我的 响应面法在药物提取工艺优化中的应用VIP

响应面法在药物提取工艺优化中的应用从药材中提取稳定的有效成分，保证药品量效关系的均一性和稳定性，是中药制造现代化的重要内容之一，因此，中药提取成为中药制造过程中极其关键的一环。提取过程中提取效率常常会受到提取溶剂、提取时间、溶液pH值等多种因素的影响，合理选用统计学方法，筛选出最佳工艺条件，已成为中药制造现代化研究的又一重要领域。实验设计与优化方法，都未能给出直观图形且不能凭直觉观察其最优化点，虽能找出最优值，但难以直观判断优化区域。响应曲面法（reponse surface methodology,RSM）是由Box等于20世纪50年代提出的1种数据分析方法，它以回归方程作为函数估算的工具，在多因子试验中，因子与响应值的相互关系用多项式拟合，把因子和响应值的关系函数化，因此，可对函数的面进行分析，研究因子与响应值之间.因子与因子之间的相互关系，并进行优化。它是一种优化反应条件和加工工艺参数的有效方法，采用RSM法可以建立连续变量曲线模型，对影响过程的因子水平及交互作用进行优化和评价，同时运用图形技术将这种函数关系显示出来，可凭直觉的观察来选择实验设计的最优化条件。该方法具有简便，结果直观，预测性好等优点。以下是响应面法在各种药物提取工艺优化中的应用。1药物中多糖的提取优化利用响应面分析法对白术多糖的提取工艺进行优化1，在单因素实验基础上选取试因素与水平，根据中心组和实验设计原理采用3因素3水平的响应面分析法，根据回归分析确定各工艺条件的影响因子，以多糖提取率为影响值作响应面和等高线。在分析各个因素显著性和交互作用后，得出白术多糖水浸提的最佳工艺条件为：料液比1：24，浸提温度94，浸提时间3.3h，浸提2次；白术多糖实际提取率可达3.13%。在用响应面法优化双孢菇菇柄多糖的提取工艺研究中，在单因素基础上,选择水料比、提取温度和提取时间为自变量,多糖提取率为响应值,根据Box-Behnken试验设计原理利用响应面法对双孢菇菇柄多糖的超声波法提取工艺进行优化研究。结果表明:超声波法提取双孢菇菇柄多糖的最佳工艺为:水料比44.941、提取温度30.53、提取时间30.58 min,在此条件下理论最大提取率为20.15%。经过3次平行验证试验,证明该模型合理可靠,能够较好的预测超声波法提取双孢菇菇柄多糖得率2。在响应面法优化蛹虫草菌丝体多糖超声波提取工艺的研究实验里，通过单因素实验对影响蛹虫草菌丝体多糖提取的主要因素提取时间、超声功率、液料比进行了探索，选取中心组合实验的范围和水平，应用响应面分析法对各因素的最佳水平范围及其交互作用进行了研究和探讨，建立了影响多糖提取率的二次多项数学模型，依据回归分析结果知，蛹虫草菌丝体多糖超声提取的最佳工艺条件为：提取时间504 s，提取功率466 W，液料比1041（mlg）。在此条件下，实际提取率7.45%，与模型理论预测值7.47%的相对误差为0.27%。该方法与传统水提法相比，提取时间缩短96%，多糖得率提高5.62%。实验结果证明，采用响应面分析法优化蛹虫草菌丝体多糖的超声波提取工艺条件准确可靠，具有实际价值。研究地木耳多糖的水提法优化工艺3。方法： 采用苯酚-硫酸法测定多糖含量，以多糖得率为考察指标，在单因素试验的基础上，利用响应面分析法对地木耳多糖提取工艺进行优化研究。结果： 提取温度、提取时间以及液料比与响应值地木耳多糖得率存在显著的相关性，最佳提取工艺条件：提取温度99，时间3.76 h，液料比52.11，提取3次，在此条件下地木耳多糖得率理论值达到7.70%，验证试验的多糖得率为7.54%，地木耳多糖样品中多糖含量为53.5%。结论： 该优化工艺的多糖得率高，可用于地木耳多糖的提取。 2 药物中总黄酮的提取优化确定小鱼仙草总黄酮的最佳提取工艺条件。方法： 以乙醇回流提取法提取小鱼仙草总黄酮，采用分光光度法测定总黄酮的含量，以总黄酮提取得率为考察指标，考察溶媒倍数、提取时间和提取次数等因素对小鱼仙草总黄酮提取得率的影响，利用响应面分析法优化提取工艺，确定总黄酮的最佳提取工艺。结果： 小鱼仙草总黄酮的最佳提取工艺为：加药材20倍量浓度为 70%乙醇，于 90水浴回流提取 3次，每次 1.9 h。此工艺下小鱼仙草总黄酮的提取得率达8.50%。结论： 该工艺提取得率高、操作简便，易于工业化大生产。研究乙醇溶剂提取三叶青中总黄酮的最佳工艺条件4。方法以乙醇溶液为提取溶剂，采用响应面设计方法，对影响总黄酮提取效果的3个因素乙醇浓度(C)、液料比(R)、提取时间(t)进行中心组合设计试验，并建立数学模型，研究这些因素对黄酮提取率的影响。结果中心组合设计试验建立总黄酮提取率与乙醇浓度(C)、液料比(R)及提取时间(t)间的数学模型显著，R2=0.950 2;最佳的提取工艺条件为28%乙醇(V/V)，液料比22(ml/g)，提取时间2.2 h;经提取次数考察，最佳提取次数为2次。该条件下三叶青总黄酮的提取率为0.30%。结论验证试验结果表明，应用响应面方法优选出的工艺稳定、合理、可行。本试验所用三叶青中总黄酮的含量测定方法经方法学考察，精密度、稳定性及加样回收试验均符合规定。三叶青总黄酮含量测定方法选用Al(NO3 ) 3 - NaNO2 - NaOH 显色后,明显生成红色絮状沉淀，此法不宜采用。而用AlCl3显色后，芦丁标准液的max = 410 nm，样品溶液的max = 400 410 nm，故选择AlCl3显色后410 nm 波长处测定吸光度。以乙醇溶液为提取溶剂，采用热回流法提取三叶青中的总黄酮，考察了乙醇浓度、液料比、提取时间等对三叶青总黄酮得率的影响,并结合BBD实验设计得到了最优提取工艺参数: 乙醇浓度为28%，液料比为22 ml/g ,提取时间为2.2 h，提取2次。在此条件下, 三叶青总黄酮提取率为0.30 %。经验证试验证明用该工艺提取三叶青总黄酮工艺简单、稳定性好。