摘要
蜜蜂巢脾是我国传统的动物中药。具有多种生物学功能和药理学价值。然而,从目前来看,对巢脾有效成分及其药理的研究还是经验多,科学验证少,定性定量少,蜜蜂蜂巢具有治疗鼻炎特别是变应性鼻炎(AR)的作用,然而药效成分和作用机理还不是十分清楚。关于蜜蜂巢脾挥发油的成分研究、药理作用研究国内外尚未报道过。本文对蜜蜂巢脾挥发油进行定性定量分析,对其提取工艺、提取动力学进行优选和数学模拟,并对其治疗AR药效学进行研究。结果如下:
1对蜜蜂巢脾挥发油提取工艺进行了优选研究,得出较佳提取工艺为蜜蜂巢脾粉碎度为Ⅱ、用8倍量水,浸泡20h、提取12h。建立并确定提取动力学方程ln(⊿A_t/⊿_t)=-0.1904t_c-2.9325(R=0.9936)和ln(0.2768-A_t)=-0.1947t-1.2934(R=0.9997)。为蜜蜂巢脾挥发油开发利用提供基础参数数据和有益参考。
2采用气相色谱、质谱联用(GC/MG)技术,首次对意大利蜜蜂巢脾挥发油组成成分进行了分析鉴定,分离鉴定出46种化合物。为蜜蜂巢脾有效成分明确化奠定基础。
3蜜蜂巢脾醇提物(15kg/kg)和蜜蜂巢脾挥发油(1mL/kg、2mL/kg、3mL/kg)均能够显著抑制小鼠二甲苯所致耳肿胀(p<0.05)、抑制小鼠醋酸所致腹腔毛细血管通透性(p<0.05),并呈良好的量效关系。揭示蜜蜂巢脾挥发油能够抑制炎性水肿、抑制渗出和血管通透性增加的急性炎症,具有抗炎作用。
4蜜蜂巢脾醇提物(15kg/kg)和蜜蜂巢脾挥发油(1mL/kg、2mL/kg、3mL/kg)均能够提高小鼠脾脏指数、胸腺指数,且呈良好的量效关系,揭示蜜蜂巢脾挥发油能够通过免疫器官发挥免疫增强作用。是其免疫增强作用机制之一。
5蜜蜂巢脾醇提物及挥发油对实验性AR有确切治疗作用:能缓减AR局部症状,改善局部粘膜水肿,减少肥大细胞和嗜酸性细胞在炎症局部的浸润、抑制AR模型大鼠血清炎性介质组胺的释放。免疫法测定血清中总IgA、总IgM和总IgG含量,表现为蜜蜂巢脾醇提物及挥发油能够显著升高血清IgA、IgG水平,揭示封闭性抗体可与进入机体的抗原物质优先结合,竞争抑制IgE抗体,调节粘膜免疫,从而对AR具有治疗作用的可能机制。
6蜜蜂巢脾挥发油成分明确,蜜蜂巢脾醇提物及挥发油具有抗炎免疫作用,对AR疗效确切,有望成为治疗此病的新型药物。
Honeybee comb ,one kind of Chinese traditional animal medicine, is valuable for its biological function and pharmacology effect. However, the study on the functional compone nts of Honeybee comb and its pharmacology effect are more experiential, less scientific validations, and less qualitative and quantitative analysis. Though honeybee comb has the effect on Allergic Rhinitis (AR), the functional components and its mechanisms are not clear. The reports about the volatile oil which is the component of honeybee comb and the effect of the volatile oil on AR are not funded all over the world. The dissertation firstly studied the components of honeybee comb volatile oil .including the qualitative and quantitative analysis, chosed a best extraction condition of honeybee comb volatile oil , made mathematics kinetics models of the extraction,and studied the effect of honeybee comb volatile oil on the AR and the its parts of immunological mechanisms. The results of the experiment showed that:
1 A best extraction condition of honeybee comb volatile oil by vapor distillation areⅡcomminution degree, 8 times water of honeybee comb, dipping for 20h, distilling for 12h; The optimizing mat hematic kinetics model for the relationship between the yield and the extraction time during the extraction process of volatile oil from honeycomb are ln(0.2768--A_t) =-0.1947t-1.2934 (R= 0.9997) and ln((?)A_t/(?)t) =-0.1904t-2.9325 (R= 0.9936) . the kinetics model is useful for controlling the process of the extraction of the volatile oil from honeycomb.
