| Andrographis paniculata, the overground parts of Andrographis paniculata (Burm. f.) Nees, is a commonly used Chinese herb medicine, and is mainly distributed in South China. It is widely used in clinical for its heat detoxification, cooling blood and anti-swelling effects. A. paniculata is one of the main constituent in many prescriptions such as tablet, capsule and injection, including Xiaoyan Lidan tablet and Fufang Chuanxinlian Pian which are the representative of excellent drugs. Since the demand of A. paniculata is increasing day by day, now it is commercially cultivated. The annual output of A. paniculata was over ten thousand tones in Guangxi, one of main production areas in China. However, the quality and quantity of this medical herbs in cultivation are unstable due to genetic variation, environmental conditions and uneven harvesting time. How to improve the content of effective components in A. paniculata and ensure the stability of the active ingredients in medicinal materials are the problems that A. paniculata quality assessment has been facing. Therefore, we carried out the experiments based on diterpene lactone and isozyme in A. paniculata. we examined the changes in the isozyme activities and the diterpene lactone content at different growing stages under the condition of natural growth in the cultivation base, and further determine the andrographolide accumulation and related enzyme activity change under the condition of phosphorus stress. It will lay the foundation for exploring the biosynthetic mechanism of diterpene lactone, and provide theoretical guide to quality evaluation.The main studies were summarized as follows:(1) Analysis of isozyme and diterpene lactone variability during ontogenesis of Andrographis paniculata (Burm. f.) NeesTo understand the relationship between isozyme activities and diterpene lactone biosynthesis of Andrographis paniculata (Burm. f.) Nees. Plants were collected during ontogeny from seeding to seed maturity, and separated into leaf and stem for determination. Morphological parameters were used to describe plant growing rules. Isozyme changes were tested by polyacrylamide gel electrophoresis. High-performance liquid chromatographic was used to develop the fingerprints as well as to determine the diterpene lactone content. 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 leaf and stem were various. In leaves, andrographolide was recorded highest (23.63 ± 1.06 mg-g-1) at the early stage of bud, whereas dexoyandrographolide was lowest (6.78± 0.27 mg·g-1) at this period and it reached the highest level at the seeding stage (26.05 ± 1.04 mg·g-1). Dehydroandrographolide and neoandrographolide 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-1), and other three diterpene lactones showed a trend of fluctuation. The aerial part biomass of total diterpene lactones reached the highest in the first flowering stage (806.71 mg·plant-1). 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.(2) study on the isozymes of Andrographis paniculata seed from different placesTen samples of A. paniculata seed were collected from Guangdong, Guangxi and Fujian during seed maturity stage. Similarity coefficient, genetic distance and clustering analysis were used to analyse the genetic diversity based on isozyme-electrophoretic results. The percentage of polymorphic loci for EST, MDH, POD, ACP were 66.7%,60.0%,33.3% and 0 respectivity. The similarity coefficient ranged from 0.667 to 1.000, and the genetic distance ranged from 0 to 0.549. Clustering analysis showed that the ten samples were grouped into two clusters. The samples from Raoping of Guangdong province and Guangxi province were close to those from Zhangpu and Longjiao of Fujian province, and the rest were grouped into the second cluster, in which the sample from Zhanjiang of Guangdong province was grouped into a subtype. The results indicated that there was a certain level of genetic variation, and obvious variation was found from the seed of Zhanjiang.(3) Phosphorus stress effects on andrographolide and enzyme activity during ontogenesis of Andrographis paniculata (Burm. f.) NeesIn order to evaluate the effects of phosphorus stress on andrographolide and enzyme activity, A. paniculata were grown in pots with four different phosphorus level treatments. These four phosphorus treatments were control level (CK,0 mg·kg-1 soil), low level (P1,6.25 mg·kg-1 soil), middle level (P2, 12.5 mg·kg-1 soil) and high level (P3,18.75 mg·kg-1 soil). The growth of plant, phosphorus accumulation, soluble protein content, POD activity, ACP activity in leaves and roots, as well as the andrographolide accumulation, 3-hydroxy-3-methylglutary-CoA-reducase and 1-deoxy-D-lxylulose-5-phosphate reductoisomerase activity in leaves during ontogeny were detected. The results showed that phosphorus stress could increase the biomass of A. paniculata, whereas the roots were thin and lateral roots were increased under CK. In leaves, the andrographolide content increased obviously as the phosphorus level increase, while the phosphorus content made no great difference. With the phosphorus level increase, the soluble protein content reduced and POD activity presented complicated change. ACP activity in leaves had the same change tendency with soluble protein content, and it kept a increase tendency as time progressed in roots. The change of 3-hydroxy-3-methylglutary-CoA-reducase and 1-deoxy-D-lxylulose-5-phosphate reductoisomerase activity was the same or complementary, but it was increased as a whole. It was obviously that phosphorus could change enzyme activity that related to promote the biosynthesis of andrographolide. |
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