夏枯草规范化种植技术及其药材质量控制
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摘要
夏枯草(Prunella vulgaris L.)为唇形科夏枯草属多年生草本植物,以干燥果穗入药,为我国常用中药材。夏枯草味辛、苦、寒,归肝、胆经,具有清肝明目,消肿散结之功效,常用于治疗甲状腺肿大、淋巴结核、乳腺增生、肺结核、急性黄疸型传染性肝炎、高血压等疾症。现代药理研究表明,夏枯草提取物具有保肝、降血糖、抗菌、抗肿瘤、抗炎等多种药理活性。以夏枯草作为主要原料开发的中成药及功能饮料多达几十种。近年来,随着市场对夏枯草需求的急剧增加,出现供不应求的局面。长期以来,夏枯草一直沿袭传统栽培模式,实际生产问题突出,已不能适应中药现代化发展要求。本课题旨在开展夏枯草规范化种植技术(GAP)及其药材质量控制研究,为提高夏枯草药材产量及其内在品质提供理论依据与技术支撑。具体研究内容如下：
1.考证历代主流本草著作,并结合实地考察进行综合分析。结果表明,历史上夏枯草分布区主要集中于我国四川省境内、淮河流域及长江中下游地区；夏枯草主治功效古今基本一致；夏枯草入药部位变化大致经历带穗全草明朝后期[(约16世纪或以前)～清末民初(约19世纪初)]、以果穗及带穗全草入药[(民国中期(约19世纪30年代)～1963年)]、单用半枯或成熟果穗(1963年～至今)三个阶段；夏枯草传统加工方法分为晒干和阴干两种,现代加工方法仅采用晒干；古代正品夏枯草基原植物主要来源于唇形科夏枯草属夏枯草P. vulgaris L.、山菠菜(长冠夏枯草)Prunella asiatica Nakai.、而云南地区将硬毛夏枯草Prunella hispida Benth混作正品药材,并一直沿用至今；夏枯草食用历史最早可追溯至明代(16世纪末)或以前。
2.采用田间小区试验,对来自全国不同居群的15份夏枯草种质的形态学性状进行相关、通径及主成分分析。结果表明,夏枯草种质中6个形态学性状存在较大遗传变异；单株穗重与单株果穗数和单株叶鲜重呈极显著正相关,与穗长呈显著正相关；主成分分析结果表明,前3个主成分对变异的贡献率达87.533%。在夏枯草丰产种质选育中应重点关注生长势强、单株果穗数较多及果穗较长的株系。
3.研究夏枯草养分吸收分配特性及其与物质积累关系。对夏枯草不同生长时期分别采样,结果表明,夏枯草植株干物质积累量与氮、磷、钾素吸收积累量呈线性增加趋势,相关系数均达显著水平。氮、磷、钾养分积累的平衡有利于干物质积累,夏枯草物质积累量随NBI(养分平衡指数)呈直线增加趋势。4月中旬至5月上旬植株干物质积累率最高,达63.71%。3月下旬至4月中旬植株干物质主要分配在叶中；5月上旬至5月下旬茎中干物质分配率最高；至6月中旬,果穗干物质分配率最大。4月中旬前,植株对氮、磷、钾积累量较少,各营养元素元素主要集中于叶片；4月下旬至5月上旬,氮、磷、钾积累量均占整个生长期最大,分别为：72.35%、59.82%、87.41%,此期营养元素主要集中于茎中；转入生殖生长期(即5月上旬后),果穗中营养元素积累量及分配率逐渐增大,并于6月中旬均达最高值。整个生长期以拔节期至现蕾期(即4月中旬至5月上旬),为夏枯草植株干物质积累量最大期,同时也是氮、磷、钾素最大效率期。拔节期至现蕾期(即4月中旬至5月上旬)为夏枯草植株生长发育最关键时期,这一时期需注意田间水肥调控,以促进植株顶端现蕾与干物质积累。每生产100kg夏枯草果穗,需吸收1.62 kg N; 0.36kg P2O5; 2.88kg K2O,各养分间吸收比例N:P2O5:K2O为1：0.22：1.77。
4.播种期对夏枯草生物量、药材产量及质量影响的研究。采用大田随机区组试验,研究夏枯草生育期进程情况,采收后随机取样,测定夏枯草植株形态、各器官生物量、果穗产量及活性成分含量。结果表明,随播种期推迟,夏枯草全生育期缩短；11月5日晚播夏枯草形态指标、各器官干物质、果穗产量显著下降；随播期推迟,夏枯草活性成分含量显著增加。综合考虑夏枯草产量及质量两因素,安徽庐江地区,夏枯草适宜播种期应设在9月5日至10月5日。
5.种植密度对夏枯草生物量、药材产量及质量影响的研究。大田试验设计6个密度处理,随机区组设计,采收后对夏枯草单株形态指标、各器官干物质积累量、果穗产量及活性成分含量进行测定与分析。结果表明,随种植密度增大,夏枯草株高显著增加,而冠幅、单株叶面积、单株果穗数及穗长显著降低；夏枯草全株干重及各器官生物量随种植密度增加而显著下降；种植密度对根干物质分配率影响较小,茎叶干物质分配率随种植密度增加而减小,果穗干物质分配率则随种植密度增加而增加；处理D果穗产量最高；水浸出物、总黄酮含量以处理C最高,多糖含量以处理D最高,熊果酸、齐墩果酸及迷迭香酸含量以处理A最高。综合考虑夏枯草果穗产量及药材品量两方面,在安徽庐江地区,株行距25 cm×25 cm为最佳种植密度。
6.施肥水平对夏枯草生物量、药材产量及质量影响的研究。田间小区试验采用四因素二次正交旋转组合设计,建立夏枯草果穗产量函数模型；并测定不同施肥处理夏枯草果穗中主要活性成分含量。模型解析结果表明,通过模型解析,氮、磷、钾及有机复合肥对夏枯草果穗产量均有显著影响,其中,氮肥对夏枯草果穗产量影响最大,其次为磷肥、再次钾肥,有机复合肥最小；在低肥力条件下,夏枯草果穗产量均随氮、磷、钾及有机复合肥施用量增加而提高；当氮、磷、钾及有机复合肥施用量过多时,会导致果穗产量下降。氮、磷、钾及有机复合肥优化配施可显著提高夏枯草果穗中主要活性成分含量。本试验条件下,夏枯草果穗产量高于722 kg·hm-2,且能有效提高药材品质的优化施肥方案：N 303.9～335.1 kg·hm-2、P2O5 432.5～500.6 kg·hm-2、K2O206.6～240.2kg·hm-2、有机复合肥2312.5～2687.5kg·hm-2。
7.水肥耦合对夏枯草生物量、药材产量及质量调控的研究。采用盆栽试验,设置2个水分梯度及3个施肥处理,随机区组设计,测定夏枯草营养(根、茎、叶)及生殖器官(果穗)生物量,并检测果穗活性成分含量。结果表明,适度干旱能显著降低夏枯草果穗生物量,但能提高果穗中熊果酸、齐墩果酸及迷迭香酸含量；正常供水的夏枯草果穗中活性成分产量显著高于干旱处理；施用适量肥料能缓解干旱胁迫对夏枯草生长及其活性成分合成的负面影响；水肥交互作用能显著影响夏枯草营养器官及生殖器官生物量、活性成分产量。在干旱或半干旱环境下,施用适量肥料有利于夏枯草生长发育；结合施肥与适时干旱胁迫可提高夏枯草果穗中活性成分产量。
8.不同采收期、不同入药部位及不同加工方法的夏枯草质量研究。分别测定夏枯草主要活性成分含量；并对其活性成分进行综合评价。结果表明,夏枯草水浸出物、醇浸出物、总黄酮、多糖、齐墩果酸、熊果酸及迷迭香酸含量均以现蕾期(5月5日)含量最高；夏枯草带穗全草水浸出物及醇浸出物含量均显著高于果穗,而果穗中熊果酸、齐墩果酸含量显著高于带穗全草,现蕾期(5月5日)至盛花期(5月20日),夏枯草果穗中总黄酮、多糖及迷迭香酸含量显著高于带穗全草,从果穗成熟期(6月15日)至果穗枯萎期(6月25日),夏枯草带穗全草中总黄酮、多糖及迷迭香酸含量则显著高于果穗；晒干及阴干对夏枯草果穗及带穗全草的水浸出物、醇浸出物、总黄酮、多糖、熊果酸及齐墩果酸等药效成分的含量影响不显著,但晒干会显著降低迷迭香酸含量；夏枯草活性成分综合函数值最高为现蕾期晒干果穗。从夏枯草产量及质量两方面考虑,应选择现蕾期(5月上旬)采收带穗全草,加工方式采用晒干。
