天然小分子诱生机体抗病毒细胞因子研究

胡小鹏，金 哲，贺震旦

深圳大学医学部医学院药学系 深圳大学生物治疗研究所 深圳天然小分子创新药物工程实验室，深圳 518060

中医药具有五千多年的历史，国内外科学家对中药、天然药、民间药和民族药物进行了大量的研究，具有抗病毒药用植物约100 余种［1］，如金银花(Lonicera japonica)、黄芩(Scutellaria baicalensis)、黄芪(Astragalus membranaceus)、五味子(Schisandra chinensis(Turcz.))、夏枯草(Prunella vulgaris)、连翘(Forsythia suspensa)等(见表1)，据迄今为止的研究表明天然小分子(Natural micromolecule)为这些抗病毒药用植物活性的物质基础。

抗病毒细胞因子(Antiviral cytokines)曾在Nasu K［2］和Fakruddin JM［3］等人发表的学术论文中提及过，但未曾对其作出具体的定义。为系统地开展细胞因子抗病毒作用及其作用机制研究的研究，有必要对抗病毒细胞因子进行明确地定义:抗病毒细胞因子是由机体细胞分泌的，具有联动机体固有免疫和特异性免疫应答，进而抑制或捕杀体内病毒的小分子蛋白。抗病毒细胞因子可分两大类:一类是可直接作用体细胞产生抗病毒蛋白，即1)干扰素(Interferon):IFN-α、IFN-β、IFN-γ 和IFN-λ(IL-28、IL-29)［4-7］;2)肿 瘤 坏 死 因 子(Tumor Necrosis Factor)［8］:TNF-α 和TNF-β;干扰素和肿瘤坏死因子可直接激活细胞内JAK(Janus activated kinase)-STAT(signal transducer and activator of transcription)信号通路［9-13］，随后引起下游信号级联反应，包括MTOR信号通路、PI3K 信号通路、MAPK 信号通路均被激活，最终启动细胞核中一些关键的蛋白基因进行转录，翻译出抗病毒蛋白进而对细菌、病毒以及癌细胞等产生防御作用［14］(见图1)。另一类是能激活巨噬细胞、自然杀伤细胞(NK 细胞)和毒性T 淋巴细胞(CTL)等细胞，进而捕杀或吞噬体内病毒的细胞因子(见图1)，即白介素(Interleukin):IL-1β、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-21、IL-27等［15-20］。

基于细胞因子对捕杀或抑制病毒方面的作用，本文综述了国内外迄今为止报道的可诱生抗病毒细胞因子的天然小分子(见图2，图3)，其中以多酚类、苷类居多，还有部分物质基础未明确的提取物，有待进一步研究。

1 多酚类化合物(Polyphenolic compounds)

白藜芦醇(1，Resveratrol，10 mol/L)能上调人外周血单核细胞IL-1β、IL-6、TNF-α，调节免疫细胞功能［21］。从姜黄(Curcuma longa)根茎分离的天然多酚姜黄素(2，Curcumin)，通过激活人细胞内MAPK以及MTOR 等信号通路，调节下游STATs 蛋白磷酸化水平，控制TNF-α、IL-1 和IL-6 的表达分泌［22］，姜黄素还可以显著的调控TRAIL(TNF 相关凋亡诱导配基)，有研究显示姜黄素(10 μmol/L 和25 μmol/L)和TRAIL(20 ng/mL)联合使用能够促进胞内半胱天冬酶的前体Procaspase-3，8，9 的分裂，并使得线粒体释放细胞色素C，导致细胞毒性的增强［23］。茶多酚复方制剂(50～150 mg/kg)可增强小鼠淋巴细胞增殖能力，提高血清溶菌酶活性，上调血清中IFN-γ 分泌［24］。番茄(Lycopersicum esculentum)的多酚提取物能够调节牛中性粒细胞TNF-α 基因的表达，加热处理的提取物可显著上调IFN-γ 的分泌［25］，具有增强动物和人类固有免疫的潜在活性。由咖啡酸(3，Caffeic acid)、阿魏酸(4，Ferulic acid)和对香豆酸(5，p-Coumaric acid)酶促聚合的多酚(20～100 μg/mL)，通过和T 细胞表面CD4+蛋白结合，激活下游信号通路后上调小鼠脾脏细胞中的IFN-γ 和GM-CSF(Granulocyte-macrophage Colonystimulating Factor)分泌量［26］，增强机体固有免疫。

