Exploring the Mechanism of Blumea balsamifera(L.)DC in Preventing and Treating Alzheimer's Disease Based on HPLC-ESI-HRMS and Network Pharmacology

[Objectives]To expose the plausible mechanism of Blumea balsamifera(L.)DC.against Alzheimer s disease via network pharmacology and HPLC-ESI-HRMS technology.[Methods]To begin with,HPLC-ESI-HRMS was employed to identify the components of B.balsamifera.Secondly,the potential targets of the components were identified and predicted based on chemical similarity and online databases.Thirdly,by way of topological analysis of a component-disease target interaction network,the primary candidate targets and potential active components were identified.Lastly,molecular docking analysis was used to confirm the interaction between active components and therapeutic targets.[Results]According to the final results,HPLC-ESI-HRMS identified 70 components.Out of these,20 components were potentially biologically active,and most of them were sesquiterpenoids.According to the molecular docking results,the primary active components were appropriately coordinated with the core targets,indicating a high level of pharmacodynamic activity.Thus,the sesquiterpenes present in B.balsamifera are considered potential active ingredients having multi-target and multi-pathway effects for treating Alzheimer s disease.[Conclusions]This research will provide a scientific reference for the future pharmacological activity and clinical application of B.balsamifera.

Effects of Calcium Element on Biomass and Effective Constituents Contents in Blumea balsamifera in Slow Growth Period of Winter

The one-year-old seedlings of Blumea balsamifera （L.） DC. were applied with CaCl2.H2O that supplied Ca in slow growth period of winter three times. The heights, ground diameters, leaf lengths, leaf widths and biomasses of B. balsamifera plants were measured. In addition, the relative contents of total flavones in different parts of B. balsamifera were determined by UV spectrophotometry, and the absolute contents of total flavones were calculated. The relative contents of L-borneol in leaves of B. balsamifera were determined by GC, and the absolute contents of L- borneol were calculated. The results showed that calcium element significantly in- creased the biomasses in leaves, stems and roots of B. balsamifera in slow growth period of winter. The leaf biomass of B. balsamifera in the 5 g/L CaCl2-H2O treat- ment group was significantly higher than those in the other three treatment groups. The leaf biomasses of B. balsamifera in the 10 and 15 g/L CaCl2.H2O treatment groups were significantly higher than that in the CK, with 3.03 and 2.65 times, re- spectively. The application of Ca inhibited the accumulation of total flavones relative contents, but significantly increased the total flavones absolute contents in different parts of B. balsamifera. The relative and absolute contents of L-borneol in the 5 g/L CaCl2 .H2O treatment group were 0.22% and 0.22 g, which were increased by 37.50%, 22.22%, 37.50% and 100%, 100%, 450%, respectively compared with those in the 0, 10 and 15 g/L CaCl2.H2O treatment groups. The Ca element could signifi- cantly promote the accumulation of biomasses in leaves, stems and roots, as well as the absolute contents of total flavones and L-borneol in B. balsamifera in slow growth period of winter.

Analysis of apigenin in <i>Blumea balsamifera</i>Linn DC. and its inhibitory activity against aldose reductase in rat lens

To investigate the therapeutic potentials of na- tural sources, stepwise polarity fractions of Blumea balsamifera were tested for their ability to inhibit aldose reductase (AR) activity in rat lenses. Of these, the ethyl acetate (EtOAc) fraction exhibited a unique AR inhibitory activity (IC50 value, 0.11 μg/mL). Apigenin was identified from the active EtOAc fraction and exhibited high AR inhibitory activity (IC50 value, 4.03 μM). The content of apigenin was measured in B. balsamifera (0.47 mg/g) by HPLC/UV analysis. Our result suggests that B. balsamifera could be a useful natural source for the development of a novel AR inhibitory agent against diabetic complications.

