A new PAT application: Optimization of processing methods for honeysuckle flower (Lonicerae Japonicae Flos) and wild honeysuckle flower (Lonicerae Flos) 新的 PAT 应用：优化金银花 （Lonicerae Japonicae Flos） 和野生金银花 （Lonicerae Flos） 的加工方法
^(a){ }^{a} School of Chinese Material Medica, Beijing University of Chinese Medicine, Beijing 100102, China ^(a){ }^{a} 北京中医药大学 中医学院， 北京 100102 ^(b){ }^{b} Engineering Research Center of Good Agricultural Practice for Chinese Crude Drugs, Ministry of Education, Beijing 100102, China ^(b){ }^{b} 中国生药良好农业规范教育部工程研究中心， 北京 100102 ^(c){ }^{c} Pharmaceutical Engineering and New Drug Development of Traditional Chinese Medicine (TCM), Ministry of Education, Beijing 100102, China ^(c){ }^{c} 中药制药工程与中药新药开发，教育部，北京100102 ^(d){ }^{d} Key Laboratory of TCM-information Engineering, State Administration of TCM, Beijing 100102, China ^(d){ }^{d} 国家中医药局 中医信息工程重点实验室， 北京 100102
Received 25 March 2017; received in revised form 28 February 2018; accepted 20 March 2018 2017 年 3 月 25 日接收;2018 年 2 月 28 日以修订后的形式接收;接受日期 2018 年 3 月 20 日
Available online 27 March 2018 2018 年 3 月 27 日在线提供
Äbstract Objective: A novel processing analytical framework of Chinese Material Medica (CMM) includes breeding process analysis, planting process analysis, producing process analysis and manufacture process analysis. In order to study this framework, we used quality control of honeysuckle flower (Lonicerae japonicae Flos) including diploid and tetraploid Lonicera japonica, and wild honeysuckle flower (Lonicerae Flos) including Lonicerae hypoglauca, Lonicerae confusa, Lonicerae fulvotomentosa and Lonicerae macranthoides as an example. Methods: A total of 360 samples were analyzed from three variables including species, growing areas, and optimization of processing methods. For quantitative analysis, chlorogenic acid (CA), isochlorogenic acid A (3, 5-DCA), and isochlorogenic acid C (4, 5-DCA) were measured with high performance liquid chromatography (HPLC). Results: The three organic acids contents are higher in wild honeysuckle flower than honeysuckle flower; LL. japonica (tetraploid) is higher than L. japonica (diploid). Modern processing methods (kill-enzyme torrefaction and fixing machine drying) are popular in growing areas and are superior to traditional methods (sun and shade drying). Äbstract 目的： 一种新的中药材 （CMM） 加工分析框架，包括育种过程分析、种植过程分析、生产过程分析和制造过程分析。为了研究这个框架，我们使用了包括二倍体和四倍体金银花在内的金银花 （Lonicerae japonicae Flos） 和野生金银花 （Lonicerae Flos） 的质量控制，包括 Lonicerae hypoglauca、Lonicerae confusa、Lonicerae fulvotomentosa 和 Lonicerae macranthoides 作为例子。方法：从物种、生长区域和加工方法优化三个变量中分析了总共 360 个样品。定量分析时，采用高效液相色谱 （HPLC） 测量绿原酸 （CA）、异绿原酸 A （3， 5-DCA） 和异绿原酸 C （4， 5-DCA）。结果： 野生金银花中 3 种有机酸含量均高于金银花; LL 。japonica（四倍体）高于 L. japonica（二倍体）。现代加工方法（杀灭酶烘烤和固定机干燥）在种植区很受欢迎，优于传统方法（阳光和阴凉干燥）。
