反相高效液相色譜法測定冠心_號(hào)湯劑中芍藥甙和阿魏酸的含量

1、基金項(xiàng)目:國家自然科學(xué)基金資助項(xiàng)目(N o.39870932,30070912;高等學(xué)校骨干教師計(jì)劃資助作者簡介:王驪麗,女,1960年12月生;高級(jí)工程師;研究方向:藥物分析及中藥臨床藥理研究反相高效液相色譜法測定冠心號(hào)湯劑中芍藥甙和阿魏酸的含量王驪麗黃熙任平張莉張航向(1.第四軍醫(yī)大學(xué)西京醫(yī)院,陜西西安710032摘要:采用反相高效液相色譜(RP -HP LC 法,用外標(biāo)法進(jìn)行了阿魏酸、芍藥甙的定性、定量分析,并測定冠心號(hào)湯劑中芍藥甙、阿魏酸含量。流動(dòng)相為甲醇水醋酸=34.8565.00.15,大連依利特ODS 色譜柱C 18(150mm 4.6mm ,5m ,實(shí)驗(yàn)結(jié)果表明,此方法分離、重

2、現(xiàn)性好,具有快速、價(jià)廉、毒性小的優(yōu)點(diǎn)。關(guān)鍵詞:反相高效液相色譜法;芍藥甙;阿魏酸;冠心號(hào)中圖分類號(hào):R286.0;R284.1文獻(xiàn)標(biāo)識(shí)碼:A 文章編號(hào):100420668(20010120051202川芎、赤芍在臨床上被廣泛應(yīng)用1。赤芍的主要化學(xué)成分為芍藥甙(Paeoniflorin ,阿魏酸(Ferulic acid 川芎的主要成分之一。關(guān)于高效液相色譜測定中成藥中芍藥甙或阿魏酸含量的方法報(bào)道很多2,也有文獻(xiàn)報(bào)道了湯劑不同煎煮條件對(duì)含量的影響3,而同時(shí)測定方劑湯液中芍藥甙和阿魏酸的方法還未見報(bào)道。我們采用反相高效液相色譜(RP 2HP LC 法,以甲醇2水2醋酸作為流動(dòng)相,通過對(duì)制劑的定性和

3、定量分析,同時(shí)測定了驗(yàn)方冠心號(hào)4(由丹參、川芎、赤芍、紅花、降香按21111配比組成的湯劑不同煎煮時(shí)間對(duì)芍藥甙、阿魏酸含量的影響,使其可以作為方劑研究的質(zhì)量控制標(biāo)準(zhǔn)和方劑治療藥物監(jiān)測5,并在進(jìn)一步改進(jìn)的基礎(chǔ)上用于方劑治療藥物監(jiān)測的體內(nèi)藥物動(dòng)力學(xué)研究6,完善在臨床中的應(yīng)用?，F(xiàn)將結(jié)果報(bào)告如下。1材料WatersHP LC 儀,600泵,996二極管陣列檢測器,7725手動(dòng)進(jìn)樣器,M32色譜工作站,Digital 計(jì)算機(jī);TG L -16臺(tái)式離心機(jī),振蕩器,0.22m 混合纖維素脂微孔濾膜(上海市新亞凈化器件廠。芍藥甙、阿魏酸對(duì)照品(中國藥品生物制品檢定所,批號(hào)0736-9608,甲醇、醋酸等均為分

4、析純(購自西安化學(xué)試劑廠。冠心號(hào)湯:中藥材丹參、赤芍、紅花、降香(購自于陜西醫(yī)藥大廈,川芎(購自于四川灌縣自制飲片,按照21111的比例配伍。采用臨床的煎煮方法,共分A 、B 、C 、D4組即煎煮時(shí)間各為15min 、30min 、45min 、60min 。將配伍比例的4等份分別第一次加8倍水浸泡30min ,在同樣條件下,火上至沸騰后用文火煎煮15min 、30min 、45min 、60min ,3層醫(yī)用紗布過濾,收集藥液;再加6倍水同前操作,兩次藥液合并作為原藥液使用。用冠心號(hào)總重量除以所得藥液的體積,其濃度分別為A :0.106g/m L (總重量為60g ,藥液體積396m L ;

