Determination of Flavonoids Compounds of Three Species and Different Harvesting Periods in Crataegi folium Based on LC-MS/MS.

Crataegi folium have been used as medicinal and food materials worldwide due to its pharmacological activities. Although the leaves of Crataegus songorica (CS), Crataegus altaica (CA) and Crataegus kansuensis (CK) have rich resources in Xinjiang, China, they can not provide insights into edible and medicinal aspects. Few reports are available on the qualitative and quantitative analysis of flavonoids compounds of their leaves. Therefore, it is necessary to develop efficient methods to determine qualitative and quantitative flavonoids compounds in leaves of CS, CA and CK. In the study, 28 unique compounds were identified in CS versus CK by qualitative analysis. The validated quantitative method was employed to determine the content of eight flavonoids of the leaves of CS, CA and CK within 6 min. The total content of eight flavonoids was 7.8-15.1 mg/g, 0.1-9.1 mg/g and 4.8-10.7 mg/g in the leaves of CS, CA and CK respectively. Besides, the best harvesting periods of the three species were from 17th to 26th September for CS, from 30th September to 15th October for CA and CK. The validated and time-saving method was successfully implemented for the analysis of the content of eight flavonoids compounds in CS, CA and CK for the first time.

Classification and phylogenetic analysis of Chinese hawthorn assessed by plant and pollen morphology.

The Chinese hawthorn (Crataegus pinnatifida Bge. var. major N.E.Br.) is uniquely originated in northern China. The ecological and horticultural importance of Chinese hawthorn is considerable and some varieties are valued for their fruit or medicine extracts. Its taxonomy and phylogeny remain poorly understood. Apart from general plant morphological traits, pollen is an important trait for the classification of plants and their evolutionary origin. However, few studies have investigated the pollen of Chinese hawthorn. Here, an analysis of plant and pollen morphological characteristics was conducted in 57 cultivars from the Shenyang region. Thirty plant morphological characters and nine pollen grain characters were investigated. The plant morphological analysis revealed that the coefficient of variation for 13 traits was >20%, which indicates a high degree of variability. We also found that the pollen grains varied greatly in size, shape (from prolate to perprolate), and exine pattern (striate-perforate predominantly). The number of apertures was typically three. Based on these findings, we suggest that pollen morphology associated with plant morphological traits can be used for classification and phylogenetic analysis of Chinese hawthorn cultivars. In sum, our results provide new insights and constitute a scientific basis for future studies on the classification and evolution of Chinese hawthorn.

Comparative genomic and phylogenetic analyses of mitochondrial genomes of hawthorn (Crataegus spp.) in Northeast China.

Hawthorn (Crataegus spp.) plants are major sources of health food and medicines. Twenty species and seven variations of Crataegus are present in China. A variety of unique Crataegus species was found in their natural distribution in northeast China. In the present study, we assembled and annotated the mitochondrial genomes of five Crataegus species from northeastern China. The sizes of the newly sequenced mitochondrial genomes ranged from 245,907 bp to 410,837 bp. A total of 45-55 genes, including 12-19 transfer RNA genes, three ribosomal RNA genes, and 29-33 protein-coding genes (PCGs) were encoded by these mitochondrial genomes. Seven divergent hotspot regions were identified by comparative analyses: atp6, nad3, ccmFN, matR, nad1, nad5, and rps1. The most conserved genes among the Crataegus species, according to the whole-genome correlation analysis, were nad1, matR, nad5, ccmFN, cox1, nad4, trnQ-TTG, trnK-TTT, trnE-TTC, and trnM-CAT. Horizontal gene transfer between organellar genomes was common in Crataegus plants. Based on the phylogenetic trees of mitochondrial PCGs, C. maximowiczii, C. maximowiczii var. ninganensis, and C. bretschneideri shared similar maternal relationships. This study improves Crataegus mitochondrial genome resources and offers important insights into the taxonomy and species identification of this genus.

Genetic relationships among some hawthorn (Crataegus spp.) species and genotypes.

The genus Crataegus is well distributed in Turkey as a wild plant, with numerous, inherently variable species and genotypes. RAPD markers were used to study 17 hawthorn genotypes belonging to Crataegus monogyna ssp. monogyna Jacq (2 genotypes), C. monogyna ssp. azarella Jacq (1), Crataegus pontica K.Koch (3), Crataegus orientalis var. orientalis Pallas Ex Bieb (3), Crataegus pseudoheterophylla Pojark (1), Crataegus aronia var. dentata Browicz (1), C. aronia var. aronia Browicz (4), and Crateagus x bornmuelleri Zabel (2). The 10 RAPD primers produced 72 polymorphic bands (88% polymorphism). A dendrogram based on Jaccard's index included four major groups and one outgroup according to taxa. The lowest genetic variability was observed within C. aronia var. aronia genotypes. The study demonstrated that RAPD analysis is efficient for genotyping wild-grown hawthorns.

