抗马链球菌马亚种组方加味优选及其体内抑菌作用研究

doi:10.16431/j.cnki.1671-7236.2022.10.041

摘要/Abstract

摘要： 【目的】筛选可抑制马腺疫主要致病菌——马链球菌马亚种的最优中药组方,为临床马腺疫的防治提供参考。【方法】选用赤芍、大黄、山豆根等27种中药,首先通过体外抑菌试验(牛津杯法、最小抑菌浓度(MIC)、最小杀菌浓度(MBC)、联合药敏试验)结合L₈(2⁶)正交试验初步筛选抗马链球菌马亚种中药组方的最佳优选因素,再通过L₁₈(3⁶)正交试验进一步筛选基础组方最佳配比;最后在基础组方的基础上,根据中兽医方剂配伍理论,添加黄芪和当归2味补虚药,通过马腺疫小鼠模型体内抑菌试验筛选加味组方配比,结合组方保护率结果及组织脏器病理变化验证加味组方的体内抑菌作用。【结果】试验筛选出了马链球菌马亚种超敏和高敏的7味中药:浙贝母、赤芍、甘草、大黄、雪白睡莲、山豆根和苦参。大黄、雪白睡莲、山豆根、甘草、苦参和浙贝母的MIC分别为0.078、0.078、0.125、0.156、0.156和0.250 mg/mL。雪白睡莲、赤芍、山豆根、甘草、大黄、苦参和浙贝母的MBC分别为0.078、0.125、0.125、0.156、0.156、0.156和0.250 g/mL。根据各单味药物的MIC和两两联合后的MIC计算得出浙贝母与雪白睡莲联合药敏指数＞2,二者存在颉颃作用,故剔删浙贝母。通过2次正交试验得出基础组方最佳配比为:赤芍∶甘草∶大黄∶雪白睡莲∶山豆根∶苦参＝4∶2∶2∶4∶4∶1;结合加味组方体内抑菌试验可知,黄芪∶当归=1∶1、配伍浓度为0.5 g/mL时,保护率可达87.5%,且中药组方对马链球菌马亚种所致的小鼠肺脏炎性损伤修复良好。【结论】本试验成功优选出1种对马腺疫主要致病菌——马链球菌马亚种具有良好抑菌、组织保护作用的中药组方,配比为:赤芍∶甘草∶大黄∶雪白睡莲∶山豆根∶苦参∶黄芪当归(1∶1)=4∶2∶2∶4∶4∶1∶1。

关键词: 马腺疫; 马链球菌马亚种; 中药组方; 正交试验; 抑菌作用

Abstract: 【Objective】 The aim of this study was to screen the optimal Chinese herbal formula that could inhibit the main pathogen of strangles Streptococcus equi subspecies equi,and provide a new reference for the prevention and treatment of strangles.【Method】 27 kinds of herbs including Radix Paeoniae Rubra,Rhubarb and Radix Sophorae Sinensis etc.were used as materials in this experiment,antibacterial Chinese herbal medicines was preliminary screened by in vitro antibacterial test (Oxford cup method,minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC),combined drug susceptibility test) and L₈(2⁶) orthogonal test,and then the formula for inhibiting Streptococcus equi subspecies equi was composed.Secondly,the optimal ratio of basic formula was further screened by L₁₈ (3⁶) orthogonal test.Finally,under the premise of basic formula,according to the theory of compatibility theory of Chinese herbal formula,two tonifying drugs were added,the ratio of modified formula was screened by in vivo bacteriostasis test of strangles mice model,and the antibacterial effect of modified formula was verified in combination with formula protection rate and pathological changes of tissues and organs.【Result】 In the experiment,seven herbs (Fritillaria thunbergii,Radix Paeoniae Licorice root,Rhubarb,Nymphaea candida,Sophora root,Sophora flavescens) were selected,which were super sensitive and highly sensitive to Streptococcus equi subspecies equi,The MIC of Rhubarb,Nymphaea candida,Sophora root,Radix Paeoniae Rubra,Sophora flavescenswere 0.078,0.078,0.125,0.156,0.156 and 0.25 mg/mL,respectively.The MBC of Nymphaea candida,Radix Paeoniae,Sophora root,Licorice root,Rhubarb,Sophora flavescens,Fritillaria thunbergii were 0.078,0.125,0.125,0.156,0.156,0.156 and 0.25 g/mL,respectively.According to the MIC values of each single herb and the MIC values after pairwise combination,it was calculated that the combined herb sensitivity index of Fritillaria thunbergii and Nymphaea candida was＞2,which was antagonistic,so Fritillaria thunbergii was deleted.Then the optimal ratio of the basic formula was obtained by two orthogonal tests as follows:Rhubarb∶Sophora flavescens∶Sophora root∶Radix Paeoniae Rubra∶Licorice root∶Nymphaea candida= 4∶2∶2∶4∶1.Combined with the in vivo bacteriostasis test of modified formula,the protection rate was 87.5% when the compatibility concentration of Astragalus membranaceus∶Angelica sinensis = 1∶1 was 0.5 mg/mL,and Chinese herbal formula could repair inflammatory injury of lung induced by Streptococcus equi subspecies equi in mice.【Conclusion】 In conclusion,the study successfully screened out a new Chinese medicine formula with good antibacterial and tissue protection effects on Streptococcus equi subspecies equi.The ratio was Rhubarb∶Sophora flavescens∶Sophora root∶Radix Paeoniae Rubra:Licorice root∶Nymphaea candida∶Astragalus-Angelica(1∶1)= 4∶2∶2∶4∶4∶1∶1.