9.不同贮藏年限及药材分级标准对夏枯草药材质量的研究。在安徽庐江夏枯草规范化种植基地内收集不同贮藏年限夏枯草果穗(当年、1年、2年),并对基地果穗药材随机取样,结合果穗外观形状与市场现行标准,将药材分为3级；并测定夏枯草药材中主要活性成分含量。结果表明,不同贮藏年限夏枯草水分含量无显著差异；总灰分及酸不溶性灰分含量随贮藏年限延长而显著增加；而水溶性浸出物、醇溶性浸出物、总黄酮、多糖、熊果酸、齐墩果酸及迷迭香酸含量随贮藏年限延长呈逐渐下降,且不同储藏年限间差异显著。夏枯草药材质量并未遵循分级标准呈现出明显规律,其中水分含量差异不显著；总黄酮含量以二级最高；多糖含量则以一级最高；总灰分、酸不溶性灰分含量呈现出从一级到三级逐渐下降趋势；而水浸出物、醇浸出物、熊果酸、齐墩果酸及迷迭香酸含量表现出从一级到三级依次上升趋势。夏枯草药材安全储藏期以当年为宜；传统分级标准未能充分体现夏枯草药材内在品质优劣,故建议取消现行夏枯草药材分级标准,以全草入药。
10.夏枯草种植基地土壤和药材中农药残和重金属含量研究。采用气相色谱法(GC)测定有机氯农药残留量,用ICP法测定铜(Cu)、铅(Pb)、铬(Cr)、砷(As)、镉(Cd)、汞(Hg)含量。结果表明,夏枯草种植土壤与药材中的有机氯农药及重金属残留因种植地点不同存在差异,但均低于国家限量标准。夏枯草种植基地土壤和药材均符合中药材GAP规定。
11.夏枯草不同部位挥发油含量及其化学组成研究。采用水蒸气蒸馏法提取鲜花、干花、茎及叶挥发油成分,采用GC-MS分析比较了不同部位间挥发油化学组成上的异同。结果表明,夏枯草鲜花、干花、叶及茎的挥发油中鉴定出24种,28种,19种,21种化合物,分别占挥发油总量的95.22%,83.47%,80.03%,79.48%；鲜花、干花及茎中挥发油主要成分为棕榈酸(58.12%,69.99%,38.44%),叶中挥发油成分则以香橙烯(55.35%)含量最高。夏枯草鲜花、干花、茎及叶挥发油成分在含量及组成上各有异同。
Prunella vulgaris L. (Labiatae), also known as the "self-heal," was a perennial herb commonly found in North Asia, Europe and North Africa. The dried spica of P. vulgaris, Prunellae Spica, a standard medicinal material in the Chinese Pharmacopoeia and occasionally as a folk medicine to treat sore throat, fever, goiter, hypertension, tuberculous lymphadenitis and mammary gland hyperplasia. Several studies on the pharmacological activities of P. vulgaris have been previously reported, including hepatoprotection, antihyperglucemia, antifugal, anti-tumor and anti-inflammatory activities. As the key raw material, there were dozens of Chinese proprietary medicine manufactured with P. vulgaris. In recent years, the demand for P. vulgaris has steadily increased in the world market. The wild population of P. vulgaris cannot meet this growing need. Long-term since, P. vulgaris was often cultured by traditional planting technology. The many problems associated with actual production process have emerged. All this shows that the cultivation techniques of P. vulgaris could not to promote the development of traditional Chinese medicine. Therefore, the good agricultural practice (GAP) and quality control of P. vulgaris were investigated, and improved the yield and quality of P. vulgaris. Specific results were as follows:
1. Textual research on medicinal works of past dynasties and field work were adopted. The results showed, the natural distributions of P. vulgaris were concentrated distribution in Sichuan Province, Huaihe River Basin, and Middle-Lower Yangtze River Valley in Chinese history; the traditional efficacy of P. vulgaris between ancient and modern was basically identical; The whole grass of P. vulgaris as medicinal parts (from late Ming dynasty to late Qing dynasty and early stage Republic of China), both whole grass and spicas as medicinal parts (from a mid-term Republic of China to 1963), and the semi-maturity or maturity of spicas as medicinal parts (from 1963 to today). The processing method for medicinal parts of P. vulgaris adopted sun drying and shady drying in ancient China, but the sun drying was only used in modern China. The original plants were originated from two species (P. vulgaris L. and Prunella asiatica Nakai.); Prunella hispida Benth.was used as a certified medicinal materials in Yunnan province from Ming Dynasty to modern time. The dietotherapy history of P. vulgaris in China could be traced back to Ming Dynasty or before.