本次实验优选出来的实验条件,对今后开发利用三叶青中的总黄酮具有一定的指导意义。研究微波提取苦丁茶总黄酮的最佳提取条件5。方法通过二次回归正交旋转组合试验设计，得到了微波提取苦丁茶中黄酮的数学回归方程；利用模型的响应曲面图及等高线图，对影响微波提取苦丁茶黄酮提取率的关键因素及相互作用进行探讨，得到最佳提取工艺条件。结果最佳提取工艺条件：乙醇浓度69%，微波功率782 W，萃取时间4.5 min，料液比115，苦丁茶黄酮提取率为9.10%。结论该工艺条件可为工业生产提供参考数据。实验表明微波功率、乙醇浓度、萃取时间、液料比对苦丁茶总黄酮得率的影响不是简单的线性关系，一次项和二次项对苦丁茶总黄酮得率影响显著，交互项影响较小。响应面法优化微波提取苦丁茶总黄酮的最佳工艺条件为：乙醇浓度69%，微波功率782 W，萃取时间4.5 min，液料比151，苦丁茶黄酮提取率为9.10%。回归分析和验证实验表明了该响应面法的合理性和可行性。响应面法优化金银花总黄酮提取工艺6，为优化金银花总黄酮的提取工艺，在单因素试验的基础上，选取乙醇浓度、时间以及液料比为自变量，黄酮含量为响应值，采用中心组合设计的方法，研究各自变量及其交互作用对黄酮含量的影响。利用响应面分析方法，模拟得到二次多项式回归方程的预测模型，并确定金银花黄酮提取工艺的最佳条件为：乙醇浓度74，提取时间2h，液料比146（mLg）,提取3次，在此条件下，提取液中的黄酮含量达到932。本研究建立的提取方法简便、可靠，可为生产工艺提供理论依据。在响应面法优化蓝莓叶黄酮的微波提取工艺7，为确定蓝莓叶中黄酮提取的最佳工艺,以蓝莓叶为主要原料,在单因素试验基础上采用Box-Behnken中心组合试验设计和响应面(RSM)分析法,建立微波提取黄酮的二次回归方程,并以黄酮提取率为响应值绘制响应面图和等高线图。考察乙醇体积分数、料液比、温度、微波功率及时间对黄酮提取率的影响。方差分析结果表明:乙醇体积分数、温度和微波功率对黄酮提取率影响显著;最佳工艺条件为提取温度72,乙醇体积分数64%,微波功率456W,在此工艺条件下黄酮提取率为4.232%。响应面法优化超声波水提马齿苋黄酮的工艺8，为优化超声波辅助水提马齿苋黄酮的提取工艺.在超声提取温度、超声提取时间、液固比3个单因素实验基础上,采用响应面法(RSM法)优化马齿苋黄酮的超声辅助提取条件.利用中心组合设计,研究以上3个自变量对响应值马齿苋黄酮得率的影响,用SAS 8.1进行结果分析.结果表明:超声波辅助提取马齿苋黄酮的最佳工艺条件为超声温度73,超声时间49,min,液固比(mLg)411;在此条件下黄酮得率为8.24,mg/g,与理论预测值的误差为2.02%,说明采用RSM法优化得到的提取条件可靠.。3 其他成分在药物中的提取优化牡丹籽油超声波辅助提取工艺的响应面法优化9，为优化牡丹籽油的超声波辅助提取工艺，在单因素试验基础上，选择液料比、超声波功率、处理时间、处理温度为自变量，牡丹籽油得率为响应值，采用BoxBehnken试验设计方法，研究各自变量及其交互作用对牡丹籽油得率的影响。利用DesignExpert软件得到回归方程的预测模型并进行响应面分析，确定超声波辅助提取牡丹籽油的最佳条件为：液料比11mLg，超声波功率300W，处理时间60min，处理温度54，提取次数为3次。在该工艺条件下。牡丹籽油得率达2412。GCMS结果表明牡丹籽油中富含不饱和脂肪酸，其中亚油酸和亚麻酸的质量分数分别为2278和6414。响应面分析法优化向日葵盘中果胶的提取工艺研究10，通过响应面分析法对向日葵盘果胶的提取工艺进行优化。利用响应面实验设计考察提取温度、提取时间、料液比和pH值4因素对果胶提取量的影响。研究发现:提取时间、温度和pH值对提取量有显著影响,向日葵盘果胶的最佳提取条件是:提取温度为79、提取时间为80min、料液比251、pH值3.20;每5g向日葵盘中果胶最大提取量为0.522g。响应面法优化超声波提取构树叶中叶绿素的工艺研究中11，为实现构树叶的高附加值利用,选取影响超声波辅助提取构树叶中叶绿素提取效果的提取温度、超声波处理时间、超声波功率、液料比4个因素,通过单因素实验选取影响因素的水平,然后在单因素实验的基础上采用四因素三水平的响应面分析法(RSA),依据回归分析优化了超声波辅助提取构树叶中叶绿素的工艺方法。结果表明:超声波辅助提取构树叶中叶绿素的优化条件为提取温度60,超声波处理时间50min,超声波功率500W,液料比10mL/g;该条件下,叶绿素的提取得率可达到1.277%。与传统的研磨法和溶剂萃取法相比,超声波辅助提取叶绿素大大缩短了提取时间,提高了有效成分的提取得率,是一种极具应用前景的方法。麦壳中原花青素的提取工艺，为充分提取中国苦荞麦壳中的活性多酚，以70% 乙醇- 水为提取剂，通过二次旋转正交试验优化提取工艺，建立了提取得率的二次旋转回归方程，通过响应面分析及岭嵴分析得到了优化组合条件。结果表明，提取温度和时间对提取得率有显著影响(P 0.05)，而料液比对提取得率的影响不显著，较优的提取工艺条件为提取温度73、提取时间118.9min、料液比1:16.9，其提取得率0.56%，理论预测值为0.59%，提取得率达到理论预测值的94.9%。响应面法优化超声提取苜蓿皂苷工艺条件12，目的:利用响应面法对超声提取苜蓿皂苷的工艺条件进行了优化。方法:研究了超声波条件下影响提取的几个因素,包括超声时间、超声温度、超声功率、固液比等,并通过响应面法优化工艺条件。结论:根据中心组合设计原理采用四因子三水平的响应面分析法,通过对各因子显著性和交互作用的分析,得出了超声提取苜蓿皂苷的最佳工艺条件为:超声时间22min,超声温度43,超声功率403W,固液比151(g/ml),此时苜蓿皂苷的得率为4.53%。响应面法优化花生根中白藜芦醇提取工艺研究，为了探索花生根白藜芦醇提取的最佳条件,在单因素试验的基础上,应用响应面法优化花生根白藜芦醇的提取条件。