| Andrographis paniculata (Chuan-Xin-Lian) is one of the specially important mecicinal herbs in China. It is world-famous for having extensive pharmacological effects and has been widely used for centuries together in Asia to treat fevers,sore throat, gastric infections, sluggish liver, diabetes, upper respiratory infection, snake bites, and many other chronic and infectious disorders. Chuanxinlian is an important constituent in majority of Chinese prescriptions, such as "Xiao Yan Li Dan Pian" which serves as a representative of star proprietary Chinese medicines in Guangdong pharmaceutical factories. Conducted researches in the past have confirmed that chuanxinlian has a surprisingly broad range of pharmacological effects such as fighting against HIV/AIDS, colon cancer and other diseases, but also it has been subjected to all sorts of studies as well in these years for possible applications in various fields such as agriculture, poultry and animal production, veterinary medicine and even for industrial applications. Consequently, the demands for AP are boosted.Andrographis paniculata (AP)populations are distributed over a broad eco-geographical range in tropical Asian countries. It grows abundantly in Southern and Southeastern Asia, which perhaps caused the diversity of the species. In China, there are mainly cultispecies and almost no wild resources. This herb has been listed among prioritized plants for development and cultivation in some southern places of China and the priority of AP is regarded by herbal industries. In another survey, AP has been presented in the list of highly traded Chinese medicinal plants with a demand of about 9000 tons in year 2013-2014 and annual growth of 12.2%. All these results imply that the absence of this plant with stable yield and good quality must be very important, so the study of germplasm resources and excellent quality-control practices must be carried out to breed high-quality germplasm and develop the cultivation of this herb as a new alternative crop in different agricultural ecosystems.Thus it will be helpful for supporting the conservation and development of AP.Therefore,we carried out a feasibility investigation and researches about AP germplasms. It will be useful for expanding and creating excellent germplasm breeding ways, improving the cultivation paractices, promoting GAP base development and construction,meantime offer a new train of thought.The main studies were summarized as follows:1.Studies on germplasm resource investigation of Andrographis paniculata(1) Distribution, morphological characteristics, production and marketing situationTo investigate and collect the germplasm resources of A. paniculata in the main areas of China, and lay a foundation for the comprehensive evaluation and the screening of excellent germplasm of A. paniculata. Field investigations of the main germplasm resources of Chuanxinlian were conducted under the premise of reviewing the relevant documents, checking the specimens and consulting the information of production and trading. Wild resources of Chuanxinlian in our country at present are extremely rare. Cultivation were mainly concentrated in Guangdong, Guangxi, Fujian, Sichuan province, and partly in Yunnan province.Meanwhile, the information of its ecological environments, cultivation and growth characteristics, Production, marketing and use was obtained. Effective measures should be taken to protect the ecological environment of A. paniculata and other medicinal plants, but also plant management system should be perfected to strengthen the sustainable development and exploitation of Medicinal plant resources of A. paniculata.(2) Datebase information construction of germplasm resourcesBased on "China Germplasm Bank of Wind Species" and "The Germplasm of South China Medicinal Plants", develop a database information platform of Andrographis paniculata underWindows system. The information of the local names, usages,pharmaceutical importance, characteristics, distribution and producing areas, habitats, pictures and so on about this herb can be displayed online and easily shared. This will be helpful for teaching, scientific researches and cultivations of Chuanxinlian.2. Study on genetic diversity of Andrographis paniculata germplasm resources(1) Evaluation of genetic diversity based on six kinds of isozymesTo understand the genetic variation and find out superior genotype for high yield and good quality of this crop, the genetic diversity of Andrographis paniculata collected from different natural habitats was investigated based on isozyme-electrophoretic analysis. Thirty samples including 3 wild species and 27 cutivars collected from 5 provinces (including Guangdong, Guangxi, Fujian, Hainan and Sichuan) of China were studied using POD, SOD, PPO, CYT, EST and CAT isozyme polymorphism. A total of 24 stable bands were observed by 6 isozymes across 30 accessions, and only one was specific band detected in Quanzhou wild AP. The percentage of polymorphic bands for POD, SOD, PPO, EST, CYT, CAT were 75.00%,40.00%,57.14%,0.00%,33.33% and 66.66%, respectively. The similarity coefficient among 30 accessions ranged from 0.708 to 1.000 and averaged at 0.863. The genetic distance ranged from 0.000 to 0.197 and averaged at 0.087. Compared with each other, the similarity coefficient values between Quanzhou wild AP and the others were relatively less, while the similarity coefficient values among cultivars and Hainan wild AP were higher. The 30 samples were grouped in three clusters at the similarity coefficient value of 0.795. The position of Quanzhou wild AP and Zhanjiang cultivars in graph of principle compotent analysis was relatively isolated which is in keeping with the clustering analysis. The results indicated that the variations in terms of isozymes were narrow.