Frankincense myrrh attenuates hepatocellular carcinoma by regulating tumor blood vessel development through multiple epidermal growth factor receptor-mediated signaling pathways

BACKGROUND In traditional Chinese medicine(TCM),frankincense and myrrh are the main components of the antitumor drug Xihuang Pill.These compounds show anticancer activity in other biological systems.However,whether frankincense and/or myrrh can inhibit the occurrence of hepatocellular carcinoma(HCC)is unknown,and the potential molecular mechanism(s)has not yet been determined.AIM To predict and determine latent anti-HCC therapeutic targets and molecular mechanisms of frankincense and myrrh in vivo.METHODS In the present study,which was based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(http://tcmspw.com/tcmsp.php),Universal Protein database(http://www.uniprot.org),GeneCards:The Human Gene Database(http://www.genecards.org/)and Comparative Toxicogenomics Database(http://www.ctdbase.org/),the efficacy of and mechanism by which frankincense and myrrh act as anti-HCC compounds were predicted.The core prediction targets were screened by molecular docking.In vivo,SMMC-7721 human liver cancer cells were transplanted as xenografts into nude mice to establish a subcutaneous tumor model,and two doses of frankincense plus myrrh or one dose of an EGFR inhibitor was administered to these mice continuously for 14 d.The tumors were collected and evaluated:the tumor volume and growth rate were gauged to evaluate tumor growth;hematoxylineosin staining was performed to estimate histopathological changes;immunofluorescence(IF)was performed to detect the expression of CD31,α-SMA and collagen IV;transmission electron microscopy(TEM)was conducted to observe the morphological structure of vascular cells;enzyme-linked immunosorbent assay(ELISA)was performed to measure the levels of secreted HIF-1αand TNF-α;reverse transcription-polymerase chain reaction(RT-qPCR)was performed to measure the mRNA expression of HIF-1α,TNF-α,VEGF and MMP-9;and Western blot(WB)was performed to determine the levels of proteins expressed in the EGFR-mediated PI3K/Akt and MAPK signaling pathways.RESULTS The results of the network pharmacology analysis showed that there were 35 active components in the frankincense and myrrh extracts targeting 151 key targets.The molecular docking analysis showed that both boswellic acid and stigmasterol showed strong affinity for the targets,with the greatest affinity for EGFR.Frankincense and myrrh treatment may play a role in the treatment of HCC by regulating hypoxia responses and vascular system-related pathological processes,such as cytokine-receptor binding,and pathways,such as those involving serine/threonine protein kinase complexes and MAPK,HIF-1 and ErbB signaling cascades.The animal experiment results were verified.First,we found that,through frankincense and/or myrrh treatment,the volume of subcutaneously transplanted HCC tumors was significantly reduced,and the pathological morphology was attenuated.Then,IF and TEM showed that frankincense and/or myrrh treatment reduced CD31 and collagen IV expression,increased the coverage of perivascular cells,tightened the connection between cells,and improved the shape of blood vessels.In addition,ELISA,RT-qPCR and WB analyses showed that frankincense and/or myrrh treatment inhibited the levels of hypoxia-inducible factors,inflammatory factors and angiogenesis-related factors,namely,HIF-1α,TNF-α,VEGF and MMP-9.Furthermore,mechanistic experiments illustrated that the effect of frankincense plus myrrh treatment was similar to that of an EGFR inhibitor with regard to controlling EGFR activation,thereby inhibiting the phosphorylation activity of its downstream targets:the PI3K/Akt and MAPK(ERK,p38 and JNK)pathways.CONCLUSION In summary,frankincense and myrrh treatment targets tumor blood vessels to exert anti-HCC effects via EGFR-activated PI3K/Akt and MAPK signaling pathways,highlighting the potential of this dual TCM compound as an anti-HCC candidate.

