银屑病治疗药物YXD09的非临床实验研究
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摘要
银屑病是一种受多种因素影响的斑块鳞屑性慢性炎症性皮肤,以表皮增生过度及角化不全为主要特征,症状的严重程度不等,从较小的局部斑块至累及全身。它具有反复性,难以根治。它影响到2%-3%的总人口,是全球最为常见的自身免疫疾病之一。它影响着全世界所有地区,在北美和欧洲,银屑病是成年人中最普遍的免疫介导性皮肤病,影响着大约2500万人。而在中国,银屑病的患病率为0.123%,其中男性多于女性,北方多于南方,城市高于农村。在非洲国家的发病率则比较低。银屑病的发病不但存在显著的种族差异、性别差异,在地理和季节上差异也很显著。高寒地区的发病率明显高于温暖地区,而冬春季病情可能加重夏季则自然减轻。
现代的研究认为银屑病是一种多基因遗传背景下的T细胞异常的自身免疫疾病,有关角质细胞、T细胞、中性粒细胞、神经细胞等在银屑病中的作用以及各种细胞之间复杂的相互作用等方面的探索和争论不断的揭示着其发病机理,推动着医学的发展,同时也促进人们寻找更有效的抗银屑病药物,为人类的健康发展提供支持。
银屑病的药物治疗按严重程度分别选用局部用药物、光疗法、传统口服药物和新型生物制剂。若患者的银屑病对局部用药物和光疗法治疗都无适当响应或疾病累及皮肤已十分广泛,则需及时改用全身用药物。传统口服用银屑病治疗药物主要有类维生素A类物质、甲氨蝶呤(methotrexate)、环孢素(ciclosporin)和阿西曲汀(acitretine)等,它们治疗银屑病有效并具有使用简便和价格低廉的优势,但对肝、肾和骨髓的毒性较大并存在致畸性,实际应用受到一定的限制。生物制剂与传统全身用药物相比,能够特异性地靶向免疫系统因而具有更好的安全性和耐受性,所以适于用作更长期的银屑病治疗药物。欧美至今已批准了6种银屑病治疗用生物制剂。其中一类靶向与银屑病发病机制相关的T细胞,但由于疗效差,目前临床应用已大为减少；另一类TNF抑制剂为治疗中至重度银屑病一线药物。生物制剂对普通患者来说,价格昂贵,安全性仍然有限。
中医治疗方法颇多,相应地出现了很多的治疗方药,青黛胶囊、雷公藤片、丹参注射液、清开灵注射液等在临床应用中得到了大力推广,并获得了良好的疗效；其它一些复方中药制剂和单味中药提取物如银杏叶内脂、氧化苦参碱、喜树果涂膜剂、去氢骆驼蓬碱软膏等治疗本病亦显示出一定的效果和发展势头。由于中医药治疗银屑病可延长复发时间,不良反应小,疗效确切,其治疗上的优势不容忽视。
但是由于中药复方有效成分不明确,并且目前中国药典中药复方质量控制大多采用单一指标成分定量的方法,该方法不能全面客观的反应中药内在质量,而且不符合中药整体性的特点,存在一定的局限性。中药制品质量和安全性评价方法还不够完善,不能实现中药质量稳定可控的要求,因此中药复方至今未得到国际主流药物市场的承认。
由于银屑病的顽固性特点及中西药在治疗上的独特缺陷,中医中药治疗银屑病具有广阔的研究空间。在基础研究方面,应进一步深化研究中医药内外治疗银屑病的作用机理,建立中医辨证分型的实验动物模型,统一实验分型标准和实验室观察指标,更深层次阐明内外用中药制剂的药理学作用机制。由此,通过完善而系统的实验研究,以中医辨证分型论治为基础,以实验室指标为依据,开发出疗效确切的中药单体或复方,探索有效的作用靶点,进而阐明其作用机制,是今后中医药治疗银屑病实验研究的主要方向。
鉴于银屑病的特征及其治疗药物的研究现状,更多地开发治疗银屑病的相关中药制剂,在完善中药研究资料、充分发挥中药毒副作用小的优势的同时,尽可能提高其质控水平、避免中药的质量评价与其作用整体性相违背的劣势,对银屑病治疗药物发展具有重要意义。
经对银屑病的特征、发病机制及银屑病药物治疗现状的认识,通过对《中药大辞典》相关组方的特性进行研究,综合国内同类制剂的疗效考察,我们进行了银屑病治疗药物的研究。
本论文通过研究银屑病治疗药物YXD09的组方,质量控制标准,生产工艺优化,运用小鼠阴道上皮细胞模型、尾部皮肤鳞片模型和离体透皮试验,丰富本药物的药效学和药代动力学,完善其非临床研究内容,为银屑病的治疗提供实验依据。
(1)银屑病治疗药物YXD09的组方研究
通过对银屑病治疗状况的认识,针对银屑病的特征及发病机制,又根据《中药大辞典》中各药味的功能特性及应用情况,建立银屑病治疗药物YXD09的组方。
(2)制备工艺优化研究
本试验选择回流时间(A)、回流次数(B)、乙醇浓度(C)、乙醇用量(D)为考察因素,以大黄素含量为评价指标,用L9(34)正交试验设计进行试验。按十分之一处方量秤取药材,共9份,按正交试验方案进行提取并浓缩至总体积的50%得YXD09,将浓缩液滤过,精密吸取滤过液20mL,以HPLC法测定YXD09中大黄素的含量。
结果表明,影响最佳工艺的因素顺序为：乙醇浓度>回流时间>回流次数>乙醇用量,且最佳工艺参数为：C3A2B2D2。方差分析表明,4种工艺因素对大黄素的含量均没有显著差异(P>0.05)。因而在实际生产中,为了降低生产成本,缩短生产周期,将回流时间定为2小时,而不用4小时。因此,最后选择的YXD09醇提优化条件为A1B2C3D2,即回流2小时,回流2次,乙醇浓度为80%,乙醇用量为8倍。
选用大黄、羊蹄和虎杖3味中药材及其余药味考察合并醇提与分开醇提对大黄素含量的影响。结果显示,合并醇提液和分开醇提液中的大黄素含量分别为162.78ug/ml和161.22ug/ml.采用EXCEL软件对其结果进行方差分析,发现P>0.05,说明两种醇提液中大黄素的含量没有显著差异。
(3)质量控制研究
采用薄层色谱法对YXD09中大黄素和当归进行了定性分析。依照薄层色谱法(中国药典2010年版一部附录VIB)试验,分别吸取供试品溶液、对照品溶液和阴性对照品溶液3种溶液各10μl,分别点于同一硅胶G薄层板上,以石油醚(30℃-60℃)-甲酸乙酯-甲酸(15：5：1)为展开剂,展开,取出,晾干,将硅胶G薄层板置于365nm紫外光灯下检视,对大黄和当归进行薄层色谱鉴别。结果显示,使用薄层色谱法鉴别YXD09中的大黄和当归,操作简单,重现性好,效果明显,可以做为YXD09的薄层鉴别标准。
通过高效液相色谱法测定了YXD09中大黄素的含量。色谱条件是色谱柱：ZORBAX Extend-C18(4.6mm×150mm,5μm)色谱柱；流动相：0-6min时,甲醇：水(40：60)；6-20min时,甲醇：水：0.1%磷酸(75：10：15),梯度洗脱；流速：1.0ml/min;检测波长：254nm；柱温：35℃。高效液相色谱法测定三个批次的YXD09中大黄素含量分别为166.83、172.52、160.43μg/ml,RSD值分别为1.7%、1.3%、1.1%。
首次采用了薄层色谱法,对YXD09中大黄和当归进行了定性分析,通过方法学考察了高效液相色谱法可以简便有效地测定YXD09中所含大黄素的总量,定性与定量相辅相成,检验方法既全面可靠又具有一定的专属性,为YXD09的质量控制及药效物质基础的研究提供了基础,尤其是对大黄素的检测及含量测定,可以作为YXD09的质量控制标准。
同时又对大黄素及蛇床子素的同时定量方法进行了研究。
(4)药效学研究