结果表明:乙醇浓度、提取温度和提取时间对花生根白藜芦醇的提取效果有显著影响,且为非线性关系,最佳提取条件为乙醇浓度65%、提取温度52、提取时间39min,在此条件下白藜芦醇的提取率为0.012%。实验证明响应面法对花生根白藜芦醇提取条件的优化是可行的,得到的白藜芦醇提取条件具有实际应用价值。响应面法优化超声辅助提取马钱子中生物碱的工艺研究，采用响应面法对超声波辅助提取马钱子中生物碱的工艺进行了优化,在单因素试验的基础上,选择提取溶剂中乙醇体积分数、超声功率、提取时间为随机因素,进行三因素三水平的Box-Behnken中心组合设计,采用响应面法(RSM)分析了3个因素对响应值的影响。结果表明,超声波法提取马钱子中生物碱的最佳工艺条件为:马钱子颗粒度为2040目,溶剂中乙醇体积分数为32.3%,溶剂pH值为5,超声功率300W,提取时间为37min,液料比为10:1。在此条件下,士的宁、马钱子碱的提取量分别达到18.75、9.24mg/g,与理论预测值基本吻合,说明该优化方法可行。响应面法优化灵芝菌丝体胞内灵芝酸的提取，利用单因子试验和响应面法优化了影响灵芝菌丝体灵芝酸的提取过程。基于3因素3水平的中心组合设计,得到了描述胞内灵芝酸提取得率与操作参数之间的二次响应面模型。在乙醇浓度为93.6%(v/v)、提取温度79.1、提取液固比42.21、提取次数2次、每次提取时间2 h的条件下,最大灵芝酸理论提取得率为28.36 mg胞内灵芝酸每克干菌丝。模型优化条件下的实际灵芝酸提取得率(28.72 mg/g干菌丝)与理论灵芝酸提取得率(28.36 mg/g干菌丝)相符。响应面法优化微波提取扶芳藤抗氧化物，用响应面法研究了微波法提取扶芳藤抗氧化物质的最佳工艺。考察了乙醇体积分数(X1)、乙醇用量(X2)、提取时间(X3)、提取温度(X4)4个因素的影响。微波提取的优化工艺条件是:乙醇体积分数49.4%、乙醇用量44 mL、提取时间9 m in、提取温度41,与模型预测值基本相符。说明微波方法可较好地应用于扶芳藤抗氧化物质的提取,通过响应面法得到一个能较好预测实验结果的模型方程。综上，我们可以了解到响应面法在药物提取工艺优化中广泛应用，与正交试验相比它的优点更为突出，正交试验设计则注重如何科学合理地安排试验，可同时考虑几种因素，寻找最佳因素水平组合；但它不能在给出的整个区域上找到因素和响应值之间的一个明确的函数表达式即回归方程，从而无法找到整个区域上因素的最佳组合和响应值的最优值因此，人们期望找到一种试验次数少、周期短，求得的同归方程精度高、能研究几种因素间交互作用的回归分析方法，响应面分析方法在很大程度上满足了这些要求。参考文献1范龚健.用响应面法优化红甘蓝色素提取工艺参数2冯涛，曹东旭，高辉等利用响应面法确定竹叶中黄酮最佳提取条件J天津轻工业学院学报，2003，18(2)：8133刘世名，董 国霞，陈靠山牛蒡叶中绿原酸的提取工艺优化J中国药学杂志，2003，38(9)：659-6614易军鹏，朱文学， 马海乐， 王易芬. 农业机械学报 2009年第6期,103-110页5段毅.蛹虫草高效培养技术M.郑州：河南科学技术出版社，2004：24. 6熊中良, 邓春生. 真菌多糖免疫活性的研究进展J. 江西农业学报, 2006, 18 (2) : 118.高丽君, 王汉忠, 崔建华, 等. 白首乌可溶性多糖提取工艺研究J.食品科学, 2004, 25(10): 1787李亚娜, 林永成, 余志刚. 响应面分析法优化羊栖菜多糖的提取工艺J.华南理工大学学报(自然科学版), 2004, 32(11) : 28.8叶红,吴涛,周春宏.马尾藻多糖提取工艺的优化J.食品研究与开发,2006,27（7）: 22.9苏宇静，贺海明，孙兆平.中国枸杞资源及在食品工业中的应用现状和开发前景J.食品科学，2002，23（8）：292.10迟国兴，田 刚.枸杞多糖保肝作用的研究J.吉林中医药，1996，2：34.11陈群力，孙江涛，马灵筱，等.有关益气养阴活血复方及枸杞多糖对糖的影响J.中华实用中西医杂志，2004，4（17）：32.12王 琦，韩晓龙.蛹虫草对老年大鼠自由基代谢影响的研究J.辽宁师范学报，2002，4（4）：104Isolation of Flavonoids, a Biscoumarin and an Amide from the Flower Buds of Daphne genkwa and the Evaluation of their Anti-complement Activity1 2 1 1 3Bo-Young Park , Byung-Sun Min , Sei-Ryang Oh , Jung-Hee Kim , Ki-Hwan Bae and 1 Hyeong-Kyu Lee *1 Laboratory of Immunomodulator, Korea Research Institute of Bioscience and Biotechnology, 52 Oundong, Yusong, Daejeo 305-333, Korea 2 College of Pharmacy, Catholic University of Daegu, Kyungbook 712-702, Korea 3 College of Pharmacy, Chungnam National University, Daejeon 305-764, KoreaAs part of an ongoing study aimed at identifying the anti-complement active compound from the flower buds of Daphne genkwa, ten flavonoids, one biscoumarin and one amide were isolated. Their