Key words: strangle; Streptococcus equi subspecies equi; herbal formula; orthogonal test; bacteriostasis test

中图分类号:

S853.74

朱怡萱, 刘诗芃, 张伟, 李金荣, 彭菲, 林娜, 王语嫣, 刘丹丹. 抗马链球菌马亚种组方加味优选及其体内抑菌作用研究[J]. 中国畜牧兽医, 2022, 49(10): 4085-4096.

ZHU Yixuan, LIU Shipeng, ZHANG Wei, LI Jinrong, PENG Fei, LIN Na, WANG Yuyan, LIU Dandan. Study on the Optimization of Modified Formula for Inhibiting Streptococcus equi subspecies equi and Its Bacteriostatic Effect in Vivo[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 4085-4096.

参考文献

[1] BOSCHWITZ J S,TIMONEY J F.Inhibition of C3 deposition on Streptococcus equi subspecies equi by M protein:A mechanism for survival in equine blood[J].Infection & Immunity, 1994,62(8):3515.
[2] 徐全圆,石明,杨夷平,等.马链球菌马亚种FNEB蛋白的截短表达及其免疫保护性研究[J].中国预防兽医学报,2015,37(2):132-135. XU Q Y,SHI M,YANG Y P,et al.Expression of truncated FNEB protein of Streptococcus equi subspecies equi and its immunoprotection[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(2):132-135.
[3] TIMONEY J F.Strangles[J].The Veterinary Clinics of North America Equine practice,1993,9(2):365-374.
[4] BRANKSTON G,ROSSI T M,OSULLIVAN T L,et al.Diagnostic testing patterns for Streptococcus equi subsp. equi in Ontario horses during the years 2008 to 2018.[J].Canadian Veterinary Journal 2021 62(6):629-636.
[5] MALIK P,KALRA S K.Prevalence of group C Streptococci amongst equines in India[J].Indian Journal of Animal Science,2009,79:459-465.
[6] PUSTERLA N,KASS P H,MAPES S,et al.Surveillance programme for important equine infectious respiratory pathogens in the USA[J].Veterinary Record,2011,169(1):12.
[7] DURHAM A E,HALL Y S,KULP L,et al.A study of the environmental survival of Streptococcus equi subspecies equi[J].Equine Veterinary Journal,2018,50(6):861-864.
[8] IJAZ M,KHAN M S,et al.Molecular identification and haemato-logical values of strangles affected mules in Pakistan[J].Pakistan Journal of Zoology,2011,43(3):587-592.
[9] 效宏儒.关于群牧马马腺疫发生规律的初步探讨[J].畜牧兽医杂志,1984,2:38-40. XIAO H R.Preliminary discussion on the occurrence rule of strangles in herd horses[J].Journal of Animal Husbandry and Veterinary Medicine,1984,2:38-40.(in Chinese)
[10] 周婷婷,冯凯,汪丽,等.马链球菌马亚种新疆分离株的分离及耐药性和进化分析[J].中国兽医科学,2019,49(6):738-745. ZHOU T T,FENG K,WANG L,et al.Isolation,drug resistance and evolutionary analysis of Streptococcus equi subsp. equi Xinjiang [J].Chinese Veterinary Science,2019,49(6):738-745.(in Chinese)
[11] 王彩蝶,苏艳,波拉提,等.新疆4县马腺疫链球菌血清抗体检测与分析[J].动物医学进展,2015,36(5):125-127. WANG C D,SU Y,BO L T,et al.Detection and analysis of serum antibody to Streptococcus equi subsp. equi in 4 counties of Xinjiang[J].Progress in Animal Medicine,2015,36(5):125-127.(in Chinese)
