药用远志的结构发育与主要药用成分积累关系的研究
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摘要
远志为我国大宗常用中药材和85种传统出口药材之一。《中国药典》中规定中药远志的原植物为远志(又名细叶远志)(Polygala tenuifolia Willd)或卵叶远志(又名宽叶远志)(Polygala sibirica L.)。细叶远志和卵叶远志隶属于远志科(Polygalaceae)远志属(Polygala),为多年生草本植物,以干燥根入药。远志的主要药用成分为皂苷、(口山)酮、寡糖酯类、脂肪油、多糖等。本文运用植物解剖学、组织化学和植物化学等方法,以药材的主流——细叶远志及最主要药用成分——皂苷为重点,系统研究了两种远志各器官的形态结构及发生发育规律,皂苷、口山酮、脂肪油和多糖在两种远志营养器官中的组织化学定位以及皂苷的细胞化学定位,皂苷在两种远志生长发育过程中含量的积累动态和变化规律,并对两种远志的药材品质进行比较,对主产区不同产地细叶远志药材的皂苷、多糖含量、和主要环境因子进行比较研究和相关性分析。研究结果如下:
细叶远志根初生结构的分化与次生结构的发生过程,包括4个阶段:即原分生组织阶段、初生分生组织阶段、初生结构和次生生长阶段。初生结构中,其初生木质部脊数为二原型,发育方式为外始式,根中无髓。根的次生结构包括周皮和次生维管组织两部分。其特点为:第一,次生韧皮部的比例远大于次生木质部,通过不同年龄远志主根的比较解剖学研究发现,次生韧皮部约占根直径的2/3-3/5,其增粗只是正常的次生生长,不存在异常的次生生长;第二,次生韧皮部的主要成分为韧皮薄壁组织细胞,储存有丰富内含物,筛管和伴胞很少,与根的储存功能相适应;第三,根中周皮发达,具有较厚的木栓层,次生木质部中导管发达,分布频率较高,是适应干旱环境的特征。
细叶远志茎的发育过程也包括四个阶段:即原分生组织阶段、初生分生组织阶段、初生生长和次生生长阶段。其成熟茎结构包括表皮、皮层、维管柱三部分。在茎的皮层与次生韧皮部之间发育出一圈排列紧密的厚壁细胞,推测这圈厚壁细胞具有质外体屏障作用,可有效地防止维管组织细胞水分丧失和离子泄漏,对于保护茎组织免受干旱的伤害具有积极的作用。茎近表皮的皮层细胞特化为同化组织,木质部发达,这些结构特点也是适应干旱环境的表现。
细叶远志叶为的结构、发生和发育类似一般被子植物。叶为典型的异面叶,栅栏组织和海绵组织分化明显。上表皮分布有表皮毛,下表皮气孔器较密集,孔下室较大,在叶肉组织中具有含晶细胞,同样表现出旱生特点。
卵叶远志营养器官的结构与细叶远志类似,所不同的是卵叶根中次生韧皮部的比例较小,卵叶远志次生韧皮部的厚度约占根直径的1/3。二者的叶在表皮毛密度、气孔器密度等方面也有差异,卵叶远志叶肉中无晶体细胞。
两种远志的花为两性花,两侧对称,萼片花瓣状,中央花瓣形状为龙骨状,顶端特化为鸡冠状附属物,雌雄蕊的结构较为特殊,8枚雄蕊的花丝部分合生,雌蕊的柱头2裂,且不等高,花部具有适应虫媒传粉的特征。子房上位,2心皮构成2室,每室1胚珠,胚珠倒生。蒴果,种子类型为双子叶有胚乳种子。
两种远志营养器官中,用5%香草醛-冰醋酸和高氯酸混合试剂进行皂苷的组织化学定位结果表明,在根的初生结构中,皂苷类物质分布于表皮、皮层及中柱内部的薄壁组织细胞内;在次生结构中,主要分布于次生韧皮部及栓内层的薄壁组织细胞中,在木质部的木射线和木薄壁细胞中也有少量分布。在茎中,主要分布于其皮层,在表皮和韧皮部细胞内也有少量分布。在叶中,主要分布于叶肉,在表皮细胞内也有少量分布。盐酸-镁粉进行组织化学定位,发现(口山)酮类化合物主要分布两种远志根的次生韧皮部和栓内层中。苏丹Ⅲ和锇酸对脂肪油的组织化学定位结果显示,在细叶远志根的次生韧皮部及栓内层具有丰富的脂肪油滴,而在卵叶远志的根中没有观察到脂肪油滴。PAS反应对多糖的组织化学定位结果显示在两种远志根的次生韧皮部和周皮中都有多糖物质的积累。因此,在两种远志根中,次生韧皮部是主要药用成分的主要积累场所。超微结构研究结果表明,积累皂苷的薄壁组织细胞中细胞器丰富,用醋酸铅沉淀的细胞化学方法进行处理,在积累皂苷的皮层细胞、韧皮薄壁细胞、筛管等细胞的液泡内及液泡膜周围观察到皂苷沉淀颗粒,进一步验证了皂苷的积累部位。
植物化学研究表明,两种远志营养器官中都有远志皂苷的积累,根中的含量高,茎叶中含量较低,与组织化学的研究结果一致。同时说明两种远志地上部分也有药用价值,提倡综合利用远志的地上茎叶。根据不同发育时期根和茎叶中远志皂苷元的动态变化趋势,建议在4-5月采收卵叶远志的比较适宜。根的不同部位的植化测定结果显示,皮部与木芯(木质部)的总皂苷和皂苷元含量差异悬殊,皮部远大于木芯,与组织化学的研究结果一致。因此,可以将皮部与木质部的比值作为判断远志品质的标准之一,根皮厚、木芯细的药材应为上品,这与传统的用药习惯是一致的。
对细叶远志在4-10月的生长发育过程中,1、2、3、4年生根中的远志皂苷元含量的动态变化研究结果表明,远志皂苷元含量均表现为:1年生根＞2年生根＞3年生根＞4年生根,表明远志皂苷在幼嫩的植株根中含量高,推测可能与远志皂苷具有化学防御的功能有关。从年平均增长率来看,根的长度、直径、次生韧皮部的厚度及根干重的增长均在药材种植第2-3年增加最快,而在第4年生长速度减慢。从根中远志皂苷元积累的总含量来看,在果后期3年生根中远志皂苷元的总含量是二年生根的2.66倍、一年生根的3.97倍,而4年生根皂苷元的总含量仅是3年生根的1.2倍,因此兼顾药材产量和质量,应该在细叶远志种植第三年的果后期(8月中旬)进行采收比较适宜。