从Cocoa 提取的黄酮类化合物，能够减少小鼠腹膜巨噬细胞活性氧和NO 的生成，上调IFN-γ 和TNF-α 的作用［27］。中药黄芪(Astragalus membranaceus)和黄芩(Scutellaria baicalensis)是传统的抗病毒药材，主含槲皮素(6，Quercetin)、黄芩素(7，Baicalein)等天然小分子。Nair［28］等人报道，槲皮素(5 μmol/L)可调节外周血单核细胞IFN-γ 和IL-4 的分泌，从而促使Th0 细胞向Th1 细胞分化;随后进行了攻毒试验，发现诱生的IFN-γ 具有很强的抗VSV(vesicular stomatitis virus)活性。Johari 等［29］报道槲皮素和黄芩素抑制JEV(Japanese Encephalitis Virus)的IC50分别为212.1、14.28 μg/mL，提示黄芩素比槲皮素具有更好的抗JEV 作用。有研究表明，黄芩素可直接抑制病毒血凝素和神经氨酸酶［30］。

桂皮醛(13，Cinnamaldehyde，1 mg/kg)［31，32］可上调小鼠血清中IL-2、IL-10 的分泌量，其诱生机制可能与Toll 样受体的激活有关。从百屈菜中提取得到 血 根 碱(14，6-acetonyl-5，6-dihydrosanguinarine)［33］，体外实验证实其可诱导巨噬细胞和树突状细胞分泌TFN-α、IL-6、IL-8 等抗病毒细胞因子，对其诱生机制的研究表明，血根碱可激活MAPK 通路中ERK，NF-kB 循环活化机制也参与其中，共同调控了下游抗病毒细胞因子的表达。朱晓新等人［34］从紫萁贯众分离得到四种多酚化合物:4＇-hydroxybenzalacetone(HBAc)，3＇，4＇-dihydroxybenzalacetone(DHBAc)、4-hydroxybenzaldehyde(HBAI)、3，4-dihydroxybenzaldehyde(DHBAI)，其中HBAI(15，10-100 ug/mL)、DHBAI(16，10-100 ug/mL)可激活胞内NF-kB 通路，可显著上调小鼠腹膜细胞分泌TNF、IL-1β、IFN-γ 等，诱生机制可能和iNOS(一氧化氮合成酶)的激活和NO 的生成有关。

2 苷类化合物(Glycosides)

研究表明苷类化合物具有抗肿瘤抗病毒活性［35］;桔梗(Platycodon grandiflorum)中分离得到的单体桔梗苷D(8，Platycodin D，0.0016～0.2 μg/mL)［36］，能够上调IL-2、IFN-γ 的mRNA 表达水平，调控Th1/Th2 细胞的转录因子T-bet 和CATA-3 表达，同时，桔梗苷D 可以提高OVA 致敏小鼠抗体水平，具有免疫调节作用。王先远等［37］研究苦瓜总皂苷对衰老小鼠免疫功能的影响，发现苦瓜总皂苷不仅可上调血清中IL-2 水平，而且促进脾脏分泌IL-2及腹腔巨噬细胞分泌TNF-α，其机制可能与DNA 多聚酶II 活性增强有关;苦瓜总皂苷促进IL-2 分泌，改变T 细胞亚群的组成，使机体免疫状态趋向年轻化。徐先祥等［38］人报道甘草皂苷(9，0.5 mg/mL)可抑制98%以上的HIV 增殖，0.125 mg/mL 时，可抑制98%以上的HIV 的增殖，并可抑制50%斑块的形成，其作用机制是诱生干扰素和增强NK 细胞的活性抵抗HIV。柚皮苷(17，naringin，20，40 and 80 mg/kg)［39］可上调Th2 型细胞分泌IL-4、IL-6、IL-10等。

表1 抗病毒药用植物Table 1 Antivial Medical Plants

注:* 已报道的可诱生抗病毒细胞因子的天然小分子Note:* These natural micromolecules have been reported to induce antiviral cytokines.

图1 天然小分子诱生细胞因子抗病毒作用机制Fig.1 The mechanisms of antiviral activity of antiviral cytokines induced by natrual micromolecule

图2 诱生抗病毒细胞因子的天然小分子1～12 的化学结构式Fig.2 The chemical structure of natural micromolecule 1-12 with the function of inducing antiviral cytokines

图3 诱生抗病毒细胞因子的天然小分子13～19 的化学结构式Fig.3 The chemical structure of natural micromolecule 13-19 with the function of inducing antiviral cytokines

另外，本课题组开展天然小分子诱生机体抗病毒细胞因子的研究中，发现天麻素(10，Gastrodin，0～1000 μmol/L)、迎春花素(11，Jasminin，1-100 μmol/L)能够显著上调RAW246.7 细胞分泌TNFα;从苦丁茶紫茎女贞(Ligustrum purpurascens)叶提取得到苯丙素总苷(crude phenylethanoid glycosides)，属多酚类化合物，活性测试表明，苯丙素总苷(0.22～1.32 g/kg)具有免疫增强作用［49］;进一步研究还发现，从苯丙素总苷分离得到的单体阿克苷(12，acteoside，0.8～200 μg/mL)，能够显著上调Balb/c 小鼠体内IFN-γ 的表达水平，动物攻毒实验证实可显著抑制小鼠体内病毒载量。