[12] 沙燕,苏战强,杜润慈,等.新疆伊犁地区部分马病血清学调查[J].畜牧与兽医,2016,48(8):91-94. SHA Y,SU Z Q,DU R C,et al.Serological survey of partial equine diseases in Yili,Xinjiang[J].Animal Husbandry & Veterinary,2016,48(8)91-94.(in Chinese)
[13] 袁贝,李海,马陈,等.新疆伊犁地区马鼻肺炎、马腺疫、马流产沙门菌病的血清学调查[J].中国预防兽医学报,2017,39(7):540-543. YUAN B,LI H,MA C,et al.Serological investigation of equine rhinopneumonia,strangles and equine abortion salmonella in Yili,Xinjiang [J].Chinese Journal of Preventive Veterinary Medicine,2017,39(7):540-543.(in Chinese)
[14] 赵亚南.马链球菌马亚种GAPDH蛋白的表达及其生物学特性的比较与分析[D].乌鲁木齐:新疆农业大学,2018. ZHAO Y N.Expression of GAPDH protein in Streptococcus equi subspecies equi and comparison and analysis of its biological characteristics [D].Urumqi:Xinjiang Agricultural University,2018.(in Chinese)
[15] JOHN P,MONICA V,LISA T,et al.Markers of long term silent carriers of Streptococcus equi subsp. equi in horses[J].Journal of Veterinary Internal Medicine,2020,34(6):2751-2757.
[16] 喻本亨,喻本元.元亨疗马集[M].北京:中国农业出版社,1957. YU B H,YU B Y.Yuanheng Therapeutic Equestrian Set[M].Beijing:China Agriculture Press,1957.(in Chinese)
[17] 代万英.马腺疫的诊断及中兽医疗法[J].中兽医学杂志,2019,4:34. DAI W Y.Diagnosis and Chinese veterinary therapy of strangles[J].Chinese Veterinary Medicine Journal,2019,4:34. (in Chinese)
[18] 胡元亮.兽医处方手册[M].北京:中国农业出版社,1999. HU Y L.Veterinary Prescription Manual[M].Beijing:China Agriculture Press,1999.(in Chinese)
[19] 阎超山.秘方治疗马腺疫[J].中兽医学杂志,2011,2:56. YAN C S.Secret recipe for the treatment of strangles[J].Journal of Traditional Chinese Veterinary Medicine,2011,2:56.(in Chinese)
[20] 陈学民.中西医结合治疗马腺疫[J].中兽医学杂志,2010,5:14-15. CHEN X M.Treatment of strangles with integrated traditional Chinese and Western medicine[J].Chinese Veterinary Medicine Journal,2010,5:14-15.(in Chinese)
[21] 唐科志.马腺疫综合防治[J].云南畜牧兽医,2007,4:35. TANG K Z.Comprehensive prevention and control of strangles[J].Yunnan Animal Husbandry Veterinary,2007,4:35.(in Chinese)
[22] 徐雨生,郭时印,申科,等.云南香叶天竺葵挥发油GC-MS分析及其体外抑菌与抗炎活性研究[J].中国生物化学与分子生物学报,2021,37(11):1502-1509. XU Y S,GUO S Y,SHEN K,et al.GC-MS analysis of volatile oil from geranium Yunnan and its antibacterial and antibacterial activities in vitro[J].Chinese Journal of Biochemistry and Molecular Biology,2021,37(11):1502-1509.(in Chinese)
[23] 苏迅.治疗奶牛乳房炎的抗菌蒙兽药成分及其复方的筛选和抑菌机理的研究[D].呼和浩特:内蒙古农业大学,2014. SU X.Screening and antibacterial mechanism of antibacterial Mongolian and veterinary drugs and their compounds for the treatment of mastitis in dairy cows[D].Hohhot:Inner Mongolia Agricultural University,2014.(in Chinese)
[24] 孙广,李小多,张海龙,等.29种中药提取物对耐甲氧西林金黄色葡萄球菌的体外抑菌活性[J].贵州医科大学学报,2020,45(10):1182-1186. SUN G,LI X D,ZHANG H L,et al.In vitro antibacterial activity of 29 Chinese herbal extracts against methicillin-resistant Staphylococcus aureus[J].Journal of Guizhou Medical University,2020,45(10):1182-1186.(in Chinese)