对两种远志根的比较结果表明,细叶远志根的长度、直径、干重、皮部厚度以及皂苷元总含量都比卵叶远志高,因此,细叶远志的产量和质量明显优于卵叶远志,故建议人工栽培时应选择细叶远志。
栽培和野生的细叶远志相比,栽培植株比较粗壮,地上部分分枝较多,根相对粗大,野生植株地上部分分枝少,根粗细不一,侧根较细。从全根和皮部皂苷元百分含量比较分析,野生品质总体要优于栽培植株。但从根中远志皂苷元积累的总含量研究分析,栽培三年细叶远志根的远志皂苷元总含量最高,远大于一、二年生根,并且也明显高于野生植株根,从而反映人工栽培远志是可行的,同时栽培三年后采收的药材产量和有效成分都比较高。
研究结果表明远志主产区山西、陕西、河北不同产地的药材中远志皂苷元、多糖的含量变化较大,差异达到显著水平(P＜0.05),反映各地远志药材的质量存在较大差异。皂苷元和多糖含量综合分析,山西闻喜、新绛的含量都处于较高水平,启示我们在今后远志品种选育时可优先考虑闻喜和新绛两地的品种。对栽培产区的主要环境因子进行分析发现,主产区具有比较相似的气候特征,气候较干旱,年积温较高;土壤的肥力不高,偏微碱性,速效氮含量低,速效钾含量高。相关性分析显示,皂苷元含量与土壤中的有机质含量呈负相关,而与土壤中锰的含量呈正相关。皂苷元与多糖的含量具有正相关性。
"Yuan-Zhi" has been used in traditional Chinese medicine. Chinese Pharmacopoeia took Polygala tenuifolia Willd and Polygala sibirica L.as two original plants of "Yuan-Zhi". P. tenuifolia Will and P. sibirica L. which belong to Polygalaceae are perennial herbaceous plants, and the main medical parts of them are dry roots. The main medicinal ingredients in its dry roots were saponin, xanthone, oligosaccharide polyesters, polysaccharide and fat oil. Anatomical, histochemical and phytochemial methods were used systematically investigated the structural features and the development of vegetative organs in plant of Polygala tenuifolia and Polygala sibirica, and the localization of saponin xanthone, fat oil and polysaccharide in vegetative organs of the two plants, and localization of saponin by cytochemistry, as well as the accumulation and the dynamic variation rule of saponin content. At the same time, the quality of crude drug of Polygala tenuifolia and Polygala sibirica were compared. The contents of saponin and polysaccharide of cultivated crude drug of Polygala tenuifolia. from different places of major producing areas were also compared and the correlation between the content of main chemical constituents and main environmental factors were analysed. The main experimental results are as follows:
The development of roots of P. tenuifolia could be divided into four stages: promeristem, primary meristem, primary structure and secondary growth. In primary structure, primary xylem was diarch; there is no pith in the center. The secondary structure included periderm and secondary vascular tissue. There were three characteristics of secondary structure of Polygala tenuifolia. Fristly, the secondary phloem made up about 3/5-2/3 of the root diameter, and the diameter of the roots increases depend on normal secondary growth not exceptional secondary