Effects of processing technology on essential oil and pharmacological action of frankincense

Background:According to the traditional Chinese medicine theory,the common processing methods for Ruxiang(frankincense)include stir-frying processing and vinegar processing.Methods:With network pharmacology and thermal analysis methods,we selected and identified the main and irritating ingredients,established the pyrolysis characteristic parameters for identifying frankincense quality,quantified the starting and optimum temperature limits for processing frankincense,and analyzed the influences of processing adjuvants(vinegar)on combustion pyrolysis characteristics of frankincense.By applying Fourier transformation infrared spectrometry,high performance liquid chromatography,gas chromatography and UV-visible spectrometry,we evaluated the processing technology developed in our study of frankincense and its processed products.Results:Based on network pharmacology,we can find that the classical compounds of essential oil are the main pharmacodynamics components of frankincense possessing the anti-inflammatory,analgesia,anti-tumor,anti-ulcer and ant-inflammatory bowel disease effects.The pyrolysis combustion rate peaks of frankincense at 285.27±7.05℃and 476.99±13.46℃were the characteristic peaks used to identify frankincense quality.The specific temperature of slow-fire processing,including the vinegar processing and stir-frying processing,was 148.72±5.09℃.Heating for 5.67±0.98 min after reaching processing temperature was needed for processing frankincense with vinegar.The kinetics analysis showed that both frankincense and its vinegar mixture exhibited good linear relationships.The feasibility and practicability for our processing technology were verified through the determination of medicinal compounds by Fourier transformation infrared spectrometry,high performance liquid chromatography,gas chromatography and UV.Conclusion:Frankincense essential oils were the main active components of frankincense,which also can be taken as the internal chemical control indexes to judge the quality control and to optimize the processing technology of frankincense.The pyrolysis combustion rate peaks of frankincense at 285.27±7.05℃and 476.99±13.46℃were the characteristic peaks that can be used to identify frankincense quality.The optimum processing conditions of vinegar-processed frankincense with were as follows:with vinegar,20%;heating temperature,148.72±5.09℃;and heating time,5.67±0.98 min.Under these conditions,frankincense essential oil could cause a moderate effect on combustion pyrolysis characteristics to exert its corresponding pharmacological effects.

Frankincense,a Special Spice Walking Along the Silk Road

Frankincense is also called“Fumigated land,”“Horsetail spice,”and“Godsend spice.”The alias“Fumigated land”shows its strong fragrance that can cover the whole land.The name of“Horsetail spice”implies its morphologic characteristics and mild effects,and another name of“Godsend spice”indicates its functions of relieving swelling and pain,healing sore,and growing muscle,as if it is the grace donated by God.The name of“Frankincense”displays its complex productive progress.With thick quality and strong fragrance,frankincense acts as daily supplies for religious activities and folk activities due to its low price and a wide range of uses in medicine and daily life.Frankincense shows the infinite charm of spice culture along the Silk Road from the distant Western Regions to the East.At the same time,it enriches the treasure house of traditional Chinese medicine and becomes one of the most popular spices in both eastern and western,nobility and common.

Mechanism of Olibanum and Myrrha for the Acute Soft Tissue Injury Based on Network Pharmacology

Objective The objective of this study was to screen the therapeutic target of olibanum and myrrha on acute soft tissue injury(ASTI)by network pharmacology and to clarify their mechanisms.Methods The main chemical constituents and the targets of olibanum and myrrha were obtained by using traditional Chinese medicine systems pharmacology database and analysis platform database.The disease targets of ASTI were searched by GeneCards.The intersection targets of herbs and diseases were selected for protein interaction analysis,protein–protein interaction network was constructed,and potential protein functional modules in the network were explored.A compound–target–disease network was constructed using Cytoscape3.8.2 software.The targets were analyzed by gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis based on the Metascape database.Results The core active components of olibanum and myrrha were quercetin,β-sitosterol,and stigmasterol.The core targets were PGR,NCOA2,PTGS2,PRKCA,and NR3C2.Pathways in cancer,AGE-RAGE signaling pathway in diabetic complications might play a potential role in olibanum and myrrha in the treatment of ASTI.Conclusion Olibanum and myrrha have the characteristics of multiple components,multiple targets,and overall regulation in the treatment of ASTI.