利用小鼠阴道上皮细胞模型探讨YXD09对雌激素周期小鼠阴道上皮细胞有丝分裂的影响。选取造模成功的健康昆明种雌性小鼠30只,随机分为3组,每组10只小鼠,分别为： YXD09组,阳性对照组,模型组；另随机取没有造模的健康昆明种小鼠10只,作为空白试验组。正式试验期为7d。第7d开始给药：YXD09组采用自制阴道灌流器,每次灌注5分钟；空白组、模型组采用同样方法灌流生理盐水10mL/kg;阳性对照组于小鼠阴道壁均匀涂抹一层0.02%的丙酸氯倍他索软膏。最后一次给药1h后,各组小鼠于腹腔注射秋水仙碱15mg/kg,使有丝分裂终止在细胞分裂中期,取阴道标本,置于10%的福尔马林溶液固定,石蜡包埋,HE染色,于普通光学显微镜下计数。结果表明,YXD09可抑制雌激素周期小鼠阴道上皮细胞的有丝分裂,效果与丙酸氯倍他索软膏相似(P>0.05)。
利用鼠尾鳞片表皮模型探讨银屑净对小鼠尾部鳞片表皮颗粒层形成的影响。选取体重相近、健康昆明种小鼠30只,随机分为3组,分别为：YXD09组,阳性对照组,空白组,每组10只小鼠,公母各半。正式试验期为7d。分别在YXD09组小鼠尾部均匀涂抹一层相应的药物,阳性对照组小鼠尾部涂抹丙酸氯倍他索软膏,空白组小鼠尾部则涂抹生理盐水。在第7天处死小鼠,取小鼠尾部距离尾根约2cm处背面皮肤一长条,置于10%的福尔马林溶液固定,石蜡包埋,HE染色,于普通光学显微镜下对每个标本逐个观察鳞片,看鳞片表皮是否有颗粒层形成。结果表明,阳性对照组和YXD09组小鼠尾部含颗粒层鳞片所占比例均极显著提高(P<0.01),分别提高70.1%、116.1%。YXD09的效果明显优于丙酸氯倍他索软膏(P<0.05)。
这些结果提示,YXD09可能通过抑制表皮细胞的过度增殖和促进表皮细胞角质化来达到抗银屑病的目的,并在抗银屑病的临床应用具有广阔的前景。
(5)药代动力学研究
运用SD大鼠腹部皮肤作为皮肤模型,采用高效液相色谱法测定了YXD09中大黄素的单位面积累积透过量和皮肤滞留量,探讨了YXD09的体外药代动力学。采用水平式Franz扩散池,扩散池体积为3.5ml,有效扩散面积为1.13cm2,恒温水浴加热,温度控制在32℃左右,转速为250rpm,接受介质为30%PEG-生理盐水,排除接受池中的气泡,向扩散池中加入3.5ml的YXD09药液。分别于1,2,4,6,8,10,12,24h时取样1ml,经0.22gm滤膜过滤,取续滤液,测定大黄素的单位面积累积透过量和皮肤滞留量。结果显示,YXD09中大黄素的体外透皮释放方程为Q=0.338t-1.411(R=0.9972),24h的累积渗透量为6.76μg/cm2,皮肤滞留量为19.10gg/cm2。表明YXD09中大黄素以零级动力学经皮肤渗透。
Psoriasis is a kind of chronic inflammation with plaques and scales, influenced by varieties of reasons. Its symptoms'severity varies from small plaques locally to areas all over body. It is repeatable and hard to cure, it influences2%-3%of the total population on earth, being one of the most common autoimmune diseases. Psoriasis affects all regions of the world, being the most common immune-mediated skin disease in adults in North America and Europe, affecting about25million people. In China, the prevalence of psoriasis is0.123%, which is more of men than women, the North than the South, the urban than rural areas. The incidence in African countries is relatively low. Psoriasis has significant difference not only in race and gender, but also in geography and season. The incidence in Alpine areas is significantly higher than in warm regions, and the Symptoms are aggravated in winter and spring, relieved in summer.
Psoriasis on modern research is considered autoimmune diseases with abnormal T cells in the polygenic background. The research and argument about the action of keratinocytes, T cells, neutrophils, and nerve cells on psoriasis and their complex reactions constantly reveal its pathogenesis, to promote the development of medical science, but also to push people to find a more effective anti-psoriasis drug to provide support for the development of human beings.
Psoriasis is treated according to its severity by four kinds of drugs, such as topical medicines, light therapy method, traditional oral medicines and new biological preparations etc. If topical medicines and light therapy method doesn't act on psoriasis, the whole-body drug should be used. Traditional oral medicines conclude vitamin A substances, methotrexate, cyclosporine and acitretine etc., they take effect and have advantages of simple using method and low price, but disadvantages of heavier toxicity in livers, kidneys and bone marrow with teratogenicity, and so restricted usage. Compared with traditional systemic drugs, biological