The guidance of processing analytical technology (PAT) for the pharmaceutical industry was issued by Food and Drug Administration (FDA) in September 2004. PAT is a system for designing, analyzing, and controlling manufacturing through timely measurements of critical quality and performance attributes in raw, in-process materials and processes with the goal of ensuring final product quality. ^(1){ }^{1} It has been successfully applied in pharmaceutical, biopharmaceutical, and food industries, which can improve the quality of products and productivity. ^(2-4){ }^{2-4} For the chemicals, within the framework of FDA’s PAT initiative, the factors of entire process analysis include raw materials, manufacturing process and final product. 美国食品药品监督管理局 （FDA） 于 2004 年 9 月发布了制药行业加工分析技术 （PAT） 指南。PAT 是一种系统，通过及时测量原材料、在制品和工艺中的关键质量和性能属性来设计、分析和控制制造，以确保最终产品质量。 ^(1){ }^{1} 它已成功应用于制药、生物制药和食品工业，可以提高产品质量和生产率。 ^(2-4){ }^{2-4} 对于化学品，在 FDA 的 PAT 倡议框架内，整个过程分析的因素包括原材料、制造过程和最终产品。
Nevertheless, due to the complexity of pharmacodynamic substrates and the natural variability of raw materials, the process analysis for Chinese Material Medica (CMM) is more different than the chemicals. ^(5){ }^{5} Therefore, a novel processing analytical framework of CMM has been proposed, which includes breeding process analysis, planting process analysis, producing process analysis and manufacture process analysis. For PAT, the concept of quality by design (QBD) indicates that the critical quality factor could be optimized. 然而，由于药效学底物的复杂性和原材料的自然可变性，中药材 （CMM） 的过程分析与化学品相比差异更大。 ^(5){ }^{5} 因此，提出了一种新的 CMM 加工分析框架，包括育种过程分析、种植过程分析、生产过程分析和制造过程分析。对于 PAT，质量源于设计 （QBD） 的概念表明关键质量因子可以优化。
The quality of CMM is influenced by many factors, in which processing method is critical. ^(6){ }^{6} However, within the process analysis of CMM, there is no report on analysis of processing methods. Thus, this is our first goal. Here, we take honeysuckle flower (Lonicerae Japonicae Flos) and wild honeysuckle flower (Lonicerae Flos) as an example and address the key issues that the optimization of processing methods. CMM 的质量受许多因素影响，其中加工方法至关重要。 ^(6){ }^{6} 但是，在 CMM 的过程分析中，没有关于加工方法分析的报告。因此，这是我们的首要目标。这里，我们以金银花 （Lonicerae Japonicae Flos） 和野生金银花 （Lonicerae Flos） 为例，解决加工方法优化的关键问题。
In the Chinese Pharmacopoeia 2015 edition, the dried buds of Lonicera japonica are called honeysuckle flower (Jinyinhua in Chinese). The dried buds of Lonicerae hypoglauca, Lonicerae confusa, Lonicerae fulvotomentosa and Lonicerae macranthoides are called wild honeysuckle flower (Shanyinhua in Chinese). ^(7){ }^{7} Honeysuckle flower and wild honeysuckle flower are commonly used traditional Chinese medicines in China. They can clear heat and detoxify. Thus, they are often used for the treatment of sores, furuncles, carbuncles and issues caused by exopathogenic wind-heat or epidemic febrile diseases. ^(8){ }^{8} With wide applications in many fields and excellent adaptability, honeysuckle flower is cultivated mainly in Shandong, Henan and Hebei province; wild honeysuckle flower is mainly distributed in southern China, such as Hunan, Guangdong and Guangxi province. 在《中国药典》2015 年版中，金银花的干燥花蕾被称为金银花（中文为金银花）。Lonicerae hypoglauca、Lonicerae confusa、Lonicerae fulvotomentosa 和 Lonicerae macranthoides 的干燥芽被称为野生金银花（中文为山音花）。 ^(7){ }^{7} 金银花和野生金银花是中国常用的中药材。它们可以清热解毒。因此，它们通常用于治疗疮、疖、痈以及由外致病性风热或流行性发热性疾病引起的问题。 ^(8){ }^{8} 金银花应用广泛，适应性极佳，主要在山东、河南和河北省种植;野生金银花主要分布在华南地区，如湖南、广东和广西省。