2. The various germplasm resources of P. vulgaris came from all over the country were investigated by analysis of correlation analysis, path analysis and principle component analysis in randomized block experiment. The results showed that the 6 morphological characteristics were greatly different from each other in various germplasm. The spicas yield per plant had a very significantly positive correlation with the number of spicas per plant and fresh leaves weight per plant, meanwhile the correlation between the spicas yield per plant and spicas length was very significantly. Three principal components which accounted for 87.533%of total variance were extracted from the principal component analysis. The strong growth ability, the number of spicas per plant and spicas length were focused on the high yield breeding and planting of P. vulgaris germplasm.
3. Through the dynamic sampling of P. vulgaris in different growing stage, and the dry weight of plant, the amount of dry matter and the content of N, P and K within each organ of the plant were measured. The results showed that the dry weight of P. vulgaris linearly increased with increase of nitrogen, phosphorus and potassium accumulation with significant correlation coefficients at whole growth period. The balance of nitrogen, phosphorus and potassium accumulation was beneficial for dry matter accumulation, which linearly increased with increase of nutrient balance index. The maximum stage accumulation rate of dry matter from mid-April to beginning of May, reached 63.71%. The amount of dry matter was mainly distribution in leaves from late March to mid-April. The distribution ratio in stem was highest from beginning of May to late May. Until mid-June, the distribution ratio of dry matter in spicas was higher than other organs. The absorption amounts of N, P and K were lower before mid-April, and the nutrient element was concentrated in plant leaves. From late April to beginning of May, the N, P and K accumulation reached maximum of total absorptive capacity, which accounted for 72.35%、59.82% and 87.41%, and the mineral elements were concentrated distribution of plant stems. When transferred to reproductive stage, both accumulation amount and distribution rate of nutrient elements in spicas was growth rapidly, which reached maximum until mid-June. From elongation stages to quaring period was the period of maximal accumulation of dry matter and showed the maximal efficiency of N, P and K nutrition. From elongation stages to quaring period is the most crucial period in the growth development stage of P. vulgaris, and should be pay more attention to the supply of mineral nutrition and moisture of plant to promote spicas formation and dry matter accumulation in this stage. The average N, P205and K2O absorption amount per 100 kg dry matter of spicas were 1.62 kg,0.36 kg,2.88 kg respectively, and the ratio of N:P2O5 : K2O was 1: 0.22: 1.77.
4. The experiment was a randomized complete block design arranged in split plot. The growth periods of P. vulgaris were investigated. The morphological characteristics, biomass of each organ, spicas yield and content of active components at harvest time were determined. The results showed, with the delay of sowing date, the growing period of P. vulgaris was shortened; the morphological characteristics, biomass of each organ and spicas yield were decreased significantly in P. vulgaris. With postpone of sowing date, the content of active components in P. vulgaris were significantly increased. Considering the two factors between spicas yield and quality in P. vulgaris, the suitable sowing period for P. vulgaris was from 5th September to 5th October in Lujiang County, Anhui Province.
5. The six planting densities were designed in the plot experiment. Morphological index per plant, dry matter accumulation in each organ, spicas yield and content of active components in P. vulgaris were determined and analyzed. The results showed that plant height of P. vulgaris significantly improved with increasing density, but crown width, leaf area per plant, number of spicas per plant, spica length of P. vulgaris significantly declined with increasing density. Dry weight of P. vulgaris and biomass of each organ significantly decreased with density increasing. Distribution ratio of dry matter in root has less effect by planting density, distribution rate of dry matter in leaf and stem decrease with density increasing, the distribution ratio of dry matter in spicas increase with increasing density. The treatment D has a highest spicas yield. The content of water-soluble extracts and total flavonoids in P. vulgaris reached the highest in treatment C, the maximum content of polysaccharides in P. vulgaris was treatment D, the highest amount of ursolic acid, oleanolic acid and rosmarinic acid in P. vulgaris were found in treatment A. Considering the two factors between spicas yield and quality in P. vulgaris, the planting density was set as 25 cm X 25 cm in Lujiang county, Anhui Province.
