應(yīng) 劍，張 波，王春波，王春玲*

（中糧營(yíng)養(yǎng)健康研究院，北京 102209）

基于網(wǎng)絡(luò)藥理學(xué)預(yù)測(cè)我國(guó)蜂膠改善代謝性疾病的生物學(xué)機(jī)制

應(yīng) 劍，張 波，王春波，王春玲*

（中糧營(yíng)養(yǎng)健康研究院，北京 102209）

為系統(tǒng)分析我國(guó)蜂膠改善2型糖尿病、肥胖等代謝性疾病的生物學(xué)機(jī)制，開發(fā)防控慢性代謝性疾病的標(biāo)準(zhǔn)化功能性食品提供科學(xué)參考，以我國(guó)蜂膠中的主要黃酮類、酚酸及酯類化合物成分為研究對(duì)象，利用結(jié)構(gòu)相似比對(duì)法預(yù)測(cè)其作用靶點(diǎn)，并利用網(wǎng)絡(luò)藥理學(xué)方法建立“成分-靶標(biāo)-疾病”的關(guān)聯(lián)，闡述蜂膠成分改善代謝性疾病的“多成分、多靶點(diǎn)”特征，分析核心作用通路。通過與現(xiàn)有文獻(xiàn)報(bào)道的對(duì)比分析，計(jì)算得到靶標(biāo)預(yù)測(cè)成功率為86.2%。PPARγ、ESR1、ESR2、SIRT1、PTPN1是蜂膠化合物總體作用概率最高的靶標(biāo)，其中PPARγ是黃酮類化合物與酚酸類化合物的共同重要靶標(biāo)。蜂膠中的黃酮類化合物是與改善糖脂代謝活性關(guān)聯(lián)最為密切的一類物質(zhì)，此外部分酚酸及酯類化合物也發(fā)揮了協(xié)同作用。網(wǎng)絡(luò)分析結(jié)果表明，蜂膠成分可能分別或者共同作用于糖脂代謝相關(guān)的多條通路，通過促進(jìn)糖攝取、促進(jìn)胰島素分泌、改善胰島素抵抗、促進(jìn)脂代謝、抑制脂肪細(xì)胞分化等途徑改善糖脂代謝。研究結(jié)果為我國(guó)蜂膠在改善代謝性疾病領(lǐng)域的應(yīng)用及相關(guān)功能性食品活性成分標(biāo)準(zhǔn)的制定提供了參考。同時(shí)，本研究證明，結(jié)構(gòu)相似比對(duì)結(jié)合網(wǎng)絡(luò)藥理學(xué)研究手段對(duì)于現(xiàn)代化功能性食品研發(fā)有重要的指引作用，可以在早期為復(fù)雜體系的物質(zhì)基礎(chǔ)及生物學(xué)機(jī)制研究提供科學(xué)證據(jù)。

結(jié)構(gòu)相似比對(duì)；網(wǎng)絡(luò)藥理學(xué)；蜂膠；代謝性疾??；黃酮類；酚酸及酯類；預(yù)測(cè)成功率

應(yīng)劍, 張波, 王春波, 等. 基于網(wǎng)絡(luò)藥理學(xué)預(yù)測(cè)我國(guó)蜂膠改善代謝性疾病的生物學(xué)機(jī)制[J]. 食品科學(xué), 2017, 38(11): 95-102. DOI:10.7506/spkx1002-6630-201711016. http://www.spkx.net.cn

YING Jian, ZHANG Bo, WANG Chunbo, et al. Systematic analysis of biological mechanisms of propolis in improving metabolic health through a network pharmacological approach[J]. Food Science, 2017, 38(11): 95-102. (in Chinese with English abstract) DOI:10.7506/spkx1002-6630-201711016. http://www.spkx.net.cn

2型糖尿病、肥胖等代謝性疾病是現(xiàn)代人面臨的重要營(yíng)養(yǎng)性疾病，并帶來了沉重的社會(huì)經(jīng)濟(jì)負(fù)擔(dān)，我國(guó)的情況尤為嚴(yán)峻。借助流行病學(xué)、藥理學(xué)等研究手段，傳統(tǒng)藥食物品改善代謝性疾病的作用逐漸被認(rèn)識(shí)，成為功能性食品開發(fā)或者藥物發(fā)現(xiàn)的重要資源。但是我國(guó)功能性食品研究水平相對(duì)較低，主要體現(xiàn)為標(biāo)準(zhǔn)化程度低、產(chǎn)品質(zhì)量不穩(wěn)定。了解功能性食品原料活性作用的物質(zhì)基礎(chǔ)和生物學(xué)機(jī)制、確定活性成分組特征，是開發(fā)質(zhì)量穩(wěn)定、功效可控的產(chǎn)品的前提。