structures wereidentified from spectroscopic and physicochemical data as genkwanin 5-O-D-primveroside(1), apigenin 7-O-D-glucuronide(2), genkwanin 5-O-D-glucopyranoside (3), apigenin 5-O-D-glucopyranoside (4), genkwanin(5), apigenin (6), luteolin 7-methyl ether (7), luteolin (8), daphnoretin (9), velutin (10), 7-methoxyacacetin (11) and aurantiamide acetate (12). Among them, compounds 9, 10 and 12 were isolated from this plant for the first time. The anti-complement activity of these compounds was tested against the classical pathway of the com-plement system. Among the compounds, daphnoretin (9) exhibited significant anti-complement activity with an IC50 value of 11.4M, whereas the other compounds were not active in the assay. Copyright (C) 2006 John Wiley & Sons, Ltd.Keywords: Daphne genkwa; anti-complement activity; daphnoretin.INTRODUCTIONGenkwa Flos (Won-hwa in Korean, Yuanhua in Chinese) is the flower buds of Daphne genkwa Sieb.et Zucc. (Thymelaeaceae), which are collected in the spring before blossoming. As a traditional medi-cine, they have been used for the treatment of ascites,edema and asthma (Lin et al., 2001a). Previous phytochemical studies on D. genkwa have led to the isolation of pharmacologically active flavonoids, diterpene orthoester and coumarins, showing inhibitory activity against xanthine oxidase and adenosine 3,5-cyclic monophosphate phosphodiesterase, as well as exhibiting antileukemic activity (Lin et al., 2001b).The complement system can be activated by a cas-cade mechanism involving the classical pathway, the alternative pathway and the MBL/MASP (mannan binding lectin/MBL-associated serine protease) path-way (Roos et al., 2001). The proteolytic cascade allows for significant amplication since each proteinase mole-cule activated at one step can generate multiple copies of activated enzyme later in the cascade. These in turn cleave non-enzymatic components such as C3, C4 and C5 (i.e. C3b, C4b and C5b), which are involved in biological effector functions, such as opsonization, phagocytosis and immunomodulation. However, the small molecules, C3a, C4a and C5a, which are known as an anaphylatoxins, induce the release of various media-tors from mast cells and lymphocytes, thereby causing a variety of diseases (Geetha and Varalakshmi, 1999).Therefore, the ability to modulate the complement sys-tem will be useful for treating inflammatory diseases.MATERIAL AND METHODSPlant material. The dried flower buds of D. genkwa were purchased from