(2) Determination of AP chloroplast genetic sequence and comparative analysis of major gene segmentThe complete chloroplast genome of Andrographis paniculata, an important medicinal plant with great economic value, has been studied in this article. The genome size is 150,249 bp in length, with 38.3% GC content. A pair of inverted repeats (IRs,25,300 bp) are separated by a large single copy region (LSC,82,459 bp) and a small single-copy region (SSC,17,190 bp).The chloroplast genome contains 114 unique genes,80 protein-coding genes,30 tRNA genes and 4 rRNA genes. In these genes,15 genes contained 1 intron and 3 genes comprised of 2 introns. Analysis of chloroplast major gene segment in different AP collections showed that the genetic diversity of different AP germplasms was low.3. Effects of environmental factors on biological characteristics of AP(1) Effect of soil factor on the quality of AP originated from different areasTo study the effects of soil factor on quality of Andrographis paniculata(Burm. f.)Nees originated from different localities. The contents of terpene lactones, flavonoids and ethanol-soluble extractives in AP gathered from different locations were compared. Physicochemical properties and mineral elements of soil were analyzed for studying the effects on quality of AP through path and correlation analysis. The quality of AP gathered from different provinces is obviously different. Soil factors have great effect on its chemical component content, available P, K affecting the lactones directly and nitrate nitogen having the greatest indirect effect on them. There is large relativity between the flavonoids and organic matter. The content of ethanol-soluble extractives have highest-level links with available K. Neutral and weak acid or weak alkaline soil are appropriate for its cultivation. Soil Taking full advantage of available K, P and nitrogen is helpful for the growth of Andrographis paniculata(Burm. f.)Nees and the accumulation of active ingredients. Studying the effects of soil factor on quality of Andrographis paniculata(Burm. f.)Nees can be used to guide planting and its GAP management.(2) Effect of fertilizers on the growth, isozymes and quality of APA pot experiment was build for studying the influence of different fertilizers including organic, chemical and micro-fertilzer on the growth, biochemistry and active components. The results showed that the difference of growth among each fertilization was low; the isozyme activities of POD and EST were stable compared to the control;the andrographolide of samples treated with micro-fertilizer was the highest.(3) Change of six isozymes in AP leaf at different growth stagesTaking the first fully developed leaf from the top as materials, six isozymes of A. paniculata leaf at different growth periods were analyzed. Results:The enzyme band number of POD, CYT and PPO isozyme and the band intensity of all the six isoenzymes in A. paniculata leaf at different periods showed obvious differences. Activities of POD, CYT, PPO and esterase isozyme are increased at flowering and fruiting time, around September. CAT and SOD are stable during its growth periods. Conclusion:It seems that changes of isoenzyme pattern can clearly reflect the growth and development specificity of Andrographis paniculata at different growth periods and may used as an physiological and chemical index of growth changes. The researched antioxidant isozyme POD may be related to the accumulation of andrographolide compounds.(4) Effect of salt stress on growth, isoenzymes and metabolites of APEfficient use of saline land for promoting the cultivation of medicinal plants is valuable for pharmaceutical and economic benefits. Relatively little is known about the physiological and biochemical basis of salt tolerance in this plant. Here, we studied the growth, isoenzymes, malondialdehyde, proline and secondary metabolites of A. paniculata in response to different salt treatments with NaCl (0,41.1,92.4,143.7 and 193.4 mM). The results indicated that growth traits were decreased and all the expression level of superoxide dismutase (SOD), catalase (CAT), cytochrome oxidase (CYT), peroxidase (POD), polyphenol oxidase (PPO) and esterase (EST) isoenzyme in leaves and roots was enhanced with increasing salinity. Malondialdehyde was positively correlated to salt levels. Proline decreased at first and then dramatically increased at 193.4 mM. The content of key secondary metabolites was continuously increased under less than 143.7 mM NaCl concentrations. Results suggest that A. paniculata has relatively salt-resistance by activating enzyme expressions both in roots and leaves, elicitation of secondary metabolites and osmoprotectant, but also reasonably regulating the allocation of resources. It is preferent to using resources for defence rather than for growth in A. paniculata and there is metabolic shift from growth to defence under salt conditions. Induction of isoenzyme activities and production of secondary metabolites are the primary defence strategies. Additionally, moderate salt treatment could promote the accumulation of bioactive phytocompounds, which is valuable for breeding plants with high quality. Thus, the cultivation of this medicinal herb in moderate saline areas could be considered as an agricultural option. Furthermore, our upshorts can provide a reference for screening better salt-tolerant cultivars of A. paniculata.