蜂膠是蜜蜂樹膠、樹脂和樹干滲出物后，混合以蜂蠟和腺體分泌物形成的膠狀物質(zhì)，用以填補(bǔ)蜂巢、抵抗病蟲害、保護(hù)蜂群。蜂膠含有多酚、黃酮、酚醛、氨基酸和維生素等成分，具有抗菌、抗炎、抗腫瘤、增強(qiáng)免疫力、調(diào)節(jié)糖脂代謝等一系列生理活性。一項(xiàng)n = 80的臨床實(shí)驗(yàn)發(fā)現(xiàn)，226.8 mg/kg巴西綠蜂膠可以有效改善2型糖尿病人胰島素抵抗的水平[1]，嚙齒動(dòng)物模型也驗(yàn)證了蜂膠及其提取物具有改善1型[2]和2型糖尿病[3-5]癥狀、減肥[6-7]等功效。蜂膠的活性作用通過促進(jìn)胰島素分泌[8]、改善胰島素抵抗[1,4,9]、抑制脂肪細(xì)胞分化[6]、促進(jìn)脂代謝[4-5,10]等方面發(fā)揮，已知最主要的機(jī)制為抗氧化[10-12]和抗炎[3,13]。因此，蜂膠可以用于開發(fā)改善代謝性疾病的功能性食品，促進(jìn)我國(guó)人民代謝健康。為開發(fā)質(zhì)量可控、功效可控的蜂膠產(chǎn)品，需要結(jié)合藥理學(xué)研究了解其發(fā)揮糖脂代謝的主要活性成分或組分及其作用機(jī)制。現(xiàn)有研究認(rèn)為黃酮類化合物是蜂膠的主要活性部位[8,14-15]；白楊素[16]、阿特匹林C[17]、咖啡酸苯乙酯[18]、山柰酚[19-20]等成分是蜂膠發(fā)揮糖脂代謝作用的主要活性成分。但是，蜂膠的活性是多成分經(jīng)不同機(jī)制協(xié)同作用的綜合結(jié)果，目前對(duì)此尚缺乏系統(tǒng)的認(rèn)識(shí)；而且由于膠源植物存在差異，不同產(chǎn)地的蜂膠存在顯著的成分差異[21-22]。對(duì)蜂膠這一復(fù)雜體系認(rèn)識(shí)的不系統(tǒng)，為標(biāo)準(zhǔn)化蜂膠產(chǎn)品的開發(fā)帶來了困擾，一定程度上阻礙了行業(yè)的發(fā)展。這也是其他功能性食品或者藥用植物的研究所存在的共性問題。

網(wǎng)絡(luò)藥理學(xué)是近年來發(fā)展起來的一種新型研究方法，適用于考察多成分復(fù)雜體系對(duì)機(jī)體多靶點(diǎn)的活性作用，因此被應(yīng)用于中藥復(fù)方的藥理學(xué)機(jī)制研究。我國(guó)的藥食同源物品通常以提取物的形式應(yīng)用于最終產(chǎn)品，因此，這種復(fù)雜體系也適宜采用網(wǎng)絡(luò)藥理學(xué)方法予以分析[23]。本研究以我國(guó)蜂膠中主要的黃酮類、酚酸及酯類成分為研究目標(biāo)[24]，通過結(jié)構(gòu)相似比對(duì)預(yù)測(cè)其可能作用的疾病靶標(biāo)，并構(gòu)建“成分-靶標(biāo)-疾病”相互作用網(wǎng)絡(luò)，分析其改善代謝性疾病活性作用的主要通路，為科學(xué)研究蜂膠、開發(fā)標(biāo)準(zhǔn)化功能性食品提供參考依據(jù)。

1 材料與方法

1.1 靶點(diǎn)預(yù)測(cè)

對(duì)于我國(guó)蜂膠中主要的黃酮類、酚酸及酯類成分，從PubChem數(shù)據(jù)庫(kù)獲取其3D結(jié)構(gòu)信息（圖1），利用開源數(shù)據(jù)庫(kù)ChemMapper預(yù)測(cè)化合物的潛在作用靶點(diǎn)。ChemMapper的算法原理為結(jié)構(gòu)相似比對(duì)[25-27]。選擇HybridScore＞1.4的靶點(diǎn)進(jìn)行進(jìn)一步分析。

圖1 蜂膠中的主要黃酮類化合物、酚酸及酯類化合物的化學(xué)結(jié)構(gòu)[24]Fig. 1 Chemical structures of major flavonoids and phenolic acids in propolis[24]

1.2 網(wǎng)絡(luò)建立及分析

利用開源數(shù)據(jù)庫(kù)DAG獲取靶標(biāo)基因與疾病的關(guān)聯(lián)[28]。用Cytoscape3.2.1分析“化合物-疾病-靶點(diǎn)”的關(guān)聯(lián)網(wǎng)絡(luò)，發(fā)現(xiàn)關(guān)鍵成分及靶點(diǎn)。基于邊數(shù)設(shè)置節(jié)點(diǎn)顏色及圖形大小。

1.3 疾病通路分析

利用開源數(shù)據(jù)庫(kù)String10.0分析靶標(biāo)蛋白的相互作用網(wǎng)絡(luò)[29]。通過KEGG pathway enrichment算法發(fā)現(xiàn)蛋白所在疾病通路，獲取P＜1×10－4的代謝性疾病相關(guān)疾病通路作為重要通路進(jìn)行進(jìn)一步分析。

1.4 靶標(biāo)預(yù)測(cè)成功率分析

對(duì)于預(yù)測(cè)有潛在作用靶標(biāo)的成分，通過PubMed文獻(xiàn)檢索獲取其在真實(shí)實(shí)驗(yàn)中的數(shù)據(jù)。被成功預(yù)測(cè)的靶標(biāo)為Gpr；未被預(yù)測(cè)到、但位于Gpr下游的靶標(biāo)為Gpdr；未被預(yù)測(cè)到且不是Gpdr的為Gr，則：

2 結(jié)果與分析

2.1 蜂膠成分改善代謝性疾病的靶標(biāo)預(yù)測(cè)

利用結(jié)構(gòu)相似比對(duì)算法，從bindingDB、PDB、ChemBL、DrugBank的數(shù)據(jù)中發(fā)現(xiàn)與蜂膠化合物可能的作用靶標(biāo)。利用DAG數(shù)據(jù)庫(kù)獲取靶標(biāo)基因與疾病的關(guān)聯(lián)，選擇與肥胖、糖尿病相關(guān)的數(shù)據(jù)進(jìn)行分析，建立“成分-靶標(biāo)-疾病”網(wǎng)絡(luò)（圖2）。根據(jù)邊數(shù)排序，PPARγ、ESR1、ESR2、SIRT1、PTPN1是所有化合物集合潛在作用概率最高的靶標(biāo)。黃酮類化合物與酚酸及酯類化合物的潛在作用靶標(biāo)有所不同（表1），PPARγ是其共同的重要靶標(biāo)，表明這兩類化合物雖然都可能有調(diào)節(jié)糖脂代謝的活性，但其作用機(jī)制有所不同，有所協(xié)同。

