Network pharmacology and preliminary cell screening studies on the anti-liver cancer activity of Nauclea Officinalis

Objective:To explore the mechanism of Nauclea Officinalis of anti-liver cancer effect based on network pharmacology,and to preliminarily verify anti-liver cancer activity of Nauclea Officinalis through cell screening.Methods:Network pharmacology was used to screen for common targets of Nauclea Officinalis and liver cancer,protein-protein interaction(PPI)network was constructed,and enrichment analysis and mechanism prediction were conductd.Molecular docking of main active ingredients of Nauclea Officinalis with core targets was made.Preliminary verification was performed by in vitro cell experiments such as CCK8,cell apoptosis,and PCR.Results:After the screening,14 active ingredients of Nauclea Officinalis were obtained,with 587 related targets.After mapping with liver cancer targets,there were 288 common targets,mainly including TP53,SRC,STAT3,and other core targets.Among them,compounds such as strictosamide,pumiloside and vincosamide may be potential active ingredients of Nauclea Officinalis of anti-liver cancer effect.They may participate in protein phosphorylation and negative regulation of the apoptosis process by mediating cancer pathways,PI3K/Akt and EGFR tyrosine kinase inhibitors resistance signaling pathways to play an anti-liver cancer role;molecular docking results showd that active ingredients of Nauclea Officinalis had a stable binding with liver cancer core targets;in vitro cell experiments showd that main ingredient strictosamide of Nauclea Officinalis had cytotoxicity against liver cancer cells,inhibited liver cancer cell proliferation(P<0.001),down-regulated gene expression of liver cancer HepG2 cells SRC,STAT3,MAPK3(P<0.05),and induced liver cancer cell apoptosis(P<0.001).Conclusion:This study preliminarily explores the potential mechanism of active ingredients of Nauclea Officinalis against liver cancer and its preliminary pharmacological effects,providing a theoretical basis for the study of Nauclea Officinalis of anti-liver cancer mechanism.

HPLC fingerprint of Nauclea officinalis stems in different harvesting period

Objective:To establish HPLC fingerprint of Nauclea officinalis stems in different harvesting periods,and analyze the effect of harvest time on the quality of the medicinal materials by combining with chemical pattern recognition.Methods:The analysis was performed on Sun Fire-C18(150 mm×4.6 mm,5μm)column.The mobile phase consisted of acetonitrile-0.1%phosphoric acid solution(gradient elution)at a flow rate of 1.0 mL/min.Results:The HPLC fingerprint of Nauclea officinalis stems was established and 12 common peaks were determined,and 3 chromatographic peaks were identified by comparison with the mixed references.There were some differences in the quality of Nauclea officinalis in different harvesting periods.The OPLS-DA analysis successfully predicted four main markers of quality difference.Conclusion:The established HPLC fingerprint could reflect the composition characteristics of Nauclea officinalis stems in different harvesting period,and the main markers that influence the composition difference of the stems could be used as key indicators for the quality control.

In vitro anti-inflammatory,anti-oxidant and in vivo anti-arthritic properties of stem bark extracts from Nauclea pobeguinii(Rubiaceae) in rats

Objective:To explore the immunomodulatory,anti-inflammatory,anti-oxidant and anti-arthritic activity of aqueous and methanolic extracts of Nauclea pobeguinii stem bark.Methods:For in vitro assays,the production of reactive oxygen species(chemiluminescence technique),the proliferation of T cells(liquid scintillation counter method),as well as the inhibition of cyclooxygenase,lipoxygenase,protein denaturation,and free radicals[DPPH,ABTS and nitric oxide(NO)inhibition methods]were evaluated.For in vivo assays,a polyarthritis model was induced by complete Freund’s adjuvant in rats.The aqueous and methanolic extracts of Nauclea pobeguinii stem bark were administered orally at 150 and 300 mg/kg.After 28 days of treatment,the total blood was taken to quantify the hematological parameters and the serum was used to evaluate the biochemical parameters(alanine aminotransferase,aspartate transaminase,phenylalnine ammonialyase,and proteins)and oxidative stress parameters(malondialdehyde,catalase,superoxide dismutase,glutathione and NO),and then the knee joint was removed for histological analysis.Results:The extracts of Nauclea pobeguinii significantly reduced the production of intra-and extracellular reactive oxygen species and decreased T cell proliferation.They had an inhibitory effect on cyclooxygenase,lipoxygenase,and protein denaturation,and both extracts had antioxidant capacity on DPPH,ABTS and NO.Both extracts alleviated joint inflammation and pain sensitivity after complete Freund’s adjuvant injection,reduced alanine aminotransferase,aspartate transaminase,alkaline phosphatase,NO and malondialdehyde levels,increased protein concentration,superoxide dismutase,catalase and glutathione activity,and restored the cytoarchitecture of the joint after complete Freund’s adjuvant injection.Conclusions:The aqueous and methanolic extracts of Nauclea pobeguinii have immunomodulatory,anti-inflammatory,anti-oxidant and anti-arthritic properties.