growth. Secondly, the secondary phloem contained mainly parenchyma with abundant storeage, the sieve vessel and companion cells were few. Thirdly, Polygala tenuifolia had developed periderm with thick cork, and secondary xylem contained developed vessels which distributed frequently. The structure features of cork and secondary xylem were adapted to the xeromorphic characteristics of Polygala tenuiflia.
The development of stems could also be divided into four stages: promeristem, primary meristem, primary structure and secondary growth. The stem composed of epidermis, cortex and vascular bundle. The ring of sclerenchymatous cells that developed between cortex and phloem might be the apoplastic protective screen which could protect the stem from drought. The cellls of cortex that close to the epidermis specialized into assimilating tissue. Xylem was developed. These characteristics adapted to arid environment.
The leaves of Polygala tenuiflia were typically bifacial, and the structure, origin and development of leaves was similar to those of ordinary dicotyledonous plants, There were epidermis hair and stomatal apparatus on the epidermis. The stoma chamber was big. There were crystal cells among the mesophyll. These characteristics of leaf also adapt to arid environment.
The structures of vegetative organs of P. sibirica were similar to that of Polygala tenuiflia. However, the secondary phloem of P. sibirica made up about 1/3 of the root diameter, the ratio was lower than that of Polygala tenuiflia. There were difference between the density of epidermis hair and stomatal apparatus of the two plants. There were not crystal cells among the mesophyll of P. sibirica.
The flower of Polygala tenuiflia and P. sibirica was bisexual, zygomorphous one. The shape of sepal differentiated into petal, and the petal in center of flower differentiated into keel, and the upper partial of petal specialized into the cristate shape. The structures of pistil and stamen were special. The filaments of eight stamens combined partially, and the stigma of pistil was split into two parts, which had unequal height. These characteristics were adapted to entomophilous pollination. The pistil consisted of two carpels. There was one anatropous ovule in the ovary which was epistasis with two loculars. The capsule was composed of pericarp and a seed. The type of seed belonged to a dicotyledonous albuminous one.