3 其他

大蒜辣素(18，Allitridin，25 mg/kg/day)［40］可调节CMV 感染小鼠体内Foxp3 蛋白表达，上调机体IFN-γ 和IL-4 分泌，恢复CMV 攻击造成的免疫系统紊乱。青蒿素(19，Artemisinin)［41］可显著刺激巨噬细胞分泌IL-12，其诱生机制和JNK 磷酸化的抑制有关。

滑菇(Pholiota nameko)提取物(20～60 mg/kg)［42］能够上调白介素、肿瘤坏死因子、干扰素等多种抗病毒细胞因子的分泌。

此外，许多中药提取物具有免疫增强作用［43］，如黄芪提取物(100～200 μg/mL)、板蓝根提取物(100～200 μg/mL)能够刺激鸡外周血淋巴细胞IFN-γ 的mRNA 的表达量，并对NDV(Newcastle disease virus)感染有抑制作用。人参叶提取物［44］可显著上调感染H5N1 小鼠和雪貂肺部IFN-γ 分泌，与空白对照组相比，动物的存活率可提高至40%～45%。五味子［45］和太子参［46］提取物，均可显著诱导正常细胞生成TNF-α。商路提取物(50 mg/kg)［47］处理小鼠，每周喂食一次，可显著刺激小鼠淋巴细胞增殖，产生IL-2，如果改变喂食频率，一周三次，IL-2 的分泌量则开始下降;此外，当剂量调整为10 mg/kg，每周三次喂食，则可以刺激巨噬细胞分泌IL-1，TNF，推测商路提取物可增强小鼠中巨噬细胞的活性，杀伤病变细胞。蓼大青叶提取物(1～100 mg/kg)［48］可显著增加小鼠脾脏IFN-γ 的分泌。

4 结语

目前，用于临床的抗病毒药物主要有化学药物和疫苗两类，而通过诱生抗病毒细胞因子途径的药物有望成为第三类新型抗病毒药物。药用植物中的天然小分子结构复杂，资源丰富，成药性强，然而目前有关天然小分子诱生抗病毒细胞因子的研究缺乏系统性，尚待进行深入研究。对抗病毒药用植物进行提取分离及纯化，进行体外细胞模型筛选，并开展动物免疫学和动物病毒学研究，进而发现活性天然小分子，通过系统的基础研究，有望研发出诱生机体抗病毒细胞因子的抗感染性疾病的临床药物，我们预计此类相关的研究将成为抗病毒药物研发的新途径。

1 Chinese Pharmacopoeia Commission(国家药典委员会).Pharmacopoeia of the People’s Republic of China(中华人民共和国药典).Beijing:China Medical Science Press，2010.Vol Ⅰ，1-362.

2 Nasu K，et al.Human oviductal epithelial cells express Tolllike receptor 3 and respond to double-stranded RNA:Fallopian tube-specific mucosal immunity against viral infection.Hum reprod，2007，22:356-361.

3 Fakruddin JM，et al.Noninfectious papilloma virus-like particles inhibit HIV-1 replication:implications for immune control of HIV-1 infection by IL-27.Blood，2007，109:1841-1849.

4 Poecka H，Ruland J.2012.From virus to inflammation:Mechanisms of RIG-I-induced IL-1β production.Eur J Cell Biol，91:59-64.

5 Brand S，et al.IL-28A and IL-29 mediate antiproliferative and antiviral signals in intestinal epithelial cells and murine CMV infection increases colonic IL-28A expression.Am J Physiol，2005，289:960-968.

6 Rossol-Voth R，et al.In vivo protective effect of tumour necrosis factor alpha against experimental infection with herpes simplex virus type 1.J Gen Virol，1991，72:143-147.

7 Sharma B，et al.Protein Kinase R as Mediator of the effects of interferon(IFN)γ and tumor necrosis factor(TNF)α on normal and dysplastic hematopoiesis.J Biol Chem，2011，286:27506-27514.

8 Xu SH(许沈华)，Zhang ZX(张宗显).The advance research of the effect of tumor necrosis factor on anti-tumor.Foreign Medical Sciences(国外医学)，1989，(3):134-138.

9 Claudio A.Bonjardim，et al.Interferons:Signaling，antiviral and viral evasion.Immunology Letters，2009，122:1-11.

10 Hsu YM，et al.The adaptor protein CARD9 is required for innate immune responses to intracellular pathogens.Nat Immunol，2007，8:198-205.

