| Zanthoxylum dissitum Hemsl is a traditional Chinese medicine plant known as Danmianzhen. It is an evergreen shrub and belong to Rutaceae family, Zanthoxylum genus. The root, stem and foliage are as medicines with function of toothache, back pain, women menorrhagia, irregular menstruation postpartum etc., It is sporadically distributed in Guangdong, Guangxi, Hunan, Hubei, Shaanxi province and other places in China. Recently, the Z.dissitum medicine factories have been expanding the production scale, while the species is in a low growth and reproduction rate, lead to threat of drug manufacturer. To seek for suitable means of propagation, and to expand available resources of drug has become inescapabe.Ethanol extract was obtained from Z. dissitum Hemsl powders, nearly neutral lipophilic compositions were gotten by removing acid and alkaline compositions. The fraction were suffered SiO2 column and the products were identified by 1H-NMR, 13C-NMR and MS. Seven components were obtained, named triacontane, 1-hexadecanol,β-sitosterol, ursolic acid, isopimpinellin, bergapten and dictamnine. Exceptβ-sitosterol and dictamnine, the others were isolated from Z. dissitum for the first time. Several components, for instance, ursolic acid, isopimpinellin, bergapten and dictamnine have significant medicinal values, the consequence provided a reference for utilizing and further development of Z. dissitum.The determination method of dictamnine by HPLC was established. The content of dictamnie in roots, stems of 1～3 years old seedlings, and crude drug of Z. dissitum were detected respectively, the result showed that there was no dictamnine could be detected in 1 and 2 year-old stem, foliage and petioles. But in the 3-year old stems,1 to 3 year-old roots, it could be detected, the content in roots was 3.89 times of stems in 3-year-old plants, and was 2.19 times of the crude drug. One year old seedlings were treated with different pH value solutions, The dictamnine content was increased with different treatments, it could be up to 0.04725μg·g-1 with pH of 2.5 solution treatment, it was 7.13 times of the reference sample; with the treatment of pH of 9.5, the content could be up to 0.03200μg·g-1, it was 4.83 times of the reference sample, but the plants in strong acidic or alkaline soil could not grow normally. The study provided a reference for ultilizing Z. dissitum efficiently and choosing the planting soil reasonablly.Ecological characteristics of Z. dissitum has been studied in this research. The result showed that Z. dissitum was mainly distributed at 500-1000m altitude, in the evergreen broad-leaved forest, it is light tolerant species grows in humid area, and often founded beside ditch stream; The pH value range of pristine habitat of Z. dissitum was 5.7-7.6, with an average of value of 6.78; the average organic content was 9.02%; C/N was in the range of 16.9-22.89, the average is 20.74; the average absolute water content in winter was 26.89%. In the high moisture, organic-rich, weak acidic soil environment, the plants grow better.The anatomical observations on the vegetative organs showed that the vegetative organs possessed the basic characters of the dicotyledon. The root's primary xylem was triarch or tetrarch, and the secondary xylem was developed; there were some epidermal hairs on the young stem, and a few secretory cavities and crystallizations in the cortex; the upper and lower epidermal cells of the leaves were significantly different, the upper epidermal cells were obviously larger than the lower epidermal cells, few secretory cavities were close to the lower epidermis. Specific chromogenic reagent was applied in study of the histochemistry localization of alkaloids of the plant. The result illustrated in the stem, alkaloids were mainly distributed in the periderm, secondary xylem, and pith; in the root, many alkaloids were deposited in the periderm, secondary phloem and secondary xylem ray cells; foliage didn't contain alkaloids. Specific chromogenic reagent was also applied in flavonoids, but the color reaction was not obvious, demonstrated the content of flavonoids was in a low level in Z. dissitum.In order to explore the underlying mechanisms relevant to the impediment in the sexual reproduction of Z. dissitum, the characteristics of the dormancy and germination of the seeds were studied. It was found that the germination inhibitors existed in the seed coat, which was believed to be a main impediment to the germination. The seeds viability was detected by TTC method, found that the vitality was up to 93.33%. The permeability of water of the seed coat was regular, though the hardness of the seed coat was not main reason to restrict its germination. When the seed coat was peeled off, it germinated, so the embryo would not induce the seed dormancy. A better method to broken the seeds dormancy was that:processed the seeds in 98% H2SO4 for 6 min., then peeled off the seed coat, dipped them in 50 mgL-1 GA3 for 5 min, finally washed them in running water and cultivated them in incubator at 25℃. The percentage of the seeds germination reached 60%. The micro-propagation technique is an effective instrument for rapid propagation of Z. dissitum. The stems with terminal bud or axillary bud were used as the explants, and were carried out by culturing on MS media with different hormones. The best media for various stages were as fellows:(1) Axially bud induction medium:MS+ 2.0 mg·L-16-BA+0.05 mg·L-1 NAA, the inducement rate was 64.0%; (2) Clump shoot induction medium:MS+2.0 mg·L-1ZT+0.2 mg·L-1 NAA, the inducement rate was 90.0%; (3) Clump shoot proliferation medium:MS+6-BA0.5 mg·L-1+NAA 0.05mg·L-1+Auxin 1.0 mg·L-1; and the multiplication quotiety can reach 4. (4) Rooting medium:1/3MS+IBA0.3mg·L-1+NAA 0.05 mg·L-1, and the rooting rate was 78%. Rooting plantlets transplanted to the medium which was matrix of yellow soil, plant ash and sand, with the proportion of 1:1:1, and disposable plastic cup as the container, sealed with transparent plastic, the transplant survival rate was 95%.In order to reveal the effect of acid and alkali stress in Z. dissitum Hemsl, the plantlets were treated in different pH values of 2,3,4,6,8,10, and 11.5. Four endogenous hormones contents of IAA, ABA, GA and ZR were assayed by ELISA The results illustrated that the plantlets could grow basic normally in the scale of pH value of 3 to 8 within 7 days, and the plantlets were suffered obviously in the treatment pH of 2,10,11.5. The IAA content in the treatment pH of 6 which as the reference sample was always higher than that of other treatment, it showed that other treatments all had different degree inhibition to the growth of the plantlets. Resemble results are found in identical endogenous hormones when treated differently, and the change rule of IAA content was just on the contrary to ABA content; the content of ZR reached peak level after having been treated 2d-3d, and the maximum content was in the treatment of pH of 6; the GA3 content change rule raised upon wave-like.
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