The histochemical localization results of Polygala tenuiflia by 5% vanillin-glacial acetic acid-perchloric acid solution showed that saponins were distributed in epidermis, cortex and the parenchyma of central cylinder in primary structure, while in secondary structure saponins mainly in secondary phloem, phelloderm, rarely in xylem ray and xylem parenchyma, and in stem mainly in phloem, rarely in epidermis and cortex, and mainly in mesophyll of leaf, rarely in epidermis. Histochemical localization results of P. sibirica were similar to the result of Polygala tenuiflia. The Mg-Hcl localization results of xanthones indicated that phelloderm parenchyma cells and secondary phloem were storage locus. The localization by SudanⅢand osmic acid indicated that there were abundant fat oil in secondary phloem, phelloderm . However, in root of P. sibirica L., fat oil could not be found. PAS reaction results showed polysaccharide were also existed in root periderm and secondary phloem of the two plants. It displayed that the secondary phloem was the main storage site of main medicinal components. The ultrastructure research result indicated that the parenchyma cell which accumulate the saponin have plentiful organelle. The cytochemistry method of PbAc—settling showed that the settleable solids of PbAc were found in vacuoles or around the tonoplast in the parenchyma cells which accumulate the saponin, such as parenchyma cells of phloem, cortex cells and sieve tube etc. It proved the location of saponin of histochemical results.
The phytochemical results also showed that the saponin accumulated in the vegetative organs of P. tenuifolia and P. sibirica, with higher content in roots and lower content in the aerial parts that included stems and leaves. This result was consistent with the findings obtained through the histochemistry experiment. Based on these results, we advocate the utilization of aerial parts of P. tenuifolia and P. sibirica facing the shortage of Yuan-Zhi" resource today. According to the dynamic variance of saponins content of P. sibirica at the developmental stage, the P. sibirica should be gathered at period from April to May. The content of total saponin and segenenin in different parts of root showed obvious variation. The content in the "skin areas" was much higher than that of xylem. The result is consistent with the histochemical localization. Accordingly, the crude drug that had thick "skin areas" and thin xylems was of top grade.
The HPLC result showed that during the growth period the senegenin percent content of roots in four different years had dynamic variant trend, the senegenin percent content had the similar result, that is annual roots'> biennial roots'> triennial roots'>quadrennial roots', it showed that the younger root had higher content. We guessed that this result came because triterpenoid in P. tenuifolia may be chemical defenses. As far as the increasing rate of roots of P. tenuifolia was concerned, the length, diameter, thickness of "skin areas" as well as dry weight content of roots increased most quickly from the second to the third growth year. The increasing rate of roots decreased at fourth growing year. In the post-fruit period, the total senegenin content of the triennial roots was 2.66 times as much as that of the biennial roots, and 3.97 times as that of the annuals. However, the total senegenin content of the quadrennial root was only 1.20 times as much as that of the triennial roots. In fact, it is the total amount of secondary metabolic product in plant that should be considered while the medicinal material is selected. So the middle ten days of August of the third growing year was optimal collecting period for the drug of Polygala tenuifolia.
The comparision of roots of two plant indicated that the diameter, thickness of "skin area", dry weight as well as senegenin contents of roots in P. tenuifolia were all higher than that of P. sibirica. So, the crude drug quality of P.tenuifolia was better than that of P. sibirica. We should choose Polygala tenuifolia as cultivated material.
The shape of cultivated plants of P. tenuifolia was more hairchested and had more branches and thicker root, comparising with the wild one. The wild plants of P. tenuifolia having less branches and the root were nonuniform, the lateral root were thiner. Judging from the senegenin contents of "skin areas" and root, the quality of wild plant was higher than that of the cultivated plant as a whole. However, analyzing from the dry weight and total senegenin contents, the roots of triennial cultivated P. tenuifolia had the highest level, which was also higher than that of the wild plant. The result indicated that the cultivated P. tenuifolia could instead of the wild, and the cultivated P. tenuifolia shoud be gathered at third growth year because of the highter total senegenin contents.