圖2 蜂膠成分-靶標(biāo)-疾病相互作用網(wǎng)絡(luò)Fig. 2 Chemical-target-disease network

表1 關(guān)鍵靶標(biāo)析Table 1 Key targets from prediction

2.2 蜂膠成分改善代謝性疾病的主要成分

表2 蜂膠化合物對(duì)應(yīng)的糖脂代謝相關(guān)靶標(biāo)數(shù)Table 2 The number of predicted targets corresponding to propolis components

由表2可知，在改善代謝性疾病方面，黃酮類化合物比酚酸類化合物有更多的潛在作用靶標(biāo)，其數(shù)目均在100以上?？Х人岜揭阴ァ-香豆酸、3,4-二羥基苯甲醛和香蘭素是主要發(fā)揮作用的酚酸類成分，而阿魏酸、異阿魏酸、苯甲酸、咖啡酸、肉桂酸、肉桂酸肉桂酯等酚酸及酯類成分未獲取潛在靶標(biāo)。對(duì)于預(yù)測(cè)有潛在作用靶標(biāo)的成分，獲取其在真實(shí)實(shí)驗(yàn)中的數(shù)據(jù)（表3），計(jì)算得到靶標(biāo)預(yù)測(cè)成功率為86.2%。未能100%預(yù)測(cè)作用靶標(biāo)的主要原因在于預(yù)測(cè)的數(shù)據(jù)來源于現(xiàn)有數(shù)據(jù)庫(kù)，數(shù)據(jù)庫(kù)的完整度直接影響方法準(zhǔn)確性。此外，現(xiàn)有疾病數(shù)據(jù)庫(kù)主要關(guān)注病理狀態(tài)，而對(duì)前驅(qū)糖尿病等準(zhǔn)疾病狀態(tài)的記錄較少。如將蜂膠開發(fā)為功能性食品，其主要目標(biāo)人群應(yīng)為準(zhǔn)疾病狀態(tài)，雖然現(xiàn)有疾病數(shù)據(jù)庫(kù)有一定參考價(jià)值，但建設(shè)準(zhǔn)疾病狀態(tài)的數(shù)據(jù)庫(kù)會(huì)大有裨益。

表3 文獻(xiàn)報(bào)道蜂膠黃酮、酚酸與酯類化合物的作用靶點(diǎn)Table 3 Targets of propolis components searched from PubMed

2.3 蜂膠改善代謝性疾病的生物學(xué)通路

利用String 10.0數(shù)據(jù)庫(kù)發(fā)現(xiàn)224 個(gè)與潛在作用靶標(biāo)對(duì)應(yīng)的人源蛋白，這些蛋白分布于214 條KEGG記載的信號(hào)通路上。經(jīng)Pathway Enrichment計(jì)算，發(fā)現(xiàn)P＜1.00×10－4且與糖脂代謝相關(guān)的通路7 條（表4），是蜂膠最可能發(fā)揮活性作用的通路。

表4 String預(yù)測(cè)的KEGG通路Table 4 Enriched KEGG pathway by String prediction

對(duì)關(guān)鍵通路進(jìn)行整合，并按照潛在作用靶標(biāo)的邊數(shù)標(biāo)注顏色（圖3）?？芍淠z成分可能通過促進(jìn)糖攝取、促進(jìn)胰島素分泌、改善胰島素抵抗、促進(jìn)脂代謝、抑制脂肪細(xì)胞分化等多種功能改善糖脂代謝過程，從而改善2型糖尿病和肥胖等代謝性疾??；其靶標(biāo)及活性作用通路根據(jù)組織的不同有所差異（表5）。在胰島素相關(guān)通路上，蜂膠成分最可能作用于胰島素受體及胰島素樣生長(zhǎng)因子受體（表6）。

黃酮類化合物和酚酸類化合物是我國(guó)蜂膠中含量較為豐富的植物化學(xué)物質(zhì)。本研究通過分子結(jié)構(gòu)相似比對(duì)和網(wǎng)絡(luò)藥理學(xué)研究發(fā)現(xiàn)，蜂膠中的黃酮類化合物改善糖脂代謝的潛在靶點(diǎn)眾多，分布于胰島素分泌、葡萄糖攝取、2型糖尿病、脂肪細(xì)胞分化、脂代謝等生理和病理通路。雖然與巴西蜂膠相比，我國(guó)蜂膠中不含萜烯類活性成分阿特比林C，但黃酮類化合物的含量是其2 倍，酚酸類化合物的含量也較高，這可能是我國(guó)蜂膠與巴西蜂膠相比，輔助降血糖、降血脂[29-82]功效略優(yōu)的一方面因素。

圖3 蜂膠成分改善代謝性疾病作用的關(guān)鍵信號(hào)通路Fig. 3 Simplified signal pathways in metabolic diseases

表5 蜂膠成分的關(guān)鍵作用靶點(diǎn)及相關(guān)功能Table 5 Predicted key targets and functions of propolis components

表6 蜂膠化合物對(duì)胰島素相關(guān)功能的影響Table 6 Effect of propolis components on insulin functions

現(xiàn)階段我國(guó)慢性代謝性疾病發(fā)病率高，處于前驅(qū)糖尿病等準(zhǔn)疾病狀態(tài)的人群眾多。功能性食品作為一種健康生活方式的選擇，有助于逆轉(zhuǎn)準(zhǔn)疾病的狀態(tài)。然而，我國(guó)功能性食品研究水平較低、生產(chǎn)過程粗放、標(biāo)準(zhǔn)化程度不足，因而無法穩(wěn)定控制終產(chǎn)品的品質(zhì)，也就難以確保其健康作用。為了使功能性食品可以有效應(yīng)用，需要確定其活性成分，了解活性成分的量效關(guān)系，基于此制定產(chǎn)品標(biāo)準(zhǔn)，并為標(biāo)準(zhǔn)化生產(chǎn)工藝的開發(fā)提供參考，以控制產(chǎn)品質(zhì)量。本研究以蜂膠為例，將一種可用于預(yù)測(cè)復(fù)雜體系活性組分及其作用機(jī)制的方法引入功能性食品研究領(lǐng)域。這一方法的優(yōu)點(diǎn)在于，在了解目標(biāo)物品化學(xué)成分組成的前提下，不需經(jīng)過分離、提純和復(fù)雜的活性評(píng)價(jià)，即能預(yù)測(cè)其潛在的功能及生物學(xué)作用機(jī)制，并提示可能的活性成分，為進(jìn)一步的功能驗(yàn)證、乃至產(chǎn)品標(biāo)準(zhǔn)的制訂提供參考。本研究利用現(xiàn)有研究文獻(xiàn)數(shù)據(jù)計(jì)算預(yù)測(cè)成功率，證明該方法有一定的參考價(jià)值。