Chemical basis of hawthorn processed with honey protecting against myocardial ischaemia.

Hawthorn including many plants from the genus Crataegus (C.) is used for traditional medicines, herbal drugs, and dietary supplements all over the world. In China, C. pinnatifida Bge. var. major N, E. Br, and C. pinnatifida Bge. are two major species that are used as hawthorn. The purpose of this study is to assay the myocardial protection of hawthorn fruit processed with honey (MSZ) and screen the chemical basis of MSZ on this effect. Firstly, ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC/Q-TOF-MS) was used to analyze the chemical constituents of the sliced dry fruit of hawthorn (SZ) and MSZ. Principal component analysis (PCA) was used to differentiate them. Orthogonal partial least squares-discriminate analysis (OPLS-DA) was applied to screen different compounds between SZ and MSZ, and 32 different compounds were selected. Then a pharmacodynamic test to investigate their protective effect against myocardial ischaemia was carried out. The results demonstrated that the protective effect of MSZ was better than that of SZ on the same dose. Finally, the chemical basis for the protective effect provided by MSZ against myocardial ischaemia was speculated based on correlation analysis. Taken together, all these results suggest that phenylpropanoids, organic acids, tannins, and flavonoids might be the chemical basis of MSZ protecting against myocardial ischaemia.

Evidence for genetic association between East Asian and western North American Crataegus L. (Rosaceae) and rapid divergence of the eastern North American lineages based on multiple DNA sequences.

Phylogeographic relationships were constructed for 72 Old and New World Crataegus species using combinations of four chloroplast and up to five nuclear regions. Maximum parsimony, maximum likelihood, and Bayesian results yield consistent relationships among major lineages. The close associations of the East Asian and western North American species point toward ancient trans-Beringian migrations. Relationships among eastern North American species are poorly resolved and few groups are identified that are congruent with existing classifications. Scant variation and short internal branches among these species suggest rapid divergence associated with polyploidy and hybridization. Incongruence between the chloroplast and nuclear data, and morphology suggest hybrid origins of three species from an extinct European lineage (the male parent) and three different North American female parents. Europe and eastern North America are suggested as the most recent common areas for Crataegus; at least four dispersal events are inferred to explain the present distribution of the genus.

Evaluation of the content and stability of the constituents of mother tinctures and tinctures: the case of Crataegus oxyacantha L. and Hieracium pilosella L.

As a part of our investigations on the content and stability of herbal drug preparations, we evaluated the content and stability of tinctures and mother tinctures of Hawthorn leaves and flowers and Hawkweed. Hawthorn preparations are mainly used by patients with cardiac diseases; Hawkweed is employed for the treatment of cellulitis and obesity due to its diuretic properties. Both tinctures (DER 1:5) and mother tinctures (DER 1:10) are herbal preparations reported in the European Pharmacopoeia. The first preparation is obtained using dried herbal drugs; the latter is a homoeopathic preparation obtained with fresh plant material, often used in substitution of tinctures. The aim of this work was to assess the qualitative and quantitative profile of the constituents of the investigated preparations and the chemical stability of their marker constituents from long-term testing using HPLC assays. Characteristic constituents of Hawthorn leaves and flowers are flavonoids such as vitexin-2''-O-rhamnoside and hyperoside and oligomeric procyanidins. Characteristic constituents of Hawkweed are caffeoyl-quinic acid derivatives, flavonoids and a coumarin:umbelliferone. Our investigation showed that Hawthorn mother tincture had a higher concentration of procyanidins with respect to the tincture but the stability of these constituents were very low in both preparations. Total flavonoidic content was 3.33 mg/ml, about 1.5 times more than the content of mother tincture and the shelf-life t(90) was about 7 months for both preparations. For Hawkweed preparations a content of caffeoyl-quinic acid derivatives (ca. 4 mg/ml) was found, but their stability was good only in the tincture. The concentrations of flavonoids and umbelliferone were two times as much in the tincture with respect to the mother tincture. Stability of these two classes of constituents was good for both preparations over a 9-month period.