Results on Polygala tenuifolia from different places of major producing areas in Shanxi, Shaanxi, Hebei province showed that there were obvious differences (p<0.05) in the contents of saponins and polysaccharide. This indicated that the quality of major producing areas were very different. Analyzing from contents of saponins and polysaccharide, the quality of crude drug of "Xin-Jiang" and "Wen-Xi" were higher. The variety of "Yuan-Zhi" from "Xin-Jiang" and "Wen-Xi" could be preferred for breeding. The analysis of major environmental factor showed that the climate of different places of major producing areas were similar, having dryclimate and higher accumulated temperature. The soil were not fertile and alkalescent, with lower content of N and highter content of K. Correlation indicated that the content of saponin had negative correlation with the organic matter of soil, but had positive correlation with the element of Mn. The content of saponin had positive correlation with content of polysaccharide.
细叶远志根初生结构的分化与次生结构的发生过程,包括4个阶段:即原分生组织阶段、初生分生组织阶段、初生结构和次生生长阶段。初生结构中,其初生木质部脊数为二原型,发育方式为外始式,根中无髓。根的次生结构包括周皮和次生维管组织两部分。其特点为:第一,次生韧皮部的比例远大于次生木质部,通过不同年龄远志主根的比较解剖学研究发现,次生韧皮部约占根直径的2/3-3/5,其增粗只是正常的次生生长,不存在异常的次生生长;第二,次生韧皮部的主要成分为韧皮薄壁组织细胞,储存有丰富内含物,筛管和伴胞很少,与根的储存功能相适应;第三,根中周皮发达,具有较厚的木栓层,次生木质部中导管发达,分布频率较高,是适应干旱环境的特征。
细叶远志茎的发育过程也包括四个阶段:即原分生组织阶段、初生分生组织阶段、初生生长和次生生长阶段。其成熟茎结构包括表皮、皮层、维管柱三部分。在茎的皮层与次生韧皮部之间发育出一圈排列紧密的厚壁细胞,推测这圈厚壁细胞具有质外体屏障作用,可有效地防止维管组织细胞水分丧失和离子泄漏,对于保护茎组织免受干旱的伤害具有积极的作用。茎近表皮的皮层细胞特化为同化组织,木质部发达,这些结构特点也是适应干旱环境的表现。
细叶远志叶为的结构、发生和发育类似一般被子植物。叶为典型的异面叶,栅栏组织和海绵组织分化明显。上表皮分布有表皮毛,下表皮气孔器较密集,孔下室较大,在叶肉组织中具有含晶细胞,同样表现出旱生特点。
卵叶远志营养器官的结构与细叶远志类似,所不同的是卵叶根中次生韧皮部的比例较小,卵叶远志次生韧皮部的厚度约占根直径的1/3。二者的叶在表皮毛密度、气孔器密度等方面也有差异,卵叶远志叶肉中无晶体细胞。
两种远志的花为两性花,两侧对称,萼片花瓣状,中央花瓣形状为龙骨状,顶端特化为鸡冠状附属物,雌雄蕊的结构较为特殊,8枚雄蕊的花丝部分合生,雌蕊的柱头2裂,且不等高,花部具有适应虫媒传粉的特征。子房上位,2心皮构成2室,每室1胚珠,胚珠倒生。蒴果,种子类型为双子叶有胚乳种子。
两种远志营养器官中,用5%香草醛-冰醋酸和高氯酸混合试剂进行皂苷的组织化学定位结果表明,在根的初生结构中,皂苷类物质分布于表皮、皮层及中柱内部的薄壁组织细胞内;在次生结构中,主要分布于次生韧皮部及栓内层的薄壁组织细胞中,在木质部的木射线和木薄壁细胞中也有少量分布。在茎中,主要分布于其皮层,在表皮和韧皮部细胞内也有少量分布。在叶中,主要分布于叶肉,在表皮细胞内也有少量分布。盐酸-镁粉进行组织化学定位,发现(口山)酮类化合物主要分布两种远志根的次生韧皮部和栓内层中。苏丹Ⅲ和锇酸对脂肪油的组织化学定位结果显示,在细叶远志根的次生韧皮部及栓内层具有丰富的脂肪油滴,而在卵叶远志的根中没有观察到脂肪油滴。PAS反应对多糖的组织化学定位结果显示在两种远志根的次生韧皮部和周皮中都有多糖物质的积累。因此,在两种远志根中,次生韧皮部是主要药用成分的主要积累场所。超微结构研究结果表明,积累皂苷的薄壁组织细胞中细胞器丰富,用醋酸铅沉淀的细胞化学方法进行处理,在积累皂苷的皮层细胞、韧皮薄壁细胞、筛管等细胞的液泡内及液泡膜周围观察到皂苷沉淀颗粒,进一步验证了皂苷的积累部位。
植物化学研究表明,两种远志营养器官中都有远志皂苷的积累,根中的含量高,茎叶中含量较低,与组织化学的研究结果一致。同时说明两种远志地上部分也有药用价值,提倡综合利用远志的地上茎叶。根据不同发育时期根和茎叶中远志皂苷元的动态变化趋势,建议在4-5月采收卵叶远志的比较适宜。根的不同部位的植化测定结果显示,皮部与木芯(木质部)的总皂苷和皂苷元含量差异悬殊,皮部远大于木芯,与组织化学的研究结果一致。因此,可以将皮部与木质部的比值作为判断远志品质的标准之一,根皮厚、木芯细的药材应为上品,这与传统的用药习惯是一致的。
对细叶远志在4-10月的生长发育过程中,1、2、3、4年生根中的远志皂苷元含量的动态变化研究结果表明,远志皂苷元含量均表现为:1年生根＞2年生根＞3年生根＞4年生根,表明远志皂苷在幼嫩的植株根中含量高,推测可能与远志皂苷具有化学防御的功能有关。从年平均增长率来看,根的长度、直径、次生韧皮部的厚度及根干重的增长均在药材种植第2-3年增加最快,而在第4年生长速度减慢。从根中远志皂苷元积累的总含量来看,在果后期3年生根中远志皂苷元的总含量是二年生根的2.66倍、一年生根的3.97倍,而4年生根皂苷元的总含量仅是3年生根的1.2倍,因此兼顾药材产量和质量,应该在细叶远志种植第三年的果后期(8月中旬)进行采收比较适宜。
对两种远志根的比较结果表明,细叶远志根的长度、直径、干重、皮部厚度以及皂苷元总含量都比卵叶远志高,因此,细叶远志的产量和质量明显优于卵叶远志,故建议人工栽培时应选择细叶远志。
栽培和野生的细叶远志相比,栽培植株比较粗壮,地上部分分枝较多,根相对粗大,野生植株地上部分分枝少,根粗细不一,侧根较细。从全根和皮部皂苷元百分含量比较分析,野生品质总体要优于栽培植株。但从根中远志皂苷元积累的总含量研究分析,栽培三年细叶远志根的远志皂苷元总含量最高,远大于一、二年生根,并且也明显高于野生植株根,从而反映人工栽培远志是可行的,同时栽培三年后采收的药材产量和有效成分都比较高。
