Pharmacokinetic Analysis of Four Bioactive Iridoid and Secoiridoid Glycoside Components of Radix Gentianae Macrophyllae and Their Synergistic Excretion by HPLC-DAD Combined with Second-Order Calibration

An HPLC-DAD method combined with second-order calibration based on the alternating trilinear decomposition(ATLD)algorithm with the aid of region selection was developed to simultaneously and quantitatively characterize the synergistic relationships and cumulative excretion of the four bioactive ingredients of Radix Gentianae Macrophyllae in vivo.Although the analytes spectra substantially overlapped with that of the biological matrix,the overlapping profiles between analytes and co-eluting interferences can be successfully separated and accurately quantified by the ATLD method on the basis of the strength of region selection.The proposed approach not only determined the content change but also revealed the synergistic relationships and the cumulative excretion in vivo of the four ingredients in urine and feces samples collected at different excretion time intervals.In addition,several statistical parameters were employed to evaluate the accuracy and precision of the method.Quantitative results were confirmed by HPLC-mass spectrometry.Satisfactory results indicated that the proposed approach can be utilized to investigate the pharmacokinetics of Radix Gentianae Macrophyllae excretion in vivo.

HPLC fingerprinting and quantification of gentiopicroside and loganic acid in Gentianae Macrophyllae Radix crude drugs

HPLC fingerprinting and quantification of gentiopicroside（GPS） and loganic acid（LA） in Gentianae Macrophyllae Radix（GMR） crude drugs were developed in this study.The samples were separated on Zorbax SB-C_（18） column（250 mm×4.6 mm, 5μm） with a linear gradient of acetonitrile and 0.04%phosphoric acid.The HPLC flow rate was 1.0 mL/min and a UV absorption was measured at 230 nm.An orthogonal L9（3^4） test was applied for the optimization of sample extraction conditions,and an aliquot of GMR sample（g） was extracted with 15-fold of 50%ethanol（mL） for 30 min by sonication.Quantitative analysis showed that the content of GPS（14.05 mg/g-74.61 mg/g） in all samples was obviously higher than that of LA（1.13 mg/g-40.46 mg/g）. Based on the content ratio of GPS over LA（1.8-11.4）,samples originated from Gentiana macrophylla（with content ratio of GPS over LA≤4.3） could be distinguished from those from G.dahurica and G.dahurica var.gracilipes（with content ratio of GPS over LA≥4.8）.The principle components analysis of the HPLC fingerprints showed that samples originated from G.macrophylla and G.dahurica（including G.dahurica var.gracilipes） could be divided into two groups.This established HPLC-DAD method could be efficiently used for the species identification and quality control of GMR crude drugs.

Chemical fingerprint analysis of Gentianae Radix et Rhizoma by high-performance liquid chromatography

Gentianae Radix et Rhizoma(also called "Longdan" in Chinese)is commonly used for eliminating damp-heat and quenching the fire of liver and gall bladder in traditional Chinese medicine.In this study,a novel and reliable method using high-performance liquid chromatography(HPLC)was developed both for quantitative analysis of four bioactive compounds(loganic acid,swertiamarin,gentiopicroside and sweroside)and chemical fingerprint analysis of "Longdan".In quantitative analysis,four compounds showed good regressions(R^(2)>40.9987)within the test ranges and the recovery of the method was in the range 97.61-102.49%.In fingerprint analysis,ten characteristic peaks were selected to evaluate the similarities of the crude drugs,and the HPLC chromatograms of twenty samples from different regions of China showed similar patterns.The results demonstrated that the combination of the quantitative and chromatographic fingerprint analyses offered an efficient way to evaluate the quality consistency of Gentianae Radix et Rhizoma.

Cryptic diversity and rampant hybridization in annual gentians on the Qinghai-Tibet Plateau revealed by population genomic analysis

Understanding the evolutionary and ecological processes involved in population differentiation and speciation provides critical insights into biodiversity formation. In this study, we employed 29,865 single nucleotide polymorphisms(SNPs) and complete plastomes to examine genomic divergence and hybridization in Gentiana aristata, which is endemic to the Qinghai-Tibet Plateau(QTP) region. Genetic clustering revealed that G. aristata is characterized by geographic genetic structures with five clusters(West, East, Central, South and North). The West cluster has a specific morphological character(i.e., blue corolla) and higher values of FSTcompared to the remaining clusters, likely the result of the geological barrier formed by the Yangtze River. The West cluster diverged from the other clusters in the Early Pliocene;these remaining clusters diverged from one another in the Early Quaternary. Phylogenetic reconstructions based on SNPs and plastid data revealed substantial cyto-nuclear conflicts. Genetic clustering and D-statistics demonstrated rampant hybridization between the Central and North clusters,along the Bayankala Mountains, which form the geological barrier between the Central and North clusters. Species distribution modeling demonstrated the range of G. aristata expanded since the Last Interglacial period. Our findings provide genetic and morphological evidence of cryptic diversity in G. aristata, and identified rampant hybridization between genetic clusters along a geological barrier.These findings suggest that geological barriers and climatic fluctuations have an important role in triggering diversification as well as hybridization, indicating that cryptic diversity and hybridization are essential factors in biodiversity formation within the QTP region.