3 結(jié) 論

綜上所述，本研究利用結(jié)構(gòu)相似比對(duì)算法、網(wǎng)絡(luò)藥理學(xué)等方法研究蜂膠改善代謝性疾病的生物學(xué)機(jī)制。結(jié)果表明，蜂膠中的黃酮類化合物和部分酚酸及酯類化合物是發(fā)揮藥理學(xué)作用的主要活性成分。這些成分可能分別或者共同作用于促進(jìn)糖攝取、促進(jìn)胰島素分泌、改善胰島素抵抗、促進(jìn)脂代謝、抑制脂肪細(xì)胞分化等糖脂代謝相關(guān)通路，發(fā)揮協(xié)同作用。PPARγ、ESR1、ESR2、SIRT1、PTPN1是蜂膠化合物總體作用概率最高的靶標(biāo)。本研究從物質(zhì)基礎(chǔ)和生物學(xué)機(jī)制兩個(gè)角度，為開發(fā)基于我國(guó)蜂膠的、輔助防控代謝性疾病的功能性食品提供了科學(xué)依據(jù)。

此外，本研究的靶標(biāo)預(yù)測(cè)成功率為86.2%，表明結(jié)構(gòu)相似比對(duì)結(jié)合網(wǎng)絡(luò)藥理學(xué)研究手段可以應(yīng)用于現(xiàn)代化功能性食品研發(fā)中對(duì)復(fù)雜體系的物質(zhì)基礎(chǔ)及生物學(xué)機(jī)制研究。借助數(shù)據(jù)庫(kù)的進(jìn)一步完善，預(yù)測(cè)成功率可進(jìn)一步提升。這一研究思路將有助于促進(jìn)我國(guó)食品行業(yè)升級(jí)，推動(dòng)傳統(tǒng)藥食用物品的再開發(fā)。

[1] FUKUDA T, FUKUI M, TANAKA M, et al. Effect of Brazilian green propolis in patients with type 2 diabetes: a double-blind randomized placebo-controlled study[J]. Biomedical Reports, 2015, 3(3): 355-360. DOI:10.3892/br.2015.436.

[2] ZHU Wei, LI yinghua, CHEN Minli, et al. Protective effects of Chinese and Brazilian propolis treatment against hepatorenal lesion in diabetic rats[J]. Human and Experimental Toxicology, 2011, 30(9): 1246-1255. DOI:10.1177/0960327110387456.

[3] KITAMURA H, NAOE y, KIMURA S, et al. Beneficial effects of Brazilian propolis on type 2 diabetes in ob/ob mice: possible involvement of immune cells in mesenteric adipose tissue[J]. Adipocyte, 2013, 2(4): 227-236. DOI:10.4161/adip.25608.

[4] LI yajing, CHEN Minli, XUAN Hongzhuan, et al. Effects of encapsulated propolis on blood glycemic control, lipid metabolism, and insulin resistance in type 2 diabetes mellitus rats[J]. Evidencebased Complementary and Alternative Medicine, 2012, 2012: 981896. DOI:10.1155/2012/981896.

[5] ZAMAMI y, FUJIWARA H, HOSODA M, et al. Ameliorative effect of propolis on insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats[J]. Journal of the Pharmaceutical Society of Japan, 2010, 130(6): 833-840.

[6] SHIN Seungho, SEO Sanggwon, MIN Soyun, et al. Caffeic acid phenethyl ester, a major component of propolis, suppresses high fat diet-induced obesity through inhibiting adipogenesis at the mitotic clonal expansion stage[J]. Journal of Agricultural and Food Chemistry, 2014, 62(19): 4306-4312. DOI:10.1021/jf405088f.

[7] WASHIO K, SHIMAMOTO y, KITAMURA H. Brazilian propolis extract increases leptin expression in mouse adipocytes[J]. Biomedical Research, 2015, 36(5): 343-346. DOI:10.2220/biomedres.36.343.

[8] 楊明, 隋殿軍, 陳文學(xué), 等. 蜂膠總黃酮對(duì)四氧嘧啶糖尿病大鼠降血糖作用[J]. 中國(guó)藥學(xué)雜志, 2014(16): 1410-1413.

[9] KANG Lijun, LEE H B, BAE H J, et al. Antidiabetic effect of propolis: reduction of expression of glucose-6-phosphatase through inhibition of y279 and y216 autophosphorylation of GSK-3alpha/beta in HepG2 cells[J]. Phytotherapy Research, 2010, 24(10): 1554-1561. DOI:10.1002/ptr.3147.

[10] HU Fuliang, HEPBURN H R, XUAN Hongzhuan, et al. Effects of propolis on blood glucose, blood lipid and free radicals in rats with diabetes mellitus[J]. Pharmacological Research, 2005, 51(2): 147-152. DOI:10.1016/j.phrs.2004.06.011.

[11] BABATUNDE I, ABDULBASIT A, OLADAyO M I, et al. Hepatoprotective and pancreatoprotective properties of the ethanolic extract of nigerian propolis[J]. Journal of Intercultural Ethnopharmacology, 2015, 4(2): 102-108. DOI:10.5455/ jice.20150202023615.

[12] El-SAyED S, ABO-SALEM O, ALy H, et al. Potential antidiabetic and hypolipidemic effects of propolis extract in streptozotocin-induced diabetic rats[J]. Pakistan Journal of Pharmaceutical Sciences, 2009, 22(2): 168-174.