11 Colonna M.TLR pathways and IFN-regulatory factors:to each its own.Eur J Immunol，2007，37:306-309.

12 Baccala R，et al.TLR-dependent and TLR-independent pathways of type I interferon induction in systemic autoimmunity.Nat Med，2007，13:543-551.

13 Schindler C，PlumLee C.Inteferons pen the JAK-STAT pathway.Semin Cell Dev Biol，2008，19:311-318.

14 Bartee E，et al.The addition of tumor necrosis factor plus beta interferon induces a novel synergistic antiviral state against poxviruses in primary human fibroblasts.J Virol，2009，83:498-511.

15 Vijay AK，et al.Inflammasomes and anti-viral immunity.J Clin Immunol，2010，30:632-637.

16 Sutlu T，et al.Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells:Implications for gene therapy.Hum Gene Ther，2012，23:1090-1100.

17 Liao Y，et al.Regulation of the p38 mitogen-activated protein kinase and dual-specificity phosphatase 1 feedback loop modulates the induction of interleukin 6 and 8 in cells infected with coronavirus infectious bronchitis virus.Virology，2011，420:106-116.

18 de Groot R，et al.Viral dsRNA-activated human dendritic cells produce IL-27，which selectively promotes cytotoxicity in naive CD8+T cells.J Leukoc Biol，2012，92:605-610.

19 Verbist KC，Lonowski KD.Functions of IL-15 in anti-viral immunity:Multiplicity and variety.Cytokine，2012，9:467-478.

20 Ryzhakov G，et al.IL-17 Boosts Proinflammatory outcome of antiviral response in human cells.J Immunol，2011，187:5357-5362.

21 Bergman M，et al.Resveratrol affects the cross talk between immune and colon cancer cells.Biomed ＆ Pharmacother，2013，67:43-47.

22 Zhou HY，et al.Targets of curcumin.Curr Drug，2011，12:332-347.

23 Deeb D，et al.Curcumin sensitizes prostate cancer cells to tumor necrosis factor-related apoptosis-inducing ligand/Apo2L by inhibiting nuclear factor-kB through suppression of IkBαphosphorylation.Mol Cancer Ther，2004，3:803-812.

24 Wang FR(王芙蓉)，et al.Effect of drug combinations from tea polyphenol on immune system of mice.China Animal Husbandry ＆ Veterinary Medicine(中国畜牧兽医)，2006，33(9):11-12.

25 Gyenai K，et al.Influence of Heirloom tomato polyphenol extracts on expression of inflammation genes In Bovine.American Journal of Animal and Veterinary Sciences，2012，7:126-135.

26 Yamanaka D，et al.The effect of enzymatically polymerised polyphenols on CD4 binding and cytokine production in murine splenocytes.Plos One，2013，7(4):e36025.

27 Ramos RS，et al.Effect of a cocoa flavonoid-enriched diet on experimental autoimmune arthritis.Br J Nutr，2012，107:523-532.

28 Nair MP，et al.The flavonoid，quercetin，differentially regulates Th-1(IFNgamma)and Th-2(IL4)cytokine gene expression by normal peripheral blood mononuclear cells.Biochimica et Biophysica Acta，2002，1593:29-36.

29 Johari J，et al.Antiviral activity of baicalein and quercetin against the japanese encephalitis virus.International journal of molecular sciences，2012，13:16785-16795.

30 Dou J，et al.Effects of baicalein on Sendai virus in vivo are linked to serum baicalin and its inhibition of hemagglutininneuraminidase.Arch Virol，2011，156:793-801.

31 Lin SS，et al.In vivo cytokine modulatory effects of cinnamaldehyde，the major constituent of leaf essential oil from Cinnamomum osmophloeum Kaneh.Phytother Res，2011，25:1511-1518.

32 Ding YY(丁媛媛)，et al.Influence of cinnamaldehyde on coxsackie virus B3 induced viral myocarditis in mice.Nat Prod Res Dev(天然产物研究与开发)2010，22:769-776.

33 Kim DH，et al.6-Acetonyl-5，6-dihydrosanguinarine(ADS)from Chelidonium majus L.triggers proinflammatory cytokine production via ROS-JNK/ERK-NF-κB signaling pathway.Food Chem Toxicol，2013，58:273-279.

34 Zhu XX，et al.Divergent immunomodulatory effects of extracts and phenolic compounds from the Osmunda japonica Thunb.Chin J Integr Med.2013，19:761-770.

35 Lee J，et al.Saponin inhibits hepatitis C virus propagation by upregulating suppressor of cytokine signaling 2.Plos One，2012，7(6):e39366.

36 Xie Y，et al.Contribution of the glycidic moieties to the haemolytic and adjuvant activity of platycodigenin-type saponins from the root of Platycodon grandi?orum.Vaccine，2008，26:3452-3460.