Gentiana macrophylla Pall.:a review of the basic source,traditional use,chemical components,pharmacological activities and quality control

Gentiana macrophylla Pall.(G.macrophylla),whose genus and family belong to the Gentianaceae and Gentiana.The main distribution centers of G.macrophylla resources were the Loess Plateau and the eastern Qinghai-Tibet Plateau in China.G.macrophylla,as a traditional medicine,has a long history and was used in different ethnic medicines.Its roots were used in traditional Chinese medicine,which had the effect of anti-inflammatory,anti-rheumatism,antiviral,promote blood circulation,eliminate swelling and pain,while its flowers were used in traditional Mongolian medicine,which had the effect of removing“Xieriwusu”(“Xieriwusu”means rheumatism),antiviral,reducing swelling.From previous studies,it could be found that there were more than forty components isolated and identified from G.macrophylla.The main chemical components were iridoids,flavonoids,triterpenoids,steroids,phenylpropanoids,and alkaloids.Iridoid terpenoid components represented by gentiopicroside and Loganic acid were the main components of the root of G.macrophylla,which had anti-inflammatory,antioxidant,hepatoprotective,analgesic,antibacterial and promote gastrointestinal tract activities.The flower mainly contains isoorientin and isovitexin as the representative of flavonoids.They have anti-tumor,liver protection,heart protection,inhibition of acetylcholinesterase and inhibition of melanin.It could be seen from previous studies that the research on G.macrophylla was mainly focused on the root,and the flower was rarely studied.It was reported that the experimental data of the anti-inflammatory and anti-tumor effects of G.macrophylla flowers show that its curative effect was very good.Therefore,the flowers of the flower of G.macrophylla can be used as potential medicinal parts for research.Given that flavonoids are mostly found in flowers and exhibit a range of functions,it is possible to investigate the flowers in order to learn more about G.macrophylla’s potential medical benefits.Based on botanical books,Chinese classic texts,medical monographs and academic search engines(Google,Google Scholar,Web of Science,SciFinder,Pubmed,CNKI,Sci-hub,Elsevier and Wanfang),the data and information on G.macrophylla in the past 20 years are inquired and summarized comprehensively.The basic source,traditional use,chemical composition,biological activity,pharmacodynamics and quality control of G.macrophylla was systematically reviewed,in order to provide reliable basis for the subsequent development and utilization of G.macrophylla.

乙醇浓度对宁夏六盘山栽培秦艽中龙胆苦苷提取率的影响

[目的]探讨乙醇浓度对宁夏六盘山栽培秦艽中龙胆苦苷提取率的影响。[方法]以室温冷浸为提取方法,采用HPLC测定秦艽中龙胆苦苷的含量,比较浓度45%、55%、65%和75%的乙醇对宁夏六盘山栽培秦艽中龙胆苦苷提取率的影响。[结果]采用浓度45%、55%、65%和75%的乙醇提取的秦艽龙胆苦苷提取率分别为12.50%、13.26%、14.53%和14.42%;当乙醇浓度为65%时,提取率最高。[结论]乙醇浓度对宁夏六盘山栽培的秦艽中龙胆苦苷的提取率有明显影响,以浓度65%的提取率最高。

植物激素对坚龙胆种子萌发的影响

[目的]探讨植物激素对坚龙胆种子萌发的影响。[方法]以坚龙胆成熟种子为试验材料,采用正交试验设计研究赤霉素(GA3)、6-苄基腺嘌吟(6-BA)、吲哚乙酸(IAA)及浸种时间对坚龙胆种子发芽情况的影响。[结果]坚龙胆种子发芽过程中以200 mg/L GA3+5mg/L 6-BA+5 mg/L IAA配比并浸种48 h处理的效果最好,种子发芽率达93%以上,种子开始发芽时间和发芽持续时间短,发芽势和发芽指数最高。[结论]经过正交试验设计优选出影响坚龙胆种子发芽率的作用大小为IAA>GA3>浸种时间>6-BA;经过植物激素处理可以提高坚龙胆种子的发芽率。

