| Seabuckthorn belongs to the genus Hippophae of family Elaeagnacea, which is a commonly used ethnic medicine in China for more than 2000 years. It has been reported has anti-tussive, cytoprotective, hepatoprotective, antihypertensive and cardio-protective properties. According to Chinses Pharmacopeia and the local standards in China, H. rhamnoides, H. gyantsensis and H. tibetana are recorded as the original plants of seabuckthorn.Due to the morphological similarities of Hippophae species, seabuckthorn are sometimes misused. Consequently, the quality control and clinical effects would ben seriously affected by the misuse. Therefore, there is an urgent need for rapid and accurate identification and authentication methods of Hippophae species.Objective:Seven species and seven subspecies of Hippophae were identified through DNA baecode and Bar-HRM analysis. Besides, the metabolomic fingerprints were established by ~1H-NMR metabolomics, and the metabolites were identified. The potential metabolic makers were discovered by chemometric analysis and quantified by internal standard method; tri-step IR macro-fingerprints were acquired by tri-step IR spectroscopy, meanwhile, the common and characteristic absorption peaks were investigated. The aforementioned investigations were aimed to provide a multidimentional identification methods and the theoretical basis for accurate choice of Tibetan medicine seabuckthorn.Methods:(1) Total genomic DNA was extracted from fruit (leaves) of Hippophae species, primer pairs ITS2 (2F/3R) and psbA-trnH(psbAY/ trnHF) were used for PCR amplification. The PCR products were visualized on agarose gels, and after electrophoresis, purified PCR products were bidirectionally sequenced by the same primers that were used for PCR in a 3730XL sequencer. Proofreading and contig assembly of sequencing peak diagrams were performed by CodonCode Aligner. All sequences were aligned by MEGA. The genetic distances were calculated according to the Kimura 2 parameter (K2P) model. The distribution of intra-vs. inter-specific variability was assessed by DNA barcoding gaps. A neighbor-joining (NJ) tree was constructed by MEGA. All these methods were applied to identify medicinal and non-medicinal Hippopahe species.(2) The universal primer pairs 2F/3R of ITS2 barcode was selected for HRM analysis to produce standard melting profiles of the Hippophae species. PCR amplification was performed using Rotor-Gene Q MDx, and the fluorescent data were acquired at the end of each extension step during the PCR, which including melting curves and melting temperature (T_m) for each sample. Different species and commercial products of seabuckthom were discriminated and authenticated according to the normalized melting curves and differential melting curves. Besides, the T_m distribution characteristics of different Hippophae species and commercial products were conducted through PCA. After HRM analysis, all the PCR products were bidirectionally sequenced and submitted to DNA Barcoding System for Identifying Herbal Medicine for Bar-HRM verification.(3) The metabolic fingerprints of Hippophae species were established by ’H-NMR metabolomics, and the major matabolites were identified. Besides, different Hippophae species were differentiated and the potential metabolic makers were discovered through PCA and PLS-DA. Some of the metabolic makers were quantified and then analyzed by one-way analysis of variance (one-way ANOVA).(4) The IR macro-fingerprints of Hippophae species fruit and four medicinal Hippophae species fruit extracts were analyzed using tri-step IR spectroscopy. The major characteristic absorption peaks were assigned based on the published references. Different Hippophae species fruits and four medicinal Hippophae species fruits extracts were discriminated by PCA. The positions, shapes and intensities of absorption peaks between medicinal materials and the extracts were compared and analyzed.Results:(1) The sequences length of ITS2 ranged from 221 to 223 bp with 43 variable sites and 23 haplotypes were identified; sequences length of psbA-trnH ranged from 300 to 313 bp with 19 variable sites among 23 haplotypes. In general, the mean inter-specific distances were higher than the mean intra-specific distances for the single-locus barcodes as well as the 2-locus barcode by the K2P model. Sequence data revealed that ITS2+psbA-trnH was the most suitable candidate barcode at the species and subspecies level. The closely related Hippophae species were effectively differentiated in the NJ tree.(2) Bar-HRM analysis method could intuitively identify different Hippopahe species by comparing the peak shapes of the melting curves and differential plot melting curves. PCA result of the T_m value of original plants and commercial products, indicated that the adulterants can be rapidly and accurately authenticated.