37 Wang XY(王先远)，et al.Effects of Momordca charantia L saponins on immune system of senile mice.Acta Nutrimenta Sinica(营养学报)，2001，23:263-265.

38 Xu XX(徐先祥)，Dai Y(戴岳).The overview of the effect of saponins from traditional Chinese medicine on anti-virus.Pharmacology and Clinics of Chinese Materia Medica(中药药理与临床)，2008，24:110-111.

39 Ahmad SF1，Zoheir KM2，Abdel-Hamied HE3，Amelioration of autoimmune arthritis by naringin through modulation of T regulatory cells and Th1/Th2 cytokines.Cell Immunol，2014 287:112-120.

40 Li YN，et al.Allium sativum-derived allitridin inhibits Treg amplification in cytomegalovirus infection.J Med Virol.2013，85:493-500.

41 Cho YC，et al.Enhanced IL-12p40 production in LPS-stimulated macrophages by inhibiting JNK activation by artemisinin.Arch Pharm Res，2012，35:1961-1968.

42 Li HP，et al.Effects of the polysaccharide from Pholiota nameko on human cytokine network in serum.Int J Biol Macromol，2012，50:164-170.

43 Qiu Y(邱妍).Studies on immune-enhancing and antivirial action and mechanism of four Chinese herbal polysaccharides.Nanjing:Nanjing Agricultural University(南京农业大学)，PhD.2007.

44 Park EH，et al.Red Ginseng-containing diet helps to protect mice and ferrets from the lethal infection by highly pathogenic H5N1 influenza virus.J Ginseng Res.2014，38:40-46.

45 Zhao T，et al.Schisandra polysaccharide evokes mmunomodulatory activityh through TLR 4-mediated activation of macrophages.Int J Biol Macromol.2014，10，65C:33-40.

46 Wong CK，et al.Tumor necrosis factor eliciting fractions separated from Pseudostellaria heterophylla.Int J Immunopharmacol，1994，16:271-277.

47 Wang HB，Zheng QY.Effects of Phytolacca acinosa polysaccharides I with different schedules on its antitumor efficiency in tumor bearing mice and production of IL-1，IL-2，IL-6，TNF，CSF activity in normal mice.Immunotoxicol，1997，19:197-213.

48 Jeong HJ，et al.Inhibition of IL-32 and TSLP production through the attenuation of caspase-1 activation in an animal model of allergic rhinitis by Naju Jjok(Polygonum tinctorium).Int J Mol Med，2014，33:142-150.

49 Song X，et al.Immunomodulatory effects of crude phenylethanoid glycosides from Ligustrum purpurascens.J Ethnopharmacol，2012，144:584-591.

50 Kim YJ，et al.Functional analysis of HMGR encoding genes in triterpene saponin-producing Panax ginseng Meyer.Plant Physiol，2014，25.［Epub ahead of print］

51 Jian YQ，et al.Two new euglobals from the leaves of Eucalyptus robusta.J Asian Nat Prod Res.2012，14:831-837.

52 Zou P，Koh HL.Determination of indican，isatin，indirubin and indigotin in Isatis indigotica by liquid chromatography/electrospray ionization tandem mass spectrometry.Rapid Commun Mass Spectrom，2007，21:1239-1246.

53 Gu YC(顾玉诚)，Tu YY(屠呦呦).Chemical constituents of Cirsium Japonicum D.C.China J Chin Mat Med(中国中药杂志)，1992，17:489-490.

54 Kim SR，et al.Trichosanthes kirilowii Ethanol Extract and Cucurbitacin D Inhibit Cell Growth and Induce Apoptosis through Inhibition of STAT3 Activity in Breast Cancer Cells.Evid Based Complement Alternat Med，2013:975350.

55 Lin RJ，et al.Cytotoxic activity of Ipomoea cairica.Nat Prod Res，2008，22:747-753.

56 Chyau CC，et al.Schisandra chinensis Peptidoglycan-Assisted Transmembrane Transport of Lignans Uniquely Altered the Pharmacokinetic and Pharmacodynamic Mechanisms in Human HepG2 Cell Model.PLoS One，2014，9:e85165.

57 Wang Z，et al.Protective effects of fractions from Pseudostellaria heterophylla against cobalt chloride-induced hypoxic injury in H9c2 cell.J Ethnopharmacol，2013，147:540-545.

58 Escobar FM，et al.Antiviral effect and mode of action of methanolic extract of Verbascum thapsus L.on pseudorabies virus(strain RC/79).Nat Prod Res，2012，26:1621-1625.

59 Huh JE，et al.Combined prescription(OAH19T)of Aralia cordata Thunb and Cimicifuga heracleifolia Komar and its major compounds inhibit matrix proteinases and vascular endothelial growth factor through the regulation of mitogen-activated protein kinase pathway.J Ethnopharmacol，2011，135:414-421.