陕西产秦艽中龙胆苦苷的提取和含量测定

[目的]采用超声技术提取秦艽中主要成分龙胆苦苷,建立高效液相色谱技术测定龙胆苦苷含量的方法。[方法]采用单因素试验,考察超声频率、超声功率、提取时间、提取剂用量对超声波提取的影响,以正交试验确定最佳提取条件;采用高效液相色谱法进行含量测定。[结果]最佳工艺条件为超声频率为80 KHz,超声功率为420 W,提取时间为20 min,提取剂用量为20 ml;在此情况下,龙胆苦苷的提取率为2.72%。[结论]该方法灵敏、准确、简便,适用于秦艽中龙胆苦苷的提取和含量测定。

青海野生与栽培麻花艽psbA-trnH序列的比较

[目的]分析野生与栽培麻花艽(Gentiana straminea Maxim.)psbA-trnH序列的差异,为麻花艽的基源鉴定和品质评价提供分子依据。[方法]采用PCR扩增纯化后直接测序的方法,测定野生与栽培麻花艽cpDNA psbA-trnH核苷酸序列并作序列同源性分析。[结果]野生与栽培麻花艽的cpDNA psbA-trnH片段长度分别为316和317 bp,两者之间有4个碱基的变异。[结论]利用psbA-trnH区序列的差异可以鉴别野生与栽培麻花艽。

四种龙胆的显微结构特征比较

为比较红花龙胆(Gentiana rhodantha)与翼萼龙胆(Gentiana pterocalyx)、滇龙胆(Gentiana rigescens)与头花龙胆(Gentiana cephalantha)的显微结构差异,用石蜡组织切片法及徒手切片法对4种龙胆的显微结构进行了研究。结果表明,红花龙胆与翼萼龙胆、滇龙胆与头花龙胆显微结构特征有明显区别。在根横切面方面,红花龙胆皮层厚,导管细胞横切口径小,无明显射线,翼萼龙胆的特征与红花龙胆的相反;滇龙胆与头花龙胆的表皮和皮层早脱,次生韧皮部发达,但滇龙胆韧皮部薄壁组织细胞较大,而头花龙胆较小。在茎横切面方面,红花龙胆中央髓约占切面的1/3,而翼萼龙胆约占切面的1/2;滇龙胆髓仅外部有散落分布的纤维束,头花龙胆髓部均可见纤维束散落分布。在叶横切面方面,红花龙胆中脉维管束较大,而翼萼龙胆较小;滇龙胆中脉维管束小,未见纤维束,头花龙胆中脉维管束大,纤维束成对位于木质部两侧。在叶表皮方面,4种植物表皮细胞径向壁波曲度与气孔指数有一定差异。根据显微结构特征可以将这4种植物区别开来。

不同树龄茶树套种滇龙胆对药材矿质元素含量的影响

采用电感耦合等离子体原子发射光谱法（ICP-AES）测定滇龙胆（Gentiana rigescens Franch.ex Hemsl.）与不同树龄（3年、4年、5年、9年、12年和15年）茶树（Camellia sinensis）套种后其根、茎、叶3个部位及生长土壤中7种矿质元素（B、Ca、Cu、Fe、Mn、Ni和Zn）的含量。结果显示,滇龙胆中矿质元素平均含量高低顺序为Ca（5 238±2 111）μg/g、Fe（622±238）μg/g、Mn（379±301）μg/g、Cu（75±9.1）μg/g、Zn（46±13）μg/g、B（17±4.9）μg/g、Ni（5.6±3.1）μg/g,与土壤矿质元素含量变化趋势基本一致。根部的B、Ca、Fe、Mn、Ni元素在树龄15年茶树-滇龙胆套种后含量最高,分别为18、3 797、1 050、254、11μg/g,Cu、Zn元素在树龄12年的茶树-滇龙胆套种中含量最高,分别为244、38μg/g。相同营养器官不同树龄的茶树-滇龙胆套种后矿质元素含量具有差异,以根部的Cu、Mn元素含量差异显著,变化范围分别为19~244μg/g和79~254μg/g,分别相差12.8倍和3.2倍;树龄15年、9年的茶树-滇龙胆套种后茎部的Cu含量分别为177、11μg/g,相差16.1倍;树龄3年、5年的茶树-滇龙胆套种后叶部的Cu含量分别为325、11μg/g,相差29.5倍。不同树龄的茶树-滇龙胆套种后根部与土壤矿质元素的相关性以及生物富集系数不同,树龄15年的茶树-滇龙胆套种后根部对Ca、Cu、Mn、Ni、Zn元素的富集能力较强,生物富集系数分别达21.0、3.3、2.2、1.8和4.7。说明茶树复合种植可改变土壤矿质元素的分布,自身生物学特性与复合种植的生态环境共同影响了滇龙胆对矿质元素吸收与运输的能力。