Differentiation of Crataegus spp. guided by nuclear magnetic resonance spectrometry with chemometric analyses.

For compliance with US Current Good Manufacturing Practice regulations for dietary supplements, manufacturers must provide identity of source plant material. Despite the popularity of hawthorn as a dietary supplement, relatively little is known about the comparative phytochemistry of different hawthorn species, and in particular North American hawthorns. The combination of NMR spectrometry with chemometric analyses offers an innovative approach to differentiating hawthorn species and exploring the phytochemistry. Two European and two North American species, harvested from a farm trial in late summer 2008, were analyzed by standard 1D 1H and J-resolved (JRES) experiments. The data were preprocessed and modelled by principal component analysis (PCA). A supervised model was then generated by partial least squares-discriminant analysis (PLS-DA) for classification and evaluated by cross validation. Supervised random forests models were constructed from the dataset to explore the potential of machine learning for identification of unique patterns across species. 1D 1H NMR data yielded increased differentiation over the JRES data. The random forests results correlated with PLS-DA results and outperformed PLS-DA in classification accuracy. In all of these analyses differentiation of the Crataegus spp. was best achieved by focusing on the NMR spectral region that contains signals unique to plant phenolic compounds. Identification of potentially significant metabolites for differentiation between species was approached using univariate techniques including significance analysis of microarrays and Kruskall-Wallis tests.

Population structure and genotypic variation of Crataegus pontica inferred by molecular markers.

Information about the natural patterns of genetic variability and their evolutionary bases are of fundamental practical importance for sustainable forest management and conservation. In the present study, the genetic diversity of 164 individuals from fourteen natural populations of Crataegus pontica K.Koch was assessed for the first time using three genome-based molecular techniques; inter-retrotransposon amplified polymorphism (IRAP); inter-simple sequence repeats (ISSR) and start codon targeted (SCoT) polymorphism. IRAP, ISSR and SCoT analyses yielded 126, 254 and 199 scorable amplified bands, respectively, of which 90.48, 93.37 and 83.78% were polymorphic. ISSR revealed efficiency over IRAP and SCoT due to high effective multiplex ratio, marker index and resolving power. The dendrograms based on the markers used and combined data divided individuals into three major clusters. The correlation between the coefficient matrices for the IRAP, ISSR and SCoT data was significant. A higher level of genetic variation was observed within populations than among populations based on the markers used. The lower divergence levels depicted among the studied populations could be seen as evidence of gene flow. The promotion of gene exchange will be very beneficial to conserve and utilize the enormous genetic variability.

[Investigation on method speliality in identifying Fructus Crataegi with vitexin rhamnoside].

The methanol extract of Fructus Crataegi was purified on macro-reticular resins(model DM130)-polyamide column. Vitexin rhamnoside(I) was identified from Fructus Crataegi by TLC on polyamide layer sheets. The false and the confused species of Fructus Crataegi and some traditional Chinese herbal medicines which contain vitexin were studied by the same method. The results indicated that compound I was special constitute of Fructus Crataegi and the identification method of Fructus Crataegi with I was special.

Comparative analysis of Tunisian wild Crataegus azarolus (yellow azarole) and Crataegus monogyna (red azarole) leaf, fruit, and traditionally derived syrup: phenolic profiles and antioxidant and antimicrobial activities of the aqueous-acetone extracts.

Quantitative and qualitative analyses of the yellow and red azarole phenolic extracts prepared from leaf, fruit peel/pulp, and syrup were comparatively investigated. The yellow azarole was found significantly richer in polyphenols than the red-fruit species. Hyperoside was the main phenolic in both yellow and red azarole leaves and only in yellow fruits, whereas procyanidin B2 was the major compound in red fruits. Yellow azarole leaf and fruit peel extracts exhibited the strongest antioxidant activities using DPPH (≈168 and 79 µmol TEAC/g fw, respectively) and FRAP (≈378 and 161 µmol Fe(2+)/g fw, respectively) assays. The highest antibacterial activities were recorded for the yellow azarole leaf and fruit peel extracts, especially against Staphylococcus aureus and Streptococcus faecalis . The low phenolic content of the syrups contrasted with their significant antioxidant and antimicrobial potentials, which were correlated to their hydroxymethylfurfural (HMF) (furan derivative amounts) content.

Reconstructing reticulation history in a phylogenetic framework and the potential of allopatric speciation driven by polyploidy in an agamic complex in Crataegus (Rosaceae).