5、依次類推得B :0.1515g/m L ;C :0.2419g/m L ;D :0.3061g/m L 。2方法與結(jié)果211色譜條件大連依利特ODS C 18色譜柱(150mm 4.6mm ,5m ;流動(dòng)相:甲醇水醋酸=34.8565.00.15,流速:F =1.0m L/min ,檢測波長:芍藥甙=230nm ,阿魏酸=320nm ;室溫,進(jìn)樣量為20L 。在此色譜條件下,分離很好(見圖1,并且本實(shí)驗(yàn)的檢測波長范圍是220nm 390nm ,芍藥甙的檢測波長為230nm ,阿魏酸為320nm ,冠心號(hào)中所含芍藥甙和阿魏酸與對(duì)照品光譜行為一致(見圖2。212煎液的預(yù)處理精確的吸取煎煮好的A 、

6、B 、C 、D 4組冠心號(hào)湯劑的原藥液10L ,加入于5m L 容量瓶中用水稀釋至刻度線,其濃度分別為A :2.1210-4g/m L ;B :3.0310-4g/m L ;C :4.8410-4g/m L ;D :6.1210-4g/m L ?；靹颉⒁?000r/min 離心,取上清液用0.22m 混合纖維素脂微孔濾膜過濾,20L 進(jìn)樣。1.芍藥甙和阿魏酸對(duì)照品色譜圖;2.冠心號(hào)中阿魏酸;3.冠心號(hào)中芍藥甙圖1芍藥甙和阿魏酸的色譜圖152001年3月第24卷第1期成都中醫(yī)藥大學(xué)學(xué)報(bào)Mar.2001,V ol.24,N o.1Journal of Chengdu University of T

7、 C M1.冠心號(hào)中阿魏酸(=230nm;2.冠心號(hào)中芍藥甙(=320nm圖2芍藥甙和阿魏酸的光譜圖213結(jié)果21311標(biāo)準(zhǔn)曲線及線性范圍法的考察精密稱取芍藥甙、阿魏酸對(duì)照品適量,用甲醇水=82配制成濃度為0.195mg/m L溶液,分別各取芍藥甙、阿魏酸對(duì)照品1、10、30、90、120、240、360、720、1440L,用甲醇定容于5m L的容量瓶中(絕對(duì)進(jìn)樣量范圍為39ng-56610ng之間,混勻,取20L進(jìn)樣。以對(duì)照品峰面積為縱坐標(biāo),進(jìn)樣量(g為橫坐標(biāo)進(jìn)行回歸,芍藥甙、阿魏酸回歸方程結(jié)果見表1。表1標(biāo)準(zhǔn)曲線各項(xiàng)參數(shù)的結(jié)果樣品名稱線性范圍(g標(biāo)準(zhǔn)曲線方程線性系數(shù)(r檢測極限(ng芍藥

8、甙0.039-56.610Y=15780X+1083.30.99512.60阿魏酸0.039-56.610Y=77612X-430730.9976 1.9521312精密度和回收率日內(nèi)精密度考察,將樣品溶液芍藥甙、阿魏酸在0.039 56.610g/m L濃度范圍內(nèi),20L進(jìn)樣(n=5,計(jì)算日內(nèi)變異系數(shù)(見表2;日間精密度考察,取同一批樣品,按不同天數(shù)(n=5測定表明,日間變異系數(shù)(見表2。測定結(jié)果本法精密度較好。以標(biāo)準(zhǔn)曲線求得理論Y 值,實(shí)際所測芍藥甙、阿魏酸的面積測定值,求得其平均相對(duì)回收率(見表3。表2精密度和回收率結(jié)果樣品名稱日內(nèi)精密度CV%日間精密度CV%回收率%芍藥甙 3.0 3.