agents can specifically target immune systems and have a better safety and tolerability, fit for a longer-term treatment of psoriasis. Six biological agents have been approved in Europe and America for psoriasis. One class of them targets to T cells related to psoriasis' mechanisms, but its usage has been decreased because of its weak effect. The other one-TNF inhibitor is the first-line drug for moderate to severe psoriasis, but it is expensive for ordinary patients and its safety is limited.
Quite a lot of therapies by traditional Chinese medicine exist, accordingly many prescriptions appear, Qingdai capsule, Tripterygium tablets, Danshen injection, Qing Kai Ling Injection have been widely used in clinical applications and got reliable good efficacy.
But, because of the active ingredients of traditional Chinese medicine are not clear, and usually one single indicator is assayed in their quality control in Chinese Pharmacopoeia, which cannot reflect the inner quality entirely and objectively, not fit for the holistic characteristics of traditional Chinese medicine, so limited to be used, and is not in line with the characteristics of Chinese medicine holistic, there are certain limitations. Chinese medicine products' quality and safety evaluation is not perfect, and can not achieve the aim of stable and controllable quality, so they have not been recognized by the international mainstream drug market.
Due to psoriasis' intractabilities and its special defects, Chinese medicine treatment of psoriasis has broad research space. In basic research, the study of Chinese medicine inside and outside action mechanisms on psoriasis should be deepened, parting of experimental animal models should be established, the classification criteria and laboratory observation indicators should be unified, and their pharmacological action mechanisms should be clarified. Thus, through a comprehensive and systematic experimental study, based on theory of differentiation of symptoms and classification of syndrome together with laboratory index, to explore monome of chinese herbs or compound preparations with definite effect, searching for useful target, and to clarify its action mechanisms is the main direction of the Experimental Study of Chinese medicine treatment of psoriasis in the future.
For psoriasis' characteristic and the research status of its drug, it is significant for psoriasis' treatment to developing more Chinese herba preparations, promoting the related data and exploiting its advantages of weak side effects, together with improving level of quality control and casting away the disadvantages of violation of herba's assessment level and integrity.
Through recognitions to psoriasis and the learning of Dictionary of Traditional Chinese Medicine, we carried out the study of drugs YXD09for psoriasis, such as its prescriptions, process, quality control method, pharmacodynamics and its pharmacokinetics.
(1) The prescriptions of YXD09
The prescriptions of YXD09are based on Dictionary of Traditiongal Chinese Medicine, and the knowledge of psoriasis' characteristics and mechanisms.