[13] MATSUSHIGE K, BASNET P, HASE K, et al. Propolis protects pancreatic beta-cells against the toxicity of streptozotocin (STZ)[J]. Phytomedicine, 1996, 3(2): 203-209. DOI:10.1016/s0944-7113(96)80037-7.

[14] 楊明, 隋殿軍, 陳文學(xué), 等. 蜂膠總黃酮對(duì)STZ誘導(dǎo)糖尿病大鼠降血糖機(jī)制研究[J]. 中藥材, 2014, 37(9): 1623-1626. DOI:10.13863/ j.issn1001-4454.2014.09.030.

[15] 張躍. 蜂膠降血糖有效部位化學(xué)成分研究[D]. 長(zhǎng)春: 吉林農(nóng)業(yè)大學(xué), 2012: 12.

[16] AHAD A, GANAI A A, MUJEEB M, et al. Chrysin, an antiinf l ammatory molecule, abrogates renal dysfunction in type 2 diabetic rats[J]. Toxicology and Applied Pharmacology, 2014, 279(1): 1-7. DOI:10.1016/j.taap.2014.05.007.

[17] CHOI S, CHA B y, IIDA K, et al. Artepillin C, as a PPARgamma ligand, enhances adipocyte differentiation and glucose uptake in 3T3-L1 cells[J]. Biochemical Pharmacology, 2011, 81(7): 925-933. DOI:10.1016/j.bcp.2011.01.002.

[18] OKUTAN H, OZCELIK N, yILMAZ R, et al. Effects of caffeic acid phenethyl ester on lipid peroxidation and antioxidant enzymes in diabetic rat heart[J]. Clinical Biochemistry, 2005, 38(2): 191-196. DOI:10.1016/j.clinbiochem.2004.10.003.

[19] RAJENDRAN P, RENGARAJAN T, NANDAKUMAR N, et al. Kaempferol, a potential cytostatic and cure for inf l ammatory disorders[J]. European Journal of Medicinal Chemistry, 2014, 86: 103-112. DOI:10.1016/j.ejmech.2014.08.011.

[20] UEDA M, HAyASHIBARA K, ASHIDA H. Propolis extract promotes translocation of glucose transporter 4 and glucose uptake through both PI3K- and AMPK-dependent pathways in skeletal muscle[J]. Biofactors, 2013, 39(4): 457-466. DOI:10.1002/biof.1085.

[21] 南垚, 郭伽, 鄭蓮香, 等. 蜂膠化學(xué)成分研究進(jìn)展[J]. 世界科學(xué)技術(shù), 2006, 8(1): 61-71; 56.

[22] 張楠楠, 吳健全, 高蔚娜, 等. 不同產(chǎn)地蜂膠改善糖尿病大鼠氧化應(yīng)激功效的比較研究[J]. 中國(guó)食品衛(wèi)生雜志, 2014(1): 23-26.

[23] ZHANG Shoude, SHAN Lei, LI Qiao, et al. Systematic analysis of the multiple bioactivities of green tea through a network pharmacology approach[J]. Evidence Based Complementary and Alternative Medicine, 2014, 2014: 512081. DOI:10.1155/2014/512081.

[24] 羅照明, 張紅城. 中國(guó)蜂膠化學(xué)成分及其生物活性的研究[J]. 中國(guó)蜂業(yè)中旬刊(學(xué)術(shù)), 2012, 62(2): 55-62.

[25] GONG Jianyu, CAI Chaoqian, LIU Xiaofeng, et al. ChemMapper: a versatile web server for exploring pharmacology and chemical structure association based on molecular 3D similarity method[J]. Bioinformatics, 2013, 29(14): 1827-1829. DOI:10.1093/ bioinformatics/btt270.

[26] LIU Xiaofeng, JIANG Hualiang, LI Honglin. SHAFTS: a hybrid approach for 3D molecular similarity calculation. 1. Method and assessment of virtual screening[J]. Journal of Chemical Information and Modeling, 2011, 51(9): 2372-2385. DOI:10.1021/ci200060s.

[27] LU Weiqiang, LIU Xiaofeng, CAO Xianwen, et al. SHAFTS: a hybrid approach for 3D molecular similarity calculation. 2. Prospective case study in the discovery of diverse p90 ribosomal S6 protein kinase 2 inhibitors to suppress cell migration[J]. Journal of Medicinal Chemistry, 2011, 54(10): 3564-3574. DOI:10.1021/jm200139j.

[28] SZKLARCZyK D, FRANCESCHINI A, WyDER S, et al. STRING v10: protein-protein interaction networks, integrated over the tree of life[J]. Nucleic Acids Research, 2015, 43: D447-D452. DOI:10.1093/ nar/gku1003.

[29] 朱威. 中國(guó)蜂膠和巴西蜂膠改善糖尿病大鼠的效果及對(duì)糖尿病腎病的作用機(jī)理[D]. 杭州: 浙江大學(xué), 2010: 12.

[30] AHN J, LEE H, KIM S, et al. The anti-obesity effect of quercetin is mediated by the AMPK and MAPK signaling pathways[J]. Biochemical and Biophysical Research Communications, 2008, 373(4): 545-549. DOI:10.1016/j.bbrc.2008.06.077.

[31] INDRA M R, KARyONO S K, RATNAWATI R, et al. Quercetin suppresses inflammation by reducing ERK1/2 phosphorylation and NF kappa B activation in Leptin-induced Human Umbilical Vein Endothelial Cells (HUVECs)[J]. BMC Research Notes, 2013, 6: 275. DOI:10.1186/1756-0500-6-275.

[32] LE N H, KIM C S, PARK T, et al. Quercetin protects against obesityinduced skeletal muscle inflammation and atrophy[J]. Mediators of Inf l ammation, 2014: 834294. DOI:10.1155/2014/834294.