9、499.41.2阿魏酸 5.2 4.299.54.1表3不同煎煮時(shí)間冠心號(hào)湯劑中芍藥甙、阿魏酸含量測定煎煮時(shí)間冠心號(hào)湯液量芍藥甙峰面積(min阿魏酸峰面積(g/m L含量(g/g.m L-1芍藥甙阿魏酸15 2.1210-4554526770.7500.361 30 3.0310-41322216940.3940.525 45 4.8410-42579013250.3090.846 60 6.1210-43246010830.3080.1083討論3.1本方法從二維、三維色譜圖觀察,與芍藥甙、阿魏酸標(biāo)準(zhǔn)品在相同條件下對(duì)照,保留時(shí)間一致,分別為4.874、6.986 (見圖1;準(zhǔn)確對(duì)其吸收峰定性

10、分析,標(biāo)準(zhǔn)品的最大吸收峰為230.1nm、320.1nm,樣品的最大吸收峰在320.1nm、392.0 nm(見圖1。采用外標(biāo)法建立了制劑中阿魏酸、芍藥甙測定的RP2HP LC法。本方法能快速、準(zhǔn)確地同時(shí)測定芍藥甙、阿魏酸,在進(jìn)行8min時(shí)間范圍內(nèi)完成測定,且保留時(shí)間無干擾。3.2芍藥甙為水溶性成分,樣品稀釋受溶劑的極性有所影響,即所用的稀釋溶劑不同,測定的結(jié)果不同;并且無論是對(duì)照品還是樣品隨時(shí)間的變化,其穩(wěn)定性變化不大。但冠心號(hào)煎液的煎煮時(shí)間對(duì)各個(gè)組分含量有所影響,采用水為稀釋溶劑的測定結(jié)果顯示,隨著不同煎煮時(shí)間的延長,從色譜圖上觀察峰面積及計(jì)算結(jié)果,芍藥甙隨著煎煮時(shí)間的延長而含量降低,15

11、min含量最高;阿魏酸在45min 含量較高,兩者都再延長時(shí)間則變化不大。在調(diào)整流動(dòng)相的配比中發(fā)現(xiàn),流動(dòng)相中酸度影響著對(duì)阿魏酸的分離。3.3陳可冀教授的驗(yàn)方冠心號(hào)1由丹參、川芎、赤芍、紅花、降香按21111配比組成,其在臨床的療效已被廣泛關(guān)注。研究冠心號(hào)與血瘀證、冠心病、藥動(dòng)學(xué)及藥物療效的相關(guān)性,都將闡明冠心號(hào)對(duì)人體的作用規(guī)律5,6。對(duì)冠心號(hào)的復(fù)方的初步研究將可能為冠心病血瘀證藥動(dòng)學(xué)的研究打下良好的基礎(chǔ)。因此我們這個(gè)研究結(jié)果將作進(jìn)一步完善的就可以廣泛用于測定動(dòng)物、人注射制劑或口服含有芍藥甙、阿魏酸的中成藥,進(jìn)行中藥復(fù)方體內(nèi)藥物監(jiān)測。參考文獻(xiàn)1劉超,王靜1赤芍總甙活血化瘀作用的研究J.中藥材,

12、2000,23(9:5572趙陸華1高效液相色譜法分析中藥成分手冊(cè)M1北京:中國醫(yī)藥科技出版社,199413李淑芝,郭亞健1不同煎煮條件對(duì)芍藥甙煎出量的影響J1中成藥,1999,(3:1434陳可冀,史載祥1實(shí)用血瘀證學(xué)M1北京:人民衛(wèi)生出版社,199912015陳可冀1邁向21世紀(jì)的中西醫(yī)結(jié)合M1北京:中國醫(yī)藥科技出版社,199112076黃熙,任平1防治高血壓病及冠心病的難點(diǎn)與突破口2方劑的治療藥物監(jiān)測J1中國中西醫(yī)結(jié)合雜志,1997,17(9:515(收稿日期:200020522525成都中醫(yī)藥大學(xué)學(xué)報(bào)2001年第24卷300C ases of Clinical Observation