醋乳香现代研究进展及质量标志物的预测

醋乳香是乳香Olibanum经醋炙法加工后的炮制品,炮制后可除去部分挥发油成分,消除其对胃黏膜的刺激性,同时能引药入经增强活血止痛、收敛生肌等功效。通过总结文献报道,从醋炙后化学成分与功效变化,刺激性研究,成分特有性、有效性、可测性,配伍环境及体内过程相关性等方面对醋乳香的质量标志物(Q-marker)进行预测分析,初步确定3-乙酰基-9,11-去氢-β-乳香酸、3-乙酰基-α-乳香酸、3-乙酰基-β-乳香酸、11-羰基-β-乳香酸、11-羰基-β-乙酰乳香酸5个三萜类成分和挥发油类成分乙酸辛酯为其可能的Q-marker,以期为醋乳香的全程质量评价、临床应用研究提供理论依据。

紫苏-乳香-没药挥发油对小鼠创口愈合的促进作用

目的探讨紫苏挥发油与乳香-没药挥发油配伍对小鼠创口愈合的促进作用。方法将ICR小鼠随机分为空白组(CON)、模型组(MOD)、表皮生长因子凝胶组(GEL)、乳香-没药挥发油组(OM,50μL/mL)、紫苏-乳香-没药挥发油组(POM,70μL/mL,V(紫苏挥发油)V(乳香-没药挥发油)=25),除CON组外,其他各组在小鼠背部造一个直径0.8 cm的开放圆形创口。除MOD组外,每只小鼠涂药1次/d,连续14 d,观察小鼠创口愈合情况、病理组织形态,比较3、7、14 d小鼠血清中TNF-α、IL-1β和IL-6的表达情况。结果POM组小鼠创口愈合率大于OM组,且显著高于MOD组(P<0.05);组织病理学检查显示,POM组创口恢复情况优于MOD组;创后3 d,POM组小鼠血清中TNF-α和IL-6水平显著低于MOD组(P<0.05)。结论紫苏-乳香-没药挥发油能减少创口前期炎症反应,促进小鼠创口愈合。

Chemical Constituents and Pharmacological Properties of Frankincense:Implications for Anticancer Therapy

The discovery of novel antitumor agents derived from natural plants is a principal objective of anticancer drug research.Frankincense,a widely recognized natural antitumor medicine,has undergone a systematic review encompassing its species,chemical constituents,and diverse pharmacological activities and mechanisms.The different species of frankincense include Boswellia serrata,Somali frankincense,Boswellia frereana,and Boswellia arabica.Various frankincense extracts and compounds exhibit antitumor,anti-inflammatory,and hepatoprotective properties and antioxidation,memory enhancement,and immunological regulation capabilities.They also have comprehensive effects on regulating flora.Frankincense and its principal chemical constituents have demonstrated promising chemoprophylactic and therapeutic abilities against tumors.This review provides a systematic summary of the mechanism of action underlying the antitumor effects of frankincense and its major constituents,thus laying the foundations for developing effective tumor-combating targets.

乳香定痛散古代文献考证

乳香定痛散是经典的清热缓痛剂,常用于治疗因毒热引起的皮肤疼痛。通过检索有关乳香定痛散的古籍文献,筛选出有效数据共22条,涉及中医古籍18部。从乳香定痛散的历史源流、药物组成、药味炮制、功能主治、处方剂量及制法用法等方面进行统计分析,发现乳香定痛散出自薛己《外科发挥》,由乳香、没药、寒水石、滑石和冰片5味药组成,有理血止痛、清热消肿之功效,主治疮疡溃烂疼痛。历代基本上对其药味组成和功效主治几无争议。在炮制上,寒水石采用煅寒水石;在剂量上,约一半的古籍继承了《外科发挥》的记载“乳香、没药均二钱,寒水石、滑石均四钱,冰片一分,为细末”;制剂几乎皆为散剂;用法上主张“搽患处”。通过对记载乳香定痛散的中医古籍文献进行挖掘整理和系统分析,为经典名方乳香定痛散的发展传承和开发利用提供了科学依据。