6. Field plot experiment with a 4-variable quadratic orthogonal rotation combination design was adopted, and a function model of spicas yield in P. vulgaris was established. Under different fertilizer treatments, the content of water-soluble extracts, total flavonoids, polysaccharides, ursolic acid, oleanolic acid and rosmarinic acid were determined. The results showed, the model analysis showed that spicas yields of P. vulgaris were significantly influenced under the N, P2O5 K2O and OF applications, among there factors, N has the greatest effects on spicas yield of P. vulgaris, secondly for P2O5, thirdly for K2O, and OF has the smallest effects on spicas yield. At the lower fertility levels, the spicas yields of P. vulgaris were improved along with the increasing of N, P2O5 K2O and OF applications, but the spicas yields decreased when N, P2O5 K2O and OF were applied too much. The main active components level in P. vulgaris spicas were significantly increased by optimized application of N, P2O5, K2O and OF. In this experiment condition, the spicas yields of P. vulgaris could reach to 722 kg·hm-2, and quality of spicas could effectively improve when the fertilizer rates were N 303.9～335.1 kg·hm-2, P2O5 432.5～500.6 kg·hm-2, K2O 206.6～240.2 kg·hm-2, OF 2312.5～2687.5 kg·hm-2.
7. The factors studied included two water conditions and three fertilizer regimes. The results showed that moderate drought stress dramatically decreases spica biomass production but increases the content of RA, UA and OA in spicas of P. vulgaris. Total RA, UA and OA yields were found to be significantly higher in well-watered plants than in drought-stressed plants. An appropriate amount of fertilizer could alleviate the negative effect of drought stress on the growth of P. vulgaris and its production of RA, UA, and OA. The interaction of water and fertilizer treatments significantly influences vegetative dry weight, reproductive dry weight and total RA, UA and OA yields in P. vulgaris. The results suggest that the application of the proper amount of fertilizer aids P. vulgaris production in arid and semi-arid regions and that the combined use of fertilizer and properly timed exposure to drought stress can enhance total RA, UA and OA yields in P. vulgaris.
8. The active compounds of P. vulgaris were measured, and the main chemical components in P. vulgaris were used for comprehensive evaluation. The results showed that the accumulation of the active constituents, including water-soluble extracts, ethanol-soluble extracts, total flavonoids, polysaccharides, ursolic acid, oleanolic acid and rosmarinic acid levels in P. vulgaris were found to highest in the squaring stage (on 5 May); The contents of water-soluble extracts and ethanol-soluble extracts in P. vulgaris whole herbs were remarkable higher than in spicas. The levels of ursolic acid and oleanolic acid in P. vulgaris spicas were significantly higher than in whole herbs. From squaring stage(on 5 May) to the flowering stage (on 20 May), the contents of total flavonoids, polysaccharides and rosmarinic acid in P. vulgaris spicas were significantly higher than in whole herbs, but from maturity stage (on 15 June) to the wilting stage (on 25 June), our results showed the reverse pattern:the total flavonoids, polysaccharides and rosmarinic acid levels of P. vulgaris whole herbs were remarkable higher than of spicas. Both sun and shade drying were found to exhibit no remarkable influence on active constituents of P. vulgaris, including water-soluble extracts, ethanol-soluble extracts, total flavonoids, polysaccharides, ursolic acid and oleanolic acid. The content of rosmarinic acid in P. vulgaris was significantly reduced by the sun drying. The highest sums of comprehensive evaluation were spicas of P. vulgaris in the squaring stage under open-air sun drying condition. The yield and quality of P. vulgaris were considered, harvest period should selecte in the squaring stage (beginning of May), whole herbs of P. vulgaris should be harvested, and sun drying method need to be adopted.
9. The spicas of P. vulgaris.in different storage period (including current year、first-year and second-year) were collected from Lujiang base in Anhui province. And the medicinal material could be dealed with srandom sampling, with the methods of morphologic characteristics and commercial size on P. vulgaris, the samples were graded into three grades; according to the methods from the Pharmacopoeia of the People's Republic of China (2005), the moisture content, water-soluble extracts, ethanol-soluble extracts, total ash and acid insoluble ash were measured, respectively, utilizing the UV spectrophotometry to detection the contents of total flavonoids and polysaccharides, the levels of ursolic acid, oleanolic acid and rosmarinic acid were adopted by HPLC. The results showed that moisture content of P. vulgaris in different storage period was showed on significant differences; the total ash and acid insoluble ash of P. vulgaris showed significantly increase during prolonged storage; however, the content of water-soluble extracts, ethanol-soluble, total flavonoids, polysaccharides, ursolic acid, oleanolic acid, rosmarinic acid showed significantly decrease during prolonged storage. The spicas of P. vulgaris exhibited no rules differences between the quality and grading standards, The moisture content of spicas in different degree showed significant differences; the second-degree belongs to high content of total flavonoids; the high content of polysaccharides was first-degree; the total ash and acid insoluble ash of spicas exhibited gradually decreased from first-degree to third-grade; and the content of water-soluble extracts, ethanol-soluble extracts, ursolic acid, oleanolic acid, rosmarinic acid showed gradually increased from first-degree to third-grade. The current year was a suitable storage time of spicas from P. vulgaris could be selected; and the traditional grading standards failed to reflect the quality of herbal medicine, therefore, we suggested that the traditional standards should be abolished.
10. The GC method was applied to determine residuals of organochlorine pesticides, the contents of Cu、Pb、Cr、As、Cd and Hg were measured by ICP. The results showed, the contents of residuals of organochlorine pesticides and heavy metals were significant differences in two planting bases, but all of the residuals between organochlorine pesticides and heavy metals were below the GB standards. Residuals of organochlorine pesticides and heavy metals in soil and P. vulgaris from two planting bases were conformed to GAP.