[33] SEO M J, LEE y J, HWANG J H, et al. The inhibitory effects of quercetin on obesity and obesity-induced inf l ammation by regulation of MAPK signaling[J]. The Journal of Nutritional Biochemistry, 2015, 26(11): 1308-1316. DOI:10.1016/j.jnutbio.2015.06.005.

[34] yOUL E, MAGOUS R, CROS G, et al. MAP Kinase cross talks in oxidative stress-induced impairment of insulin secretion. Involvement in the protective activity of quercetin[J]. Fundamental and Clinical Pharmacology, 2014, 28(6): 608-615. DOI:10.1111/fcp.12078.

[35] NOH H, KIM C, KANG J, et al. Quercetin suppresses MIP-1alphainduced adipose inf l ammation by downregulating its receptors CCR1/ CCR5 and inhibiting inf l ammatory signaling[J]. Journal of Medicinal Food, 2014, 17(5): 550-557. DOI:10.1089/jmf.2013.2912.

[36] OVERMAN A, CHUANG C C, MCINTOSH M K. Quercetin attenuates inflammation in human macrophages and adipocytes exposed to macrophage-conditioned media[J]. International Journal of Obesity, 2011, 35(9): 1165-1172. DOI:10.1038/ijo.2010.272.

[37] CHEN Pin, CHEN Jingbo, CHEN Wenyu, et al. Effects of quercetin on nuclear factor-kappaB p65 expression in renal ubiquitin-proteasome system of diabetic rats[J]. Chinese Journal of Internal Medicine, 2012, 51(6): 460-465.

[38] KOBORI M, TAKAHASHI y, SAKURAI M, et al. Quercetin suppresses immune cell accumulation and improves mitochondrial gene expression in adipose tissue of diet-induced obese mice[J]. Molecular Nutrition and Food Research, 2015, 60(2): 300-312. DOI:10.1002/mnfr.201500595.

[39] MAHMOUD M, HASSAN N, El-BASSOSSy H, et al. Quercetin protects against diabetes-induced exaggerated vasoconstriction in rats: effect on low grade inf l ammation[J]. PLoS ONE, 2013, 8(5): e63784. DOI:10.1371/journal.pone.0063784.

[40] PANCHAL S, POUDyAL H, BROWN L. Quercetin ameliorates cardiovascular, hepatic, and metabolic changes in diet-induced metabolic syndrome in rats[J]. The Journal of Nutrition, 2012, 142(6): 1026-1032. DOI:10.3945/jn.111.157263.

[41] CHUANG C, MARTINEZ K, XIE G X, et al. Quercetin is equally or more effective than resveratrol in attenuating tumor necrosis factor-{alpha}-mediated inflammation and insulin resistance in primary human adipocytes[J]. The American Journal of Clinical Nutrition, 2010, 92(6): 1511-1521. DOI:10.3945/ajcn.2010.29807.

[42] DONG Jing, ZHANG Xian, ZHANG Lei, et al. Quercetin reduces obesity-associated ATM infiltration and inflammation in mice: a mechanism including AMPKalpha1/SIRT1[J]. Journal of Lipid Research, 2014, 55(3): 363-374. DOI:10.1194/jlr.M038786.

[43] EID H, NACHAR A, THONG F, et al. The molecular basis of the antidiabetic action of quercetin in cultured skeletal muscle cells and hepatocytes[J]. Pharmacognosy Magazine, 2015, 11(41): 74-81. DOI:10.4103/0973-1296.149708.

[44] ZHAO Lirong, DU yujun, CHEN Lei, et al. Quercetin protects against high glucose-induced damage in bone marrow-derived endothelial progenitor cells[J]. International Journal of Molecular Medicine, 2014, 34(4): 1025-1031. DOI:10.3892/ijmm.2014.1852.

[45] ESEBERRI I, MIRANDA J, LASA A, et al. Doses of quercetin in the range of serum concentrations exert delipidating effects in 3T3-L1 preadipocytes by acting on different stages of adipogenesis, but not in mature adipocytes[J]. Oxidative Medicine and Cellular Longevity, 2015, 2015: 480943. DOI:10.1155/2015/480943.

[46] JUNG C, CHO I, AHN J, et al. Quercetin reduces high-fat diet-induced fat accumulation in the liver by regulating lipid metabolism genes[J]. Phytotherapy Research, 2013, 27(1): 139-143. DOI:10.1002/ptr.4687.

[47] SEO y, KANG O, KIM S, et al. Quercetin prevents adipogenesis by regulation of transcriptional factors and lipases in OP9 cells[J]. International Journal of Molecular Medicine, 2015, 35(6): 1779-1785. DOI:10.3892/ijmm.2015.2185.

[48] KOBORI M, MASUMOTO S, AKIMOTO y, et al. Chronic dietary intake of quercetin alleviates hepatic fat accumulation associated with consumption of a Western-style diet in C57/BL6J mice[J]. Molecular Nutrition and Food Research, 2011, 55(4): 530-540. DOI:10.1002/ mnfr.201000392.

[49] ALAM M, MEERZA D, NASEEM I. Protective effect of quercetin on hyperglycemia, oxidative stress and DNA damage in alloxan induced type 2 diabetic mice[J]. Life Sciences, 2014, 109(1): 8-14. DOI:10.1016/j.lfs.2014.06.005.

[50] SHIMIZU M, LI J, INOUE J, et al. Quercetin represses apolipoprotein B expression by inhibiting the transcriptional activity of C/EBPbeta[J]. PLoS ONE, 2015, 10(4): e0121784. DOI:10.1371/journal.pone.0121784.

[51] KIM C, KWON y, CHOE S, et al. Quercetin reduces obesity-induced hepatosteatosis by enhancing mitochondrial oxidative metabolism via heme oxygenase-1[J]. Nutrition and Metabolism, 2015, 12: 33. DOI:10.1186/s12986-015-0030-5.

[52] BUMKE-VOGT C, OSTERHOFF M A, BORCHERT A, et al. The fl avones apigenin and luteolin induce FOXO1 translocation but inhibit gluconeogenic and lipogenic gene expression in human cells[J]. PLoS ONE, 2014, 9(8): e104321. DOI:10.1371/journal.pone.0104321.