[25] VAARA M,SIIKANEN O,APAJIALAHTI J,et al.Susceptibility of carbapenemase-producing strains of Klebsiella pneumoniae and Escherichia coli to the direct antibacterial activity of NAB739 and to the synergistic activity of NAB7061 with rifampicin and clarithromycin[J]. Journal of Antimicrobial Chemotherapy,2010,65(5):942.
[26] IAN R.小动物药物手册[M].7版.北京:中国农业出版社,2014. IAN R.Handbook of Small Animal Drugs[M].7th ed.Beijing:China Agriculture Press,2014.(in Chinese)
[27] 徐淑云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社,2002. XU S Y,BIAN R L,CHEN X.Pharmacological Experimental Methodology[M].Beijing:People’s Medical Publishing House,2002.(in Chinese)
[28] 陈奇.中药药理研究方法学[M].北京:人民卫生出版社,1993. CHEN Q.Pharmacological Research Methodology of Traditional Chinese Medicine[M].Beijing:People’s Medical Publishing House,1993.(in Chinese)
[29] 王宁.马链球菌马亚种感染模型的建立及其噬菌体的分离鉴定[D].北京:中国农业科学院,2020. WANG N.Establishment of Streptococcus equi subspecies equi infection model and isolation and identification of its phage[D].Beijing:Chinese Academy of Agricultural Sciences,2020(in Chinese)
[30] 王雨朦.马链球菌马亚种的分离鉴定、致病性检测和抗菌药物筛选[D].乌鲁木齐:新疆农业大学,2017. WANG Y M.Isolation and identification,pathogenicity detection and antimicrobial screening of Streptococcus equi subspecies equi[D].Urumqi:Xinjiang Agricultural University,2017.(in Chinese)
[31] 李成应.治疗奶牛乳房炎的复方中药制剂的开发研究[D].呼和浩特:内蒙古农业大学,2008. LI C Y.Development study of compound Chinese herbal preparation for treating mastitis in dairy cows[D].Hohhot:Inner Mongolia Agricultural University,2008.(in Chinese)
[32] ARAFA A A,HEDIA R H,ATA N S,et al.Vancomycin resistant Streptococcus equi subsp. equi isolated from equines suffering from respiratory manifestation in Egypt[J].Veterinary World,2021,14(7):1808-1814.
[33] 杨美翠.马腺疫的临床诊断及防治[J].吉林畜牧兽医,2019,40(5):52. YANG M C.Clinical diagnosis,prevention and treatment of strangles[J].Jilin Animal Husbandry Veterinary,2019,40(5):52.(in Chinese)
[34] 金梅,许梅花,贺希格吉日勒·巴依尔太等.马腺疫病的诊断治疗[J].中国畜禽种业,2019,15(4):162. JIN M,XU M H,BAYIERTAI H,et al.Diagnosis and treatment of strangles[J].China Livestock and Poultry Breeding Industry,2019,15 (4):162.(in Chinese)
[35] 雷定学.马腺疫综合防治[J].中国畜禽种业,2019,15(3):169. LEI D X.Comprehensive prevention and control of strangles[J].China Livestock and Poultry Breeding Industry,2019,15(3):169.(in Chinese)
[36] 孙丹岚,买占海,佟盼盼,等.北疆地区马链球菌分离鉴定及耐药性和毒力基因分析[J].中国动物传染病学报,2022,5:1-9. SUN D L,MAI Z H,TONG P P,et al.Isolation and identification of Streptococcus equi and analysis of drug resistance and virulence genes in Northern Xinjiang[J].Chinese Journal of Zoonotic Diseases,2022,5:1-9.(in Chinese)
