School of Chinese Materia Medica, Guangdong Pharmaceutical University, Guangzhou, China.
School of Chinese Materia Medica, Guangdong Pharmaceutical University, Guangzhou, China; Key Laboratory of Digital Quality Evaluation of Traditional Chinese Medicine, National Administration of Traditional Chinese Medicine, Guangzhou, China; Traditional Chinese Medicine Quality Engineering and Technology Research Center of Guangdong Universities, Guangzhou, China.
J Pharm Biomed Anal. 2023 Oct 25;235:115619. doi: 10.1016/j.jpba.2023.115619. Epub 2023 Aug 1.
Typhae Pollen (TP) and its carbonized product (carbonized Typhae Pollen, CTP), as cut-and-dried herbal drugs, have been widely used in the form of slices in clinical settings. However, the two drugs exhibit a great difference in terms of their clinical efficacy, for TP boasts an effect of removing blood stasis and promoting blood circulation, while CTP typically presents a hemostatic function. Since the active ingredients of CTP, so far, still remain unclear, this study aimed at identifying the active ingredients of CTP by spectrum-effect relationship approach coupled with multi-block partial least squares (MBPLS), partial least squares (PLS), and support vector machine (SVM) algorithms. In this study, the chemical profiles of a series of CTP samples which were stir-fried for different duration (denoted as CTP0∼CTP9) were firstly characterized by UHPLC-QE-Orbitrap MS. Then the hemostatic effect of the CTP samples was evaluated from the perspective of multiple parameters-APTT, PT, TT, FIB, TXB, 6-keto-PGF1α, PAI-1 and t-PA-using established rat models with functional uterine bleeding. Subsequently, MBPLS, PLS and SVM were combined to perform spectrum-effect relationship analysis to identify the active ingredients of CTP, followed by an in vitro hemostatic bioactivity test for verification. As a result, a total of 77 chemical ingredients were preliminarily identified from the CTP samples, and the variations occurred in these ingredients were also analyzed during the carbonizing process. The study revealed that all the CTP samples, to a varying degree, showed a hemostatic effect, among which CTP6 and CTP7 were superior to the others in terms of the hemostatic effect. The block importance in the projection (BIP) indexes of MBPLS model indicated that flavonoids and organic acids made more contributions to the hemostatic effect of CTP in comparison to other ingredients. Consequently, 9 bioactive ingredients, including quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, quercetin, kaempferol, isorhamnetin, 2-methylenebutanedioic acid, pentanedioic acid, benzoic acid and 3-hydroxybenzoic acid, were further identified as the potential active ingredients based on PLS and SVM models as well as the in vitro verification. This study successfully revealed the bioactive ingredients of CTP associated with its hemostatic effect, and also provided a scientific basis for further understanding the mechanism of TP processing. In addition, it proposed a novel path to identify the active ingredients for Chinese herbal medicines.
蒲黄（TP）及其炭化产物（炭化蒲黄，CTP）作为干药材，以切片形式广泛应用于临床。然而，这两种药物在临床疗效上存在很大差异，因为 TP 具有活血化瘀的作用，而 CTP 通常具有止血作用。由于 CTP 的活性成分至今仍不清楚，本研究旨在采用谱效关系方法结合多块偏最小二乘（MBPLS）、偏最小二乘（PLS）和支持向量机（SVM）算法，鉴定 CTP 的活性成分。本研究首先采用 UHPLC-QE-Orbitrap MS 对一系列炒制时间不同的 CTP 样品（分别记为 CTP0∼CTP9）的化学成分进行了表征。然后，采用建立的功能性子宫出血大鼠模型，从 APTT、PT、TT、FIB、TXB、6-酮-PGF1α、PAI-1 和 t-PA 等多个参数的角度评价了 CTP 样品的止血作用。随后，将 MBPLS、PLS 和 SVM 结合进行谱效关系分析，以鉴定 CTP 的活性成分，并通过体外止血生物活性试验进行验证。结果，从 CTP 样品中初步鉴定出 77 种化学成分，分析了炭化过程中这些成分的变化。研究表明，所有 CTP 样品均表现出不同程度的止血作用，其中 CTP6 和 CTP7 在止血作用方面优于其他样品。MBPLS 模型的投影重要性指标（BIP）表明，与其他成分相比，类黄酮和有机酸对 CTP 的止血作用贡献更大。因此，根据 PLS 和 SVM 模型以及体外验证，进一步鉴定出 9 种生物活性成分，包括槲皮素-3-O-葡萄糖苷、山柰酚-3-O-芦丁、槲皮素、山柰酚、异鼠李素、2-亚甲基丁二酸、戊二酸、苯甲酸和 3-羟基苯甲酸，作为潜在的活性成分。本研究成功揭示了与 CTP 止血作用相关的生物活性成分，为进一步了解 TP 炮制机制提供了科学依据。此外，它为鉴定中草药的活性成分提供了一条新途径。
