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关键词 HPLC-UV 甘辛喷雾剂 豚鼠 超敏性鼻炎模型 药效学
Determination of glycyrrhizin acid in compound nasal spray ofglycyrrhizin
and flower flos magnoliae using HPLC-UV and therapeutic
effect of the formulation on guinea pig’s hypersensitive rhinitis
Chen Wen，Liu Qinghua，Lv Wanliang
College of Pharmaceutical Sciences，Shihezi University，Shihezi832002.
【Abstract】 Objective To establish HPLC-UV method for determination of glycyrrhizin acid in compound nasal spray of glycyrrhizin and flower flos magnoliae，the chief active ingredient of formulation，glycyrrhizin acid，and to observethe therapeutic effect of this formulation on guinea pig’s hypersensitive rhinitis.Methods A compound nasal spray formulation was prepared using extracts of glycyrrhizin and measured by HPLC-UV method.Mobile phase conˉsisted of methanol，0.2mol/L ammonium acetate and glacial acetic acid（67:33:1，v/v），analytical column was Zorbax ODS C 18 and wavelength250nm.Guinea pig’s hypersensitive rhinitis was induced using toluene-2，4-diisocyanate（TDI）.Fifty guinea pigs were equally divided into5groups among which guinea pigs of4groups were of hypersensiˉtive rhinitis and of1group were healthy（normal animals）.High and low doses of formulation（contained710and355μg of glycyrrhizin acid）were administered by nasal spraying to guinea pigs at every12h and given continuously for8days.Commercial nasal drop（DIKANG nasal drop）and olive oil were given as a positive and a blank control，respecˉtively.In addition，olive oil was given to healthy animals as a normal control.After8days，animals were sacrificed and nasal mucosa of each animal was removed for phathological examination.Results Peak of glycyrrhizin acid appeared at4.5min in HLPC-UV chromatogram.Other peaks did not disturb the measurement.A linear relationship range was from0.2to1.0μg/ml（r=0.9999）.The mean recovery was（100.67±4.44）%and inter-day relative standard deviation1.63%.In comparison with blank control group，infiltration of eosinophilic granulocyte，neutrophilic granuloˉcyte and lymphocyte arising from rhinitis were significantly reduced（P<0.01）by administration of high and low dosˉes offormulation，and gland hyperplasia and secretion were obviously inhibited（P<0.05）.The therapeuticeffect was similar to that of positive control group.Conclusion The HPLC-UV is simple and stable for determination glycyrrhizin acid in compound nasal spray of glycyrrhizin and flower flos magnoliae.This compound formulation significantly inhibits the course of the typeI allergic reaction and protects the structure of nasal mucosa.The possible mechanism may derive from regulation of the body’s situation of immunological function，improvement and rehabilitation of the cell’s structure.
Key words HPLC-UV compound nasal spray glycyrrhizin and hypersensitive rhinitis model pharmacodynamics flower flosmagˉnoliae guinea pig
过敏性鼻炎（allergic rhinitis）是一种反复发作性喷嚏、水样清涕、鼻塞为主要特征的鼻粘膜炎症。过敏性鼻炎是鼻腔粘膜的变态反应性疾病，根据不同临床特点，分为突发型和慢发型两种。突发型，患者病情会突然发作，感觉鼻痒眼痒，连续不停地打喷嚏、鼻塞、流清鼻涕，常在早上或环境气候急剧变化时发生。症状持续时间短，鼻腔内粘膜肿胀，有大量水状分泌物，发作过后，患者一切如常。慢发型，鼻痒和连续喷嚏等症状不明显，主要症状是间歇性鼻塞，鼻分泌物增多并向鼻咽部排放;分泌物中有大量嗜酸性粒细胞，鼻腔中鼻甲肿胀、湿润、颜色苍白、表面光滑;病情严重的患者鼻甲水肿或息肉样病变，鼻腔底部有水状分泌物滞留。过敏性鼻炎属于中医鼻鼽的范畴，历代医家均认为本病与风寒入侵密切相关，而风寒入侵又与卫外阳气不足有关。甘草甜素，主要成分为甘草酸，与肝脏胆固醇代谢酶有较强的亲和力，从而阻碍皮质醇与醛固酮的灭活，使用后显示明显的皮质激素样效应，如抗炎作用、抗过敏及保护膜结构等作用;可诱生γ-干扰素，增强NK细胞活性;能减轻肝细胞变性坏死、降低谷丙转氨酶，防止脂肪变性，促进胆红素代谢。辛夷具有收敛作用而保护鼻粘膜，并能促进粘膜分泌物的吸收，减轻炎症，并有利于鼻腔通畅，辛夷浸剂或煎剂均有浸润麻醉作用，辛夷水或醇提取物有降压作用，水煎剂对横纹肌有乙酰胆碱样作用，并能兴奋子宫平滑肌、亢奋肠蠕动、对多种致病菌有抑制作用，辛夷花化学成份含挥发油，主要为桉油精（cineole）、α-蒎烯（α-pinene）、α-松油醇（α-terpineol）等 ［1～4］ 。目前，国内外对过敏性鼻炎尚无特异性治疗药物。甘草甜素的抗过敏作用已为现代医学体内外实验所证实，但目前国内外尚无此药用于过敏性鼻炎治疗的公开报道。在原有辛夷滴鼻液的基础上加入甘草甜素制成鼻用喷雾剂，本文的研究目的是建立测定复方甘草和辛夷喷雾剂（甘辛喷雾剂）的含量的方法，考查其对过敏性鼻炎的治疗效果。
1 材料与方法
1.1 受试动物和药物 选用健康无鼻炎的豚鼠50只，雌雄各半，体重为300～350g，新疆维吾尔自治区流行病研究所提供。甘草甜素（由甘草中提取制得）;迪康-迪通鼻炎水为市售，成都迪康制药有限公司生产，批号010301;甲苯-2，4-二异氰酸酯（TDI），上海试剂一厂生产。
1.2 甘辛喷雾剂的制备与含量测定 称取辛夷花500g，置于蒸馏容器中加适量水浸渍，蒸馏并浓缩后得到辛夷液 ［5］ ，将适量制得的甘草甜素加入辛夷液中，加入防腐剂等制成浅黄色的澄明液体，装入定量喷瓶中即得。用反相高效液相色谱法测定制剂中甘草酸的含量 ［6］ ，检测波长250nm，Zorbax ODS C 18 分析柱，流动相为甲醇:0.2mol/L醋酸铵:冰醋酸（67:33:1）。以甘草酸单铵为标准品（根据药典换算方法，甘草酸单铵0.2mg相当于甘草酸0.1959mg）进行线性试验;甘草酸单铵为标准品配成高、中、低3种不同浓度，加入已知浓度的制剂样品，进行回收率试 验;同一批号样品分别取样5份测定含量，计算精密度。结果表明重现性良好，RSD为1.63%。分别精密吸取甘草酸单铵标准品