There are various processing methods for honeysuckle flower and wild honeysuckle flower including kill-enzyme torrefaction, fixing machine drying, simple oven drying, sun drying, shade drying, etc. Due to the different processing methods and geographical origins, the quality of 金银花和野生金银花有多种加工方法，包括杀灭酶烘烤、定影机干燥、简易烤箱干燥、晒干、阴凉干燥等。由于加工方法和产地不同，质量
honeysuckle flower and wild honeysuckle flower varies widely regardless of the appearance of dried buds and the content of the main active components. ^(9-12){ }^{9-12} In this situation, it is critical to perform process analysis on the quality assessment and then select the optimal processing methods for honeysuckle flower and wild honeysuckle flower in different growing areas. 金银花和野生金银花无论干芽的外观和主要活性成分的含量如何，差异很大。 ^(9-12){ }^{9-12} 在这种情况下，对质量评估进行过程分析，然后为不同生长区域的金银花和野生金银花选择最佳加工方法至关重要。
Quality control of honeysuckle flower and wild honeysuckle flower usually uses high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), mass spectrometry (MS), etc. Most active components focus on organic acids and flavonoids such as chlorogenic acid (CA), isochlorogenic acids, luteoloside, and saponins (macranthoidin B and dipsacoside B). ^(13-16){ }^{13-16} In this study, organic acids were selected as an optimized indicator. Organic acids are the main antibacterial components in honeysuckle flower and wild honeysuckle flower, which include CA and the dicaffeoylquinic acids (DCA) with dicaffeoyl at different substitution sites including isochlorogenic acid B (3, 4-DCA), isochlorogenic acid AA (3,5-DCA) and isochlorogenic acid C(4,5-DCA).^(17)C(4,5-D C A) .{ }^{17} Furthermore, DCA exhibits significant activities such as anti-oxidation, anti-inflammation, enzyme inhibition and anti-PAF effects. ^(18){ }^{18} Besides, CA, 3,5-DCA and 4,5-DCA have a higher content in honeysuckle flower and wild honeysuckle flower. Therefore, CA, 3,5-DCA and 4,5-DCA were chosen as active components. 金银花和野生金银花的质量控制通常采用高效液相色谱（HPLC）、超高效液相色谱（UPLC）、质谱（MS）等。大多数活性成分集中在有机酸和类黄酮上，例如绿原酸 （CA）、异绿原酸、木犀草苷和皂苷（大杂烩素 B 和二沙谷苷 B）。 ^(13-16){ }^{13-16} 在本研究中，有机酸被选为优化指标。有机酸是金银花和野生金银花的主要抗菌成分，其中包括 CA 和二咖啡酰奎宁酸 （DCA），在不同取代位点与二咖啡酰基，包括异绿原酸 B （3,4-DCA）、异绿原酸 AA （3,5-DCA） 和异绿原酸 C(4,5-DCA).^(17)C(4,5-D C A) .{ }^{17} 此外，DCA 具有抗氧化、抗炎、酶抑制和抗 PAF 等显著活性。 ^(18){ }^{18} 此外，CA、3,5-DCA 和 4,5-DCA 在金银花和野生金银花中的含量较高。因此，选择 CA、3,5-DCA 和 4,5-DCA 作为活性组分。
Supported by Engineering Research Center of Good Agricultural Practice (GAP) for Chinese Crude Drugs of Ministry of Education, 8 provinces and 25 villages were investigated in 2012. These are regarded as the genuine growing areas of honeysuckle flower and wild honeysuckle flower. We collected 360 samples including different species, growing areas and processing methods. Natural honeysuckle flower is diploid, and tetraploid honeysuckle flower was induced successfully by the colchicine. Some studies show that the tetraploid Lonicerae japonicae is better than the diploid one in many respects sunch as yield, quality, and resistance. ^(19,20){ }^{19,20} 2012 年，在教育部中药生药良好农业规范工程研究中心 （GAP） 的支持下，对 8 个省份和 25 个村进行了调查。这些被认为是金银花和野生金银花的真正生长区。我们收集了 360 个样本，包括不同的物种、生长区域和加工方法。天然金银花是二倍体的，四倍体金银花被秋水仙碱成功诱导。一些研究表明，四倍体 Lonicerae japonicae 在许多方面都优于二倍体 Sunch，无论是产量、质量和抗性。 ^(19,20){ }^{19,20}