Two new indole alkaloids from Nauclea officinalis

Two new indole alkaloids,4-oxo-4,12-dihydroindolo[2,3-a]quinolizine-3-carbaldehyde(1)and 1,6,7-trihydro-indolo-[2,3- a]furan[3,4-g]quinolizine-3,4(13H)-dione(2),were isolated from Nauclea officinalis.Their structures were determined on the basis of 1D and 2D NMR spectral data.

Natural Distribution of <i>Nauclea diderrichii</i>(Rubiceae) in Semi Deciduous Forest of Togo (West Africa) and Implementation of Integrated Silviculture

West African tropical forests spread from Senegal to Togo. They are considered as one of the world biodiversity hotspots. But these forests are disappearing very quickly because of human activities. In Togo, tropical forests degradation has been increased since 1980 because of improved coffee cultivation;about 67% of forests in the sub-humid mountains zone disappeared between 1978 and 2000. Facing this fast deterioration of the forests, populations begin to plant teak (an exotic species) that is the only choice proposed by local forest service. This survey consists in valuing the potentialities to restore the Togolese tropical forest using the local commercial species, recognized on the wood market. The first species retained is Nauclea diderrichii (De Wild. & Th. Dur.) Merrill, that spreads in the African tropical forest from the Sierra Leonne to Uganda and through the Congo basin. In Togo, this plant species colonises the plain of the Litimé. The objective of the present study is to analyze the distribution of the natural populations in the plain of Litimé forest but also natural and artificial regeneration of the species. The perspective is to repeat this experiment with other local species in other regions of Togo in order to diversify the plantations and to protect the local forest biodiversity.

Survival, Growth and <i>Orygmophora mediofoveata</i>Shoot Borer Attack of <i>Nauclea diderrichii</i>Progenies Established in Three Ecological Zones in Ghana

Nauclea diderrichii is a tropical African hardwood species and a suitable candidate for plantation development. However, attack by the Orygmophora mediofoveata, Hamps shoot borer threatens establishment of the species in plantations. A genotype * environment assessment of 15 N. diderrichii progenies from Ghana and Togo was conducted in the Wet Evergreen, Moist Semi-deciduous and Dry Semi-deciduous forest zones. Progeny performance (Attack intensity, survival and growth) varied significantly between sites, and marginally within sites after 2.7 years. Overall, incidence of shoot borer attack was lower at the wet zone than at the moist or dry zones. Percent survival was higher at the wet (79.5%) than at the moist (50.8%) or dry (55.0%) forest zones. Mean height across the 15 progenies was 5.40 m, 4.30 m, and 2.73 m at the wet, dry and moist forests, respectively. Similarly, mean diameter was 5.31 cm, 4.58 cm, and 2.83 cm at the wet, dry and moist zones, respectively. The relatively low growth rate recorded at the moist zone was attributed to the paucity of soil conditions at the experimental site. Three wet forest zone progenies (BS9, BS3 and BS2) and two moist forest zone progenies (BE2 and GA1) performed better than average and have been recommended for planting.

Study on the chemical composition of Nauclea officinalis in different months of the same year and different growth years

Objective:Based on the quality analysis of Nauclea officinalis in different growth years and different months in the same year,the most suitable harvest year and the specific month in the same year were determined according to the evaluation of the active components in the stem of medicinal parts of Nauclea officinalis.Methods:LC-MS/MS method was used to investigate the quality of Nauclea officinalis from different growth years and different months in the same year,mainly including the content determination of the active ingredients in the medicine.Results:With the prolongation of production life,the content of active ingredients in stem of Nauclea officinalis increased continuously.After reaching a certain number of years,the content of active ingredients increased little;In the middle of the same year,from mid-October to mid-June of the next year,the content of iso-vincin lactam was relatively high,especially between the end of March and the end of May.The content was high and stable,and the highest point was the stem sample collected in May.Conclusion:The content of each component in the stem of Nauclea officinalis with a growth life of more than 5 years has reached a relatively high and stable level.Nauclea officinalis with a growth life of more than 5 years should be collected as raw materials for medicinal materials.The month with high and stable content of isocyanoside lactam,namely May of each year,was taken as the best harvest time of gallbladder in one year.The best harvest time was from mid-October to mid-June of the next year,or from March to May of each year.