[37] 孙静,冶冬阳,王旭荣,等.防治奶牛乳房炎中药组方的筛选及其抑菌杀菌作用研究[J].中国畜牧兽医,2018,45(7):1990-2000. SUN J,YE D Y,WANG X R,et al.Screening of Chinese herbal formula for prevention and treatment of mastitis in dairy cows and study on its bacteriostatic and bactericidal effects[J].China Animal Husbandry & Veterinary Medicine,2018,45(7):1990-2000.(in Chinese)
[38] 国家药典委员会.中华人民共和国药典[M].北京:中国医药科技出版社,2015. CHINESE PHARMACOPOEIA COMMISSION.Chinese Pharmacopoeia[M].Beijing:China Medical Science and Technology Press,2015.(in Chinese)
[39] 刘丹丹,罗洁,向薇薇,等.雪白睡莲花粗提物对奶牛乳房炎主要致病菌体外抑菌和抗炎作用[J].动物医学进展,2021,42(7):17-21. LIU D D,LUO J,XIANG W W,et al.In vitro antibacterial effect of crude extract of Nymphaea candida on main pathogenic bacteria of cow mastitis[J]. Progress in Animal Medicine,2021,42(7):17-21.(in Chinese)
[40] 沈思钰,胡兵,蔡辉.正交试验设计在中药方剂学研究中的应用[A].中国中西医结合学会第七次全国实验医学学术研讨会论文汇编[C].2004. SHEN S Y,HU B,CAI H.Application of orthogonal experimental design in the study of traditional Chinese medicine formula[A].Paper Collection of the Seventh National Symposium on Experimental Medicine of Chinese Association of Integrative Medicine[C].2004.(in Chinese)
[41] 朴喜航,艾红佳.中药抗菌成分及其抗菌机制的研究进展[J].吉林医药学院学报,2017,38(6):445-447. PIAO X H,AI H J.Research progress of antimicrobial components and antimicrobial mechanism of traditional Chinese medicine [J].Journal of Jilin Medical College,2017,38(6):445-447.(in Chinese)
[42] 孔祥英.黄芪与当归配伍的免疫实验研究[J].山东中医药大学学报,1997,4:27-30. KONG X Y.Immunological experimental study on the compatibility of Radix Astragali and Radix Angelicae Sinensis[J].Journal of Shandong University of Traditional Chinese Medicine,1997,4:27-30.(in Chinese)
[43] 毛文点,庹玲玲,齐文续,等.黄芪-当归不同配伍对其6种主要有效成分溶出的影响[J].湖南中医药大学学报,2021,41(3):350-354. MAO W D,TUO L L,QI W X,et al.Effect of different compatibility of Radix Astragali and Radix Angelicae Sinensis on the dissolution of 6 main active ingredients[J].Journal of Hunan University of Traditional Chinese Medicine,2021,41(3):350-354.(in Chinese)
[44] 刘兰椿,刘超,毛启远,等.基于网络药理学探讨当归补血汤治疗化疗所致心肌损伤的机制[J].中西医结合心脑血管病杂志,2021,19(20):3441-3447. LIU L C,LIU C,MAO Q Y,et al.To explore the mechanism of Danggui Buxue decoction in treating myocardial injury induced by chemotherapy based on network pharmacology[J].Journal of Cardiovascular and Cerebrovascular Diseases of Integrated Traditional Chinese and Western Medicine,2021,19(20):3441-3447.(in Chinese)
[45] 孙璐,张静,尹苗,等.4种清热解毒复方中草药体外抑菌及抗PRRSV活性研究[J].中国畜牧兽医,2022,49(2):746-754. SUN L,ZHANG J,YIN M,et al.Study in the antibacterial and anti-PRRSV activities of four heat clearing and detoxifying compound Chinese herbal medicine in vitro[J].China Animal Husbandry & Veterinary Medicine,2022,49(2):746-754.(in Chinese)