Morphological Development of Sambong (Blumea balsamifera (L.) DC.) Leaf Studied by Frozen Section and Thin Section

The sambong (Blumea balsamifera (L.) DC.) leaf was investigated by applying frozen section and thin section technology, and observed through optical microscope. The results showed that the sambong leaf was firstly originated nearby the apical bud at the stem tip. The first leaf grows fast, that the mitosis happens frequently and the cytoplasm manufacture rapidly. The cells in the first few leaves have not yet differentiated into the spongy parenchyma and palisade parenchyma. In the mature leaf, the spongy parenchyma and palisade parenchyma were fully developed. The epidermal hairs were firstly developed on the abaxial side of the first leaf.

Microscopic Structural Comparison between Epidermal Trichomes in Blumea balsamifera (L.) DC. and Blumea laciniata (Roxb.) DC

Immature leaves and stems, which were about one to three centimeters nearby the stem tips, of Blumea balsamifera (L.) DC. (BB) and Blumea laciniata (Roxb.) DC. (BL) were cut into sections under ?18?C with a frozen section machine, and observed under an optical microscope. Results show that the most significant difference is that the BB has only one kind of glandular trichomes, while the BL has two. The glandular trichomes found on BB terms were all short glandular hairs (SGH), which were not longer than 100 μm. On BL stems, besides the SGH, the long glandular hairs (LGH), which were longer than 200 μm, were also found. By the factors pointed out in present study, the BB and BL and be distinguished from each other.

The Effect of Magnesium on Accumulations of Primary Metabolites and Yield of Blumea balsamifera

[Objectives]This study was conducted to investigate the effects of magnesium on the yield of Blumea balsamifera(L.)DC.and the accumulation of primary metabolites that affect yield of the medicinal material.[Methods]The annual seedlings of B.balsamifera were selected as experimental materials.The treatment concentrations of magnesium(Mg)were set as 0,1.5,15 and 150 mg/ml supplied by MgSO4·7H2O.The yield of the medicinal material was measured dynamically.And the content of total sugar was determined by 3,5-dinitrosalicylic acid colorimetry;the content of crude protein was determined by the Kjeldahl method;the ash content was determined by the high-temperature burning method;the crude fat content was determined with a crude fat instrument;and the crude fiber content was determined by the acid-base washing and weighing method.[Results]Mg significantly increased the yield of B.balsamifera medicinal material,especially 15 mg/ml Mg.It was found that in September,October and November,1.5 mg/ml and 15 mg/ml Mg significantly increased the contents of primary metabolites including total sugar,ash,crude protein,crude fat and crude fiber,and 150 mg/ml of Mg increased the accumulation of total sugar,ash,crude protein and crude fiber to different degrees,but had certain inhibitory effect on the accumulation of crude fat.In December,the application of Mg inhibited the accumulation of total sugar,ash and crude protein to different degrees,but significantly promoted the accumulation of crude fat and fiber.[Conclusions]This study provides a theoretical basis for clarifying the effects of different concentrations of magnesium on plant growth.

Study on the Variation in Main Chemical Composition of Blumea balsamifera in Different Growth Stages

[Objectives]To study the variation in main chemical composition of Blumea balsamifera in different growth stages,and to provide a theoretical basis to determine appropriate harvest period.[Methods]The l-borneol of the plant was determined by gas chromatography( GC)with methyl salicylate as internal standard,and total flavonoids were determined by the Aluminum nitrate colorimetry method of Ultraviolet-Visible spectrophotometry. The analysis of variance was used for statistical analysis of the l-borneol and total flavonoids of B. balsamifera in different months and different plant age.[Results]There were differences in chemical ingredients of B. balsamifera in different months of the year.The content of l-borneol in October was the highest,but there was no significant difference from August to December in l-borneol content( P >0. 05). l-borneol content of B. balsamifera in three ages was the highest compared with other ages,and there was a significant difference with B. balsamifera in one year or less( P < 0. 05). Relatively speaking,total flavonoids in June,August and November were higher; total flavonoids content of B. balsamifera in two ages was the highest compared with other ages,and there was a significant difference with other ages( P <0. 05).[Conclusions]If l-borneol is taken as an indicator,for extracting Aipan( l-borneol),it can be harvested from September to midDecember,but October is the best. And it can be harvested at least three years. If the content of total flavonoids is taken as an indicator,it should be harvested in November. And it can be harvested 2-3 years.