乌檀化学成分研究

目的:研究乌檀Nauclea officinalis(Pierre ex Pitard)Merr.et Chun茎干的化学成分。方法:采用硅胶、RP-18和Sephadex LH-20葡聚糖凝胶等柱色谱进行分离纯化,运用现代波谱学技术进行化合物结构鉴定。结果:从乌檀茎干乙醇提取物的二氯甲烷相中分离纯化得到17个化合物,分别鉴定为:19-乙氧基牛眼马钱托林碱(1)、3,14-二氢狭花马钱碱(2)、安枯斯特定碱(3)、3,14-二氢-19-乙氧基牛眼马钱托林碱(4)、4-(3-氨丙基)-2-甲氧基苯酚(5)、松脂素(6)、(-)-去甲氧基木兰素(7)、(+)-去甲氧基木兰素(8)、右旋杜仲树脂酚(9)、(+)-南烛木树脂酚(10)、猫眼草酚D(11)、3,4-二甲氧基苯酚(12)、顺式-5-羟基亚铁酸(13)、3-(4-羟基-3,5-二甲氧基苯基)丙烷-1,2-二醇(14)、(+)-赤式-7-乙氧基愈创木甘油(15)、(-)-赤式-7-乙氧基愈创木甘油(16)和苏式-3-(4-羟基-3,5-二甲氧基苯基)-3-乙氧基丙烷-1,2-二醇(17)。结论:其中,化合物5和13为新天然产物,化合物5、7~13和15~17为首次从乌檀属植物中分离得到。

不同采收期胆木茎干HPLC指纹图谱研究

目的:建立不同采收期胆木药材茎干HPLC指纹图谱,结合化学模式识别分析采收期对药材质量的影响。方法:以Sun Fire C18(150 mm×4.6 mm,5μm)为色谱柱;乙腈⁃0.1%磷酸水溶液进行梯度洗脱;流速1.0 mL/min。结果:建立了不同采收期胆木茎干HPLC指纹图谱,确定12个共有峰,指认出3个色谱峰。不同采收期的胆木药材质量存在一定差异,OPLS⁃DA预测了4个主要质量差异标志物。结论:建立的HPLC指纹图谱能够反映不同采收期胆木茎干的组分特征,预测的主要质量差异标记物可作为胆木药材质量控制的关键指标。

胆木树干乙酸乙酯部位化学成分研究

目的:研究胆木Nauclea officinalis Pierre ex Pitard树干的化学成分。方法:采用硅胶柱、Sephadex LH-20葡聚糖凝胶和制备型高效液相等色谱技术对胆木树干95%乙醇提取物的乙酸乙酯部位进行分离纯化,根据理化性质及波谱数据鉴定化合物的结构。结果:从胆木树干乙酸乙酯部位中分离得到14个化合物,分别鉴定为:(6R,7E,9R)-9-hydroxy-4,7-megastigmadien-3-one(1)、(9S)-9-hydroxymegastigma-4,6E-dien-3-one(2)、blumenol C(3)、9-epi-blumenol C(4)、松柏醛(5)、3,5-二甲氧基-4-羟基苯甲醛(6)、芥子醛(7)、3,4-二甲氧基苯酚(8)、3,4,5-三甲氧基苯酚(9)、2-oxopomolic acid(10)、覆盆子酸(11)、桔皮晶(12)、(E)-3,3′-二甲氧基-4,4′-二羟基二苯乙烯(13)、fraxidin(14)。结论:其中,化合物2~8、10、11、13、14为首次从乌檀属植物中分离得到,化合物1、9、12为首次从胆木中分离得到。