60 Yim SH，et al.Cytotoxic caffeic acid derivatives from the rhizomes of Cimicifuga heracleifolia.Arch Pharm Res，2012，35:1559-1565.

61 Lai HY，et al.Blechnum orientale Linn-a fern with potential as antioxidant，anticancer and antibacterial agent.BMC Complement Altern Med，2010，10:15.

62 Cui CW(崔传文)，et al.Preliminary study on chemical constituents seperated from Cayratia japonica(中国中药杂志)，2012，37:2906-2909.

63 Jia SS，et al.Studies on flavonoid constituents isolated from the leaves of Glycyrrhiza uralensis Fisch.Acta Pharmaceutica Sinica，1990，25:758-762.

64 Wang N(王楠)，et al.Chemical constituents of Pyrrosia petiolosa.Journal of Shenyang Pharmaceutical University(沈阳药科大学学报)，2003，20:425-438.

65 Xuan SQ，et al.Polymorphic microsatellite loci for the genetic analysis of Lycoris radiata(Amaryllidaceae)and cross-amplification in other congeneric species.Genet Mol Res，2011，10:3141-3145.

66 Piacente S，et al.Constituents of Ardisia japonica and their in vitro anti-HIV activity.J Nat Prod，1996，59:565-569.

67 Tang X，et al.Screening anti-HIV Chinese materia medica with HIV and equine infectious anemic virus reverse transcriptase.J Tradit Chin Med，1994，14:10-13.

68 Zhang J，et al.Paeoniflorin abrogates DSS-induced colitis via a TLR4-dependent pathway.Am J Physiol Gastrointest Liver Physiol，2014，306:27-36.

69 Ye XY，et al.Dolichin，a new chitinase-like antifungal protein isolated from field beans(Dolichos lablab).Biochem Biophys Res Commun，2000，269:155-159.

70 Zhang XM(张雪梅)，et al.Simultaneous determination of five constituents in Scrophularia ningpoensis by HPLC.China Journal of Chinese Materia Medica(中国中药杂志)，2011，36:709-711.

71 Nagao T，et al.Studies on the constituents of Luffa acutangula Roxb.I.Structures of acutosides A-G，oleanane-type triterpene saponins isolated from the herb.Chem Pharm Bull(Tokyo)，1991，39:599-606.

72 Cheng HB，et al.Chemical constituents of ethyl acetate extract from Polygonum perfoliatum.J Chin Med Mater，2012，35:1088-1090.

73 Zhou XW(周兴旺)，et al.Chemical constituents in the roots of Lycium chinense Mill.China Journal of Chinese Materia Medica(中国中药杂志)，1996，21:675-676.

74 Szymańska G，et al.Field cultivation and in vitro cultures，root-forming callus cultures and adventitious root cultures，of Panax quinquefolium as a source of ginsenosides.Z Naturforsch C，2013，68:482-488.

75 Yang XZ，et al.Alkaloids from roots of Stemona sessilifolia and their antitussive activities.Planta Med，2009，75:174-177.

76 Hai LQ(海力茜)，et al.Studies on chemical constituents from root of Hedysarum polybotrys.China Journal of Chinese Materia Medica(中国中药杂志)，2004，29:432-434.

77 Qi HY，et al.Chemical constituents of Polygonum cillinerve.The 3～(rd)International Conference on Natural Products:Natural Products-a Must for Human Survival，Nanjing，China，2004，10:190.

78 Kan S，et al.Chemical constituents from the roots of Xanthium sibiricum.Nat Prod Res，2011，25:1243-1249.

79 Wang K，et al.Phenolic constituents from Brainea insignis.Chem Pharm Bull(Tokyo)，2010，58:868-871.

80 Fang X，et al.Microwave-assisted extraction followed by RPHPLC for the simultaneous extraction and determination of forsythiaside A，rutin，and phillyrin in the fruits of Forsythia suspensa.J Sep Sci，2013，36:2672-2679.

81 Yuan Y，et al.Functional diversity of genes for the biosynthesis of paeoniflorin and its derivatives in Paeonia.Int J Mol Sci，2013，14:18502-18519.

82 Schühly W，et al.Chemical characterization of Magnolia biondii(Flos Magnoliae，Xin Yi).Nat Prod Commun，2009，4:231-234.

83 Li HY，et al.Antioxidant activities of extracts and fractions from Lysimachia foenum-graecum Hance.Bioresour Technol，2009，100:970-974.

84 Chen QZ(陈秋竹)，et al.Studies on chemical constituents of the extract of Lonicera japonica.Journal of Chinese Medicinal Materials(中国中药杂志)，2010，33:920-922.

85 Chen CX(陈常兴).Studies on the chemical constituents from the fruit of Aristolochia debilis J Chin Med Mater(中国中药杂志)，2010，33:1260-1261.