进口乳香药材的检测方法研究

目的 :分析进口索马里乳香与埃塞俄比亚乳香挥发油的化学成分 ,并建立其相应主成分的含量测定方法。方法 :GC MS联用鉴定乳香挥发油成分 ;GC法进行含量测定 ,SE 5 4填充柱 (2 .1m× 3.2mm) ,柱温为程序升温 ,初始温度 80℃ ,保持 1min ,终止温度 170℃ ,每分钟升温 15℃。结果 :分别鉴定了索马里乳香挥发油 40种成分与埃塞俄比亚乳香挥发油 2 2种成分 ,其中索马里乳香挥发油主成分为α 蒎烯 ,埃塞俄比亚乳香挥发油主成分为乙酸辛酯 ;对 17批次不同样品进行了测定 ,α 蒎烯线性范围 0～ 10 .80 μg ,r =0 .9995 ,加样回收率为 98.16 % ,RSD 1.83% ;乙酸辛酯线性范围为 0～ 10 .32 μg ,r =0 .9996 ,加样回收率为 99.5 6 % ,RSD 1.36 %。 结论 :通过本项研究 ,为乳香进口药材质量标准的制订奠定基础。

乳香、制乳香含量测定研究

目的建立HPLC法测定乳香及制乳香中11-羰基-β-乙酰乳香酸的含量。方法采用Agilent TC-C18柱,流动相为乙腈-0.1%冰醋酸溶液(80∶20),流速为1.0mL.min-1,检测波长为250nm,柱温为30℃。结果 11-羰基-β-乙酰乳香酸在0.1535～2.4560μg范围内线性良好,r=0.99999。平均回收率为97.62%,RSD=1.36%。结论本法专属、快速、准确,可用于乳香的含量测定。

乳香-没药配伍前后汤液理化参数变化与化学成分的关联分析

目的探讨乳香-没药水提液(汤液)理化性质与化学成分变化之间的关系。方法测定汤液的理化参数(电导率、盐度、浊度、黏度、pH值);采用UPLC-MS/MS分析汤液中的化学成分组成;采用逐步回归分析方法对汤液的理化参数与化学成分的相关性进行分析,并进行验证试验。结果汤液中的化学成分组成及其含有量变化影响着汤液的浊度、黏度等理化参数;其中五环三萜酸酯类化合物与汤液的浊度呈显著正相关,倍半萜类化合物与汤液的黏度呈显著负相关。结论为深入研究乳香-没药配伍协同增效的物质基础提供了一定的依据。

用镇痛与刺激实验优选乳香最佳炮制工艺的研究

目的优选乳香烘制法的最佳炮制工艺。方法以小鼠的痛阈值和家兔眼睛充血、水肿数为评价指标,采用正交试验优选乳香的最佳炮制工艺。结果乳香烘制的最佳工艺为120℃烘制3 h、药物放置厚度1 cm、药物直径1 cm。结论优选出的炮制工艺简单、实用、条件可控、成品质量好。

麝香配伍乳香对趋化激活单核细胞触发炎性级联反应相关蛋白表达的影响

目的:观察麝香、乳香含药血浆对前列腺抗原刺激诱导的单核细胞炎症因子释放及前列腺细胞或内皮细胞炎症反应相关蛋白表达的影响。方法:采用SD大鼠制备麝香、乳香含药血浆,BALB/c小鼠制备单核细胞与前列腺抗原。单核细胞以梯度浓度(0,2.5%,5%,10%,20%)的麝香、乳香含药血浆预处理1 h后,前列腺抗原诱导活化,培养96 h时ELISA法检测炎症因子释放水平;前列腺细胞RWPE-1与内皮细胞EA.hy926共培养,梯度浓度DP-M&O预处理1 h后,加入经PAgs诱导活化的单核细胞,培养96 h时Western印迹法检测RWPE-1或EA.hy926细胞炎症反应相关蛋白表达水平。结果:麝香、乳香含药血浆随浓度增加,降低TNF-α、IL-1β、IL-6、IL-8及升高IL-10作用逐渐明显,并呈剂量依赖性,20%麝香、乳香含药血浆预处理后,单核细胞释放TNF-α、IL-1β、IL-6、IL-8、IL-10[(70.8±22.3)pg/ml,(277.5±22.6)pg/ml,(232.7±62.7)pg/ml,(227.3±79.2)pg/ml,(640.2±201.2)pg/ml]水平与前列腺抗原组[(277.1±65.5)pg/ml,(630.4±89.7)pg/ml,(994.2±182.3)pg/ml,(769.3±284.1)pg/ml,(271.1±55.8)pg/ml]比较差异有统计学意义(P均<0.01);10%与20%浓度麝香、乳香含药血浆预处理,RWPE-1细胞MCP-1/CCL2(0.56±0.11,0.34±0.08)及EA.hy926细胞VCAM-1(0.52±0.17,0.38±0.12)表达降低,与前列腺抗原组(MCP-1/CCL2:1.12±0.34,VCAM-1:0.94±0.22)比较,差异有统计学意义(P均<0.05)。结论:麝香配伍乳香可通过抑制前列腺上皮细胞MCP-1/CCL2产生,降低血管内皮细胞VCAM-1表达,从而阻断白细胞粘附的关键步骤达到减弱炎症反应。