[53] SUH K S, OH S, WOO J T, et al. Apigenin attenuates 2-deoxy-D-ribose-induced oxidative cell damage in HIT-T15 pancreatic betacells[J]. Biological and Pharmaceutical Bulletin, 2012, 35(1): 121-126.

[54] KIM M A, KANG K, LEE H J, et al. Apigenin isolated from Daphne genkwa Siebold et Zucc. inhibits 3T3-L1 preadipocyte differentiation through a modulation of mitotic clonal expansion[J]. Life Sciences, 2014, 101(1/2): 64-72. DOI:10.1016/j.lfs.2014.02.012.

[55] LI Rui, ZANG Aihua, ZHANG Lei, et al. Chrysin ameliorates diabetes-associated cognitive deficits in Wistar rats[J]. Neurological Sciences, 2014, 35(10): 1527-1532. DOI:10.1007/s10072-014-1784-7. [56] FENG Xiujing, QIN Haohan, SHI Qian, et al. Chrysin attenuates inflammation by regulating M1/M2 status via activating PPARgamma[J]. Biochemical Pharmacology, 2014, 89(4): 503-514. DOI:10.1016/j.bcp.2014.03.016.

[57] LEE J, JUNG E, JIENNy L, et al. Isorhamnetin represses adipogenesis in 3T3-L1 cells[J]. Obesity (Silver Spring), 2009, 17(2): 226-232. DOI:10.1038/oby.2008.472.

[58] ByUN Miran, JEONG Hana, BAE Sujung, et al. TAZ is required for the osteogenic and anti-adipogenic activities of kaempferol[J]. Bone, 2012, 50(1): 364-372. DOI:10.1016/j.bone.2011.10.035.

[59] FANG Xiankang, GAO Jie, ZHU Danni. Kaempferol and quercetin isolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activity[J]. Life Sciences, 2008, 82(11/12): 615-622. DOI:10.1016/j.lfs.2007.12.021.

[60] LEE y, CHOI H, SEO M, et al. Kaempferol suppresses lipid accumulation by inhibiting early adipogenesis in 3T3-L1 cells and zebraf i sh[J]. Food and Function, 2015, 6(8): 2824-2833. DOI:10.1039/ c5fo00481k.

[61] PARK U, JEONG J, JANG J, et al. Negative regulation of adipogenesis by kaempferol, a component of Rhizoma Polygonati falcatum in 3T3-L1 cells[J]. Biological and Pharmaceutical Bulletin, 2012, 35(9): 1525-1533.

[62] LUO Cheng, yANG Hui, TANG Chengyong, et al. Kaempferol alleviates insulin resistance via hepatic IKK/NF-kappaB signal in type 2 diabetic rats[J]. International Immunopharmacology, 2015, 28(1): 744-750. DOI:10.1016/j.intimp.2015.07.018.

[63] ZHANG yanling, LIU Dongmin. Flavonol kaempferol improves chronic hyperglycemia-impaired pancreatic beta-cell viability and insulin secretory function[J]. European Journal of Pharmacology, 2011, 670(1): 325-332. DOI:10.1016/j.ejphar.2011.08.011.

[64] ZHANG yanling, ZHEN Wei, MAECHLER P, et al. Small molecule kaempferol modulates PDX-1 protein expression and subsequently promotes pancreatic beta-cell survival and function via CREB[J]. The Journal of Nutritional Biochemistry, 2013, 24(4): 638-646. DOI:10.1016/j.jnutbio.2012.03.008.

[65] ALKHALIDy H, MOORE W, ZHANG y L, et al. Small molecule Kaempferol promotes insulin sensitivity and preserved pancreatic betacell mass in middle-aged obese diabetic mice[J]. Journal of Diabetes Research, 2015, 2015: 532984. DOI:10.1155/2015/532984.

[66] CHOI H, KANG M J, LEE S, et al. Ameliorative effect of myricetin on insulin resistance in mice fed a high-fat, high-sucrose diet[J]. Nutrition Research and Practice, 2014, 8(5): 544-549. DOI:10.4162/ nrp.2014.8.5.544.

[67] KANDASAMy N, ASHOKKUMAR N. Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin-cadmium induced diabetic nephrotoxic rats[J]. Toxicology and Applied Pharmacology, 2014, 279(2): 173-185. DOI:10.1016/j.taap.2014.05.014.

[68] LIU I, TZENG T, LIOU S, et al. Improvement of insulin sensitivity in obese Zucker rats by myricetin extracted from Abelmoschus moschatus[J]. Planta Medica, 2007, 73(10): 1054-1060. DOI:10.1055/ s-2007-981577.

[69] LIU I, LIOU S, CHENG J. Mediation of beta-endorphin by myricetin to lower plasma glucose in streptozotocin-induced diabetic rats[J]. Journal of Ethnopharmacology, 2006, 104(1/2): 199-206. DOI:10.1016/j.jep.2005.09.001.

[70] FENG Jianfang, CHEN Xiaonan, WANG yuanyuan, et al. Myricetin inhibits proliferation and induces apoptosis and cell cycle arrest in gastric cancer cells[J]. Molecular and Cellular Biochemistry, 2015, 408(1/2): 163-170. DOI:10.1007/s11010-015-2492-1.

[71] BEZERRA R M N, VEIGA L F, CAETANO A C, et al. Caffeic acid phenethyl ester reduces the activation of the nuclear factor kappaB pathway by high-fat diet-induced obesity in mice[J]. Metabolism, 2012, 61(11): 1606-1614. DOI:10.1016/j.metabol.2012.04.006.

[72] CELIK S, ERDOGAN S. Caffeic acid phenethyl ester (CAPE) protects brain against oxidative stress and inf l ammation induced by diabetes in rats[J]. Molecular and Cellular Biochemistry, 2008, 312(1/2): 39-46. DOI:10.1007/s11010-008-9719-3.