Chilsung pharmaceutical company,Daejeon, Korea. A voucher specimen (PB3906.2)was deposited at the herbarium of Plant Extract Bank, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. General instrumental equipment. Melting point was measured on a Yanagimoto micro hot-stage melting point apparatus. Optical rotations were measured with a Jasco DIP-370 automatic polarimeter in CHCl3.1 13H- and C-NMR spectra were recorded on Bruker DMX 300 or 600 spectrophotometers, the chemical shifts being represented as ppm. EI and ESI-MS were measured on a HP5989A DIP mass spectrometer.General instrumental equipment. Melting point was measured on a Yanagimoto micro hot-stage melting point apparatus. Optical rotations were measured with a Jasco DIP-370 automatic polarimeter in CHCl3. 1 13 H- and C-NMR spectra were recorded on Bruker DMX 300 or 600 spectrophotometers, the chemical shifts being represented as ppm. EI and ESI-MS were measured on a HP5989A DIP mass spectrometerExtraction and isolation. The dried flower buds of D. genkwa (11.7kg) were extracted with MeOH at room temperature to yield a dark-green extract (1.5 kg). The MeOH extract was suspended in H2O and then parti- tioned with hexane to give the hexane-soluble fraction(323.0 g). The resulting H2O layer was further parti-tioned with CHCl3 and EtOAc to yield the CHCl3-soluble fraction (150.0g) and EtOAc-soluble fraction(164.0 g), respectively. The CHCl3-soluble fraction was kept overnight at room temperature to yield yellow precipitates (52.0g). A portion of this precipitate (50.0g) was chromatographed on a sephadex LH-20 column eluted with a stepwise gradient of MeOHH2O to give eight fractions (Fr. AH). Repeated column chromatography of Fr. B (600.0mg) on a RP C-18 silica gel (60%, MeOH in H2O) and a sephadex LH-20 (50%, MeOH in H2O) afforded compounds 1 (22.8mg) and 2 (79.2mg). From Fr. C (1.0 g), compounds 3 (34.5 mg) and 4 (7.0 mg) were obtained by silica column chroma- tography using CHCl3MeOHH2O (7:1:0.1) as an eluent. Fr. E (145.5 mg) was rechromatographed on aRP C-18 silica gel column eluted with a stepwise gradi-ent of MeOHH2O gradient to give compound 5(2.7 mg). Fr. F (747.0mg) was chromatographed on a RP C-18 silica gel column (MeOHH2O gradient sys-tem) to afford compounds 6 (24.2mg) and 7 (10.0 mg).Fr. G (1.7g) was rechromatographed on a RP C-18silica gel column using a stepwise gradient system of MeOHH2O to obtain compound 8 (12.0 mg). Also, a portion of CHCl3-soluble fraction (74.0g) was