4种方法提取乳香化学成分及其GC-MS研究

目的对4种方法提取乳香得率和化学成分进行分析,并测定各提取物主要成分含量。方法GC—MS鉴定各提取物中成分;GC法含量测定。结果不同提取方法的得率和各提取物中醋酸辛酯含量差异很大,各得率和醋酸辛酯含量分别为:水蒸气蒸馏,0.69%和57.00%;超临界CO2萃取,20.40%和3.23%;索氏提取,15.60%和0.73%;超声提取,13.00%和0.80% 结论超临界CO2萃取得率和萃取物中醋酸辛酯含量较高,且其中除含有挥发油外还有其他成分,综合评价,该法优于其他3 种提取方法,更适合乳香的提取。

基于气质联用技术的乳香醋炙前后挥发油成分变化分析

目的比较乳香炮制前后挥发油中主要成分组成及含量的差异,研究不同炮制条件对挥发油成分的影响。方法采用水蒸气蒸馏法提取挥发油,控制炮制温度和时间得到不同炮制条件醋乳香。利用气相色谱质谱联用技术(gas chromatography-mass spectrometry,GC-MS)分析乳香醋炙前后挥发油,通过质谱数据库检索和对照品对照确定化学成分结构,采用色谱峰峰面积归一化法计算各成分的相对百分含量。结果乳香与醋乳香的主要成分均为乙酸辛酯、正辛醇、α-蒎烯、柠檬烯,乳香中主要成分的相对含量范围分别为51.92%~79.59%、2.09%~13.50%、0.16%~3.73%、0.10%~1.82%,醋乳香中主要成分的相对含量范围分别为66.72%~80.94%、4.33%~12.2%、0.39%~2.43%、0.25%~4.15%。萜类成分含量随醋炙温度的升高及醋炙时间的延长而增加,醇类及有机酸酯类成分随醋炙温度的升高及醋炙时间的延长而减少,大部分成分随醋炙程度加深而含量增加,而己酸乙酯、α-蒎烯等在乳香中含量较少且炮制后含量减少。结论乳香挥发油含量较醋乳香高,各挥发油成分含量在炮制后发生改变。

乳香中三萜类成分的薄层色谱鉴别和含量测定

目的:以乳香中主要三萜类成分为指标,建立和完善乳香的定性定量分析方法。方法:用TLC鉴别法,以三萜酸类成分11-羰基-β-乙酰-乳香酸(AKBA)为对照品,采用硅胶GF254薄层板,以环己烷-乙酸乙酯-冰醋酸(10:2:0.2)为展开剂,鉴别乳香药材中AKBA;用HPLC测定方法,AgilentTC-C18色谱柱(250mm×4.6mm,5μm),乙腈-0.05%磷酸溶液(80:20)为流动相,流速:0.8mL·min-1,检测波长:250nm,测定不同来源乳香药材中AKBA和11-羰基-β-乳香酸(KBA)含量。结果:乳香TLC图谱中,AKBA斑点清晰、分离效果好;HPLC含量测定法中,AKBA和KBA分别在0.02463～0.2463mg·mL-1和0.01004～0.1004mg·mL-1,线性关系良好。AKBA和KBA的回收率分别为96.1%(RSD=3.1%)和96.4%(RSD=3.5%)。结论:所建立的TLC和HPLC法操作简便、结果准确、重复性好,可作为乳香定性、定量的质量控制方法。