11. The essential oils from the fresh flowers, dried flowers leaves and stems of P. vulgaris were isolated by hydrodistillation. The collected oils were analyzed by capillary GC and GC-MS. The results showed that a total of 24,28,19 and 21 compounds were identified, accounting for 95.22%,83.47%,80.03%and 79.48%of fresh flowers, dried flowers, leaves and stems oil, respectively. The main constituent of the essential oil of fresh flowers, dried flowers and stem oils, respectively, was hexadecanoic acid (58.12%,69.99% and 38.44%). Aromadendrene (55.35%) was identified as the highest main constituent for leaves. The composition of essential oil from fresh flowers, dried flowers, leaves and stems of P. vulgaris were discrepant.
1.考证历代主流本草著作,并结合实地考察进行综合分析。结果表明,历史上夏枯草分布区主要集中于我国四川省境内、淮河流域及长江中下游地区；夏枯草主治功效古今基本一致；夏枯草入药部位变化大致经历带穗全草明朝后期[(约16世纪或以前)～清末民初(约19世纪初)]、以果穗及带穗全草入药[(民国中期(约19世纪30年代)～1963年)]、单用半枯或成熟果穗(1963年～至今)三个阶段；夏枯草传统加工方法分为晒干和阴干两种,现代加工方法仅采用晒干；古代正品夏枯草基原植物主要来源于唇形科夏枯草属夏枯草P. vulgaris L.、山菠菜(长冠夏枯草)Prunella asiatica Nakai.、而云南地区将硬毛夏枯草Prunella hispida Benth混作正品药材,并一直沿用至今；夏枯草食用历史最早可追溯至明代(16世纪末)或以前。
2.采用田间小区试验,对来自全国不同居群的15份夏枯草种质的形态学性状进行相关、通径及主成分分析。结果表明,夏枯草种质中6个形态学性状存在较大遗传变异；单株穗重与单株果穗数和单株叶鲜重呈极显著正相关,与穗长呈显著正相关；主成分分析结果表明,前3个主成分对变异的贡献率达87.533%。在夏枯草丰产种质选育中应重点关注生长势强、单株果穗数较多及果穗较长的株系。
3.研究夏枯草养分吸收分配特性及其与物质积累关系。对夏枯草不同生长时期分别采样,结果表明,夏枯草植株干物质积累量与氮、磷、钾素吸收积累量呈线性增加趋势,相关系数均达显著水平。氮、磷、钾养分积累的平衡有利于干物质积累,夏枯草物质积累量随NBI(养分平衡指数)呈直线增加趋势。4月中旬至5月上旬植株干物质积累率最高,达63.71%。3月下旬至4月中旬植株干物质主要分配在叶中；5月上旬至5月下旬茎中干物质分配率最高；至6月中旬,果穗干物质分配率最大。4月中旬前,植株对氮、磷、钾积累量较少,各营养元素元素主要集中于叶片；4月下旬至5月上旬,氮、磷、钾积累量均占整个生长期最大,分别为：72.35%、59.82%、87.41%,此期营养元素主要集中于茎中；转入生殖生长期(即5月上旬后),果穗中营养元素积累量及分配率逐渐增大,并于6月中旬均达最高值。整个生长期以拔节期至现蕾期(即4月中旬至5月上旬),为夏枯草植株干物质积累量最大期,同时也是氮、磷、钾素最大效率期。拔节期至现蕾期(即4月中旬至5月上旬)为夏枯草植株生长发育最关键时期,这一时期需注意田间水肥调控,以促进植株顶端现蕾与干物质积累。每生产100kg夏枯草果穗,需吸收1.62 kg N; 0.36kg P2O5; 2.88kg K2O,各养分间吸收比例N:P2O5:K2O为1：0.22：1.77。
4.播种期对夏枯草生物量、药材产量及质量影响的研究。采用大田随机区组试验,研究夏枯草生育期进程情况,采收后随机取样,测定夏枯草植株形态、各器官生物量、果穗产量及活性成分含量。结果表明,随播种期推迟,夏枯草全生育期缩短；11月5日晚播夏枯草形态指标、各器官干物质、果穗产量显著下降；随播期推迟,夏枯草活性成分含量显著增加。综合考虑夏枯草产量及质量两因素,安徽庐江地区,夏枯草适宜播种期应设在9月5日至10月5日。
5.种植密度对夏枯草生物量、药材产量及质量影响的研究。大田试验设计6个密度处理,随机区组设计,采收后对夏枯草单株形态指标、各器官干物质积累量、果穗产量及活性成分含量进行测定与分析。结果表明,随种植密度增大,夏枯草株高显著增加,而冠幅、单株叶面积、单株果穗数及穗长显著降低；夏枯草全株干重及各器官生物量随种植密度增加而显著下降；种植密度对根干物质分配率影响较小,茎叶干物质分配率随种植密度增加而减小,果穗干物质分配率则随种植密度增加而增加；处理D果穗产量最高；水浸出物、总黄酮含量以处理C最高,多糖含量以处理D最高,熊果酸、齐墩果酸及迷迭香酸含量以处理A最高。综合考虑夏枯草果穗产量及药材品量两方面,在安徽庐江地区,株行距25 cm×25 cm为最佳种植密度。
6.施肥水平对夏枯草生物量、药材产量及质量影响的研究。田间小区试验采用四因素二次正交旋转组合设计,建立夏枯草果穗产量函数模型；并测定不同施肥处理夏枯草果穗中主要活性成分含量。模型解析结果表明,通过模型解析,氮、磷、钾及有机复合肥对夏枯草果穗产量均有显著影响,其中,氮肥对夏枯草果穗产量影响最大,其次为磷肥、再次钾肥,有机复合肥最小；在低肥力条件下,夏枯草果穗产量均随氮、磷、钾及有机复合肥施用量增加而提高；当氮、磷、钾及有机复合肥施用量过多时,会导致果穗产量下降。氮、磷、钾及有机复合肥优化配施可显著提高夏枯草果穗中主要活性成分含量。本试验条件下,夏枯草果穗产量高于722 kg·hm-2,且能有效提高药材品质的优化施肥方案：N 303.9～335.1 kg·hm-2、P2O5 432.5～500.6 kg·hm-2、K2O206.6～240.2kg·hm-2、有机复合肥2312.5～2687.5kg·hm-2。
7.水肥耦合对夏枯草生物量、药材产量及质量调控的研究。采用盆栽试验,设置2个水分梯度及3个施肥处理,随机区组设计,测定夏枯草营养(根、茎、叶)及生殖器官(果穗)生物量,并检测果穗活性成分含量。结果表明,适度干旱能显著降低夏枯草果穗生物量,但能提高果穗中熊果酸、齐墩果酸及迷迭香酸含量；正常供水的夏枯草果穗中活性成分产量显著高于干旱处理；施用适量肥料能缓解干旱胁迫对夏枯草生长及其活性成分合成的负面影响；水肥交互作用能显著影响夏枯草营养器官及生殖器官生物量、活性成分产量。在干旱或半干旱环境下,施用适量肥料有利于夏枯草生长发育；结合施肥与适时干旱胁迫可提高夏枯草果穗中活性成分产量。
8.不同采收期、不同入药部位及不同加工方法的夏枯草质量研究。分别测定夏枯草主要活性成分含量；并对其活性成分进行综合评价。结果表明,夏枯草水浸出物、醇浸出物、总黄酮、多糖、齐墩果酸、熊果酸及迷迭香酸含量均以现蕾期(5月5日)含量最高；夏枯草带穗全草水浸出物及醇浸出物含量均显著高于果穗,而果穗中熊果酸、齐墩果酸含量显著高于带穗全草,现蕾期(5月5日)至盛花期(5月20日),夏枯草果穗中总黄酮、多糖及迷迭香酸含量显著高于带穗全草,从果穗成熟期(6月15日)至果穗枯萎期(6月25日),夏枯草带穗全草中总黄酮、多糖及迷迭香酸含量则显著高于果穗；晒干及阴干对夏枯草果穗及带穗全草的水浸出物、醇浸出物、总黄酮、多糖、熊果酸及齐墩果酸等药效成分的含量影响不显著,但晒干会显著降低迷迭香酸含量；夏枯草活性成分综合函数值最高为现蕾期晒干果穗。从夏枯草产量及质量两方面考虑,应选择现蕾期(5月上旬)采收带穗全草,加工方式采用晒干。