黎药胆木内生细菌群落结构多样性研究及其基因功能预测分析

以海南省5个产区采集的胆木为材料,采用高通量16SrRNA测序技术分析了胆木的根、茎、叶和表皮4个组织的内生细菌群落多样性,并通过PICRUSt分析其内生细菌的潜在功能。结果表明,胆木在16SrRNAV3-V4区高质量序列片段共得到3 759 378条有效序列,划分为3 475个OUTs,内生细菌分为30门、82纲、183目、326科、764属,具有丰富的菌群多样性,组间差异显著。对内生细菌群落结构分析可知,共有优势菌属为Chloroplast_unclassified(25.11%)、芽孢杆菌属Bacillus(15.16%)、伯克霍尔德氏菌属Paraburkholderia(7.06%)、罗尔斯通菌属Ralstonia(5.03%)、马赛菌属Massilia(4.60%)。其中,根优势菌属为芽孢杆菌属(Bacillus)、伯克霍尔德氏菌属(Paraburkholderia)、寡养单胞菌属(Stenotrophomonas)、固氮根瘤菌(Azorhizobium)、根瘤菌属(Rhizobium);茎优势菌属为Chloroplast_unclassified、罗尔斯通菌属(Ralstonia)、马赛菌属(Massilia)、Mitochondria_unclassified;叶优势菌属为短小杆菌属(Curtobacterium);表皮优势菌属为赖氨酸芽胞杆菌属(Lysinibacillus)、类芽孢杆菌属(Paenibacillus)。PICRUSt分析显示,胆木不同组织内生细菌主要涉及6个生物代谢通路及36个子功能。不同组织的内生细菌基因二级功能层预测基因种基本无差异,但基因丰度具有显著差异,代谢为影响胆木植株内生细菌多样性和丰富度的重要因素。通过全面揭示胆木不同组织内生细菌多样性的变化趋势和规律,能为其内生细菌的进一步应用、充分挖掘其蕴含的丰富微生物资源奠定理论基础。

胆木抗肝癌活性的网络药理学及初步细胞筛选研究

目的:基于网络药理学探究胆木抗肝癌的作用机制,并通过细胞筛选初步验证胆木抗肝癌活性。方法:利用网络药理学筛选胆木与肝癌的共同靶点,构建蛋白互作网络以及进行富集分析及作用机制预测,并将胆木的主要活性成分与核心靶点进行分子对接。利用CCK8、细胞凋亡及PCR等体外细胞实验进行初步验证。结果:经筛选获得胆木活性成分14种,相关的靶点587个,与肝癌靶点映射后共同靶点有288个,主要有TP53、SRC、STAT3等核心靶点。其中异长春花苷内酰胺、短小蛇根草苷、喜果苷等化合物可能是胆木抗肝癌的潜在活性成分,可能通过介导癌症通路、PI3K/Akt和EGFR酪氨酸激酶抑制剂抵抗等信号通路,参与蛋白质磷酸化、凋亡过程的负调控等过程,发挥抗肝癌作用;分子对接结果显示胆木活性成分与肝癌核心靶点产生稳定的结合;体外细胞实验结果表明,胆木中主要成分异长春花苷内酰胺对肝癌细胞具有细胞毒性,抑制肝癌细胞增殖(P<0.001),内在机制是下调肝癌HepG2细胞SRC、STAT3、MAPK3的基因表达(P<0.05),并诱导肝癌细胞凋亡(P<0.001)。结论:本研究初步探讨了胆木活性成分抗肝癌的潜在机制及初步药效作用,为胆木抗肝癌作用机制研究提供理论依据。

胆木茎中一个新的吲哚生物碱苷

目的研究中药胆木水溶性部位的化学成分。方法采用大孔吸附树脂,硅胶柱色谱和Sephadex LH20凝胶柱色谱分离纯化,通过光谱分析鉴定化合物的结构。结果从胆木水溶性部位中分离得到7个化合物,分别鉴定为:nauc leam ide A-10-O--βD-glucopyranoside(I),5--βcarboxystrictosid in(II),獐牙菜苷(III),番木鳖苷A(IV),3,4-d im ethoxyphenyl--βD-glucopyranoside(V),3,4,5-trim ethoxyphenyl--βD-glucopyranoside(V I),2-phenethylrutinoside(V II)。结论化合物I为新的吲哚类生物碱苷,化合物I^V II均为首次从该植物中分得。

中药胆木的化学成分

AIM: To investigate the chemical constituents of Nauclea officinalis, an anti-inflammatory and antibacterial traditional Chinese medicine. METHOD: The compounds were isolated and purified by silica gel and Sephadex LH20 column chromatography repeatedly, and their structures were identified by spectral analysis. RESULT: Eight compounds were isolated from the bark and wood of Nauclea officinalis, and their structures were identified as β-sitosterol(1), sitosterone (2), 3-O-acetylerythrodiol (3), vanillin (4), oleanlic acid(5), veratric acid(6), 3,4-dimethoxycinnamic acid(7), glycosmisic acid(8). CONCLUSION: Compounds 2～8 were isolated from this plant for the first time.