(3) A total of 36 metabolites, which including sterols, pentacyclic triterpenoids, fatty acids, amino acids, saccharides and flavonoids were identified. Combined with PCA and PLS-DA, different Hippophae species were clearly differentiated. Besides, oleanolic acid, alanine, fatty acids, malic acid, choline, quinic acid, L-quebrachitol, β-D-glucose, and a-D-glucose were demonstrated to be the potential metabolic makers. Quantitive analysis showed that H. tibetana and H. gyantsensis contain more oleanolic acid; H. litangensis and H. neurocarpa subsp. neurocarpa contain more alanine; the contents of fatty acids were higher in H. salicifolia, H. gyantsensis and H. tibetana than other Hippophae speceis; H. rhamnoides subsp. yunnanensis and H. rhamnoides subsp. wolongensis contained more malic acid; there’s more choline in H. rhamnoides subsp. mongolica and H. rhamnoides subsp. turkestanica; higher quinic acid content was determined in H. rhamnoides subsp. turkestanica; H. rhamnoides subsp. mongolica contains more β-D-glucose and a-D-glucose; more L-quebrachitol were detected in H. rhamnoides subsp. mongolica, H. rhamnoides subsp. turkestanica and H. tibetana. These contents differences were proved statistically significant through One-way ANOVA.(4) The FT-IR, SD-IR and 2D-IR macro-fingerprints of different Hippophae species fruits, and four medicinal Hippophae species fruits extracts were acquired. The main characteristic absorption peaks were assigned, which were O-H, C-O, C=C, =C-O-C and C-H groups of flavonoids, fatty acids and saccharides. The intensities of main characteristic absorption peaks of four medicinal Hippophae species fruits extracts were enhanced compared with the raw materials. PCA results illustrated that differnet Hippophae species, as well as the corresponding extracts can be distinguished by tri-step IR method.Conclusions:(1) DNA barcode is applicable for the identification of different Hippophae species. The suitable bacode is selectable according to the tested samples in practice. The successful application of this method has promising future for the identification of Tibetan medicine seabuckthorn and its closely related species.(2) As a universal and data visualized identification method, Bar-HRM identified medicinal materials rapidly, and solved the tough problem of subspecies identification. The method offers a rapid and reliable method for the authentication of the ethnic medicinal plant and the commercial products.(3) The potential metabolic markers of malic acid, choline, quinic acid, L-quebrachitol, β-D-glucose, and a-D-glucose can be used as new indicators for species discrimination. The metabolites can be qualitatively and quantitatively measured by ~1H-NMR metabolomics. This approach can be used as novel method for species identification of Tibetan medicine seabuckthorn and other ethnic medicine.(4) The characteristic absorption peak positions and intensities elucidated the similarities and differences among different Hippophae species, which could be used as evidences for the chemical classification. The enhanced peka intensities of four medicinal Hippophae species fruit extracts demonstrated the rationality and scientificity of the traditional drug application forms of seabuckthorn. The rapid, non-destructive, accurate technique provides a new way for monitoring the scientificity of Tibetan meidicine seabuckthorn and the extracts.Innovations:(1) The ITS2 and psbA-trnH sequences based DAN barcoding was applicable for the identification of all Hippophae species distributed in China, and a mini database of the corresponding sequences was established. Besides, Bar-HRM was firstly developed for Hippophae species identification, especially at subspecies level. In addition, counterfeited, substituted and adulterated products were found by investigating the commercial products of "Shaji". The successful application of DNA barcoding and Bar-HRM offers new detection mean for medicinal materials authentication of Tibetan meidicine seabuckthorn.(2) This study established the metabolic fingerprints of all Hippophae species which distributed in China, and 36 metabolites were identified. In addition, the potential inter and intra specific metabolic markers were investigated for the first time through the combination of ~1H-NMR metabolomics and chemometrics analysis.(3) Tri-step IR spectroscopy was utilized to acquire the macro-fingerprints of Hippophae species and the four medicinal Hippophae species fruit extracts. The main characteristic absorption peaks were assigned, the similarities and differences among different Hippophae species were discovered. Coupled with PCA, different Hippophae species were discriminated.(4) In this study, the molecular biological identification methods,’H-NMR based metabolomics and tri-step IR spectroscopy were conjunctly used for species identification of Tibetan medicine seabuckthom. This integrated efficient method has a promising future for the identification and quality evaluation of seabuckthom and other ethnic medicine. |
PDF Full Text Request