9.不同贮藏年限及药材分级标准对夏枯草药材质量的研究。在安徽庐江夏枯草规范化种植基地内收集不同贮藏年限夏枯草果穗(当年、1年、2年),并对基地果穗药材随机取样,结合果穗外观形状与市场现行标准,将药材分为3级；并测定夏枯草药材中主要活性成分含量。结果表明,不同贮藏年限夏枯草水分含量无显著差异；总灰分及酸不溶性灰分含量随贮藏年限延长而显著增加；而水溶性浸出物、醇溶性浸出物、总黄酮、多糖、熊果酸、齐墩果酸及迷迭香酸含量随贮藏年限延长呈逐渐下降,且不同储藏年限间差异显著。夏枯草药材质量并未遵循分级标准呈现出明显规律,其中水分含量差异不显著；总黄酮含量以二级最高；多糖含量则以一级最高；总灰分、酸不溶性灰分含量呈现出从一级到三级逐渐下降趋势；而水浸出物、醇浸出物、熊果酸、齐墩果酸及迷迭香酸含量表现出从一级到三级依次上升趋势。夏枯草药材安全储藏期以当年为宜；传统分级标准未能充分体现夏枯草药材内在品质优劣,故建议取消现行夏枯草药材分级标准,以全草入药。
10.夏枯草种植基地土壤和药材中农药残和重金属含量研究。采用气相色谱法(GC)测定有机氯农药残留量,用ICP法测定铜(Cu)、铅(Pb)、铬(Cr)、砷(As)、镉(Cd)、汞(Hg)含量。结果表明,夏枯草种植土壤与药材中的有机氯农药及重金属残留因种植地点不同存在差异,但均低于国家限量标准。夏枯草种植基地土壤和药材均符合中药材GAP规定。
11.夏枯草不同部位挥发油含量及其化学组成研究。采用水蒸气蒸馏法提取鲜花、干花、茎及叶挥发油成分,采用GC-MS分析比较了不同部位间挥发油化学组成上的异同。结果表明,夏枯草鲜花、干花、叶及茎的挥发油中鉴定出24种,28种,19种,21种化合物,分别占挥发油总量的95.22%,83.47%,80.03%,79.48%；鲜花、干花及茎中挥发油主要成分为棕榈酸(58.12%,69.99%,38.44%),叶中挥发油成分则以香橙烯(55.35%)含量最高。夏枯草鲜花、干花、茎及叶挥发油成分在含量及组成上各有异同。
Prunella vulgaris L. (Labiatae), also known as the "self-heal," was a perennial herb commonly found in North Asia, Europe and North Africa. The dried spica of P. vulgaris, Prunellae Spica, a standard medicinal material in the Chinese Pharmacopoeia and occasionally as a folk medicine to treat sore throat, fever, goiter, hypertension, tuberculous lymphadenitis and mammary gland hyperplasia. Several studies on the pharmacological activities of P. vulgaris have been previously reported, including hepatoprotection, antihyperglucemia, antifugal, anti-tumor and anti-inflammatory activities. As the key raw material, there were dozens of Chinese proprietary medicine manufactured with P. vulgaris. In recent years, the demand for P. vulgaris has steadily increased in the world market. The wild population of P. vulgaris cannot meet this growing need. Long-term since, P. vulgaris was often cultured by traditional planting technology. The many problems associated with actual production process have emerged. All this shows that the cultivation techniques of P. vulgaris could not to promote the development of traditional Chinese medicine. Therefore, the good agricultural practice (GAP) and quality control of P. vulgaris were investigated, and improved the yield and quality of P. vulgaris. Specific results were as follows:
1. Textual research on medicinal works of past dynasties and field work were adopted. The results showed, the natural distributions of P. vulgaris were concentrated distribution in Sichuan Province, Huaihe River Basin, and Middle-Lower Yangtze River Valley in Chinese history; the traditional efficacy of P. vulgaris between ancient and modern was basically identical; The whole grass of P. vulgaris as medicinal parts (from late Ming dynasty to late Qing dynasty and early stage Republic of China), both whole grass and spicas as medicinal parts (from a mid-term Republic of China to 1963), and the semi-maturity or maturity of spicas as medicinal parts (from 1963 to today). The processing method for medicinal parts of P. vulgaris adopted sun drying and shady drying in ancient China, but the sun drying was only used in modern China. The original plants were originated from two species (P. vulgaris L. and Prunella asiatica Nakai.); Prunella hispida Benth.was used as a certified medicinal materials in Yunnan province from Ming Dynasty to modern time. The dietotherapy history of P. vulgaris in China could be traced back to Ming Dynasty or before.