乌檀的化学成分研究

从珍稀药用植物乌檀(Naucleaofficinalis)的枝叶中分离得到1个甾醇类、3个三萜类、2个酚类和1个生物碱类化合物。通过光谱分析,分别鉴定为豆甾-4-烯-3-酮(1)、铁冬青酸(2)、常春藤皂苷元(3)、3-羰基奎诺瓦酸(4)、2,5-二甲氧基苯甲酸(5)、3,4,5-三甲氧基苯甲酸(6) 和Strictosamide (7)。7个化合物均是首次从乌檀中得到。

胆木生物碱成分研究

目的研究胆木Nauclea officinalis的生物碱成分。方法95％乙醇提取胆木带皮茎枝的化学成分，采用反复硅胶柱色谱、Sephadex LH20凝胶柱色谱和C-18反相硅胶柱色谱分离纯化，通过光谱分析鉴定化合物的结构。结果从醋酸乙酯部位分离得到11个成分，其中9个为吲哚类生物碱，2个喹啉酮类生物碱，分别鉴定为：牛眼马钱托林碱（angustoline，Ⅰ）、19-乙氧基牛眼马钱托林碱（19-O-ethylangustoline，Ⅱ）、3-S-3，4-二氢牛服马钱托林碱（3-S-3，4-dihydroangustoline，Ⅲ）、3-R-3，4-二氢牛眼马钱托林碱（3-R-3，4-dihydroangustoline，Ⅳ）、naucleamide A（Ⅴ）、异长春花苷内酰胺（strictosamide，Ⅵ）、喜果苷（vincosamide，Ⅵ）、6′-异乙酰基长春花苷内酰胺（6′-acetylstrictosamide，Ⅷ、2′-异乙酰基长春花苷内酰胺（2′acetyl-strictosamide，Ⅸ）、短小蛇根草苷（pumiloside，Ⅹ）、3-表短小蛇根草苷（3-epi-pumiloside，Ⅺ）。结论化合物Ⅰ～Ⅺ均为首次从该植物中分离得到。

狄氏黄胆木干燥工艺的初步研究

通过 1 0 0℃干燥试验法的试验 ,获得了狄氏黄胆木木材的基本干燥特性数据。狄氏黄胆木木材的干燥特性是 :初期开裂与截面变形的干燥等级为 3级 ,内部开裂的干燥等级为 2级。根据这些特性因子编制了狄氏黄胆木板材的干燥基准。表 6参

胆木中两个新生物碱甙的结构研究

从胆木(Nauclea officinalis Pierre ex Pitard)树干中,分得两个新生物碱甙,分别命名为胆木碱庚(nauclecoside)和胆木碱辛(nauclecosidine)。根据它们的理化性质,光谱数据和化学反应,推断胆木碱庚的结构为Ⅰa,胆木碱辛为Ⅱa。并且还得到一已知碱胆木碱壬,鉴定为喜果甙(vincoside laetam,Ⅲ)。

胆木不同部位异长春花苷内酰胺的含量研究

目的:获得胆木茎木、皮及叶中有效成分异长春花苷内酰胺含量高低的研究结果,为挖掘胆木新的药用部位奠定科学基础。方法:采用薄层色谱法,对胆木的茎木、皮、叶中异长春花苷内酰胺化学成分进行定性鉴别;同时采用高效液相色谱技术对该3个部位中异长春花苷内酰胺含量高低进行检测。结果:胆木茎木、皮及叶的薄层色谱图中均出现异长春花苷内酰胺蓝色荧光斑点;且茎木、皮、叶中异长春花苷内酰胺含量值分别为1.22%,1.08%,1.54%。结论:定性鉴别和含量测定的实验结果均表明:胆木茎木、皮、叶中均含有效成分异长春花苷内酰胺,且叶中含量相对较高、皮中含量相对较低。

HPLC法同时测定胆木及其制剂中酚酸和生物碱类成分

目的建立胆木药材及其制剂中原儿茶酸,短小舌根草苷,3-表短小舌根草苷,异长春花苷内酰胺的测定方法。方法采用HPLC法,X-SELECT CSHTMC18色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈-0.05%甲酸水,梯度洗脱;检测波长258 nm(原儿茶酸),243 nm(短小舌根草苷、3-表短小舌根草苷),226 nm(异长春花苷内酰胺);体积流量1 mL/min;柱温30℃。结果原儿茶酸、短小舌根草苷、3-表短小舌根草苷和异长春花苷内酰胺分别在0.966～154.56μg/mL、0.944～151.04μg/mL、0.954～152.64μg/mL和0.916～146.56μg/mL内具有良好的线性关系(R2≥0.999 8);4种成分在胆木药材中的平均加样回收率分别为98.2%、101.3%、97.4%和99.2%,RSD≤3.62%。在胆木注射液中的平均加样回收率为99.7%,103.0%,98.9%,100.7%,RSD≤2.44%。结论本法操作简便,结果可靠,重现性好,可作为胆木药材及其制剂质量控制的方法。