Polyploidy plays a prominent role in the speciation process in plants. Many species are known to be part of agamic complexes comprising sexual diploids and more or less exclusively asexual polyploids. However, polyploid formation has been studied in very few cases, primarily because of the challenges in examining these cases phylogenetically. In this study, we demonstrate the use of a variety of phylogenetic approaches to unravel origins and infer reticulation history in a diploid-polyploid complex of black-fruited Crataegus. The tree approaches are shown to be useful in testing alternative hypotheses and in revealing genealogies of nuclear genes, particularly in polyploid organisms that may contain multiple copies. Compared to trees, network approaches provide a better indication of reticulate relationships among recently diverged taxa. Taken together, our data point to both the autopolyploid and allopolyploid origins of triploids in natural populations of Crataegus suksdorfii, whereas tetraploids are formed via a triploid bridge, involving the backcross of allotriploid offspring with their diploid C. suksdorfii parent, followed by gene introgression from sympatric C. douglasii. Our findings provide empirical evidence for different pathways of polyploid formation that are all likely to occur within natural populations and the allopatric establishment of neopolyploids subsequent to their formation.

Pharmacokinetic study of isoquercitrin in rat plasma after intravenous administration at three different doses

The aim of this study is to develop a simple and specific HPLC method using vitexin as the internal standard to investigate the pharmacokinetics of isoquercitrin (ISOQ) after three different doses administrated intravenously to rats. The pharmacokinetic parameters were calculated by both compartmental and non-compartmental approaches. The results showed that ISOQ fitted a three-compartment open model. The values of AUC increased proportionally within the range of 5-10 mg·kg-1. Moreover, a half-life, b half-life, ªCL, MRT0-t and MRT0→∞ of ISOQ in rats showed significant differences between 20 mg·kg-1 and other doses, indicating that ISOQ presented dose-dependent pharmacokinetics in the range of 5-10 mg·kg-1 and non-linear pharmacokinetics at higher doses.

O objetivo deste estudo é desenvolver um método simples e específico de HPLC usando vitexina como padrão interno para investigar a farmacocinética do isoquercitrina (ISOQ) após três doses diferentes administradas por via intravenosa a ratos. Os parâmetros farmacocinéticos foram calculados pelas abordagens compartimental e não compartimental. Os resultados mostraram que ISOQ se encaixa no modelo de três compartimentos. Os valores de AUC aumentaram proporcionalmente na faixa de 5-10 mg·kg-1. Além disso, a meia-vida, b meia-vida, ªCL, MRT0-t and MRT0→∞ de ISOQ em ratos mostraram diferenças significativas entre 20 mg·kg-1 e outras doses, o que significa que ISOQ apresenta farmacocinética dose-dependente no intervalo de 5-10 mg·kg-1 e farmacocinética não linear em doses mais elevadas.

Variability of total flavonoids in Crataegus--factor evaluation for the monitored production of industrial starting material.

European Hawthorn species (Crataegus spp.) are traditionally used for their demonstrated cardioprotective benefits. Most products are based on the compendial leaf and flower drug (LFD), which is standardised on the total flavonoid content (TF). In order to reduce variability associated with the wild plant origin and to ease product standardisation, we set out to develop a sustainable source of high quality raw material. Firstly, the LFD of wild trees in Germany was screened in terms of TF content (spectrophotometric analysis, calculated as hyperoside) according to the current pharmacopoeia requirements. Secondly, eleven high value provenances were selected, propagated, cultivated and the grafted clones were reanalysed. Thirdly, major environmental and sourcing influences were assessed: the year of harvest, the growing location, exposure to sunlight, the harvest time and the portion of leaf, flower and wood within the LFD. We found the TF in LFD of 150 wild grown Crataegus ranged between 0.28% and 1.92% (average 1.15%). Pure single styled Crataegus monogyna and hybrid trees represented the major portion (57%) of all screened trees. The hybrids with mainly two-styled flowers (pure Crataegus laevigata and hybrids) showed slightly higher TF values. The selected clones proved to maintain a high TF profile in cultivation, although superiority was attenuated when not only O-glycosides, but also C-glycosides were covered by the assay. Environmental influence surpassed in part the genetic variation of the selection in this study. In conclusion, cultivated high performance trees under central European conditions produced LFD exceeding the Pharmacopoeia standards. Careful monitoring of production--particularly in terms of the harvest time and actual LFD location and composition--has decisive impact to guarantee consistent high TF values.