.

Genome-wide identification of WRKY gene family and expression analysis under abiotic stresses in Andrographis paniculata

Andrographis paniculata(A.paniculata)is a Chinese herbal medicine that clears away heat,reduces inflammation,protects the liver,and promotes choleretics.The WRKYs of A.paniculata are still not well characterized,although many WRKYs have been identified in various plant species.In the present study,59 A.paniculata WRKY(ApWRKY)genes were identified and renamed on the basis of their respective chromosome distribution.These ApWRKYs were divided into three groups via phylogenetic analysis according to their WRKY domains and combined with WRKY of Arabidopsis.The 59 identified ApWRKY transcription factors were nonuniformity distributed on 23 chromosomes of A.paniculata.From the structural analysis of the conserved motifs,different ApWRKYs structures showed different biological functions,and the ApWRKY transcription factor had certain species-specificity in the evolutionary process.The expression patterns of the 41 ApWRKYs were examined through quantitative real-time PCR(qRT-PCR)in various tissues and under abiotic stresses(salt).The results showed that most of the ApWRKY had different reactions to salt treatment.In addition,the content of the four main secondary metabolites in A.paniculata leaves was determined under salt stress.The results show that under a low concentration of salt treatment,the synthesis of andrographolide can be improved.

Zebrafish facilitates drug screening: potential of 3-deoxy-andrographoside from Chuanxinlian(Herba Andrographitis Paniculatae) as an anti-inflammatory agent

OBJECTIVE: To systematically evaluate the in vivo antiinflammatory potential of diterpene lactones from Chuanxinlian(Herba Andrographitis Paniculatae)(AP). METHODS: We firstly adopted zebrafish, a novel and ideal animal model for high-throughput drug screening, to investigate the in vivo anti-inflammatory activities of 17 diterpene lactones isolated from AP.RESULTS: The results showed that most of diterpene lactones displayed significant anti-inflammatory effects in lipopolysaccharide microinjection-, copper sulfate exposure-or tail transection-induced zebrafish inflammation models. Moreover, diterpene lactone 3-deoxy-andrographoside(AP-5) was firstly found to attenuate inflammatory responses, which was closely associated with the myeloid differentiation primary response 88/nuclear factor-kappa B and signal transducer and activator of transcription 3 pathways. CONCLUSION: Our research sheds light on the inestimable roles of zebrafish in high-throughput drug screening, elucidates the potent inhibitory effects of diterpene lactones against inflammation and indicates that AP-5 may serve as a potential alternative agent for the treatment of inflammatory diseases.