(2) The preparation optimization of YXD09
We choose orthogonal test on L9(34) design with reflow time (A), reflow cycles (B), ethanol concentration (C), the amount of ethanol (D) for factors, emodin for indicator. Taken one-tenth of prescriptions'weight,9shares, after orthogonal test, the extract by ethanol is filtrated and the emodin content is measured by HPLC.
The results indicate that the range of factors that affect the optimum process is: ethanol concentration> reflux time> reflux times> the amount of ethanol, and the optimum parameters:C3A2B2D2. Analysis of variance showed that the four process factors were not significantly different (P>0.05). Thus in the practical production, in order to reduce production costs, shorten the production cycle, the reflux time is set at2hours instead of4hours. Thus, the final choice of YXD09's optimization condition is A1B2C3D2, which is refluxing for2hours,2times, with80%ethanol in8times of the drugs' amount.
Choose rhubarb, Rumex and Polygonum cuspidatum and other herbs to examine the influence of the combined and separate extract to emodin content. The result of the combined and separate one is shown to be162.78ug/ml and161.22ug/ml respectively. Analysis of variance with EXCEL software shows P>0.05, the two extract methods have no significant difference in content of emodin.
(3) The quality control method study
By the thin-layer chromatography, qualitative analysis of emodin and Angelica in YXD09is done. According to thin-layer chromatography (Chinese Pharmacopoeia2010edition is an appendix VI B), drawing10μl of the test solution, reference solution and negative control solution respectively, applying them on the same silica G plate with petroleum ether (30℃-60℃)-ethyl formate-formic acid (15:5:1) as developing agent, after spreading, it is taken under the365nm UV lamp to examine. The method shows the advantages of simple operation, good reproducibility, obvious effect, so can be used as TLC standard of YXD09.
Emodin in YXD09is measured by high-performance liquid chromatography. HPLC conditions are like that, Column:ZORBAX Extend-C18(4.6mm×150mm,5um) column; mobile phase:0-6min, methanol:water (40:60);6-20min, methanol: water:0.1%phosphoric acid (75:10:15), gradient elution; flow rate:1.0ml/min; detection wavelength:254nm; column temperature:35℃. HPLC determination value of emodin of three batches of YXD09is166.83μg/ml,172.52μg/ml,160.43μg/ml, and the RSD value is1.7%,1.3%,1.1%, respectively.
The above-mentioned method first use thin-layer chromatography to qualitatively analyze rhubarb and angelica of YXD09, and adopt an effective method which is high-performance liquid chromatography to determine the emodine in rhubarb, with advantages of the qualitative and quantitative analysis supplementing each other, comprehensive, reliable and exclusive, and can be the quality control standards of YXD09.
On the other hand, the simultaneous assaying of Emodine and Osthole has been studied.
(4) The pharmacodynamic study
The test is to use mouse vaginal epithelial cells as the model to study the effects of YXD09on mitosis of it.30health female mice from Kunming after successful modeling are randomly divided into three groups, with each group of10mice, respectively:YXD09group, the positive control group, model group; another randomly selected10without modeling as blank test group. The test period is7days. On the7th day, the drugs are administered:YXD09group is perfused5minutes each; the blank group, model group, by the same method is perfused with lOmL/kg of saline each; the positive control group is evenly coated with a layer of0.02%propionate clobetasol ointment in its vaginal wall.1h after the last dose, the mice in each group is treated by intraperitoneal injection of colchicine of15mg/kg to stop its mitosis at the mid age of cell division. Samples are fixed in10%formalin solution, paraffin-embedded, HE stained, counted under the ordinary optical microscope. The results show that YXD09can inhibit mitosis of mice vaginal epithelial cells in estrogen cycle, the effect close to propionate clobetasol ointment (P>0.05).
The test also uses mouse-tail scaly epidermis as model to examine the effect of YXD09on forming of its granular layer.30health female mice with similar weight, are randomly divided into three groups, with each group of10mice, respectively: YXD09group, the positive control group, blank group, male and female half and half. The test period is7days. Smearing correspondent drug to YXD09group, while the positive control group treated with propionate clobetasol ointment,and the blank by saline. Mice are sacrificed on the7th day, take the tail's back-skin2cm away from the root, fixed in10%formalin solution, paraffin-embedded, HE stained, placed under ordinary optical microscope to examine forming of granular layer on scales. The results showed that the granular layer proportion of positive control group, YXD09group are significantly increased (P<0.01), respectively,70.1%,116.1%. Effect of YXD09is significantly superior to that of propionate clobetasol ointment (P<0.05).
These results suggest that the the YXD09may inhibit the excessive proliferation of epidermal cells and to promote epidermal cells cutin to achieve the purpose of inhibiting psoriasis, and has broad prospects in the clinical application.
(5) The pharmacokinetic study
Using SD rat's abdominal skin as model, by HPLC to determine cumulative transit and skin retention rate of emodin in YXD09per unit area, the vitro pharmacokinetic is studied. Adopting Franz horizontal diffusion cell with volume of3.5ml, the effective diffusion area of1.13cmz, heating by constant temperature water bath to control the temperature at about32℃, the rotational speed of250rpm, accept medium of30%PEG-saline after bubbles' excluded,3.5ml YXD09solution is added to the diffusion cell. Sampling lml respectively in1,2,4,6,8,10,12,24h after0.22μm membrane filtration, the filtrate obtained, determination of cumulative transit and skin retention rate of emodin per unit area. The results show equation of vitro transdermal delivery of emodin is Q=0.338t-1.411(R=0.9972), with24h cumulative permeation amount of6.76μg/cm2, skin retention of19.10μg/cm2, indicating that emodin zero-order kinetics in YXD09's penetration through the skin.
