Discrimination of Lonicera japonica THUNB. from Different Geographical Origins Using Restriction Fragment Length Polymorphism Analysis

Abstract

Lonicera japonica THUNB. (Caprifoliaceae) is commonly used as an anti-inflammatory herbal medicine in China. 1,2) We found that L. japonica produced in Henan and Shandong Provinces has the most potent anti-inflammatory effect.3) The herbs grown in these two provinces are recognized as geo-authentic and possess a higher content of therapeutically effective components in the flowers.4) The ecological factors for the cultivation of geo-authentic L. japonica have been studied.5) We attempted to classify the geo-authentic L. japonica by microscopic and chemical characteristics, but because these factors are not well-established, it was difficult to identify specific criteria to distinguish the geo-and non-authentic varieties of L. japonica. 6)Standardization of herbal products is important for safe and effective use. 7) Because herbal medicines are extracted from raw botanical materials, control of the sources of botanical material is the first step in quality assurance of herbal medicines. 8,9) Recent developments in biological techniques, such as molecular genetic markers, allow for the identification of plant species through the detection of species-specific alleles. Compared to chemical constituents, genomic characters are stable for a certain plant species or population, and can be used as a marker for the discrimination of plant species or populations. 10) Our group has established speciesspecific PCR and PCR-RFLP methods to classify Fritillaria cihhosa and Fritillaria pallidiflora.11,12) However, authentication of the geographic origin of plants is difficult with molecular methods, because distinguishing sites in DNA sequences are hard to find.In the present study, we found that a mutation site in the nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) region from geo-authentic L. japonica can be recognized by the restriction endonuclease EcoN I. Since the PCR products from geo-authentic L. japonica cannot be digested completely and some PCR products from non-geo-authentic L. japonica can be digested partially, a quantitative PCR-RFLP method to discriminate between the two populations is developed. The cleavage rate of PCR products by EcoN I was determined and correlated remarkably with the geographical origin of L. japonica. This method has been used successfully in distinguishing commercial herbs. (Fig. 1). The flowers of L. hypoglauca (L2002028) were collected in Jiujian, Jiangxi Prov.; L. confuse (L2002031) was collected in Xupu, Hunan Prov.; L. dasystyla (L2002034) was collected in Guilin, Guangxi Prov. Crude drugs of L. japonia were purchased from three different pharmacies in Nanjing, Jiangsu Prov. (L2002046, L2002047, L2002048), Wulong, Chongqing Prov. (L2002049) and Guiyang, Guizhou Prov. (L2002050). Each specimen was morphologically identified by the authors and voucher specimens were deposited in the University Herbarium at the China Pharmaceutical University, Nanjing, China. MATERIALS AND METHODS Plant MaterialsPCR Amplification and Sequence Determination of nrDNA ITS Region Three fresh ...

Stability and Accuracy Assessment of Identification of Traditional Chinese Materia Medica Using DNA Barcoding: A Case Study on Flos Lonicerae Japonicae

“…Several criteria and techniques have been used in the identification of Flos Lonicerae Japonicae to ensure its quality and therapeutic effects [16–19]. Li et al introduced the morphologic and microstructural methods for identifying L. japonica and Lonicerae [20].…”

Identification ofLonicera japonicaand its Related Species Using the DNA Barcoding Method

“…To ensure the quality and therapeutic effects of Chinese medicines, differentiating the species that serves as medicinal materials for many traditional Chinese medicine products is critical. Previous studies on the identification of L. japonica and its related species have been focused mainly on traditional classification, chemical methods [3,4], and various genetic molecular markers, such as RFLP and the diagnostic PCR [5,6]. However, it is usually hard to identify the closely re-Abstract !…”

Identification ofLonicera japonicaby PCR-RFLP and Allele-Specific Diagnostic PCR Based on Sequences of Internal Transcribed Spacer Regions

“…are widely used in traditional Chinese medicine for their heat-clearing, detoxifying and anti-inflammatory properties [1]. However, some closely related species of L. japonica with similar morphology but weaker biological activity are also used medicinally [2], including Lonicera macranthoides Hand.-Mazz., Lonicera confusa DC., Lonicera hypoglauca Miq., Lonicera similis Hemsl., and Lonicera fulvotomentosa P. S.Hsu [3,4]. However, species discrimination methods based on morphological and chemical fingerprinting often fail to distinguish the adulterants because these approaches rely on features of the overall appearance, histological structure, and chemical components.…”