Thus, in this study, L. japonica (diploid and tetraploid) and LL. hypoglauca, LL. confusa, L. fulvotomentosa and L. macranthoides of different growing areas and processing methods were regarded as study objects. Active multicomponents, namely CA, 3, 5-DCA, 4,5-DCA, were measured by HPLC. Based on the concept of PAT and the characteristics of CMM, species, growing areas and processing methods were studied. As a result, we optimized processing methods in different growing areas. This is a critical point for process analysis of honeysuckle flower and wild honeysuckle flower. The results have great value in honeysuckle flower and wild honeysuckle flower production. This insight can provide guidance for the processing analytical framework of CMM. 因此，在这项研究中，L. japonica（二倍体和四倍体）和 LL .Hypoglauca， LL .将不同生长区域和加工方式的 confusa、L. fulvotomentosa 和 L. macranthoides 视为研究对象。通过 HPLC 测量活性多组分，即 CA、3、5-DCA、4,5-DCA。基于 PAT 的概念和 CMM 的特性，研究了物种、生长区域和加工方法。因此，我们优化了不同种植区域的加工方法。这是金银花和野生金银花过程分析的关键点。结果对金银花和野生金银花的生产具有重要价值。这种洞察力可以为 CMM 的加工分析框架提供指导。
Materials and methods 材料和方法
Materials 材料
A total of 220 samples of wild honeysuckle flower and 140 samples of honeysuckle flower were collected in genuine growing areas from July to August in 2012. These include Shandong, Guangdong, Guangxi, Guizhou province and so on (Table 1). All samples were identified by Dr. Weidong Li (School of Chinese Materia Medica, Beijing University of Chinese Medicine). 2012 年 7 月至 8 月，我们在真正的种植区共收集了 220 个野生金银花样本和 140 个金银花样本。这些省份包括山东、广东、广西、贵州省等（表 1）。所有样本均由 Weidong Li 博士（北京中医药大学中药学院）鉴定。
Three reference compounds, CA (121226), 3,5-DCA (121209), 4,5-DCA (121207), were supplied by Shanghai Winherb Medical Technology Co. Ltd. The purity of three organic acids was above 98%98 \%. HPLC-grade acetonitrile from Fisher (Massachusetts, America) and deionized water from Wahaha (Hangzhou, China) were purchased. The other solvents of analytical grade were obtained from Beijing Chemical Works (Beijing, China). 三种参比化合物 CA （121226）、3,5-DCA （121209）、4,5-DCA （121207） 由上海永热医疗科技有限公司提供。三种有机酸的纯度在 98%98 \% 以上。采购了 Fisher 公司（美国马萨诸塞州）的 HPLC 级乙腈和娃哈哈公司（中国杭州）的去离子水。其他分析级溶剂购自北京化工厂（中国北京）。
Preparation of standard solutions 标准溶液的制备
The stock solutions of CA, 3,5-DCA, and 4,5-DCA with 70% ethanol were 0.2200,0.22640.2200,0.2264 and 0.2830mg//mL0.2830 \mathrm{mg} / \mathrm{mL} respectively. All solutions were stored at 4^(@)C4^{\circ} \mathrm{C}. 含 70% 乙醇的 CA、3,5-DCA 和 4,5-DCA 的储备溶液分别为 0.2200,0.22640.2200,0.2264 和 0.2830mg//mL0.2830 \mathrm{mg} / \mathrm{mL} 。所有溶液均储存在 。 4^(@)C4^{\circ} \mathrm{C}
Extraction 萃取