小麦、蚕豆和油菜根系分泌物对麻花艽种子萌发的影响

分别用去离子水和乙酸乙酯萃取小麦、蚕豆和油菜根系分泌物刺激麻花艽(Gentiana straminea Maxim)种子,再进行麻花艽种子的发芽试验,考察小麦、蚕豆和油菜的根系分泌物对麻花艽种子萌发及胚根生长的影响。结果表明,小麦的根系分泌物水相浸提液对麻花艽种子萌发及胚根生长有轻微的促进作用,且随着水相浸提次数的增多,促进作用减弱。小麦的根系分泌物酯相浸提液对麻花艽种子萌发的影响不明显;蚕豆根系分泌物水相和酯相浸提液对麻花艽种子萌发及胚根生长抑制作用较弱;油菜根系分泌物水相和酯相浸提液对麻花艽种子萌发及胚根生长有明显的抑制作用,且随着水相和酯相浸提液浓度的降低,抑制作用减弱;油菜根系分泌物对麻花艽种子萌发及胚根生长的抑制作用最强,蚕豆根系分泌物对麻花艽种子萌发及胚根生长抑制作用较弱,小麦根系分泌物对麻花艽种子发芽有一定的促进作用。

贵州产坚龙胆组织培养体系的建立

以贵州野生坚龙胆(Gentiana rigescens Franch)带有顶芽或腋芽的茎段作为外植体,建立坚龙胆的组织培养体系。结果表明,适宜的不定芽诱导培养基为MS+2.0 mg/L 6-BA+0.2 mg/L NAA,最佳的增殖培养基为MS+1.0 mg/L 6-BA+0.10 mg/L NAA,最适生根培养基为1/2MS+0.1 mg/L IBA。在组织培养过程中还观察到了瓶苗开花的现象。

Prevention and Control of Gentiana macrophylla Pall Root Rot Disease

The root rot is a major disease affecting yield of Gentiana macrophyl a pal , reducing yield or even causing total crop failure. This research introduced the occurrence characteristics and various control techniques of root rot of Gentiana macrophyl a pal , in order to provide references for farmers.

Study on Chloroplast psbA-trnH Nucleotide Variation and Genetic Differentiation in Cultivated Plants of Gentiana officinalis from Qinghai

[Objective] The aim was to investigate the genetic diversity and analyze the genetic differentiation of germplasm resources of Gentiana officinalis H.Smith from Qinghai.[Method] The cpDNA of chloroplast psbA-trnH gene was sequenced to analyze the genetic diversity of six populations of G.officinalis.[Result] A total of 10 distinct haplotypes were detected in the studied populations,and seven variable sites were found by comparing their sequences.G.officinalis with high-level genetic diversity（h=0.771）.Genetic diversity was largely varied within populations,ranging from 0.563 to 0.857 for haplotype diversity,and from 0.002 43 to 0.005 83 for nucleotide diversity,respectively.Genetic differentiation among populations（Gst） of G.officinalis was 0.196 0;gene flow（Nm） was 2.05;80.40% of the genetic variability occurred within population.[Conclusion] The cultivated G.officinalis in Qinghai showed rich genetic diversity,which is beneficial for the production of high-quality herb medicine.

Study on the Flower Bud Differentiation Process of Gentiana triflora Pall.

[Objective] This study was to obseve the flower bud differentiation process of Gentiana triflora Pall., and to clarify the flower bud development law and the correlation between flower buds differentiation and external morphology. [Method] The paraffin section method was used to observe the flower bud of Gentiana triflora un- der microscope. [Result] The flower bud differentiation process of Gentiana triflora could be divided into six stages： the undifferentiated stage, initial differentiation stage, sepal primordia differentiation stage, petal primordia differentiation stage, stamen pri- mordia differentiation stage and the pistil primordia differentiation stage. The flower bud differentiation started from early May, when the plant had 10-11 stem nodes, and completed in early June, lasted about 40 d. The effect of plant height on flower bud differentiation was not significant. [Conclusion] The study provided theoretical basis for the high-yield cultivation, variety selection and flowering regulation of Gentiana triflora.