镉积累对艾纳香内生菌群落结构和共发生网络的影响

为探究器官镉(Cd)积累对艾纳香内生菌的影响,该文采用16S rRNA基因高通量测序技术结合分子生态网络分析,研究不同外源镉处理(0、2.0 mg·kg^(-1))下艾纳香根、茎、叶Cd积累对内生菌群落结构特征的影响。结果表明:(1)与未添加外源Cd(0 mg·kg^(-1),Cd0)相比,Cd(2.0 mg·kg^(-1),Cd2)处理促进了植株生长,根、茎、叶中Cd积累量顺序为叶(16.75 mg·kg^(-1))>茎(11.99 mg·kg^(-1))>根(3.96 mg·kg^(-1))。(2)α多样性分析表明,各器官内生菌丰富度(Sobs指数、Ace指数和Chao1指数)和多样性(Shannon指数和Simpson指数)均以根最高,茎次之,叶最低,并且Cd2处理各器官内生菌丰富度和多样性均高于Cd0处理。(3)在门水平上,两个处理根、茎、叶内生菌均以变形菌门、放线菌门和厚壁菌门为优势菌群;在属水平上,代尔夫特菌是Cd0和Cd2处理各器官的主要菌属,相对丰度分别为53.0%~92.7%和57.1%~89.2%;艾纳香根、茎、叶内生菌群落结构有一定的相似性,Cd2处理提高了根、茎、叶共有内生菌属的比例及各器官(根除外)独有内生菌属比例。(4)线性判别分析(LEfSe)表明,相同处理不同器官间及不同处理相同器官间内生菌属存在差异。(5)冗余分析(RDA)发现,根际土壤Cd含量、器官Cd含量与内生菌群落结构组成有明显的相关性。(6)共发生网络分析结果显示,Cd积累使艾纳香根、叶内生菌共发生网络变得复杂且增强了根、茎物种间的竞争作用和叶物种间的协同共生作用。综上认为,外源Cd处理影响了艾纳香各器官内生菌群落结构及相互作用模式。

艾纳香油通过NF-κB非经典信号影响花生四烯酸代谢缓解LPS所致巨噬细胞的炎症反应

【目的】本文旨在探讨艾纳香油(BBO)通过核转录因子κB(NF-κB)非经典通路影响花生四烯酸(AA)代谢通路发挥抗炎作用。【方法】采用豚鼠离体回肠法检测BBO对慢反应物质(SRS-A)生成的影响,ELISA法检测BBO对脂多糖(LPS)诱导巨噬细胞前列腺素E_(2)(PGE_(2))及白三烯B_(4)(LTB_(4))产生的影响,qRT-PCR检测BBO对LPS诱导巨噬细胞COX-2、5-LOX、FLAP和RelB表达的影响,Western blot检测BBO对NF-κB非经典通路蛋白肿瘤坏死因子受体作用因子3(TRAF3)、肿瘤坏死因子受体作用因子2(TRAF2)、NF-κB诱导激酶(NIK)、p100和RelB浓度的影响。【结果】在1mg·mL^(-1)的BBO药物浓度下减弱了豚鼠回肠的收缩张力(P<0.001),对SRS-A的生成抑制率达到65.34%;与LPS模型组相比,BBO在40-80μg·mL^(-1)浓度下降低AA代谢通路中PGE_(2)(P<0.05)和LTB_(4)(P<0.05)的浓度,降低COX-2(P<0.05)、5-LOX(P<0.05)和FLAP(P<0.05)的表达;此外,40-80μg·mL^(-1)浓度下BBO还降低LPS导致的NF-κB非经典通路中TRAF3(P<0.05)、TRAF2(P<0.05)和NIK(P<0.05)的浓度,进一步抑制p100蛋白的磷酸化(P<0.05),同时抑制转录因子RelB的表达(P<0.05)和RelB蛋白的水平(P<0.05),而BBO自身不引起这些基因与蛋白的变化。【结论】BBO可能通过抑制NF-κB非经典信号中调节蛋白TRAF3和TRAF2,转录因子RelB的浓度,造成NIK蛋白的诱导激酶作用受到抑制,进一步抑制了p100蛋白的磷酸化及其与转录因子RelB的结合,从而影响到下游AA通路中重要限速酶COX-2、5-LOX和FLAP的表达与炎症介质PGE_(2)和LTB_(4)的水平发挥抗炎作用。