Electronic scales (Sartorius BP211D, Gottingen, Germany) and an ultrasonic cleaner (KQ500DE, Kunshan, China) were used in the sample preparation. First, samples were milled into powder with a grinder, and passed through an 80-mesh sieve (particle size <= 0.2mm\leq 0.2 \mathrm{~mm} ). Then, 0.5 g of powdered plant material was extracted with 50 mL of 70%70 \% ethanol using an ultrasonic extraction method (45^(@)C,250(W),35KHz:}\left(45^{\circ} \mathrm{C}, 250 \mathrm{~W}, 35 \mathrm{KHz}\right. ) for 45 minutes. After cooling to room temperature, the extracted solution was filtered through a 0.45 mum0.45 \mu \mathrm{~m} membrane filter before final injection. 样品制备使用电子秤（Sartorius BP211D，德国哥廷根）和超声波清洗机（KQ500DE，中国昆山）。首先，用研磨机将样品研磨成粉末，然后通过 80 目筛子（粒度 <= 0.2mm\leq 0.2 \mathrm{~mm} ）。然后，使用超声提取法 (45^(@)C,250(W),35KHz:}\left(45^{\circ} \mathrm{C}, 250 \mathrm{~W}, 35 \mathrm{KHz}\right. 将 0.5 g 粉末状植物材料与 50 mL 70%70 \% 乙醇一起提取 45 分钟。冷却至室温后，提取的溶液在最终进样前通过 0.45 mum0.45 \mu \mathrm{~m} 膜过滤器过滤。
Table 2 The main parameters of the calibration curves in honeysuckle flower and wild honeysuckle flower. 表 2 金银花和野生金银花中校准曲线的主要参数。
An HPLC method described previously was used for HPLC analysis on a Waters HPLC system (Milford, USA) equipped with a binary gradient pump (model 1525), an ultraviolet detector (model 2489), an auto-sampler (model 2707) and a Breeze-2 system for data processing. ^(9){ }^{9} 使用前面描述的HPLC方法在配备二元梯度泵（1525型）、紫外检测器（2489型）、自动进样器（2707型）和Breeze-2系统（用于数据处理）的Waters HPLC系统（美国米尔福德）上进行HPLC分析。 ^(9){ }^{9}
The mobile phases consisted of eluent A [0.1% ( v//v\mathrm{v} / \mathrm{v} ) phosphoric acid in water] and B (acetonitrile). The linear gradient elution program was set as follows: 0 minute rarr\rightarrow12%12 \% B, 10 minutes rarr13%,26\rightarrow 13 \%, 26 minutes rarr26%\rightarrow 26 \%, 31 minutes rarr47%,33\rightarrow 47 \%, 33 minutes rarr63%,35\rightarrow 63 \%, 35 minutes rarr70%\rightarrow 70 \%, 40 minutes rarr12%\rightarrow 12 \% maintained at 12%12 \% at 40-4540-45 minutes. The flow rate was 1.0mL//min1.0 \mathrm{~mL} / \mathrm{min}, and the loading volume was 10 muL10 \mu \mathrm{~L}. The detection wavelength and column temperature were set at 355 nm and 25^(@)C25^{\circ} \mathrm{C}. HPLC analysis was carried out on an Agilent ZORBAX SB-phenyl ( 4.6mmxx250mm,5mum4.6 \mathrm{~mm} \times \mathbf{2 5 0} \mathrm{mm}, 5 \mu \mathrm{~m} ). 流动相由洗脱液 A [0.1% （ v//v\mathrm{v} / \mathrm{v} ） 磷酸水溶液] 和 B （乙腈） 组成。线性梯度洗脱程序设置如下：0 min rarr\rightarrow12%12 \% B、10 min rarr13%,26\rightarrow 13 \%, 26rarr26%\rightarrow 26 \% 、31 min rarr47%,33\rightarrow 47 \%, 33 min rarr63%,35\rightarrow 63 \%, 35rarr70%\rightarrow 70 \% 、40 min rarr12%\rightarrow 12 \% ，维持在 12%12 \% at 40-4540-45 分钟。流速为 1.0mL//min1.0 \mathrm{~mL} / \mathrm{min} ，加载体积为 10 muL10 \mu \mathrm{~L} 。检测波长和柱温设置为 355 nm 和 25^(@)C25^{\circ} \mathrm{C} 。在 Agilent ZORBAX SB-苯基 （ ） 上进行 HPLC 分析 4.6mmxx250mm,5mum4.6 \mathrm{~mm} \times \mathbf{2 5 0} \mathrm{mm}, 5 \mu \mathrm{~m} 。
Results 结果
Calibration curves and chromatography 校准曲线和色谱