2 Used the the combination technologis of gas chromatography and mass spectrum (GC/MS), 46 compounds were identified.
3 Both the ethanolic extracts from honeybee comb (15kg/kg) and the honeybee comb volatile oil (1mL/kg^ 2mL/kgx 3mL/kg)can restrain the mice ear edema caused by dimethylbenzene and the mice capillary permeability caused by acetic acid, the honeybee comb volatile oil has the good relationship between the dosages and the effect.the results showed that both of them have the effect of antiinflammation by controlling the acute inflammation which concluding the edema ,the capillary permeability and so on.
4 Both the ethanolic extracts from honeybee comb (15kg/kg) and the honeybee comb volatile oil (1mL/kg、2mL/kg、3mL/kg)can increase the index of mice' s thymus gland and the index of mice's spleen (p<0.05) .the honeybee comb volatile oil has the good relationship between the dosages and the effect, the results showed that strengthening the immunity apparatuses such as thymus gland and spleen may be one of the mechanisms of the compounds and the honeybee comb volatile oil increasing the capacity of immunity.
5 The therapy effect of Both the ethanolic extracts from honeybee comb and the honeybee comb volatile oil on AR was significant and clear : improving the symptoms of rats suffered from AR ; reducing the number of mast cell,the number of eosinophil and other inflammation cells ; reducing the level of rat's histamine of blood serum ; Both the ethanolic extracts from honeybee comb and the honeybee comb volatile oil can increase the levels of IgA and IgG of blood serum , It can be one of the immunology mechanisms of the effect on AR that IgA and IgG may combine with the antigen first in body, complete with IgE,restrain IgE action.and regulate the immunity of nasal mucosal.
6 In conclusion, the compounds of honeybee comb volatile oil is clear.The ethanolic extracts and the volatile oil from honeybee comb , have the effect of antiinflammation, can increase the capacity of immunity, both of which effect on AR is valid, and will be new medicines to AR.
引文
[1] 闫亚美,吴珍红,缪晓青.蜜蜂巢脾及其开发利用.山东中医杂志,2006,25(8):555.
[2] 刘元帛.养蜂蜂巢的生药鉴定.中药材,1996,19(2):76-77.
[3] Angela R. Cnstodio, Máircia M. C. Ferreira,, Giuseppina Negri, etc. Clustering of Comb and Propolis Waxes Based on the Distribution of Aliphatic Constituents J. Braz. Chem. Soc. 2003, 14(3): 354-357.
[4] 徐颖,雷明吉,程诚.蜂胶与蜂胶黄酮[J].食品工业,2005,26(3):18-20.
[5] 魏振满,李惠敏,张苏刚等.复方巢脾制—复方扶正饮对脾淋巴细胞增殖反应的影响.中医药研究,1996,5:64.
[6] 陈福生.蜂蜡在医学上的用途.养蜂科技,1994,6:33.
[7] 吴国栋,施建民,俞永裕等.蜜蜂巢脾浸膏对鸡的增重及成活率试验.中国禽业导刊,1994,10(5):26.
[8] 胡福良,玄红专.蜂胶的生物学活性及毒性和过敏反应.科技通报.2003,19(2):166-169.
[9] Harish Z, Rubinstein A, Golodner M, etal. Suppression of HIV -1 replication by propolis and its immunoregulatory effect. [J]. Drug Exp Clin Res, 1997, 23: 89~96.
[10] 王开发.花粉药用进展综述.世界科学技术,2000,2(2):51-55.
[11] 王钰,金光明,郑艺梅等.蜂花粉对动物免疫器官发育的影响.中国中药杂志,2005,30(19):1532-1536.
[12] 朱俊彦,喻庆禄,邓必麟,等.蜂巢药效学研究.时珍国医国药.1999,3(10):168-169.
[13] 匡邦郁.蜜蜂产品成分及其在医疗上的应用.浙江中医学院学报,1979,3(5):38.
[14] Ozturk F, Kurt E, Inan U U, et al. The effects of acetylcholine and propolis extract on corneal epithelial wound healing in rats [J]. Comer, 1999, 18: 466~471.
[15] 韩旭,王新建,许奇伟.蜂蜡软膏对治疗缺损性慢性皮肤溃疡大鼠的实验观察,大连医科大学学报,2006,28(2):109—110.
[16] Assegid Garedew, Erik Schmolz, Ingolf Lamprecht. Microbiological and calorimetric investigations on the antimicrobial actions of different propolis extracts: an in vitro approach Thermochimica Acta 2004, 422(9): 115-124.
[17] 梁崇礼,李海山,李树荣,等.蜂胶的乙醇提取物对小鼠体表螨虫的净化研究(J).中国实验动物学报,1999,7(1):52~54.