现代的研究认为银屑病是一种多基因遗传背景下的T细胞异常的自身免疫疾病,有关角质细胞、T细胞、中性粒细胞、神经细胞等在银屑病中的作用以及各种细胞之间复杂的相互作用等方面的探索和争论不断的揭示着其发病机理,推动着医学的发展,同时也促进人们寻找更有效的抗银屑病药物,为人类的健康发展提供支持。
银屑病的药物治疗按严重程度分别选用局部用药物、光疗法、传统口服药物和新型生物制剂。若患者的银屑病对局部用药物和光疗法治疗都无适当响应或疾病累及皮肤已十分广泛,则需及时改用全身用药物。传统口服用银屑病治疗药物主要有类维生素A类物质、甲氨蝶呤(methotrexate)、环孢素(ciclosporin)和阿西曲汀(acitretine)等,它们治疗银屑病有效并具有使用简便和价格低廉的优势,但对肝、肾和骨髓的毒性较大并存在致畸性,实际应用受到一定的限制。生物制剂与传统全身用药物相比,能够特异性地靶向免疫系统因而具有更好的安全性和耐受性,所以适于用作更长期的银屑病治疗药物。欧美至今已批准了6种银屑病治疗用生物制剂。其中一类靶向与银屑病发病机制相关的T细胞,但由于疗效差,目前临床应用已大为减少；另一类TNF抑制剂为治疗中至重度银屑病一线药物。生物制剂对普通患者来说,价格昂贵,安全性仍然有限。
中医治疗方法颇多,相应地出现了很多的治疗方药,青黛胶囊、雷公藤片、丹参注射液、清开灵注射液等在临床应用中得到了大力推广,并获得了良好的疗效；其它一些复方中药制剂和单味中药提取物如银杏叶内脂、氧化苦参碱、喜树果涂膜剂、去氢骆驼蓬碱软膏等治疗本病亦显示出一定的效果和发展势头。由于中医药治疗银屑病可延长复发时间,不良反应小,疗效确切,其治疗上的优势不容忽视。
但是由于中药复方有效成分不明确,并且目前中国药典中药复方质量控制大多采用单一指标成分定量的方法,该方法不能全面客观的反应中药内在质量,而且不符合中药整体性的特点,存在一定的局限性。中药制品质量和安全性评价方法还不够完善,不能实现中药质量稳定可控的要求,因此中药复方至今未得到国际主流药物市场的承认。
由于银屑病的顽固性特点及中西药在治疗上的独特缺陷,中医中药治疗银屑病具有广阔的研究空间。在基础研究方面,应进一步深化研究中医药内外治疗银屑病的作用机理,建立中医辨证分型的实验动物模型,统一实验分型标准和实验室观察指标,更深层次阐明内外用中药制剂的药理学作用机制。由此,通过完善而系统的实验研究,以中医辨证分型论治为基础,以实验室指标为依据,开发出疗效确切的中药单体或复方,探索有效的作用靶点,进而阐明其作用机制,是今后中医药治疗银屑病实验研究的主要方向。
鉴于银屑病的特征及其治疗药物的研究现状,更多地开发治疗银屑病的相关中药制剂,在完善中药研究资料、充分发挥中药毒副作用小的优势的同时,尽可能提高其质控水平、避免中药的质量评价与其作用整体性相违背的劣势,对银屑病治疗药物发展具有重要意义。
经对银屑病的特征、发病机制及银屑病药物治疗现状的认识,通过对《中药大辞典》相关组方的特性进行研究,综合国内同类制剂的疗效考察,我们进行了银屑病治疗药物的研究。
本论文通过研究银屑病治疗药物YXD09的组方,质量控制标准,生产工艺优化,运用小鼠阴道上皮细胞模型、尾部皮肤鳞片模型和离体透皮试验,丰富本药物的药效学和药代动力学,完善其非临床研究内容,为银屑病的治疗提供实验依据。
(1)银屑病治疗药物YXD09的组方研究
通过对银屑病治疗状况的认识,针对银屑病的特征及发病机制,又根据《中药大辞典》中各药味的功能特性及应用情况,建立银屑病治疗药物YXD09的组方。
(2)制备工艺优化研究
本试验选择回流时间(A)、回流次数(B)、乙醇浓度(C)、乙醇用量(D)为考察因素,以大黄素含量为评价指标,用L9(34)正交试验设计进行试验。按十分之一处方量秤取药材,共9份,按正交试验方案进行提取并浓缩至总体积的50%得YXD09,将浓缩液滤过,精密吸取滤过液20mL,以HPLC法测定YXD09中大黄素的含量。
结果表明,影响最佳工艺的因素顺序为：乙醇浓度>回流时间>回流次数>乙醇用量,且最佳工艺参数为：C3A2B2D2。方差分析表明,4种工艺因素对大黄素的含量均没有显著差异(P>0.05)。因而在实际生产中,为了降低生产成本,缩短生产周期,将回流时间定为2小时,而不用4小时。因此,最后选择的YXD09醇提优化条件为A1B2C3D2,即回流2小时,回流2次,乙醇浓度为80%,乙醇用量为8倍。
选用大黄、羊蹄和虎杖3味中药材及其余药味考察合并醇提与分开醇提对大黄素含量的影响。结果显示,合并醇提液和分开醇提液中的大黄素含量分别为162.78ug/ml和161.22ug/ml.采用EXCEL软件对其结果进行方差分析,发现P>0.05,说明两种醇提液中大黄素的含量没有显著差异。
(3)质量控制研究
采用薄层色谱法对YXD09中大黄素和当归进行了定性分析。依照薄层色谱法(中国药典2010年版一部附录VIB)试验,分别吸取供试品溶液、对照品溶液和阴性对照品溶液3种溶液各10μl,分别点于同一硅胶G薄层板上,以石油醚(30℃-60℃)-甲酸乙酯-甲酸(15：5：1)为展开剂,展开,取出,晾干,将硅胶G薄层板置于365nm紫外光灯下检视,对大黄和当归进行薄层色谱鉴别。结果显示,使用薄层色谱法鉴别YXD09中的大黄和当归,操作简单,重现性好,效果明显,可以做为YXD09的薄层鉴别标准。
通过高效液相色谱法测定了YXD09中大黄素的含量。色谱条件是色谱柱：ZORBAX Extend-C18(4.6mm×150mm,5μm)色谱柱；流动相：0-6min时,甲醇：水(40：60)；6-20min时,甲醇：水：0.1%磷酸(75：10：15),梯度洗脱；流速：1.0ml/min;检测波长：254nm；柱温：35℃。高效液相色谱法测定三个批次的YXD09中大黄素含量分别为166.83、172.52、160.43μg/ml,RSD值分别为1.7%、1.3%、1.1%。