研究结果表明远志主产区山西、陕西、河北不同产地的药材中远志皂苷元、多糖的含量变化较大,差异达到显著水平(P＜0.05),反映各地远志药材的质量存在较大差异。皂苷元和多糖含量综合分析,山西闻喜、新绛的含量都处于较高水平,启示我们在今后远志品种选育时可优先考虑闻喜和新绛两地的品种。对栽培产区的主要环境因子进行分析发现,主产区具有比较相似的气候特征,气候较干旱,年积温较高;土壤的肥力不高,偏微碱性,速效氮含量低,速效钾含量高。相关性分析显示,皂苷元含量与土壤中的有机质含量呈负相关,而与土壤中锰的含量呈正相关。皂苷元与多糖的含量具有正相关性。
"Yuan-Zhi" has been used in traditional Chinese medicine. Chinese Pharmacopoeia took Polygala tenuifolia Willd and Polygala sibirica L.as two original plants of "Yuan-Zhi". P. tenuifolia Will and P. sibirica L. which belong to Polygalaceae are perennial herbaceous plants, and the main medical parts of them are dry roots. The main medicinal ingredients in its dry roots were saponin, xanthone, oligosaccharide polyesters, polysaccharide and fat oil. Anatomical, histochemical and phytochemial methods were used systematically investigated the structural features and the development of vegetative organs in plant of Polygala tenuifolia and Polygala sibirica, and the localization of saponin xanthone, fat oil and polysaccharide in vegetative organs of the two plants, and localization of saponin by cytochemistry, as well as the accumulation and the dynamic variation rule of saponin content. At the same time, the quality of crude drug of Polygala tenuifolia and Polygala sibirica were compared. The contents of saponin and polysaccharide of cultivated crude drug of Polygala tenuifolia. from different places of major producing areas were also compared and the correlation between the content of main chemical constituents and main environmental factors were analysed. The main experimental results are as follows:
The development of roots of P. tenuifolia could be divided into four stages: promeristem, primary meristem, primary structure and secondary growth. In primary structure, primary xylem was diarch; there is no pith in the center. The secondary structure included periderm and secondary vascular tissue. There were three characteristics of secondary structure of Polygala tenuifolia. Fristly, the secondary phloem made up about 3/5-2/3 of the root diameter, and the diameter of the roots increases depend on normal secondary growth not exceptional secondary growth. Secondly, the secondary phloem contained mainly parenchyma with abundant storeage, the sieve vessel and companion cells were few. Thirdly, Polygala tenuifolia had developed periderm with thick cork, and secondary xylem contained developed vessels which distributed frequently. The structure features of cork and secondary xylem were adapted to the xeromorphic characteristics of Polygala tenuiflia.
The development of stems could also be divided into four stages: promeristem, primary meristem, primary structure and secondary growth. The stem composed of epidermis, cortex and vascular bundle. The ring of sclerenchymatous cells that developed between cortex and phloem might be the apoplastic protective screen which could protect the stem from drought. The cellls of cortex that close to the epidermis specialized into assimilating tissue. Xylem was developed. These characteristics adapted to arid environment.
