Development of 1 H-NMR methods for quantitative determination of alkylamides in Zanthoxylum bungeanum and quality evaluation based on its fingerprint.

Alkylamides, as the representative hemp flavor and active ingredients, can reflect the quality of Zanthoxylum bungeanum Maxim. However, conjugated triene structure exists in alkylamides, which is easy to be oxidized and decomposed in air, making it difficult to quantify. In this study, a method for the quantitative determination of alkylamides by 1 H-NMR technology was developed with 85% ethanol as the best extraction solvent, CDCl3 as the best deuterium dissolution reagent, pyrazine as the internal standard, and triple peaks of hydrogen protons on the amide bond at δ 6.33 ppm as the quantitative signal peak. Meanwhile, methodological verification was carried out to prove the reliability and effectiveness of the method. On this basis, the contents of alkylamides in nine germplasms of Zanthoxylum with monthly dynamics were obtained. The results showed that the alkylamides of Hancheng stingless Z. bungeanum (HC) exhibited the highest content in August (51.92 ± 0.96 mg/g), while the lowest was FG in June (1.23 ± 0.21 mg/g). The results of 1 H-NMR corresponded to those of HPLC, and the effectiveness of this method was verified. Accumulation dynamic results show that the best harvest period of Z. bungeanum is July and August. Moreover, the quality of nine varieties of Zanthoxylum bungeanum from a common garden was evaluated by the established 1 H-NMR fingerprint and chemometric analyses. The results showed that Hancheng stingless Z. bungeanum was the best germplasm. This study provides a new strategy for the quantitative determination of alkylamides in Z. bungeanum and improves the quality evaluation system of Z. bungeanum. PRACTICAL APPLICATION: The results provide a new research idea for the analysis of important chemical components of Z. bungeanum. Meanwhile, the study provides a scientific basis for the quality evaluation and high-quality germplasm resources of Z. bungeanum.

Comparative Transcriptome Analysis and Expression of Genes Reveal the Biosynthesis and Accumulation Patterns of Key Flavonoids in Different Varieties of Zanthoxylum bungeanum Leaves.

Zanthoxylum bungeanum (Rutaceae), a popular food flavoring and traditional Chinese medicine ingredient, is an important cash crop. Its leaves are rich in flavonoids with multiple bioactivities. However, the transcriptional sequencing has not been investigated, and the molecular basis for the flavonoid biosynthesis remains unclear in this plant. This paper, the key flavonoids (epicatechin, rutin, hyperoside, trifolin, quercitrin, and afzelin) contents were determined in the leaves of 10 Z. bungeanum varieties from a common garden. Results show the leaves of Z. bungeanum mainly contained hyperoside (11.410-21.721 mg/g) and quercitrin (9.401-18.016 mg/g). The total content of these key components was the highest in Fengxian Dahongpao (66.012 mg/g) and the lowest in Fugu (32.223 mg/g). Three varieties (Hancheng stingless, Fugu, and Fengxian Dahongpao) with significant differences in the total content of key flavonoids were selected for transcriptome analysis to obtain flavonoid biosynthesis-related genes. In total, 83 522 unigenes were obtained, 40 668 (48.69%) unigenes were annotated, and 6656 differentially expressed genes (DEGs) were identified. Comparison of the other two varieties, Fugu had many differentially expressed genes indicating the particularity of its variety. Flavonoid-related DEGs of 22 structural genes, including three PALs, one CYP73A, three 4CLs, six CHSs, one CHI, one F3H, one DFR, two ANSs, one ANR, one FLS, and two CYP75B1s, as well as nine MYBs were obtained. These structural genes had different expression patterns in different Z. bungeanum varieties. It is worth noting that the genes expressing the flavonoid 3'5' hydroxylase are absent in Z. bungeanum. Furthermore, quantitative real-time PCR experiment showed consistent results in transcriptome analysis. The RNA-Seq data set of this study sheds lights on the molecular mechanism of flavonoid biosynthesis in Z. bungeanum, provides valuable information for the metabolic regulation of flavonoids, and may serve as a guide for future breeding programs.

Metabolite Profiles, Bioactivity, and HPLC Fingerprint of Different Varieties of Eucommia ulmoides Oliv.: Towards the Utilization of Medicinal and Commercial Chinese Endemic Tree.

Eucommia ulmoides Oliv. is widely regarded in China as a precious medicinal and commercial endemic tree. Due to cross-breeding or natural variation of E. ulmoides, the metabolite composition may vary significantly, making control of the medical quality difficult. In order to improve the rational development and utilization, the quality of seven varieties of E. ulmoides were evaluated based on metabolite profiles (total phenolic, total flavonoid, gutta-percha, aucubin, geniposidic acid, chlorogenic acid, geniposide, pinoresinol diglucoside, rutin, hyperoside, and astragalin), bioactivities (in vitro, in vivo antioxidant activities, and antibacterial activities) and HPLC fingerprint combined with chemometrics analysis. On this basis, the differences of medicinal parts (leaf and bark) were further carried out. For the traditional use of bark, Purple-leaf E. ulmoides was the most suitable. For the use of leaf, Qinzhong 1 and Purple-leaf E. ulmoides were appropriate. HPLC fingerprint analysis showed that significant differences in metabolite profiles exist among seven varieties of E. ulmoides. Combined with chemometrics analysis, seven varieties of E. ulmoides were divided into three groups from the use of leaf and bark. The analysis not only evaluated quality of seven varieties of E. ulmoides, but also could distinguish different varieties and different regions of origin. The results can provide theoretical basis for E. ulmoides resources utilization and cultivation of fine varieties.

Phenolic Compounds and Antioxidant Activity of Different Organs of Potentilla fruticosa L. from Two Main Production Areas of China.

This report compared the phenolic compounds and antioxidant activity of the leaves, flowers, and stems of Potentilla fruticosa L. collected from two main production areas of P. R. China (Taibai Mountains and the Qinghai Huzhu Northern Mountains). The results indicated that there were significant differences in the phenol contents and antioxidant activities among the different organs and between the two productions. High-performance liquid-chromatography analysis indicated that hyperoside, (+)-catechin, ellagic acid, and rutin were the primary compounds in leaves and flowers; for stems, the content of six phenolic compounds, from two productions, were the lowest. The 1,1-diphenyl-2-picryl hydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) di-ammonium salt (ABTS), ferric reducing power (FRAP), lipid peroxidation assays, and microbial test system (MTS) were used to evaluate the antioxidant activity. The results demonstrated that the leaves from two productions exhibited powerful antioxidant activity than other organs, which did not significantly differ from that of the positive control (rutin), followed by the flowers and stems. The correlation between the content of phytochemicals and the antioxidant activities of different organs showed that the total phenol, tannin, hyperoside, and (+)-catechin contents may influence the antioxidant activity, and these compounds can be used as markers for the quality control of P. fruticosa.