chroma-tographed on a silica gel column using a CHCl3MeOH gradient system as an eluent to afford ten fractions (Fr.110). Of these, column chromatography of Fr. 3 (20.0g) on RP C-18 silica gel (70% MeOH in H2O) gave ten sub-fractions (Fr. 3AJ). Fr. 3A (3.1 g), 3B (1.6 g)and 3C (1.1g) were crystallized from MeOH at room temperature to yield compounds 9 (30.4mg; white powder), 10 (310.5 mg; yellow needles) and 11 (62.4mg;yellow needles), respectively. Fraction 3B was further chromatographed on a silica gel column (hexaneEtOAc, 5:1) to yield ten subfractions (3B-13B-10).Subfraction 3B-9 was crystallized from hexaneEtOAc(10:1) to yield compound 12 (20.3 mg; white powder)Daphnoretin (9). White powder; mp 244245C; ESI- +MS m/z: 351.4 M - H , 375.5 M + Na (C19H12O7);1H-NMR (300MHz, DMSO-d6) : 3.80 (s, -OCH3), 6.36(d, J = 9.6 Hz, H-3), 6.87 (s, H-8), 7.12 (dd, J = 2.4,8.7Hz, H-6), 7.18 (d, J = 2.4 Hz, H-8), 7.22 (s, H-5),7.71 (d, J = 8.7 Hz, H-5), 7.87 (s, H-4), 8.04 (d, J =9.6Hz, H-4).Velutin (10). Yellow needles; mp 225226C; EI-MS+ 1m/z: 314 M (C17H14O6); H-NMR (600 MHz, DMSO-d6) : 3.87 (s, -OCH3), 3.90 (s, -OCH3), 6.37 (d. J =2.4 Hz, H-6), 6.80 (d, J =2.4 Hz, H-8), 6.84 (s, H-3),6.94 (d, J = 8.4 Hz, H-5) 7.58 (br s, H-6), 7.60 (d, J =1.8 Hz, H-2).Aurantiamide acetate (12). White powder, mp 186- +187 C; ESI-MS m/z: 443.7 MH ,467.7 M+Na1(C27H28N2O4); H-NMR (600MHz, CDCl3) : 2.04 (s,H-4), 2.75 (2H, ddd, J = 6.6, 7.2, 14.4 Hz, H-), 3.07. (dd, J = 8.4, 13.8 Hz, H-a), 3.23 (dd, J = 5.8, 13.8 Hz,H-b), 3.83 (dd, J = 4.2, 11.4Hz, H-2a), 3.94 (dd, J =5.1, 11.4Hz, H-2b), 4.36 (m, H-), 4.77 (dd, J = 7.2,13.8Hz, H-), 5.93 (d, J = 7.2 Hz, -NH), 6.73 (d, J =5.4Hz, -NH), 7.077.20 (5H, m, H-), 7.237.31(5H, m, H-), 7.45 (2H, t, J = 7.8Hz, H-4, 6), 7.53(t,J = 7.8Hz, H-5), 7.72 (2H, d, J = 7.8Hz, H-3, 7).Anti-complement activity through the classical path-way. Anti-complement activity was examined by amodified method of Mayer as described previously (Minet al., 1989). Each sample was dissolved in DMSO and used as a blank. Anti-complement activity was deter-mined as a mean of triplicate measurements and ex-pressed as the IC50 values from complement-dependenthemolysis of the control. Tiliroside was used as a posi-tive control (Jung et al., 1998).RESULTS AND DISCUSSIONThe ability to modulate the complement activity might be important for the treatment of inflammation(Cimanga et al., 1995). As part of an ongoing study aimed at identifying novel active anti-complement com-pounds from natural