Three stock solutions were diluted at a series of different gradient concentration respectively. 10 muL10 \mu \mathrm{~L} was injected for HPLC analysis. A calibration curve was obtained by plotting 将三种储备液分别以一系列不同的梯度浓度稀释。 10 muL10 \mu \mathrm{~L} 进样进行 HPLC 分析。通过绘制获得校准曲线
Table 1 Honeysuckle flower and wild honeysuckle flower samples used for this study. 表 1 本研究使用的金银花和野生金银花样品。
Species 物种
Number 数
Geographical sources 地理来源
L. japonica (diploid) L. japonica （二倍体）
18
Julu & Mancheng county in Hebei 河北省的巨鹿县和满城县
16
Fengqiu county in He'nan 河南市枫丘县
32
Pingyi & Fei county in Shandong 山东平邑县和费县
L. japonica (tetraploid) L. japonica （四倍体）
4
Yunnan, Guangdong, Guangxi & Zhejiang 云南、广东、广西和浙江
23
Pingyi, Weihai, Jinan & Jining in Shandong 平邑、威海、济南和济宁在山东
8
Shucheng & Wuhu city in Anhui 安徽省的舒城和芜湖市
6
Kunming & Xucheng in Yunnan 云南的昆明和徐城
7
Taizhou & Taixing in Yunnan 云南的泰州和泰兴
L. hypoglauca L. hypoglauca 低叶乳杆菌
26
Hebei, He'nan, Hunan, Sichuan and so on 河北、河南、湖南、四川等
33
Xincheng county in Guangxi 广西新城县
32
Mashan county in Jiangsu 江苏省马山县
2
Baise & Du'an in Guangxi 广西百色和都安
L. confusa 孔氏乳杆菌
3
Zhejiang & Fujian 浙江 & 福建
L. fulvotomentosa 黄毛乳杆菌
25
Shaoguan city in Guangdong 广东省韶关市
L. macranthoides L. macranthoides 麦兰特乳杆菌
55
Xingyi in Guizhou 兴义 在贵州
26
Xiushan county in Chongqing 重庆市秀山县
44
Longhui & Xupu county in Hu'nan 胡隆惠县和徐浦县
Species Number Geographical sources
L. japonica (diploid) 18 Julu & Mancheng county in Hebei
16 Fengqiu county in He'nan
32 Pingyi & Fei county in Shandong
L. japonica (tetraploid) 4 Yunnan, Guangdong, Guangxi & Zhejiang
23 Pingyi, Weihai, Jinan & Jining in Shandong
8 Shucheng & Wuhu city in Anhui
6 Kunming & Xucheng in Yunnan
7 Taizhou & Taixing in Yunnan
L. hypoglauca 26 Hebei, He'nan, Hunan, Sichuan and so on
33 Xincheng county in Guangxi
32 Mashan county in Jiangsu
2 Baise & Du'an in Guangxi
L. confusa 3 Zhejiang & Fujian
L. fulvotomentosa 25 Shaoguan city in Guangdong
L. macranthoides 55 Xingyi in Guizhou
26 Xiushan county in Chongqing
44 Longhui & Xupu county in Hu'nan| Species | Number | Geographical sources |
| :--- | :--- | :--- |
| L. japonica (diploid) | 18 | Julu & Mancheng county in Hebei |
| | 16 | Fengqiu county in He'nan |
| | 32 | Pingyi & Fei county in Shandong |
| L. japonica (tetraploid) | 4 | Yunnan, Guangdong, Guangxi & Zhejiang |
| | 23 | Pingyi, Weihai, Jinan & Jining in Shandong |
| | 8 | Shucheng & Wuhu city in Anhui |
| | 6 | Kunming & Xucheng in Yunnan |
| | 7 | Taizhou & Taixing in Yunnan |
| L. hypoglauca | 26 | Hebei, He'nan, Hunan, Sichuan and so on |
| | 33 | Xincheng county in Guangxi |
| | 32 | Mashan county in Jiangsu |
| | 2 | Baise & Du'an in Guangxi |
| L. confusa | 3 | Zhejiang & Fujian |
| L. fulvotomentosa | 25 | Shaoguan city in Guangdong |
| L. macranthoides | 55 | Xingyi in Guizhou |
| | 26 | Xiushan county in Chongqing |
| | 44 | Longhui & Xupu county in Hu'nan |
Corresponding author. 通讯作者。
E-mail address: liweidong2005@126.com (W. Li). 电子邮件地址：liweidong2005@126.com （W. Li）。
Peer review under responsibility of Beijing University of Chinese Medicine. 由北京中医药大学负责的同行评审。 ^(1){ }^{1} These authors contributed equally to this study. ^(1){ }^{1} 这些作者对这项研究做出了同样的贡献。