[18] Banskota A H, Tezuka Y, Midorikawa K, etal. Two novel cytotoxic benzofuran derivatives from Brazilian propolis [J]. J Nat Prod, 2000, 63: 1277~1279.
[19] Dong Wang, De-Bing Xiang, Yu-Jun He, etal. Effect of caffeic acid phenethyl ester on proliferation and apoptosis of colorectal cancer cells in vitro World. [J] Gas troenterol 2005, 11(26): 4008-4012.
[20] Jeng SN, Shih MK, Kao CM, etal. Anti mutagenicity of ethanol extracts of bee glue against environmental mutagetts. [J]. Food Chem Toxicol, 2000, 38: 893~897.
[21] Mitamura T, Matsuno T, Sakamoto S, et al. Effects of a new clerodane diterpenoid isolated from propolis on chemically induced skin tumors in mice [J]. Anticancer Res, 1996, 16: 2669~2672.
[22] Kimoto T, Arai S, Kohguchi M, etal. Apoptosis and suppression of tumor growth by artepillin C extracted from Brazilian propolis [J]. Cancer Detect Prevent, 1998, 22: 506~515.
[23] Kimoto T, Koya S, Hino K, et al. Renal carcinogenesis induced by ferric nitrilotriacetate in mice, and protection from it by Brzailian propolis and atrepillin C[J]. Pathol Int, 2000, 50: 679~689.
[24] Ozkul Y, Silici S, Eroglu E. The anticarcinogenic effect of propolis in human lymphocytes culture Phytomedicine, 2005, 12: 742-747.
[25] Scheller S, Krol W, Swiacik J, etal. Antitumoral property of ethanolic extract of propolis in mice bearing Ehrlich carcinoma, as compared to bleomycin [J]. Z Naturforsch C, 1989, 44: 1063~1065.
[26] Kujumgiev A, Tsvetkova I, Serkedjieva Y, etal. Antibacterial, antifungal and antiviral activity of propolis of different geographic origin [J]. J Ethopharmacol, 1999, 64: 235~240.
[27] 骆尚骅译.蜂胶—化学组成、生物学特性和治疗作用[J].中国养蜂,1997,(2):29~30.
[28] 刘法锦,孙冬梅.中药蜂蜡降血脂有效成分的研究.中国中药杂志,1996,21(9)574—576.
[29] 廖芳,张小娜.新型降胆固醇药物蜂蜡素胶囊.中国药师,2005,8(12):1039—1040.
[30] 曾志将,汪礼国,饶波等.蜂花粉多糖对大鼠降血脂效果研究.江西农业大学学报,2004,2(3):406-408.
[31] 聂增义.蜂胶的质量评价(1):蜂胶和露蜂房在清除自由基作用方面的比较.国外医学.中医中药分册,1996,18(2):41-42.
[32] Fang S, Shoko H, Sakiko H, etal. In vivo antioxidative activity ofpropolis evaluated by the interaction with vitamins C and E and the level of lipid [J]. J Agric Food Chem, 2000, 48: 1462~1465..
[33] 丛涛,赵霖,鲍善芬等.松花粉对大鼠前列腺增生治疗作用的研究.军医进修学院学报,2005,26(5):395-397.
[34] 耿文奎,文振乾,钟进义.蜂巢浆致畸胎毒性研究.广西医学,1990,12(1):24-25.
[35] 宋心仿.蜂巢临床验方集锦.中国养蜂,1997,2:12.
[36] [加]S.O.Freedman等.陈泽仪译.临床免疫学.上海科学技术出版社出版.1982,10:105-108.
[37] 张罗,韩得民.变应性鼻炎的治疗进展.中华耳鼻喉头颈外科杂志.40(3):230—231.
[38] BousquetJ. Allerglc rhinitis as a global heath problem. ACIInt. 2001, 13(4): 13.
[39] 周郁,何江,王瑞.变应性鼻炎的研究进展.中国优生与遗传杂志.2006,14(4):124-125.
[40] 程靖,彭贵阳,张奇峰等.变应性鼻炎与食物因素相关性的初步研究.临床耳鼻咽喉科杂志.2002,6(8):393-395.
[41] Bonsquet J, Van cauwenberge P, Khalter N. Allergic rhinitis and its impact on asthma. J Allergy Clin Immunol. 2001, 108(suppl5): 147—147.
[42] Salib RJ, Drake Lee A. Howarth PH. Allergic rhinitis: past, present and the future[J]. Clin Otolaryngol Allied Sci, 2003, 28(4): 291-303.
[43] 殷明德.变应性鼻炎:全球性健康问题.临床耳鼻咽喉科杂志.2002,16(5):195—195.