sources, the MeOH extract of the flower buds from D. genkwa was found to exhibit appreciable anti-complement activity with an IC50 value of 14.2 g/mL. The MeOH extract was further parti-tioned with CHCl3, EtOAc and H2O, successively. Of these fractions, the CHCl3-soluble fraction exhibited strong activity with an IC50 value of 10.13g/mL (Table 1).Table 1. Anti-complement activity of extracts and compounds(112) isolated from D.genkwa against complement system in vitro. Experimental values are expressed as mean SD from three independent experimentsa No effect.b Used as a positive control.* IC50 represents the 50% inhibitory concentration from complement-dependent hemolysis of the control.Figure 1. Chemical structures of compounds from the flowers of Daphne genkwa.Repeated column chromatography of the CHCl3-precipitate and CHCl3-soluble fractions on sephadex LH-20, silica gel and RP C-18 silica gel columns using a bioassay-guided separation procedure led to the iso-lation of ten flavones (18, 1011), one biscoumarin (9)and an amide (12). These compounds were identified by comparing their spectroscopic data with those reported in the literature as genkwanin 5-O-D-pimveroside (1), apigenin 7-O-D-glucuronide (2),genkwanin 5-O-D-glucopyranoside (3), apigenin 5-O-D-glucopyranoside (4), genkwanin (5), apigenin (6),luteolin 7-methyl ether (7), luteolin (8), daphnoretin(9), velutin (10), 7-methoxy acacetin (11) and aurantiamide acetate (12) (Fig. 1). Of these compounds,9, 10 and 12 were isolated from the flower buds of D.genkwa for the first time.The compounds (112) were bioassayed for their clas-sical pathway complement inhibitory activity in vitro using the protocol previously reported(Jung et al., 1998).The results (IC50 values) are summarized in Table 1. Of these, compound 9 had a significant effect on the clas-sical pathway of the complement system with an IC50 value of 11.40M (Fig. 2), when compared with tiliroside(IC50 = 78.56M), which was used as a positive control.On the other hand, the other compounds (18, 1011) were completely inactive. Biological studies of daphnoretin (9) from this family (Thymelaeaceae) have reported that it inhibits DNA polymerase lyase, pro-tein kinase C activation (Li et al., 2004) and exhibitsanti-fungal activity (Hu et al., 2000). To our knowledge,this is the first report of the anti-complement activity of daphnoretin (9). However, more detailed studies will be needed to clarify the structure-activity relationship with other bisco