[73] HASSAN N, El-BASSOSSy H, MAHMOUD M, et al. Caffeic acid phenethyl ester, a 5-lipoxygenase enzyme inhibitor, alleviates diabetic atherosclerotic manifestations: effect on vascular reactivity and stiffness[J]. Chemico-Biological Interactions, 2014, 213: 28-36. DOI:10.1016/j.cbi.2014.01.019.

[74] JUMAN S, yASUI N, IKEDA K, et al. Caffeic acid phenethyl ester suppresses the production of pro-inflammatory cytokines in hypertrophic adipocytes through lipopolysaccharide-stimulated macrophages[J]. Biological and Pharmaceutical Bulletin, 2012, 35(11): 1941-1946.

[75] CELIK S, ERDOGAN S, TUZCU M. Caffeic acid phenethyl ester (CAPE) exhibits significant potential as an antidiabetic and liver-protective agent in streptozotocin-induced diabetic rats[J]. Pharmacological Research, 2009, 60(4): 270-276. DOI:10.1016/ j.phrs.2009.03.017.

[76] PARK S, MIN T. Caffeic acid phenethyl ester ameliorates changes in IGFs secretion and gene expression in streptozotocin-induced diabetic rats[J]. Life Sciences, 2006, 78(15): 1741-1747. DOI:10.1016/ j.lfs.2005.08.011.

[77] KIM J, PARK S, yU M, et al. Effect of Ganoderma applanatum mycelium extract on the inhibition of adipogenesis in 3T3-L1 adipocytes[J]. Journal of Medicinal Food, 2014, 17(10): 1086-1094. DOI:10.1089/jmf.2013.3036.

[78] KIM y S, KIM N H, LEE S W, et al. Effect of protocatechualdehyde on receptor for advanced glycation end products and TGF-beta1 expression in human lens epithelial cells cultured under diabetic conditions and on lens opacity in streptozotocin-diabetic rats[J]. European Journal of Pharmacology, 2007, 569(3): 171-179. DOI:10.1016/j.ejphar.2007.05.054.

[79] CHENG J T, LIU I M, TZENG T F, et al. Release of beta-endorphin by caffeic acid to lower plasma glucose in streptozotocin-induced diabetic rats[J]. Hormone and Metabolic Research, 2003, 35(4): 251-258. DOI:10.1055/s-2003-39482.

[80] NATARELLI L, RANALDI G, LEONI G, et al. Nanomolar caffeic acid decreases glucose uptake and the effects of high glucose in endothelial cells[J]. PLoS ONE, 2015, 10(11): e0142421. DOI:10.1371/journal.pone.0142421.

[81] CHAO C, MONG M, CHAN K, et al. Anti-glycative and antiinflammatory effects of caffeic acid and ellagic acid in kidney of diabetic mice[J]. Molecular Nutrition and Food Research, 2010, 54(3): 388-395. DOI:10.1002/mnfr.200900087.

[82] 王雪梅, 王飛飛, 田海霞, 等. 巴西蜂膠和國(guó)產(chǎn)蜂膠乙醇濃縮液對(duì)2型糖尿病大鼠糖、脂代謝的影響研究[J]. 糖尿病新世界, 2015(17): 56-59. DOI:10.3969/j.issn.1672-4062.2015.17.025.

Systematic Analysis of Biological Mechanisms of Propolis in Improving Metabolic Health through a Network Pharmacological Approach

YING Jian, ZHANG Bo, WANG Chunbo, WANG Chunling*
(COFCO Nutrition and Health Research Institute, Beijing 102209, China)

Chronic metabolic diseases, such as type 2 diabetes and obesity, have brought a huge burden to our society. Certain functional foods could bring health benef i ts to people with metabolic abnormalities, and sometimes help delay the onset of metabolic diseases. Propolis is a traditional Chinese medicine, and has been used as a raw material of functional foods for quite a long time. Pharmacological studies and clinical trials have provided evidence that propolis and its active components could be promising candidates for improving metabolic health. In order to develop standardized functional foods with consistent quality and functions, a systemic view of the mechanism of action is required. We should also be aware that propolis is a combination of multiple active components. Network pharmacology is a recently developed method as an integrative system which enables a systemic investigation of interactions between multiple components and multiple targets. The method of network pharmacology has been used in studies of traditional Chinese medicine. In order to survey the molecular mechanisms of propolis components in treating metabolic diseases, we use structural similarity search to predict therapeutic targets of propolis fl avonoids, phenolic acids and esters, and to construct a ‘component-target-disease’ network. Comparing with published data, we calculated that the success ratio of prediction was 86.2%. Based on the network, we concluded that PPARγ, ESR1, ESR2, SITR1 and PTPN1 are key targets of propolis. PPARγ is the most important target for both fl avonoids and phenolic acids. Propolis fl avonoids and some phenolic acids and esters contribute to the regulation of glucose and lipid metabolism by propolis through various pathways such as lipid metabolism, adipocyte differentiation, insulin secretion and insulin resistance. Based on the results of our study, we introduced a new research tool that can be used in the early stage of functional food development. We found that network pharmacology could provide information forformulating product standards for functional foods, which is important for innovation and upgrading of food industry.

structural similarity search; network pharmacology; propolis; metabolic diseases; fl avonoids; phenolic acids and esters; prediction ratio

10.7506/spkx1002-6630-201711016

S896

A

1002-6630（2017）11-0095-08引文格式：

2016-04-08

國(guó)家高技術(shù)研究發(fā)展計(jì)劃（863計(jì)劃）項(xiàng)目（2014AA021503）；北京市科技計(jì)劃課題（Z161100000616012）

應(yīng)劍（1984—），女，高級(jí)工程師，博士，研究方向?yàn)樗幨秤弥参镅芯颗c開發(fā)。E-mail：yingjian@cofco.com

*通信作者：王春玲（1972—），女，高級(jí)工程師，博士，研究方向?yàn)闋I(yíng)養(yǎng)科學(xué)。E-mail：wangchunling@cofco.com