86 Agatonovic-Kustrin S，Loescher CM.Qualitative and quantitative high performance thin layer chromatography analysis of Calendula officinalis using high resolution plate imaging and artificial neural network data modelling.Anal Chim Acta，2013，798:103-108.

87 Scragg AH，Allan EJ.Production of the triterpenoid quassin in callus and cell suspension cultures of Picrasma quassioides Bennett.Plant Cell Rep，1986，5:356-359.

88 Patel R，et al.Analgesic and antipyretic activities of Momordica charantia Linn.fruits.J Adv Pharm Technol Res，2010;1:415-418.

89 Shao H，et al.Matrine effectively inhibits the proliferation of breast cancer cells through a mechanism related to the NFκB signaling pathway.Oncol Lett，2013，6:517-520.

90 Chang SJ，et al.Antiviral Activity of Isatis indigotica Extract and Its Derived Indirubin against Japanese Encephalitis Virus.Evid Based Complement Alternat Med，2012，925830.

91 Jin S，et al.Biotransformation of polydatin to resveratrol in Polygonum cuspidatum roots by highly immobilized edible Aspergillus niger and Yeast.Bioresour Technol，2013，136:766-770.

92 Li N(李娜)，et al.Studies on anti-inflammation chemical constitutes of Patrinia villosa.Journal of Chinese Medicinal Materials(中国中药杂志)，2008，31:51-53.

93 Wang Y，et al.The genus Anemarrhena Bunge:A review on ethnopharmacology，phytochemistry and pharmacology.J Ethnopharmacol，2014，S0378-8741(14)00114-7.

94 Jiang HX，et al.Benzofuran derivatives from Eupatorium fortunei.Nat Prod Res，2008;22:937-941.

95 Zhang HB，et al.Flavonoids from Inula britannica reduces oxidative stress through inhibiting expression and phosphorylation of p47(phox)in VSMCs.Pharm Biol，2011，49:815-820.

96 Xiong Y，et al.Quality control of Lonicera japonica stored for different months by electronic nose.J Pharm Biomed Anal，2014，91:68-72.

97 Zhao RZ，et al.Determination of isofraxidin and astilbin by HPLC in rat plasma and its application after orally administration the extract of Sarcandra glabra.Pak J Pharm Sci，2013，26:1-6.

98 Yang ZN，et al.Quality evaluation of Houttuynia cordata Thunb.by high performance liquid chromatography with photodiode-array detection(HPLC-DAD).Pak J Pharm Sci，2014，:223-231.

99 Zomorodian K，et al.Chemical composition and antimicrobial activities of essential oil of nepeta cataria L.Against common causes of oral infections.J Dent(Tehran)，2013，10:329-337.

100 Lee LS，et al.Metabolomic analysis of the effect of shade treatment on the nutritional and sensory qualities of green tea.J Agric Food Chem，2013，61:332-338.

101 Nugroho A，et al.vivo sedative and gastroprotective activities of Salvia plebeia extract and its composition of polyphenols.Arch Pharm Res，2012，35:1403-1411

102 Hou HC(侯惠婵)，Liang SZ(梁少珍).Determination of indirubin and indigo in Baphicacanthus cusia(Nees)Bremek by HPLC.J Chin Med Mater(中国中药杂志)，2006，29:681-682.

103 Ogata S，et al.Apoptosis induced by the flavonoid from lemon fruit(Citrus limon BURM.f.)and its metabolites in HL-60 cells.Biosci Biotechnol Biochem，2000，64:1075-1078.

104 Zhao L，et al.Phytochemical and Biological Activities of an Anticancer Plant Medicine:Brucea javanica.Anticancer Agents Med Chem，2014，14:440-458.

105 Xie C，et al.Flavonol glycosides from Lysimachia capillipes.J Asian Nat Prod Res，2002，4:17-23.

106 Yu H，et al.Protective effects of the total Saponins from Dioscorea nipponica Makino against carbon tetrachloride-induced liver injury in mice through suppression of apoptosis and inflammation.Int Immunopharmacol，2014，19:233-244.

107 Sulaiman CT，Balachandran I.Total phenolics and total flavonoids in selected Indian medicinal plants.Indian J Pharm Sci，2012，74:258-260.

108 Park NI，et al.Anthocyanin accumulation and expression of anthocyanin biosynthetic genes in radish(Raphanus sativus).J Agric Food Chem，2011，59:6034-6039.

109 Kocevski D，et al.Antifungal effect of Allium tuberosum，Cinnamomum cassia，and Pogostemon cablin essential oils and their components against population of Aspergillus species.J Food Sci，2013，78:731-737.

110 Feng L，et al.Antioxidant activities of total phenols of Prunella vulgaris L.in vitro and in tumor-bearing mice.Molecules，2010，15:9145-9156.