首次采用了薄层色谱法,对YXD09中大黄和当归进行了定性分析,通过方法学考察了高效液相色谱法可以简便有效地测定YXD09中所含大黄素的总量,定性与定量相辅相成,检验方法既全面可靠又具有一定的专属性,为YXD09的质量控制及药效物质基础的研究提供了基础,尤其是对大黄素的检测及含量测定,可以作为YXD09的质量控制标准。
同时又对大黄素及蛇床子素的同时定量方法进行了研究。
(4)药效学研究
利用小鼠阴道上皮细胞模型探讨YXD09对雌激素周期小鼠阴道上皮细胞有丝分裂的影响。选取造模成功的健康昆明种雌性小鼠30只,随机分为3组,每组10只小鼠,分别为： YXD09组,阳性对照组,模型组；另随机取没有造模的健康昆明种小鼠10只,作为空白试验组。正式试验期为7d。第7d开始给药：YXD09组采用自制阴道灌流器,每次灌注5分钟；空白组、模型组采用同样方法灌流生理盐水10mL/kg;阳性对照组于小鼠阴道壁均匀涂抹一层0.02%的丙酸氯倍他索软膏。最后一次给药1h后,各组小鼠于腹腔注射秋水仙碱15mg/kg,使有丝分裂终止在细胞分裂中期,取阴道标本,置于10%的福尔马林溶液固定,石蜡包埋,HE染色,于普通光学显微镜下计数。结果表明,YXD09可抑制雌激素周期小鼠阴道上皮细胞的有丝分裂,效果与丙酸氯倍他索软膏相似(P>0.05)。
利用鼠尾鳞片表皮模型探讨银屑净对小鼠尾部鳞片表皮颗粒层形成的影响。选取体重相近、健康昆明种小鼠30只,随机分为3组,分别为：YXD09组,阳性对照组,空白组,每组10只小鼠,公母各半。正式试验期为7d。分别在YXD09组小鼠尾部均匀涂抹一层相应的药物,阳性对照组小鼠尾部涂抹丙酸氯倍他索软膏,空白组小鼠尾部则涂抹生理盐水。在第7天处死小鼠,取小鼠尾部距离尾根约2cm处背面皮肤一长条,置于10%的福尔马林溶液固定,石蜡包埋,HE染色,于普通光学显微镜下对每个标本逐个观察鳞片,看鳞片表皮是否有颗粒层形成。结果表明,阳性对照组和YXD09组小鼠尾部含颗粒层鳞片所占比例均极显著提高(P<0.01),分别提高70.1%、116.1%。YXD09的效果明显优于丙酸氯倍他索软膏(P<0.05)。
这些结果提示,YXD09可能通过抑制表皮细胞的过度增殖和促进表皮细胞角质化来达到抗银屑病的目的,并在抗银屑病的临床应用具有广阔的前景。
(5)药代动力学研究
运用SD大鼠腹部皮肤作为皮肤模型,采用高效液相色谱法测定了YXD09中大黄素的单位面积累积透过量和皮肤滞留量,探讨了YXD09的体外药代动力学。采用水平式Franz扩散池,扩散池体积为3.5ml,有效扩散面积为1.13cm2,恒温水浴加热,温度控制在32℃左右,转速为250rpm,接受介质为30%PEG-生理盐水,排除接受池中的气泡,向扩散池中加入3.5ml的YXD09药液。分别于1,2,4,6,8,10,12,24h时取样1ml,经0.22gm滤膜过滤,取续滤液,测定大黄素的单位面积累积透过量和皮肤滞留量。结果显示,YXD09中大黄素的体外透皮释放方程为Q=0.338t-1.411(R=0.9972),24h的累积渗透量为6.76μg/cm2,皮肤滞留量为19.10gg/cm2。表明YXD09中大黄素以零级动力学经皮肤渗透。
Psoriasis is a kind of chronic inflammation with plaques and scales, influenced by varieties of reasons. Its symptoms'severity varies from small plaques locally to areas all over body. It is repeatable and hard to cure, it influences2%-3%of the total population on earth, being one of the most common autoimmune diseases. Psoriasis affects all regions of the world, being the most common immune-mediated skin disease in adults in North America and Europe, affecting about25million people. In China, the prevalence of psoriasis is0.123%, which is more of men than women, the North than the South, the urban than rural areas. The incidence in African countries is relatively low. Psoriasis has significant difference not only in race and gender, but also in geography and season. The incidence in Alpine areas is significantly higher than in warm regions, and the Symptoms are aggravated in winter and spring, relieved in summer.
