草药的分子定量(Herb-Q):基于热测序的方法及其在半夏中的应用。

IF 4 2区 医学 Q1 INTEGRATIVE & COMPLEMENTARY MEDICINE Chinese Journal of Natural Medicines Pub Date : 2024-07-01 DOI:10.1016/S1875-5364(24)60636-9
LIU Kui, PANG Tao
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引用次数: 0

摘要

由于品种掺假和剂量不准确造成的草药剂量变化会严重影响临床安全性和疗效。准确的物种定量仍然具有挑战性,因为目前的方法往往得出不一致的结果。本研究介绍了一种基于热测序的新型技术,称为草药分子定量(Herb-Q),旨在对草药产品进行精确定量。我们使用半夏及其五种掺杂物对其有效性进行了评估。首先,我们利用公共数据库中的序列对常用的 DNA 条形码进行了评估,确定了两个候选区域,即成熟酶 K(matK)和内部转录间隔 2(ITS2),用于筛选特定的单核苷酸多态性(SNP)位点,从而进行物种特异性鉴定。通过对采集样本的基因组材料进行扩增和测序,对这些位点进行了验证。我们的验证研究表明,Herb-Q 具有出色的线性、准确性、重复性和检测限。我们建立的定量标准曲线具有很高的 R2 值(> 0.99),可实现精确的物种定量,结合外部标准,可提供清晰准确的视觉定量结果。在对 P. ternata 块茎进行定量时,平均偏差为 2.38%,这证实了 Herb-Q 能够准确鉴定和定量草药产品成分。此外,整个定量过程用时不到 4 小时。该研究提出了一种新颖、快速的方法来准确定量草药产品中的物种,并推进了 DNA 条形码从物种鉴定到定量检测的应用。
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Molecular quantification of herbs (Herb-Q): a pyrosequencing-based approach and its application in Pinellia ternata

Variations in herb dosage due to species adulteration and dosing inaccuracies can substantially affect clinical safety and efficacy. Accurate species quantification remains challenging, as current methods often yield inconsistent results. This study introduces a novel pyrosequencing-based technique, termed herb molecular quantification (Herb-Q), designed to precisely quantify herbal products. We evaluated its effectiveness using Pinellia ternata and five of its adulterants. Initially, we assessed commonly used DNA barcodes with sequences from a public database, identifying two candidate regions, Maturase K (matK) and internal transcribed spacer 2 (ITS2), for screening specific single nucleotide polymorphism (SNP) loci, allowing for species-specific identification. These loci were validated by amplifying and sequencing genomic material from collected samples. Our validation studies showed that Herb-Q demonstrated excellent linearity, accuracy, repeatability, and detection limits. We established quantitative standard curves with high R2 values (> 0.99) to enable precise species quantification, which were combined with external standards to provide clear and accurate visual quantification results. The average bias in quantifying the tuber of P. ternata was 2.38%, confirming that Herb-Q can accurately identify and quantify herbal product constituents. Moreover, the entire quantification process took less than 4 h. This study presents a novel, rapid method for accurately quantifying species in herbal products and advances the application of DNA barcoding from species identification to quantitative detection.

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来源期刊
Chinese Journal of Natural Medicines
Chinese Journal of Natural Medicines INTEGRATIVE & COMPLEMENTARY MEDICINE-PHARMACOLOGY & PHARMACY
CiteScore
7.50
自引率
4.30%
发文量
2235
期刊介绍: The Chinese Journal of Natural Medicines (CJNM), founded and sponsored in May 2003 by China Pharmaceutical University and the Chinese Pharmaceutical Association, is devoted to communication among pharmaceutical and medical scientists interested in the advancement of Traditional Chinese Medicines (TCM). CJNM publishes articles relating to a broad spectrum of bioactive natural products, leading compounds and medicines derived from Traditional Chinese Medicines (TCM). Topics covered by the journal are: Resources of Traditional Chinese Medicines; Interaction and complexity of prescription; Natural Products Chemistry (including structure modification, semi-and total synthesis, bio-transformation); Pharmacology of natural products and prescription (including pharmacokinetics and toxicology); Pharmaceutics and Analytical Methods of natural products.
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