111 Lu CN，et al.Saikosaponin a and its epimer saikosaponin d exhibit anti-inflammatory activity by suppressing activation of NF-κB signaling pathway.Int Immunopharmacol，2012，14:121-6.

112 Jin A，et al.Four new compounds from the bulbs of Lycoris aurea with neuroprotective effects against CoCl2 and H 2O 2-induced SH-SY5Y cell injuries.Arch Pharm Res，2014 Mar;37:315-323.

113 Jun HJ1，et al.Iristectorigenin B isolated from Belamcanda chinensis is a liver X receptor modulator that increases ABCA1 and ABCG1 expression in macrophage RAW 264.7 cells.Biotechnol Lett，2012，34:2213-2221.

114 Xue Z(薛智)，et al.Studies on the minor alkaloids of Cephalotaxus hainanensis Li(author＇s transl).Acta Pharmaceutica Sinica(药学学报)，1981，16:752-756.

115 Kristanti RA，et al.Accelerated biodegradation of nitrophenols in the rhizosphere of Spirodela polyrrhiza.J Environ Sci(China)，2012，24:800-807.

116 Lee WJ，Choi SW.Quantitative Changes of Polyphenolic Compounds in Mulberry(Morus alba L.)Leaves in Relation to Varieties，Harvest Period，and Heat Processing.Prev Nutr Food Sci，2012，17:280-285.

117 Li L，et al.Antibacterial activity of Lansiumamide B to tobacco bacterial wilt(Ralstonia solanacearum).Microbiol Res，2014，S0944-5013(13)00196-1.

118 Wangchuk P，et al.Antimalarial alkaloids from a Bhutanese traditional medicinal plant Corydalis dubia.J Ethnopharmacol，2012，143:310-313.

119 Zhang Z，et al.Baicalein Reduces the Invasion of Glioma Cells via Reducing the Activity of p38 Signaling Pathway.PLoS One，2014，9:e90318.

120 Liu R，et al.Expression profile of a PAL gene from Astragalus membranaceus var.Mongholicus and its crucial role in flux into flavonoid biosynthesis.Plant Cell Rep，2006，25:705-710.

121 Chen L(陈亮)，et al.Non-alkaloid chemical constituents from Coptis chinensis.China Journal of Chinese Materia Medica(中国中药杂志)，2012，37:1241-1244.

122 Li XC，et al.Steroid saponins from Polygonatum kingianum.Phytochemistry，1992，31:3559-3563.

123 Mishra MR，et al.Antidiabetic and Antioxidant Activity of Scoparia dulcis Linn.Indian J Pharm Sci，2013，75:610-614.

124 Zang X，et al.A-type proanthocyanidins from the stems of Ephedra sinica(Ephedraceae)and their antimicrobial activities.Molecules，2013，18:5172-5189.

125 Li YB(李遇伯)，et al.Chemical constituents from herb of Epimedium brevicornum.China Journal of Chinese Materia Medica(中国中药杂志)，2005，30:586-588.

126 Lim DW，et al.Anti-inflammatory effects of total isoflavones from Pueraria lobata on cerebral ischemia in rats.Molecules，2013，18:10404-10412.

127 Duelund L，et al.Influence of the active compounds of Perilla frutescens leaves on lipid membranes.J Nat Prod，2012，75:160-166.

128 Numata A，et al.Plant constituents biologically active to insects.VI.Antifeedants for larvae of the yellow butterfly，Eurema hecabe mandarina，in Osmunda japonica.(2).Chem Pharm Bull(Tokyo)，1990，38:2862-2865.

129 Fu GM(浮光苗)，et al.Studies on the chemical constituents from the aerial parts of Breynia fruticosa.China Journal of Chinese Materia Medica(中国中药杂志)，2004，29:1052-1054.

130 Oh HA，et al.Evaluation of the effect of kaempferol in a murine allergic rhinitis model.Eur J Pharmacol，2013，718(1-3):48-56.

131 Rubio-Pina J，et al.Pharmaceutical applications of the benzylisoquinoline alkaloids from Argemone mexicana L.Curr Top Med Chem，2013，13:2200-2207.

132 He ZD，et al.Antioxidant activity of phenylethanoid glycosides from Brandisia hancei.J Ethnopharmacol，2000，71:483-486.

133 Dong W，et al.A novel near-infrared spectroscopy and chemometrics method for rapid analysis of several chemical components and antioxidant activity of mint(Mentha haplocalyx Briq.)samples.Appl Spectrosc，2014，68:245-254.

134 Wang D(王大海)，et al.Nonvolatile chemical constituentsfrom Pogostemon cablin.China Journal of Chinese Materia Medica(中国中药杂志)，2010，35:2704-2707.