响应面法优化艾纳香多糖的超声提取工艺

本文旨在研究艾纳香多糖超声提取的最佳工艺条件。以艾纳香多糖成分的得率作为考察指标,采用单因素及响应面法优化提取工艺。考察温度、提取时间、超声功率和液料比对艾纳香多糖提取率的影响,采用Box-Behnken实验设计的四因素三水平响应面分析方法优化提取工艺。结果表明,艾纳香多糖的最佳提取工艺条件为:提取温度61℃、提取时间32min、超声功率64W、液料比32∶1(mL∶g),此条件下艾纳香多糖得率为2.55%。该提取工艺优化结果合理可行,可为艾纳香多糖的提取提供参考。

Effect of volatile oil from Blumea Balsamifera(L.) DC. leaves on wound healing in mice

OBJECTIVE: To assess the effectiveness of violateoil from Blumea Balsamifera(L.) DC. leaves(BB oil)on wound healing in mice.METHODS: Undiluted BB oil and its diluted solutions with olive oil to 1/5 and 1/10 to yield BB oil1/5and BB oil1/10 were applied to the wounded skin before wound healing conditions were assessed by healing rate, histopathology, and contents of collagen, hydroxyproline, and Neuropeptide Substance P(SP). All above results were compared with the efficacies of the control, pure olive oil, basic fibroblast growth factor(BFGF), and cream of Jing Wan Hong(JWH).RESULTS: BB oil1/5and BB oil1/10 improved wound contraction and closure. Histopathology study further confirmed a desirable histological organization of wound tissues. BB oil1/5and BB oil1/10 reduced the number of inflammatory cells, increased wound-healing rates, and significantly increased the hydroxyproline content. Both BB oil1/5and BB oil1/10 improved formation of collagen, and reduced the frequency of fibroblasts. Moreover, BB oil1/5and BB oil1/10 markedly promoted SP expression. However, undiluted BB oil may induce skin thickening and hardening, inhibite collagen synthesis and delay complete skin wound healing.CONCLUSION: The BB oil1/5and BB oil1/10 promoted capillary regeneration, blood circulation, collagen deposition, granular tissue formation, epithelial deposition, and wound contraction. The mechanism underlying the action might be related to induction of SP secretion, and the proliferation and differentiation of mesenchymal cells.

Antihyperglycemic Effect of Various Fractions from Residues of Blumea balsamifera

Objective To evaluate the antihyperglycemic activities of various fractions from theresidues of Blumea balsamifera（BB）,and to properly utilize the waste resource.MethodsThe antihyperglycemic activities were evaluated by the suppression on serum glucose level in vivo andα-glucosidase inhibition assay in vitro.The high-,mid-,and low-dose（1,0.5,and 0.25 g/kg of the herb）fractions were ig given to mice for 8 d.The serum glucose was monitored at 1 and 12 h after feeding.Results The fasting and postprandial serum glucose levels of mice treated with high-dose petroleum ether fraction,ethyl acetate fraction,butyl alcohol fraction,methanol fraction,and water extract from BB were 4.45,4.39,4.43,4.15,3.74 mmol/L and 6.98,6.23,6.45,6.26,5.88 mmol/L,respectively,while those in vehicle control group were 5.63 and 7.50 mmol/L.There are four different inhibiting manners by the results ofα-glucosidase inhibition assay.Conclusion The residues of BB have anti-diabetes activities after steam distillation.

Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae

Borneol is a precious monoterpenoid with two chiral structures,(-)-borneol and(+)-borneol.Bornyl diphosphate synthase is the key enzyme in the borneol biosynthesis pathway.Many(+)-bornyl diphosphate synthases have been reported,but no(-)-bornyl diphosphate synthases have been identified.Blumea balsamifera leaves are rich in borneol,almost all of which is(-)-borneol.In this study,we identified a high-efficiency(-)-bornyl diphosphate synthase(BbTPS3)from B.balsamifera that converts geranyl diphosphate(GPP)to(-)-bornyl diphosphate,which is then converted to(-)-borneol after dephosphorylation in vitro.BbTPS3 exhibited a K m value of 4.93±1.38μM for GPP,and the corresponding k cat value was 1.49 s−1.Multiple strategies were applied to obtain a high-yielding(-)-borneol producing yeast strain.A codon-optimized BbTPS3 protein was introduced into the GPP high-yield strain MD,and the resulting MD-B1 strain produced 1.24 mg⋅L^(-1)(-)-borneol.After truncating the N-terminus of BbTPS3 and adding a Kozak sequence,the(-)-borneol yield was further improved by 4-fold to 4.87 mg⋅L^(-1).Moreover,the(-)-borneol yield was improved by expressing the fusion protein module of ERG20 F96W-N127W-YRSQI-t14-BbTPS3K2,resulting in a final yield of 12.68 mg⋅L^(-1) in shake flasks and 148.59 mg⋅L^(-1) in a 5-L bioreactor.This work is the first reported attempt to produce(-)-borneol by microbial fermentation.

艾纳香BbSVP基因的克隆与表达分析

SVP转录因子在调控植物生长发育以及应答非生物胁迫过程中发挥着重要作用。本研究通过同源克隆和RACE技术在艾纳香(Blumea balsamifera)中获得了一个SVP-like基因,并命名为BbSVP。生物信息学分析表明,BbSVP基因的编码区长度为681 bp,编码226个氨基酸,属于MADS-box蛋白家族,蛋白内部无明显信号肽和跨膜结构域。同源性比对显示,BbSVP与同为菊科植物向日葵HaSVP X1的亲缘关系最近。进一步研究发现,BbSVP集中分布于细胞核。BbSVP基因在艾纳香中具有组织表达特异性,在叶片中表达量最高,在花中最低。对艾纳香叶片进行非生物胁迫处理后,RT-qPCR分析显示,BbSVP在转录水平上能够应答PEG、NaCl和4℃低温胁迫。研究表明,BbSVP基因在序列和功能上具有一定的保守性,其可能参与调控艾纳香响应非生物胁迫的过程。

一标多测法同时测定艾纳香药材中6种成分的含量

目的建立一标多测法同时测定艾纳香药材中β-蒎烯、樟脑、芳樟醇、β-石竹烯、α-石竹烯和龙脑6种主要活性成分含量。方法采用气相色谱法,色谱柱为PEG-20M(30 m×0.32 mm,10μm)石英毛细管柱,升温程序:起始温度60℃,维持2 min,以5℃·min^(-1)升至140℃,维持2 min,以2℃·min^(-1)升至150℃。高纯氮气为载气(99999%),火焰离子化检测器和进样口温度均设定为240℃。以龙脑为内参物,建立其与β-蒎烯、樟脑、芳樟醇、β-石竹烯和α-石竹烯的相对校正因子,计算各待测组分的含量,并将测定结果与用内标法测定结果进行比对。结果β-蒎烯、樟脑、芳樟醇、β-石竹烯、α-石竹烯和龙脑分别在2.01~80.4μg·mL^(-1)、6.03~241.2μg·mL^(-1)、2.00~80.0μg·mL^(-1)、6.06~242.4μg·mL^(-1)、4.04~161.6μg·mL^(-1)和80.1~3204μg·mL^(-1)浓度范围内具有良好线性关系,方法平均回收率(n=9)分别为98.4%(RSD=06%)、99.1%(RSD=1.2%)、98.9%(RSD=1.2%)、99.0%(RSD=1.3%),98.8%(RSD=1.5%)和98.5%(RSD=1.1%),与常规内标法比较,一标多测法所得结果无显著性差异。结论该方法简便快速,准确可靠,可用于艾纳香药材的质量控制。