2. The various germplasm resources of P. vulgaris came from all over the country were investigated by analysis of correlation analysis, path analysis and principle component analysis in randomized block experiment. The results showed that the 6 morphological characteristics were greatly different from each other in various germplasm. The spicas yield per plant had a very significantly positive correlation with the number of spicas per plant and fresh leaves weight per plant, meanwhile the correlation between the spicas yield per plant and spicas length was very significantly. Three principal components which accounted for 87.533%of total variance were extracted from the principal component analysis. The strong growth ability, the number of spicas per plant and spicas length were focused on the high yield breeding and planting of P. vulgaris germplasm.
3. Through the dynamic sampling of P. vulgaris in different growing stage, and the dry weight of plant, the amount of dry matter and the content of N, P and K within each organ of the plant were measured. The results showed that the dry weight of P. vulgaris linearly increased with increase of nitrogen, phosphorus and potassium accumulation with significant correlation coefficients at whole growth period. The balance of nitrogen, phosphorus and potassium accumulation was beneficial for dry matter accumulation, which linearly increased with increase of nutrient balance index. The maximum stage accumulation rate of dry matter from mid-April to beginning of May, reached 63.71%. The amount of dry matter was mainly distribution in leaves from late March to mid-April. The distribution ratio in stem was highest from beginning of May to late May. Until mid-June, the distribution ratio of dry matter in spicas was higher than other organs. The absorption amounts of N, P and K were lower before mid-April, and the nutrient element was concentrated in plant leaves. From late April to beginning of May, the N, P and K accumulation reached maximum of total absorptive capacity, which accounted for 72.35%、59.82% and 87.41%, and the mineral elements were concentrated distribution of plant stems. When transferred to reproductive stage, both accumulation amount and distribution rate of nutrient elements in spicas was growth rapidly, which reached maximum until mid-June. From elongation stages to quaring period was the period of maximal accumulation of dry matter and showed the maximal efficiency of N, P and K nutrition. From elongation stages to quaring period is the most crucial period in the growth development stage of P. vulgaris, and should be pay more attention to the supply of mineral nutrition and moisture of plant to promote spicas formation and dry matter accumulation in this stage. The average N, P205and K2O absorption amount per 100 kg dry matter of spicas were 1.62 kg,0.36 kg,2.88 kg respectively, and the ratio of N:P2O5 : K2O was 1: 0.22: 1.77.
4. The experiment was a randomized complete block design arranged in split plot. The growth periods of P. vulgaris were investigated. The morphological characteristics, biomass of each organ, spicas yield and content of active components at harvest time were determined. The results showed, with the delay of sowing date, the growing period of P. vulgaris was shortened; the morphological characteristics, biomass of each organ and spicas yield were decreased significantly in P. vulgaris. With postpone of sowing date, the content of active components in P. vulgaris were significantly increased. Considering the two factors between spicas yield and quality in P. vulgaris, the suitable sowing period for P. vulgaris was from 5th September to 5th October in Lujiang County, Anhui Province.
5. The six planting densities were designed in the plot experiment. Morphological index per plant, dry matter accumulation in each organ, spicas yield and content of active components in P. vulgaris were determined and analyzed. The results showed that plant height of P. vulgaris significantly improved with increasing density, but crown width, leaf area per plant, number of spicas per plant, spica length of P. vulgaris significantly declined with increasing density. Dry weight of P. vulgaris and biomass of each organ significantly decreased with density increasing. Distribution ratio of dry matter in root has less effect by planting density, distribution rate of dry matter in leaf and stem decrease with density increasing, the distribution ratio of dry matter in spicas increase with increasing density. The treatment D has a highest spicas yield. The content of water-soluble extracts and total flavonoids in P. vulgaris reached the highest in treatment C, the maximum content of polysaccharides in P. vulgaris was treatment D, the highest amount of ursolic acid, oleanolic acid and rosmarinic acid in P. vulgaris were found in treatment A. Considering the two factors between spicas yield and quality in P. vulgaris, the planting density was set as 25 cm X 25 cm in Lujiang county, Anhui Province.
6. Field plot experiment with a 4-variable quadratic orthogonal rotation combination design was adopted, and a function model of spicas yield in P. vulgaris was established. Under different fertilizer treatments, the content of water-soluble extracts, total flavonoids, polysaccharides, ursolic acid, oleanolic acid and rosmarinic acid were determined. The results showed, the model analysis showed that spicas yields of P. vulgaris were significantly influenced under the N, P2O5 K2O and OF applications, among there factors, N has the greatest effects on spicas yield of P. vulgaris, secondly for P2O5, thirdly for K2O, and OF has the smallest effects on spicas yield. At the lower fertility levels, the spicas yields of P. vulgaris were improved along with the increasing of N, P2O5 K2O and OF applications, but the spicas yields decreased when N, P2O5 K2O and OF were applied too much. The main active components level in P. vulgaris spicas were significantly increased by optimized application of N, P2O5, K2O and OF. In this experiment condition, the spicas yields of P. vulgaris could reach to 722 kg·hm-2, and quality of spicas could effectively improve when the fertilizer rates were N 303.9～335.1 kg·hm-2, P2O5 432.5～500.6 kg·hm-2, K2O 206.6～240.2 kg·hm-2, OF 2312.5～2687.5 kg·hm-2.