Psoriasis on modern research is considered autoimmune diseases with abnormal T cells in the polygenic background. The research and argument about the action of keratinocytes, T cells, neutrophils, and nerve cells on psoriasis and their complex reactions constantly reveal its pathogenesis, to promote the development of medical science, but also to push people to find a more effective anti-psoriasis drug to provide support for the development of human beings.
Psoriasis is treated according to its severity by four kinds of drugs, such as topical medicines, light therapy method, traditional oral medicines and new biological preparations etc. If topical medicines and light therapy method doesn't act on psoriasis, the whole-body drug should be used. Traditional oral medicines conclude vitamin A substances, methotrexate, cyclosporine and acitretine etc., they take effect and have advantages of simple using method and low price, but disadvantages of heavier toxicity in livers, kidneys and bone marrow with teratogenicity, and so restricted usage. Compared with traditional systemic drugs, biological agents can specifically target immune systems and have a better safety and tolerability, fit for a longer-term treatment of psoriasis. Six biological agents have been approved in Europe and America for psoriasis. One class of them targets to T cells related to psoriasis' mechanisms, but its usage has been decreased because of its weak effect. The other one-TNF inhibitor is the first-line drug for moderate to severe psoriasis, but it is expensive for ordinary patients and its safety is limited.
Quite a lot of therapies by traditional Chinese medicine exist, accordingly many prescriptions appear, Qingdai capsule, Tripterygium tablets, Danshen injection, Qing Kai Ling Injection have been widely used in clinical applications and got reliable good efficacy.
But, because of the active ingredients of traditional Chinese medicine are not clear, and usually one single indicator is assayed in their quality control in Chinese Pharmacopoeia, which cannot reflect the inner quality entirely and objectively, not fit for the holistic characteristics of traditional Chinese medicine, so limited to be used, and is not in line with the characteristics of Chinese medicine holistic, there are certain limitations. Chinese medicine products' quality and safety evaluation is not perfect, and can not achieve the aim of stable and controllable quality, so they have not been recognized by the international mainstream drug market.
Due to psoriasis' intractabilities and its special defects, Chinese medicine treatment of psoriasis has broad research space. In basic research, the study of Chinese medicine inside and outside action mechanisms on psoriasis should be deepened, parting of experimental animal models should be established, the classification criteria and laboratory observation indicators should be unified, and their pharmacological action mechanisms should be clarified. Thus, through a comprehensive and systematic experimental study, based on theory of differentiation of symptoms and classification of syndrome together with laboratory index, to explore monome of chinese herbs or compound preparations with definite effect, searching for useful target, and to clarify its action mechanisms is the main direction of the Experimental Study of Chinese medicine treatment of psoriasis in the future.
For psoriasis' characteristic and the research status of its drug, it is significant for psoriasis' treatment to developing more Chinese herba preparations, promoting the related data and exploiting its advantages of weak side effects, together with improving level of quality control and casting away the disadvantages of violation of herba's assessment level and integrity.
Through recognitions to psoriasis and the learning of Dictionary of Traditional Chinese Medicine, we carried out the study of drugs YXD09for psoriasis, such as its prescriptions, process, quality control method, pharmacodynamics and its pharmacokinetics.
(1) The prescriptions of YXD09
The prescriptions of YXD09are based on Dictionary of Traditiongal Chinese Medicine, and the knowledge of psoriasis' characteristics and mechanisms.
(2) The preparation optimization of YXD09
We choose orthogonal test on L9(34) design with reflow time (A), reflow cycles (B), ethanol concentration (C), the amount of ethanol (D) for factors, emodin for indicator. Taken one-tenth of prescriptions'weight,9shares, after orthogonal test, the extract by ethanol is filtrated and the emodin content is measured by HPLC.