[44] 周兵,王向东,魏均民.2004年全国变应性鼻炎与哮喘关系专题学术会议纪要.中华耳鼻咽喉头颈外科杂.2005,40(3):181—181.
[45] Juniper EF. Health elated quality of life inrhnitis In: Naclerio RM, Durham SR, Mygind N. Rhinitis mechanism and management. NewYork: Marcel Dekker Inc, 1999.5(3): 135-146.
[46] 林学颜.免疫学基础.福建科学技术出版社.1980,4:27-45.
[47] Grayson MH, Korenblat PE. The emerging role of leukotriene modi —lets in allergic rhinitis. Am J Respir Med, 2003, 2(6): 441—450.
[48] 刘学兵,刘钢.变应性鼻炎与细胞因子免疫学研究.天津医药.2006,34(2):140-141.
[49] Nakamura H, Miyagawa K, Ogino K, etal. High contribution contrast between the genes of eosinophil peroxidase and IL—4 recptoralpha —chain in Japanese cedar pollinosis. [J] Allergy Clin Immunol, 2003, 112(6): 1127—113.
[50] Hirschberg A, Jokuti A, Darvas Z, elal. Tbe pathogenesis of nasal poly—posis by immunoglobulin and interleukin— 5 is completed by trans— forming growth factor— betal. Laryngoscope, 2003, 113: 120—124.
[51] Dewson G, Cohen GM, Ward law AJ. Interleukin—5 inhibits transloca—tion of bax to the mitochondria, cytochrome c release, and activation of cas pases in human eosinophils Blood, 2001, 98: 2239—2247.
[52] Finotto S, Neurath MF, Glickman JN, etal. Development of spontaneous airway changes consistent with human asthma in mice lacking T—bet. Scienee, 2002, 295(1): 336—338.
[53] 熊天琴.辛夷挥发油治疗过敏性鼻炎的分子机理研究.成都中医药大学2003届博士论文:9—10.
[54] 樊忠,王天铎主编.实用耳鼻喉科学.山东科学技术出版社.1998年第1版第二次印刷:341-341.
[55] D. S. Postma, E. R. Bleeker, Genetics of asthma, in: P. J. Barnes, M. M. Grunstein, A. R. Leff, A. J. Woolcock (Eds.), Asthma. Lippincott-Raven, Philadelphia, 1997, 72(7): 145-154.
[56] Postma DS, Koppelman GH, Mayers DA. The genetics of atopy and airway hyper responsiveness[J]. Am J Respir Crit Care Med, 2000, 162(3Pt2): 118-123.
[57] Wrighi AL, Holberg CJ, Martinez FD, et al. Epidemiology of physican diagnosed allergic rhinitis in childhood[J]. Pediatrics. 1994, 94(4): 895-900.
[58] 余少卿,章如新.变应性鼻炎相关基因的研究进展[J].国外医学.2003,27(2):71-75.
[59] Strachan DP. Hay fever, hygiene and house hold size [J]. BMJ, 1989, 299 (5): 1259-1260.
[60] 席斌.变应性鼻炎与微量元素关系研究.广东微量元素科学.2006,13(3):26-29.
[61] Strachan D, Sibbald B, Weiland S, etal. World wide variation sinpreal ence of symptom so failer gicrhinoconjunctivitisin children [J]. Pediatr Allergy Immunol, 1997, 8(2): 161.
[62] 谢华,何韶衡,陈萍等.沈阳农村哮喘与其他变态反应性疾病相关性的研究[J].实用预防医学.2003,10(3):280-282..
[63] 邹红云,李力,仇东辉等.乌鲁木齐市变应性鼻炎患病率调查及发病因素分析[J].新疆预防医学.2000,16(3):141-143.
[64] 殷明德.变应性鼻炎:全球性健康问题.临床耳鼻咽喉科杂志.2002,16(5):195-196.
[65] 谭宁宁,王荣珍.过敏性鼻炎与支气管哮喘的相关性分析.江苏医药.2004,30(2):135-136.
[66] Simons FER. Allergic rhino bronchitis: The asthma allergic rhinitis link. J. Allergy ClinImmunol. 1999, 104 (3): 534-540.
[67] 殷明德.鼻窦疾病与支气管哮喘.临床耳鼻咽喉科志.2000,14(11):526-527..
[68] 殷明德.变应性鼻炎药物治疗进展.江苏医药杂志.2002,28(9):796-797.
[69] 胥科,梁传余,周光耀.药物治疗变应性鼻炎的研究进展.华西医学.2006,21(1):210-211.
[70] 洪钱江.中西医治疗变应性鼻炎的进展.中国中西医结合耳鼻咽喉科杂志[J].2006,14(1):63-68.
