摘要
为保障中药产品质量,研究了中药复杂提取物的转晶现象及固态形式对理化性质的影响。以葛根总黄酮提取物为模型药物,通过不同条件下放样使其转晶,并进行特性溶出试验。发现环境应力条件下湿度是诱导葛根总黄酮转晶的关键因素,湿度越高,转晶越快;转晶后,葛根总黄酮溶出速率最高下降了96.51%。进一步模拟葛根总黄酮的制剂工艺后发现,引入了水分后的湿法制粒过程也将导致转晶行为并降低溶出速率。转晶样品的溶出速率与转晶量之间存在反比例关系。中药提取物在储存和制剂过程中均存在转晶风险,其转晶行为会对溶出速率产生显著影响,进而影响中药产品质量。本研究发现了无定形态中药复杂提取物的转晶现象,并系统研究了转晶现象对溶出行为的影响,为保障中药产品质量、促进中药制剂水平的提高提供了新的研究思路。
具有复杂化学成分组成的中药提取物是中药制剂的主要制剂中间体,长期以来,人们对中药复杂提取物的固体存在形态极少予以关注。中药复杂提取物的固化多采用喷雾干燥工艺,由于该工艺去除溶剂时间极短,化合物来不及成核生长,且中药多以复杂提取物入药,共存的复杂化学组分相互之间存在抑晶作用,使得提取物的固态形式多为无定形
葛根总黄酮(批号:20200106,含量:约77.7%,绵阳膳意生物科技有限公司);葛根素(纯度99.98%,浙江震元制药有限公司);甲醇(色谱纯,上海安谱科学仪器有限公司;实验用水(Milli-Q水纯化系统自制,美国Millipore公司);无水乙醇 (分析纯,上海泰坦公司)。
采用Hypersil BDS C18柱(250 mm × 4.6 mm, 5 μm)对葛根总黄酮进行洗脱,检测波长设置为 250 nm,流速为0.7 mL/min,柱温为30 ℃,进样体积为10 μL,流动相选用甲醇(A)和水(B),进行梯度洗脱,洗脱程序为:0 ~ 30 min,A:25% ~ 45%;30 ~ 40 min,A:45% ~ 60%;40 ~ 50 min,A:60% ~ 75%;50 ~ 60 min,A:75% ~ 55%;60 ~ 70 min,A:55% ~ 35%;70 ~ 80 min,A:35% ~ 25%。
分别称取葛根总黄酮样品1 g,薄铺于表面皿中,在92.5% RH高湿环境中放置3、6、10、15、20 d。上述样品取出后放置在真空干燥箱中干燥,干燥样品过3遍100目筛,分别记作葛根总黄酮转晶样品1、2、3、4、5,用于后续的固态表征和溶出行为考察。
在葛根总黄酮粉末1 g中分别加入无水乙醇、50%乙醇水溶液和纯水1 mL作润湿剂,搅拌均匀,制成湿颗粒,然后将湿颗粒放置在真空干燥箱中干燥,干燥样品过3遍100目筛,分别记作葛根总黄酮转晶样品6、7、8,用于后续的固态表征和溶出行为考察。
取少量待观察样品薄铺于载玻片上并滴加少量液体石蜡分散均匀后置于载物台上,在5 × 10倍偏光显微镜下观察固体形态。
对葛根总黄酮提取物进行紫外全波长扫描,选取最大吸收波长250 nm作为检测波
按照《中华人民共和国药典》(2020 年版)通则0931溶出度与释放度测定第二法 (桨法),取未转晶葛根总黄酮样品以及葛根总黄酮转晶样品1 ~ 8各250 mg,使用液压机将样品在112.5 MPa下压制成直径为13 mm,表面完好的圆形片,封入蜂蜡,投入纯水中进行特性溶出试验。溶出介质体积为900 mL, 温度为37 ℃, 转速为50 r/min,分别于1、3、5、10、15、30、45、60、90、120 min 时取样5 mL,经0.45 μm PTFE水相滤膜过滤,同时补充相同体积的介质。将续滤液稀释至适宜浓度,采用上述紫外分光光度法测定葛根总黄酮浓度。每个样品平行测定3份。
本研究以相对转晶百分数定义葛根总黄酮的转晶量,结合PXRD技
(1) |
对葛根总黄酮提取物进行HPLC分析,由

Figure 1 Chromatograms of total flavonoids from Pueraria lobata (A) and puerarin reference (B)
无定形态处于热力学不稳定的高能态,在储存过程中往往会受到环境应力的影响而向热力学稳定的晶态转变。因此,为探索葛根总黄酮在储存过程中是否有发生转晶行为的可能性,进行了温度和湿度条件下的放样试验,并利用偏光显微镜观察其固态形式的变化,判断转晶现象。
由

Figure 2 Polarizing light microscope (PLM) diagrams of total flavonoids from Pueraria lobata under different temperature conditions (5 × 10)
由