The results indicate that the range of factors that affect the optimum process is: ethanol concentration> reflux time> reflux times> the amount of ethanol, and the optimum parameters:C3A2B2D2. Analysis of variance showed that the four process factors were not significantly different (P>0.05). Thus in the practical production, in order to reduce production costs, shorten the production cycle, the reflux time is set at2hours instead of4hours. Thus, the final choice of YXD09's optimization condition is A1B2C3D2, which is refluxing for2hours,2times, with80%ethanol in8times of the drugs' amount.
Choose rhubarb, Rumex and Polygonum cuspidatum and other herbs to examine the influence of the combined and separate extract to emodin content. The result of the combined and separate one is shown to be162.78ug/ml and161.22ug/ml respectively. Analysis of variance with EXCEL software shows P>0.05, the two extract methods have no significant difference in content of emodin.
(3) The quality control method study
By the thin-layer chromatography, qualitative analysis of emodin and Angelica in YXD09is done. According to thin-layer chromatography (Chinese Pharmacopoeia2010edition is an appendix VI B), drawing10μl of the test solution, reference solution and negative control solution respectively, applying them on the same silica G plate with petroleum ether (30℃-60℃)-ethyl formate-formic acid (15:5:1) as developing agent, after spreading, it is taken under the365nm UV lamp to examine. The method shows the advantages of simple operation, good reproducibility, obvious effect, so can be used as TLC standard of YXD09.
Emodin in YXD09is measured by high-performance liquid chromatography. HPLC conditions are like that, Column:ZORBAX Extend-C18(4.6mm×150mm,5um) column; mobile phase:0-6min, methanol:water (40:60);6-20min, methanol: water:0.1%phosphoric acid (75:10:15), gradient elution; flow rate:1.0ml/min; detection wavelength:254nm; column temperature:35℃. HPLC determination value of emodin of three batches of YXD09is166.83μg/ml,172.52μg/ml,160.43μg/ml, and the RSD value is1.7%,1.3%,1.1%, respectively.
The above-mentioned method first use thin-layer chromatography to qualitatively analyze rhubarb and angelica of YXD09, and adopt an effective method which is high-performance liquid chromatography to determine the emodine in rhubarb, with advantages of the qualitative and quantitative analysis supplementing each other, comprehensive, reliable and exclusive, and can be the quality control standards of YXD09.
On the other hand, the simultaneous assaying of Emodine and Osthole has been studied.
(4) The pharmacodynamic study
The test is to use mouse vaginal epithelial cells as the model to study the effects of YXD09on mitosis of it.30health female mice from Kunming after successful modeling are randomly divided into three groups, with each group of10mice, respectively:YXD09group, the positive control group, model group; another randomly selected10without modeling as blank test group. The test period is7days. On the7th day, the drugs are administered:YXD09group is perfused5minutes each; the blank group, model group, by the same method is perfused with lOmL/kg of saline each; the positive control group is evenly coated with a layer of0.02%propionate clobetasol ointment in its vaginal wall.1h after the last dose, the mice in each group is treated by intraperitoneal injection of colchicine of15mg/kg to stop its mitosis at the mid age of cell division. Samples are fixed in10%formalin solution, paraffin-embedded, HE stained, counted under the ordinary optical microscope. The results show that YXD09can inhibit mitosis of mice vaginal epithelial cells in estrogen cycle, the effect close to propionate clobetasol ointment (P>0.05).
The test also uses mouse-tail scaly epidermis as model to examine the effect of YXD09on forming of its granular layer.30health female mice with similar weight, are randomly divided into three groups, with each group of10mice, respectively: YXD09group, the positive control group, blank group, male and female half and half. The test period is7days. Smearing correspondent drug to YXD09group, while the positive control group treated with propionate clobetasol ointment,and the blank by saline. Mice are sacrificed on the7th day, take the tail's back-skin2cm away from the root, fixed in10%formalin solution, paraffin-embedded, HE stained, placed under ordinary optical microscope to examine forming of granular layer on scales. The results showed that the granular layer proportion of positive control group, YXD09group are significantly increased (P<0.01), respectively,70.1%,116.1%. Effect of YXD09is significantly superior to that of propionate clobetasol ointment (P<0.05).
These results suggest that the the YXD09may inhibit the excessive proliferation of epidermal cells and to promote epidermal cells cutin to achieve the purpose of inhibiting psoriasis, and has broad prospects in the clinical application.
(5) The pharmacokinetic study
Using SD rat's abdominal skin as model, by HPLC to determine cumulative transit and skin retention rate of emodin in YXD09per unit area, the vitro pharmacokinetic is studied. Adopting Franz horizontal diffusion cell with volume of3.5ml, the effective diffusion area of1.13cmz, heating by constant temperature water bath to control the temperature at about32℃, the rotational speed of250rpm, accept medium of30%PEG-saline after bubbles' excluded,3.5ml YXD09solution is added to the diffusion cell. Sampling lml respectively in1,2,4,6,8,10,12,24h after0.22μm membrane filtration, the filtrate obtained, determination of cumulative transit and skin retention rate of emodin per unit area. The results show equation of vitro transdermal delivery of emodin is Q=0.338t-1.411(R=0.9972), with24h cumulative permeation amount of6.76μg/cm2, skin retention of19.10μg/cm2, indicating that emodin zero-order kinetics in YXD09's penetration through the skin.
引文
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