13、of H ysteromyoma T reated with ”Gong 2liu 2qing ”C apsuleYANG Jia 2lin ,YAN Xiao 2ping et al (楊家林,等(A ffiliated H ospital ,Chengdu University of TC M ,Chengdu ,610072,China ABSTRACT :Objective :T o obserced the curative effect of ”Gong 2liu 2qing ”Capsule to hysteromy oma.Methods :300cases of hyster

14、omy oma were treated with ”Gong 2liu 2qing ”Capsule and other 120cases were treated with ”Gui 2zhi 2fu 2ning ”Capsule ,and the curative effects of tw o groups were compared.Results :the overall effective rates of treated group and control group were 47.67%and 22.50%,and the excellent effective rates

15、 of the tw o groups were 86.67%and 57.50%respectively (P 0.05.Conclusion :”Gong 2liu 2qing ”Capsule had a g ood ef 2fect for hysteromy oma.KE Y WOR DS :”Gong 2liu 2qing ”Capsule ;hysteromy oma(Original article on page 3E ffect of Sodium Ferulate to Left V entricular Diastolic Function of Diabetic Me

16、llitusLI Xiao 2miao ,QIAO Xue 2feng ,LI Yuan ,et al (李曉苗,等(X ijing H ospital ,F ourth Military Medical University ,X i an ,710032,China ABSTRACT :Objective :T o study the evaluation of s odium Ferulate in the tratment of left ventricular diastolic function in dvabets mellitus.Methods :left ventricul

17、ar diastolic function be measured by echocardiography with before treatment &after treatment.Results :C O ,VE andE DT of treatment at 1/2m onth were m ore than that of before treatment ;VA and E AT of treatment at 1/2m onth were lower than that of before treatment.Conclusion :S odium Ferulate can im

18、prove left ventricular diastolic in diabetes mellitus.KE Y WOR DS :S odium ferulate ;diabetes mellitus ;left ventricular diastolic(Original article on page 21Experimental Study on Changes of E nteroendocrine Cells of Somatostain ,Motilin and Cholecystokinin in Splenic Asthenia R at ModelREN Ping ,LI

19、U F ang ,HUANG Xi ,et al (任平,等(X ijing H ospital ,F ourth Military Medical University ,X i an ,710032,China ABSTRACT :Objective :T o investigate the relationship of ”dys function of the spleen in transport ”with gastrointestinal enterocrine cells.Methods :The short -term and long -term rat m odels o

20、f spleen -asthenia syndrome (RMS A I and RMS A II group were treated by reserpine for seven days and fourteen days respectively.S i -jun -zi Decoction was used as a remedy to verify the avilability of the m odel.Immunohisto 2chemical S ABC method was employed to stain D cell of gastric antrum ,I cel

21、l and M o cell of duodenum.The variation of appearance and quan 2tity of endocrine cells were observed by light microscope.Results :In the rat m odels of seven days ,as compared with controls ,the number of D cell increased (P 0.01,the number of M o cell reduced (P 0.01,the percentage of strong posi

22、tive D cell reduced (P 0.01,the percentage of strong positive I cell and M o cell increased (P 0.01,P 0.05.In the rat m odels of fourteen days ,as compared with con 2trols ,the number of the number of cell increased (P 0.01,the percentage of strong positive D cell reduced (P 0.01,the percentage of s

23、trong positive D cell and cell increased (P 0.01,the percentage of strong positive M o cell reduced (P 0.01.Conclusion :The synthetic and secretory function of the enteroendocrinr cells varyed with the development of spleen -asthenia syndrome.KE Y WOR DS :S pleen -asthenia syndrome ;S omatostain ;M otilin ;Cholecystokinin ;enteroendocrinr cell (Origi