艾纳香种子萌发特性研究

为了解艾纳香种子的生物学特性,探讨硝酸钾、氯化钙、IAA、温度和贮藏时间等因素对艾纳香种子萌发的影响,以艾纳香种子为材料,测定其千粒重、大小及含水量,比较分析不同硝酸钾、氯化钙、IAA、温度、贮藏时间等条件下种子发芽势、发芽率和根长。结果表明:艾纳香种子细小,无休眠现象;25℃条件下,贮藏时间超过70 d,发芽率快速降低,4℃条件下贮藏340 d种子发芽率不降低;500、1000 mg/L硝酸钾溶液和200 mg/L氯化钙溶液浸种能显著提高艾纳香种子发芽势、发芽率、根长;40℃温水浸种能够显著提高种子发芽率,超过60℃种子失去发芽能力。因此,艾纳香种子最佳贮藏温度为4℃,在此条件下,以500 mg/L硝酸钾浸泡种子12 h能有效提高发芽率。

不同基质配比对艾纳香幼苗生长的影响

为筛选适宜艾纳香幼苗生长的基质及组成,以黄土、泥炭土、珍珠岩和椰糠为原料,研究不同基质配比对艾纳香幼苗成活率、地上部生长、根生长和叶绿素含量的影响,并进行综合评价。结果表明,不同基质配比对艾纳香幼苗生长的影响差异显著,以黄土、珍珠岩、椰糠为基质的T10处理幼苗存活率、株高、茎粗、植株干重、壮苗指数、主根长和平均根长等指标显著高于其他处理;较适宜的基质组成为T3(黄土、泥炭土、珍珠岩)和T4(黄土、泥炭土、椰糠)处理,基质为黄土的处理艾纳香幼苗生长最差。营养钵种植条件下,最适宜艾纳香幼苗生长的基质配比为黄土∶珍珠岩∶椰糠=46∶27∶27。

苗药艾纳香艾片提取工艺优化及不同产地的品质评价

目的:优化艾片提取工艺并对5个不同产地10个采集点的栽培及野生艾片主要品质指标进行初步评价。方法:采用超声辅助水蒸气蒸馏法,通过正交实验设计提取艾片,以料液比、超声时间、浸泡时间、提取时间为考察指标,艾片提取率为评价指标,采用气相色谱法(GC)对艾片进行含量测定及成分分析。结果:艾片最佳提取条件为料液比1∶12(g∶mL)、超声25 min、浸泡时间75 min、提取时间90 min;野生和栽培艾纳香在艾片提取率上均存在显著差异。结论:本实验建立的艾片提取工艺稳定可行,人工种植艾片质量优于野生艾片。

苗药艾纳香挥发油提取工艺优化及不同产地的品质评价

目的:优化艾纳香挥发油提取工艺并对5个不同产地10个采集点的栽培及野生艾纳香挥发油主要品质指标进行初步评价。方法:采用超声辅助水蒸气蒸馏法,通过正交实验提取艾纳香挥发油,以提取温度、提取时间、药材粉碎度、浸泡时间、料液比、超声时间为考察指标,挥发油提取率为评价指标;采用气相色谱法(GC)对艾纳香挥发油进行含量测定及成分分析。结果:挥发油最佳提取温度为115℃、提取时间为3 h、药材粉碎度为3号筛(50目)、浸泡时间为1 h、料液比为1∶16(g∶mL)、超声为20 min;野生和栽培艾纳香在挥发油含量上无显著差异,栽培药材提取的艾纳香挥发油中L-龙脑、β-蒎烯、芳樟醇、β-石竹烯的含量均高于野生艾纳香挥发油中的含量。结论:所建立的挥发油提取工艺稳定可行,人工种植艾纳香挥发油质量优于野生艾纳香挥发油。

艾纳香油中花椒油素的分离及含量测定

建立了艾纳香油中花椒油素的中压制备分离方法,采用甲醇水为流动相,梯度洗脱;流速为8 mL/min;柱温30℃;进样量100μL;λ=254/360 nm。艾纳香油样品中花椒油素在该条件下分离度好,结合GC-MS进行验证并测定其纯度达99.335%。并通过HPLC检测方法测定了6批次艾钠香油中的花椒油素的含量,结果显示不同批次或不同产家的艾纳香油中花椒油素的含量不一致,且相差较大。该法的建立与含量测定可为艾纳香油的质量控制提供理论基础。