Figure 3 PLM diagrams of total flavonoids from Pueraria lobata under different relative humidity (RH) conditions (5 × 10)
通过对葛根总黄酮进行转晶影响因素考察可知,温度对葛根总黄酮的转晶行为几乎没有影响,而湿度是影响葛根总黄酮发生固态转变的重要因素。由于存在鞣质、糖类等易吸湿成
考虑到中药制剂研发过程中,储存和制剂条件下可能引入水分,造成转晶风险,因此接下来的实验中,模拟了葛根总黄酮在储存条件放样和湿法制粒后的样品,用PLM和PXRD表征样品固态形式,判断转晶程度,并考察了转晶样品的溶出行为。
为缩短实验周期,将葛根总黄酮在剧烈的92.5% RH高湿条件下放置3、6、10、15、20 d来模拟储存条件,以得到不同转晶量样品,分析固体形态及其对成药性的影响。
由

Figure 4 PLM diagrams of total flavonoids from Pueraria lobata under 92.5% RH with different times (5 × 10)
A: 92.5% RH 3 d; B: 92.5% RH 6 d; C: 92.5% RH 10 d; D: 92.5% RH 15 d; E: 92.5% RH 20 d
通过PXRD对湿度放样的葛根总黄酮样品进行检测,结果如

Figure 5 PXRD patterns of sample of total flavonoids from Pueraria lobata without crystallization and sample-1-sample-4
Sample-1: 92.5% RH 3 d; Sample-2: 92.5% RH 6 d; Sample-3: 92.5% RH 10 d; Sample-4: 92.5% RH 15 d
采取紫外分光光度法对葛根提取物中的总黄酮进行定量检测,结果如

Figure 6 UV-visible absorption spectra of puerarin (A) and total flavonoids from Pueraria lobata (B)
为探索储存条件下的转晶对中药提取物成药性的影响,进行了储存条件下葛根总黄酮转晶样品的特性溶出试验,结果如

Figure 7 Intrinsic dissolution profiles of sample of total flavonoids from Pueraria lobata without crystallization and sample-1-sample-5 ()
Sample-1: 92.5% RH 3 d; Sample-2: 92.5% RH 6 d; Sample-3: 92.5% RH 10 d; Sample-4: 92.5% RH 15 d; Sample-5: 92.5% RH 20 d
在考虑过储存过程中可能存在的转晶风险后,进一步探索了中药复杂提取物制剂工艺中湿法制粒过程引入的润湿剂对转晶及溶出行为的影响。
由偏光显微镜观察结果(

Figure 8 PLM diagrams of total flavonoids from Pueraria lobata samples with different adhesives (5 × 10)
A: Pure ethanol as adhesive; B: 50% ethanol as adhesive; C: Pure water as adhesive

Figure 9 PXRD patterns of total flavonoids from Pueraria lobata sample-6-sample-5
Sample-6: Pure ethanol as adhesive; Sample-7: 50% ethanol as adhesive; Sample-8: Pure water as adhesive
葛根总黄酮不同润湿剂湿法制粒样品的特性溶出试验结果如

Figure 10 Intrinsic dissolution profiles of total flavonoids from Pueraria lobata sample-6-sample-8 ()
因此,不仅储存过程会影响葛根总黄酮的固态形式,在制剂工艺的湿法制粒过程中,润湿剂的选用也会影响葛根总黄酮的固态形式,若要避免溶出降低,应尽量控制制剂工艺过程中水分的加入。
对上述未转晶葛根总黄酮样品以及转晶样品1 ~ 8的相对转晶百分数进行了计算,并将其与特性溶出速率之间进行了数学模型的相关性分析,结果如

Figure 11 Correlation between the relative crystallization transformation percentage and dissolution rate of total flavonoids from Pueraria lobata
药物的固体存在形式对药物的溶解与溶出、稳定性、制剂成型性等有着重要影响,与成药性息息相关。长期以来,在中药领域人们忽略了对中药复杂提取物固体形态以及固体形态与成药性之间关系的研究。本研究首次关注到中药复杂提取物的固体形态,发现葛根总黄酮在储存环境以及制剂工艺过程中均会发生固体形态的变化,即存在转晶现象,同时葛根总黄酮的溶出速率显著降低。通过对环境应力条件考察,发现就葛根总黄酮而言,湿度是影响葛根总黄酮转晶最主要的因素,吸湿越多,体系转晶越多。进一步模拟湿法制粒工艺过程中润湿剂对葛根总黄酮固态形式的影响,发现润湿剂为水或者有水参与时,将促使葛根总黄酮发生显著转晶。
本研究结果提示,在进行中药制剂研究过程中,应充分考虑提取、纯化、浓缩、干燥及制剂成型过程(如制粒、压片等)和贮存条件(温度、湿度等)等操作可能带来的对中药提取物固体形态的影响,及时关注中药提取物固体存在形态的变化情况,提升中药制剂产品质量,保证患者用药的安全性、有效性。关于中药复杂提取物的转晶现象及机制的分析,值得进一步深入探索,以为中药现代化发展提供更多理论指导。
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