The leaves of Polygala tenuiflia were typically bifacial, and the structure, origin and development of leaves was similar to those of ordinary dicotyledonous plants, There were epidermis hair and stomatal apparatus on the epidermis. The stoma chamber was big. There were crystal cells among the mesophyll. These characteristics of leaf also adapt to arid environment.
The structures of vegetative organs of P. sibirica were similar to that of Polygala tenuiflia. However, the secondary phloem of P. sibirica made up about 1/3 of the root diameter, the ratio was lower than that of Polygala tenuiflia. There were difference between the density of epidermis hair and stomatal apparatus of the two plants. There were not crystal cells among the mesophyll of P. sibirica.
The flower of Polygala tenuiflia and P. sibirica was bisexual, zygomorphous one. The shape of sepal differentiated into petal, and the petal in center of flower differentiated into keel, and the upper partial of petal specialized into the cristate shape. The structures of pistil and stamen were special. The filaments of eight stamens combined partially, and the stigma of pistil was split into two parts, which had unequal height. These characteristics were adapted to entomophilous pollination. The pistil consisted of two carpels. There was one anatropous ovule in the ovary which was epistasis with two loculars. The capsule was composed of pericarp and a seed. The type of seed belonged to a dicotyledonous albuminous one.
The histochemical localization results of Polygala tenuiflia by 5% vanillin-glacial acetic acid-perchloric acid solution showed that saponins were distributed in epidermis, cortex and the parenchyma of central cylinder in primary structure, while in secondary structure saponins mainly in secondary phloem, phelloderm, rarely in xylem ray and xylem parenchyma, and in stem mainly in phloem, rarely in epidermis and cortex, and mainly in mesophyll of leaf, rarely in epidermis. Histochemical localization results of P. sibirica were similar to the result of Polygala tenuiflia. The Mg-Hcl localization results of xanthones indicated that phelloderm parenchyma cells and secondary phloem were storage locus. The localization by SudanⅢand osmic acid indicated that there were abundant fat oil in secondary phloem, phelloderm . However, in root of P. sibirica L., fat oil could not be found. PAS reaction results showed polysaccharide were also existed in root periderm and secondary phloem of the two plants. It displayed that the secondary phloem was the main storage site of main medicinal components. The ultrastructure research result indicated that the parenchyma cell which accumulate the saponin have plentiful organelle. The cytochemistry method of PbAc—settling showed that the settleable solids of PbAc were found in vacuoles or around the tonoplast in the parenchyma cells which accumulate the saponin, such as parenchyma cells of phloem, cortex cells and sieve tube etc. It proved the location of saponin of histochemical results.