(5) Effect of glycine betaine on growth, andrographolide and isoenzymes in salt-damaged APGlycine betaine (GB), a natural wetting agent, is reported to be involved in the defense mechanisms of plants against abiotic stresses such as salinity, water deficiency and so on. Elements involved in growth rate, activity of enzyme have therefore been studied in A. paniculata. It found that GB can anesis the growth inhibition and keep the high content of andrographolides.(6) Water stress effects on growth, peroxidase, esterase and andrographolides of APThe response of A. paniculata to water stress was investigated in order to explore the adaptive changes in plant growth, enzymes and accumulation of andrographolides.Pot experiments were conducted. Three water stress treatments were applied:100% of water holding capacity (WHC),40% of WHC,20% of WHC. Water stress reduced plant growing.The activities of peroxide(POD) and esterase(EST) were decreased, especially the POD in root and the EST in leaf. Drought induced a little concentration of andrographolide content (a 30% increase under 40% condition) and the other compounds decreased. AP showed a weak plasticity in its adaptation to water stress. Interesting, the little increase in andrographolide may contribute to its self-protection.4. Study on the quality evaluation of AP germplasm resources(1) HPTLC analysisIn this section, to build a systematic quality evaluation method, the composition of mobile phase, extract solvent were optimized. As a result, a simple, clear and visualized HPTLC for quantitative analysis and an accurate and reliable method involving HPLC for the simultaneous determination of four bioactive diterpenoids were developed. Then, the established methods were successfully applied to determine above four diterpenoids in AP germplasms and samples from different parts, processing methods, markets. The results of this part would play an important role on the quality evaluation and on the guidance of standard setting for quality of AP.(2) HPLC analysisThe HPLC fingerprint analysis pointed out a significant quantitative and qualitative variability of phytocomponents. Promising accessions having high content of andrographolides were distributed in Guangdong province. The germplasm of Guangdong is potentially important sources for breeding and improving of cultivars to obtain higher medicinal yield. The growth conditions in Guangdong are fit for this valuable crop and can be exploited for large-scale cultivation. The chemical diversity mostly depends on environmental factors such as illumination.The content changes of andrographolides in different solvents were detected by HPLC-UV. Then investigated their thermostability. The results showed methanol had the better extracting efficiency than ethyl alcohol, ethyl acetate and chloroform. Addition of suitable amount of water is helpful for extracting andrographolides.50% methanol solution was especially good for andrographolide and dehydroandrographolide extraction.75% ethanol solution could increase the efficacy by 39.5% with comparison to pure methanol and 85% ethanol solution took the second place. But the addition of water could reduce the stability of andrographolides.Andrographolide and deoxyandographolide were increased after 30 min 100℃ heating treatment then decreased after heating for 2h; But dehydroandrographolide was still increased after 100℃ heating for 2 h. X1 and X2 were strikingly dropped after heating; All of the five compounds barely changed after -80℃ freezing.85% ethyl alcohol is the best extracting solution for andrographolides based on efficiency, stability and safety; Andrographolide have not a good thermal resistance but freezing tolerance, so monitoring the temperature environment in manufacturing process ought to be payed attention to.(3) Determination of 19 trace elements,16 organochlorine and 6 pyrethroid pesticide residues in APTo establish an ICP-MS method for simultaneous determination of 19 trace elements, i. e. Na, Mg, Al, Si, K, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Ag, Cd, Sn, Hg, and Pb, in Andrographis herba. After microwave digestiontreatment, samples were directly determined by inductively coupled plasma mass spectrometry based in octopole reaction system(ORS).The validated method indicated that the correlative coefficients (r) for all elements were above 0.9990. The limits of detections were in the range of 0.001-0.051μ g· kg-1. The reproducibility and stability were satisfactory with all RSDs below 10%. The spiked recoveries for Andrographis paniculata were between 88.64% and 102.98%. The method is simple, rapid and sensitive, which meets the requirement of trace analysis and can be used for the quality control of Andrographis herba.To develop a simultaneous analytical method on 16 organochlorine and 6 pyrethroid pesticides in crude Andrographis crude materials by GC-ECD method. Using florisil solid-phase extraction for the clean-up of the samples and GC-ECD for determination. LODs of the method were 0.1-3.0lg·kg-1, the mean recoveries for 22 pesticides were 80.97%-113.45%(RSD=0.68-3.41%, n=6). The developed method could simultaneously determined 22 organochlorine and pyrethroid pesticides in Andrographis herba, it can also be used as a regular method for other crude Chinese medicinal materials. |
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