[71] 张怀璋,何宗德.温阳化饮汤治疗变应性鼻炎的临床研究.中国中西医结合耳鼻咽喉科杂志.1995,3(2):8—10.
[72] 张芯诚,田素华,梁树兰等.鼻敏康治疗过敏性鼻炎60例疗效观察[J].新中医.2000,32(4):30.
[73] 熊大经,郑军,杨安华等.中成药“鼻窦炎合剂”配合玉屏风散治疗过敏性鼻炎及其对IgE影响的观察[J].上海中医药杂志.1992,(2):20~21.
[74] 卢国华,孙玉英,李锋杰等.鼻炎敏滴鼻剂治疗变应性鼻炎的实验研究[J].潍坊医学院学报.2003,25(3):207—208.
[75] 郑湘瑞,姚忆,贯剑等.敏康片对变应性鼻炎大鼠一般情况及血浆组胺的影响[J].上海中医药大学学报.2005,19(2):45—47.
[76] 胡锡元,吴正红.抗鼻炎中药液治疗过敏性鼻炎的实验研究[J].中医外治杂志,2003,12(3):6—7.
[77] 余彦,杜俊蓉,况国成等.辛芩片剂治疗过敏性鼻炎的主要药效学研究[J].中成药.2004,26(5):401—404.
[78] 张蓉,余彦,杜俊蓉等.辛芩片对豚鼠鼻超敏反应的影响[J].中国中药杂志.2005, 30(10):785—788.
[79] 梁晓阳,王士贞,邱宝珊等.变应性鼻炎证型与免疫关系的初步探讨.实用医学杂志,2006,22(5):593.
[80] 谭敬书,徐绍勤.敷贴疗法治疗变应性鼻炎60例[J].中国中西医结合杂志,1994,14(6):342.
[81] 唐代屹,熊大经.鼻敏舒对过敏性鼻炎红细胞免疫功能影响的实验研究[J].云南中医中药杂志,1996,17(4):34—35.
[82] 王大海,李凡成.鼻炎喷雾剂的药效学研究.湖南中医学院学报.1999,19(2):2-3.
[83] 郭群,苏玮.辛夷体外抗菌作用的试验研究.中国中医药科技.1999,6(1):18.
[84] 钱彦方,李炳文,周正谋等.克鼻敏消肿止痒的实验研究.河北医科大学学报,1998,19(5):280-282.
[85] 中华人民共和国药典[S]一部.1995,附录62.
[86] 成都科技大学化工原理编写组.化工原理[M].下册.成都:成都科技大学出版社,2000,4.
[87] 何兵,田吉.挥发油提取过程动力学模型及其参数的确定.泸州医学院学报,2003,26(6):478—479
[88] 王茜.NZ中抗菌、抗炎、抗癌有效成分研究及化学结构特征.武汉大学2001年博士论文:4—26.
[89] 徐叔云,卞如濂,陈修,主编.药理实验方法学(第三版)[M].北京:人民卫生出版社,2002:1408—1409,1409,911..
[90] 窦淑筠,符云峰.血及组织中组织胺的荧光测定法[J].中华医学检验杂志,1981,4(2):100—102.
[91] 刘介眉等.编著病理组织染色的理论和应用.人民卫生出版社,1983,12:21—29.
[92] 孟运莲主编,现代组织学与细胞学技术.武汉大学出版社,2004,9:25—28.
[93] 赵宇,C.Andrew van Hassel,吴港生等.卵白蛋白经鼻致敏建立变应性鼻炎动物模型.中华耳鼻咽喉头颈外科杂志,2005,40(3):671—672.
[94] 景雅曼.辛芩颗粒治疗变应性鼻炎治疗观察[J].华西药学杂志,1999,14(3):199.
[95] 郭裕.辛芩颗粒治疗过敏性鼻炎的疗效观察[J].华西药学杂志.1999,14(5 6):416..
[96] 王锦歧,闫浩.辛芩颗粒治疗过敏性鼻炎的疗效观察[J].华西药学杂志,2000,15(2):145..
[97] 王海英,慈军,方英等.尼多考米钠治疗兔变应性鼻炎的病理实验研究.山东大学耳鼻喉眼学报,2006,20(4):316.
[98] 唐代屹,郭赛珊,梁晓春.中药复方防治变应性鼻炎机制研究进展.山东中医杂志,2000,19(1):55-57.
[99] 金光明,杨倩,刘胜兵.动物粘膜免疫与分泌型免疫球蛋白A.安徽技术师范学院学报,2003,17(2):107-111