The phytochemical results also showed that the saponin accumulated in the vegetative organs of P. tenuifolia and P. sibirica, with higher content in roots and lower content in the aerial parts that included stems and leaves. This result was consistent with the findings obtained through the histochemistry experiment. Based on these results, we advocate the utilization of aerial parts of P. tenuifolia and P. sibirica facing the shortage of Yuan-Zhi" resource today. According to the dynamic variance of saponins content of P. sibirica at the developmental stage, the P. sibirica should be gathered at period from April to May. The content of total saponin and segenenin in different parts of root showed obvious variation. The content in the "skin areas" was much higher than that of xylem. The result is consistent with the histochemical localization. Accordingly, the crude drug that had thick "skin areas" and thin xylems was of top grade.
The HPLC result showed that during the growth period the senegenin percent content of roots in four different years had dynamic variant trend, the senegenin percent content had the similar result, that is annual roots'> biennial roots'> triennial roots'>quadrennial roots', it showed that the younger root had higher content. We guessed that this result came because triterpenoid in P. tenuifolia may be chemical defenses. As far as the increasing rate of roots of P. tenuifolia was concerned, the length, diameter, thickness of "skin areas" as well as dry weight content of roots increased most quickly from the second to the third growth year. The increasing rate of roots decreased at fourth growing year. In the post-fruit period, the total senegenin content of the triennial roots was 2.66 times as much as that of the biennial roots, and 3.97 times as that of the annuals. However, the total senegenin content of the quadrennial root was only 1.20 times as much as that of the triennial roots. In fact, it is the total amount of secondary metabolic product in plant that should be considered while the medicinal material is selected. So the middle ten days of August of the third growing year was optimal collecting period for the drug of Polygala tenuifolia.
The comparision of roots of two plant indicated that the diameter, thickness of "skin area", dry weight as well as senegenin contents of roots in P. tenuifolia were all higher than that of P. sibirica. So, the crude drug quality of P.tenuifolia was better than that of P. sibirica. We should choose Polygala tenuifolia as cultivated material.
The shape of cultivated plants of P. tenuifolia was more hairchested and had more branches and thicker root, comparising with the wild one. The wild plants of P. tenuifolia having less branches and the root were nonuniform, the lateral root were thiner. Judging from the senegenin contents of "skin areas" and root, the quality of wild plant was higher than that of the cultivated plant as a whole. However, analyzing from the dry weight and total senegenin contents, the roots of triennial cultivated P. tenuifolia had the highest level, which was also higher than that of the wild plant. The result indicated that the cultivated P. tenuifolia could instead of the wild, and the cultivated P. tenuifolia shoud be gathered at third growth year because of the highter total senegenin contents.
Results on Polygala tenuifolia from different places of major producing areas in Shanxi, Shaanxi, Hebei province showed that there were obvious differences (p<0.05) in the contents of saponins and polysaccharide. This indicated that the quality of major producing areas were very different. Analyzing from contents of saponins and polysaccharide, the quality of crude drug of "Xin-Jiang" and "Wen-Xi" were higher. The variety of "Yuan-Zhi" from "Xin-Jiang" and "Wen-Xi" could be preferred for breeding. The analysis of major environmental factor showed that the climate of different places of major producing areas were similar, having dryclimate and higher accumulated temperature. The soil were not fertile and alkalescent, with lower content of N and highter content of K. Correlation indicated that the content of saponin had negative correlation with the organic matter of soil, but had positive correlation with the element of Mn. The content of saponin had positive correlation with content of polysaccharide.
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