7. The factors studied included two water conditions and three fertilizer regimes. The results showed that moderate drought stress dramatically decreases spica biomass production but increases the content of RA, UA and OA in spicas of P. vulgaris. Total RA, UA and OA yields were found to be significantly higher in well-watered plants than in drought-stressed plants. An appropriate amount of fertilizer could alleviate the negative effect of drought stress on the growth of P. vulgaris and its production of RA, UA, and OA. The interaction of water and fertilizer treatments significantly influences vegetative dry weight, reproductive dry weight and total RA, UA and OA yields in P. vulgaris. The results suggest that the application of the proper amount of fertilizer aids P. vulgaris production in arid and semi-arid regions and that the combined use of fertilizer and properly timed exposure to drought stress can enhance total RA, UA and OA yields in P. vulgaris.
8. The active compounds of P. vulgaris were measured, and the main chemical components in P. vulgaris were used for comprehensive evaluation. The results showed that the accumulation of the active constituents, including water-soluble extracts, ethanol-soluble extracts, total flavonoids, polysaccharides, ursolic acid, oleanolic acid and rosmarinic acid levels in P. vulgaris were found to highest in the squaring stage (on 5 May); The contents of water-soluble extracts and ethanol-soluble extracts in P. vulgaris whole herbs were remarkable higher than in spicas. The levels of ursolic acid and oleanolic acid in P. vulgaris spicas were significantly higher than in whole herbs. From squaring stage(on 5 May) to the flowering stage (on 20 May), the contents of total flavonoids, polysaccharides and rosmarinic acid in P. vulgaris spicas were significantly higher than in whole herbs, but from maturity stage (on 15 June) to the wilting stage (on 25 June), our results showed the reverse pattern:the total flavonoids, polysaccharides and rosmarinic acid levels of P. vulgaris whole herbs were remarkable higher than of spicas. Both sun and shade drying were found to exhibit no remarkable influence on active constituents of P. vulgaris, including water-soluble extracts, ethanol-soluble extracts, total flavonoids, polysaccharides, ursolic acid and oleanolic acid. The content of rosmarinic acid in P. vulgaris was significantly reduced by the sun drying. The highest sums of comprehensive evaluation were spicas of P. vulgaris in the squaring stage under open-air sun drying condition. The yield and quality of P. vulgaris were considered, harvest period should selecte in the squaring stage (beginning of May), whole herbs of P. vulgaris should be harvested, and sun drying method need to be adopted.
9. The spicas of P. vulgaris.in different storage period (including current year、first-year and second-year) were collected from Lujiang base in Anhui province. And the medicinal material could be dealed with srandom sampling, with the methods of morphologic characteristics and commercial size on P. vulgaris, the samples were graded into three grades; according to the methods from the Pharmacopoeia of the People's Republic of China (2005), the moisture content, water-soluble extracts, ethanol-soluble extracts, total ash and acid insoluble ash were measured, respectively, utilizing the UV spectrophotometry to detection the contents of total flavonoids and polysaccharides, the levels of ursolic acid, oleanolic acid and rosmarinic acid were adopted by HPLC. The results showed that moisture content of P. vulgaris in different storage period was showed on significant differences; the total ash and acid insoluble ash of P. vulgaris showed significantly increase during prolonged storage; however, the content of water-soluble extracts, ethanol-soluble, total flavonoids, polysaccharides, ursolic acid, oleanolic acid, rosmarinic acid showed significantly decrease during prolonged storage. The spicas of P. vulgaris exhibited no rules differences between the quality and grading standards, The moisture content of spicas in different degree showed significant differences; the second-degree belongs to high content of total flavonoids; the high content of polysaccharides was first-degree; the total ash and acid insoluble ash of spicas exhibited gradually decreased from first-degree to third-grade; and the content of water-soluble extracts, ethanol-soluble extracts, ursolic acid, oleanolic acid, rosmarinic acid showed gradually increased from first-degree to third-grade. The current year was a suitable storage time of spicas from P. vulgaris could be selected; and the traditional grading standards failed to reflect the quality of herbal medicine, therefore, we suggested that the traditional standards should be abolished.
10. The GC method was applied to determine residuals of organochlorine pesticides, the contents of Cu、Pb、Cr、As、Cd and Hg were measured by ICP. The results showed, the contents of residuals of organochlorine pesticides and heavy metals were significant differences in two planting bases, but all of the residuals between organochlorine pesticides and heavy metals were below the GB standards. Residuals of organochlorine pesticides and heavy metals in soil and P. vulgaris from two planting bases were conformed to GAP.
11. The essential oils from the fresh flowers, dried flowers leaves and stems of P. vulgaris were isolated by hydrodistillation. The collected oils were analyzed by capillary GC and GC-MS. The results showed that a total of 24,28,19 and 21 compounds were identified, accounting for 95.22%,83.47%,80.03%and 79.48%of fresh flowers, dried flowers, leaves and stems oil, respectively. The main constituent of the essential oil of fresh flowers, dried flowers and stem oils, respectively, was hexadecanoic acid (58.12%,69.99% and 38.44%). Aromadendrene (55.35%) was identified as the highest main constituent for leaves. The composition of essential oil from fresh flowers, dried flowers, leaves and stems of P. vulgaris were discrepant.
引文
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