乳香、没药药对配伍挥发油成分的GC-MS分析

目的考察乳香、没药药对配伍对挥发油化学成分的影响。方法采用气-质联用系统对乳香、没药药材以及药对配伍中的挥发油进行分析,评价配伍对挥发油成分及含量的影响。结果采用GC-MS对乳香、没药药材和药对挥发油的成分进行分析比较,发现乳香挥发油中检出的trans-3,5,6,8 a-四氢-2,5,5,8 a-四甲基-yl-2H-1-苯并吡喃、荜澄茄-1(10),4-二烯等成分和没药挥发油中检出的2-(1,2-二甲基-2-环戊烯-1-yl)-乙酸苯酯、(1S-cis)-1,2,3,4-四氢-1,6-二甲基-4-(1-甲基乙基)-萘、1,4-二甲基-7-(1-甲基乙基)-Azulen-2-醇、(-)-匙叶桉油烯醇等成分在药对挥发油中未检出,而相对于单煎所得的挥发油,药对挥发油中新增成分有1-乙烯基-1-甲基-2,4-二异丙基-环己烷、1,2,3,4-四氢-1-甲基-8-异丙基萘等。结论乳香、没药药对配伍对所提挥发油的成分和含量都有较大的影响。

乳香与醋乳香对2型糖尿病大鼠改善作用的对比研究

目的:探讨中药乳香醋炙前后对链脲佐菌素(streptozotocin,STZ)联合高脂饲料诱导2型糖尿病大鼠的改善作用,为扩展中医药的应用和阐明炮制增效机理奠定基础。方法:除正常组外将24只2型糖尿病模型大鼠随机分为模型组、乳香组、醋乳香组及阳性药组,以腹腔注射STZ的方法辅以高脂饲料诱导糖尿病模型。正常组及模型组给予0.5%羧甲基纤维素钠(carboxymethylcellulose sodium,CMC-Na),阳性药组给予盐酸二甲双胍的0.5%CMC-Na混悬液,乳香组和醋乳香组分别给予临床等效剂量乳香及醋乳香的0.5%CMC-Na混悬液进行灌胃给药,共给药2周,给药期间每周进行体质量及空腹血糖的监测。末次给药后,采用ELISA法测定血中胰岛素、糖化血红蛋白(glycosylated hemoglobin,GHb)、一氧化氮(nitric oxide,NO)和内皮素-1(endothelin-1,ET-1)水平并比较胰腺病理切片。结果:与模型组比较,乳香和醋乳香在给药2周后,体质量和胰岛素水平显著提升(P<0.05或P<0.01),而空腹血糖值、GHb、NO及ET-1水平显著降低(P<0.05或P<0.01)。胰腺病理切片显示,醋乳香组糖尿病大鼠胰岛萎缩和胰泡细胞变性程度较乳香组降低,且醋乳香的改善作用优于乳香。结论:乳香醋炙后对糖尿病大鼠的改善作用增强。

乳香中化学成分和药理作用的研究进展

乳香中主要含有五环三萜、四环三萜和大环二萜等化合物,具有良好的抗炎、抗肿瘤、抗氧化等作用,临床上具有广泛的应用价值。现对乳香的化学成分、药理作用和临床应用等方面的最新进展作一综述,以冀为乳香的研究开发提供参考。

利用TGA-DTA热分析法快速鉴别中药乳香及质量分析研究

目的:利用TGA-DTA热分析法快速鉴别中药乳香,并进行质量分析。方法:收集市场上乳香药材共计25份,通过考察升温范围、升温速率、粉末粒度三个重要因素对TGA-DTA热分析实验的影响,利用TGA-DTA特征图谱快速鉴别药材真伪;分别采用一阶中点法和连线法进行热重分析与热焓计算,对乳香药材进行定量分析。结果:确定了乳香TGA-DTA实验的最佳条件:升温范围选择50-750℃;升温速率选择20℃·min-1;粉末过100目筛;在该条件下,通过特征峰位(T1=447±5℃、T2=549±5℃、T3=350±5℃)、热重分析(TV-max、ΔW2+ΔW3)、热焓分析(ΔH)可以很好的区分正品乳香、伪品乳香以及乳香的掺伪品。结论:TGA-DTA技术操作简单,是一种快速、准确、简单的乳香鉴别与质量分析新方法。