滇龙胆漂浮育苗基质的优选

以滇龙胆(Gentiana rigescens Franch)成熟种子为试材,采用正交试验设计分析育苗基质珍珠岩、蛭石、草炭、腐殖土4个因素对滇龙胆漂浮育苗种子萌发和幼苗生长的影响。结果表明,滇龙胆漂浮育苗种子萌发的最佳基质配比是珍珠岩∶蛭石∶草炭∶腐殖土=3∶1∶3∶4,种子萌发率达93.4%,珍珠岩和腐殖土对种子萌发率有显著影响,蛭石和草炭对种子萌发作用不显著;滇龙胆漂浮育苗幼苗生长的最佳基质配比是珍珠岩∶蛭石∶草炭∶腐殖土=2∶1∶3∶4,幼苗高、根长及茎叶干重均显著高于其他基质配比,幼苗根干重极显著高于其他基质配比,为(0.281 9±0.001 4)g。

林药复合种植的滇龙胆中元素的化学计量特征研究

药用植物复合种植是对传统中药材种植模式的优化,在一定程度上可缓解中药材与作物争地的矛盾,对中药资源的保护与可持续发展具有重要意义。试验通过采集单一种植模式的滇龙胆（Gentiana rigescens Franch.ex Hemsl.）样品以及滇龙胆与茶树[Camellia sinensis（L.）O.Ktze]、大叶桉（Eucalyptus robusta Smith）、木瓜[Chaenomeles sinensis（Thouin）Koehne]、旱冬瓜（Alnus nepalensis D.Don）、核桃（Juglans regia L.）、杉木[Cunninghamia lanceolata（Lamb.）Hook.]、木果石栎[Lithocarpus xylocarpus（Kurz）Markg.]等林药复合种植模式的滇龙胆样品,用微波消解-ICP-AES法测定各滇龙胆样品中的Ca、Mg、Fe、Zn、P、Cu、Mn、Cd、Pb、Ni、Sr、Ti等元素含量,以探明不同种植模式下滇龙胆植株中元素的化学计量特征。结果表明,该方法的加标回收率在95.40%~108.25%,相对标准偏差在0.26%~2.08%。其中滇龙胆根中的元素含量排序为Ca、Fe、Mg、P、Mn、Zn、Ti、Sr、Ni,茎、叶中的为Ca、Mg、P、Fe、Mn、Zn、Sr、Ti、Ni。以滇龙胆-茶树复合种植模式的P元素含量最高,其在根、茎、叶中分别为1 384.73、1 640.12、2 399.04μg/g;滇龙胆-旱冬瓜复合种植模式的Fe和Ti元素含量最高;滇龙胆-大叶桉复合种植模式的Mg元素含量最高,其根、茎、叶中分别为1 486.08、2 770.99、4 673.24μg/g;滇龙胆-核桃复合种植模式的Sr元素含量最高,其根、茎、叶中分别为37.02、52.80、34.63μg/g。重金属元素Cd与Ca、Fe、Ni元素含量之间呈显著或极显著正相关,相关系数分别为0.741、0.929、0.893;Ni与Fe、Ti含量之间呈显著或极显著正相关,与Pb含量之间呈显著负相关;Mg与Cu含量之间呈显著正相关,Mn与Sr含量之间呈极显著正相关。

层积和赤霉素协同作用对滇龙胆种子漂浮育苗的影响

以滇龙胆(Gentiana rigescens Franch)成熟种子为试验材料,采用L9(34)正交设计方法研究层积方式、层积时间、赤霉素稀释液浓度和浸种时间对滇龙胆种子漂浮育苗的萌发及成苗的影响。结果表明,滇龙胆种子漂浮育苗促进萌发的最佳处理组合是湿沙层积方式+层积60 d+赤霉素稀释液浓度200 mg/L+浸种24 h,其中层积时间对滇龙胆种子萌发率有极显著的影响(P<0.01),赤霉素稀释液浓度对滇龙胆种子萌发率有显著的影响(P<0.05),而层积方式和浸种时间对滇龙胆种子的萌发作用不显著;滇龙胆种子漂浮育苗促进成苗的最佳处理组合是一般层积方式+层积30 d+赤霉素稀释液浓度50 mg/L+浸种24 h,其中赤霉素稀释液浓度对滇龙胆种子的成苗率有显著的影响(P<0.05),而层积方式、层积时间和浸种时间对滇龙胆种子的成苗作用不显著。综合考虑各种因素的影响,滇龙胆种子漂浮育苗促进萌发及成苗的最佳处理组合是一般层积方式+层积30 d+赤霉素稀释液浓度50 mg/L+浸种24 h,其种子萌发率为90.23%、成苗率高达97.50%。

秦艽高效育苗技术

介绍了秦艽(Gentiana macrophylla)高效育苗技术,为秦艽规模化育苗提供相应技术参考。