Chemical composition analysis of branch bark of different Eucommia ulmoides clones under short cycle leaf forest mode
Eucommia ulmoides Oliv. is a single genus of relict tree species that survived in China after the Quaternary glacial invasion. It is also an important medicinal tree species in China, known for its bark extraction and medicinal use since ancient times. Eucommia ulmoides bark is rich in compounds such as lignans, cyclohexene ether terpenes, phenylpropanoids, flavonoids, etc. For example, geniposide has the effects of lowering and regulating blood pressure, geniposide has anti-tumor activity and anti complement properties, and aucubin has strong analgesic, antibacterial, and anti-inflammatory effects. And lignans such as turpentine diglucosides are known active ingredients in Eucommia ulmoides bark that have blood pressure lowering effects. Therefore, Eucommia ulmoides bark plays a very important role as a traditional Chinese medicine in healthcare and other fields.

With the proposal of the comprehensive development and utilization concept of Eucommia ulmoides, the cultivation mode of Eucommia ulmoides has also developed from the traditional medicinal cultivation mode to various management modes such as fruit Eucommia ulmoides cultivation mode, male flower cultivation mode, wood medicine dual-use cultivation mode, and leaf cultivation mode. The forest cultivation mode of Eucommia ulmoides leaves utilizes the strong sprouting ability of Eucommia ulmoides, shortens the management cycle, greatly improves the yield and quality of Eucommia ulmoides leaves and bark, and has significant economic benefits. A comparative analysis was conducted on the chemical composition of Eucommia ulmoides leaves between traditional deciduous forest and leaf forest models, and it was found that secondary metabolites can effectively distinguish deciduous forest from leaf forest. In addition, the content of chlorogenic acid in Eucommia ulmoides leaves and bark in the leaf forest model is higher than that in the deciduous forest model, indicating that leaf forest cultivation is the optimal cultivation mode. Another study suggests that the application of exogenous hormones can significantly increase the gum content of Eucommia ulmoides leaves and bark in short-term leaf forests. However, research on the chemical composition of bark between different Eucommia ulmoides clones under the cultivation mode of leaf forest has not been carried out, which limits the development and utilization of bark resources under the cultivation mode of leaf forest.

Headspace solid-phase microextraction (HS-SPME) is a sample pretreatment technique that integrates extraction, concentration, and desorption. This method is easy to operate, requires minimal sample size, and has no complicated pretreatment process. It can also avoid the oxidation and decomposition of unstable components in the sample. In recent years, the combination of gas chromatography-mass spectrometry (GC-MS) has been widely used in the study of plant volatile components. Research has shown that many volatile compounds can not only be directly used for insect resistance, medicinal applications, or as lead components in organic synthesis, but also serve as important intermediates in various metabolic pathways of plants, providing guidance for analyzing plant metabolic networks and studying plant self-defense responses. Fourier transform infrared spectroscopy (FTTR) is also a commonly used analytical method in quality evaluation, which can qualitatively analyze samples as a whole and has the characteristics of being fast, accurate, and non-destructive. Therefore, in this study, high-performance liquid chromatography (HPLC) was used to determine the content of seven main active ingredients in the bark of eight Eucommia ulmoides clones under short cycle leaf forest cultivation mode. Combined with headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) technology, the volatile components were analyzed to identify the differential volatile substances in the bark of Eucommia ulmoides clones, and infrared spectroscopy was used for comparative analysis. Preliminary determination of the chemical characteristics of each clone, in order to provide scientific basis for quality control, targeted cultivation, and resource development and utilization of branch bark under the short cycle leaf forest model of Eucommia ulmoides.

 

In this study, HPLC and HP-SPME-GC-MS were used to quantitatively detect and analyze the chemical components of different Eucommia ulmoides clones’ bark under short period leaf forest mode. At the same time, infrared spectroscopy was used for comparison, and fingerprint spectra of 8 clones’ bark were extracted. The relationship between chemical components and infrared spectra was preliminarily analyzed. It was found that there were differences in the chemical components of the bark of 8 Eucommia ulmoides clones. Among the 7 main active ingredients, iridoid compounds such as aucubin, geniposide, and geniposide were found to have higher contents in the bark of different Eucommia ulmoides clones. Circene terpenes have various biological activities, so they can be utilized in the bark of different Eucommia ulmoides clones under the leaf forest model. Zhang et al. found that the content of sitosterol diglucoside in Eucommia ulmoides bark increases with tree age, indicating that the content of sitosterol diglucoside is higher in old bark and lower in new bark. The average content of the characteristic compound turpentine diglucoside in Eucommia ulmoides bark from Yeyonglin is relatively low, with only asexual strains EU1, EU4, and EU7 having a content greater than 0.1%, which meets the requirements of the 2020 edition of the Chinese Pharmacopoeia. Therefore, when using turpentine diglucoside as a medicinal quality indicator, these three asexual Eucommia ulmoides bark should be selected for utilization.

Plant specific volatile compounds play an important signaling role in the transmission of plant information. This study utilized the strategy of plant metabolomics to investigate the types and quantities of volatile substances released from Eucommia ulmoides bark. The data obtained from plant metabolomics needs to be preprocessed by peak alignment, cutting, and noise filtering. Therefore, MS-DIAL software was selected to perform feature peak matching, alignment, and extraction on multiple sample mass spectrometry peaks. Combined with the database, 70 volatile substance components were detected in seven major categories: esters, alcohols, aldehydes, terpenes, ketones, lactones, and alkenes. Wu Tingting et al. found that aldehydes and lipid compounds are the main components of total volatile compounds in tea trees under monoculture and peanut+tea intercropping modes, mainly playing a role in repelling pests and attracting natural enemies. Alcohols, aldehydes, and esters are the main volatile compounds in the bark of different Eucommia ulmoides clones, which may play a major role in the defense against herbivorous insects and require further research. Isophytol is a twenty carbon unsaturated tertiary alcohol of diterpenes composed of four isoprene molecules connected end-to-end. It is the main raw material for the production of vitamin E. The terpenoid substance isophytol has the highest relative content in the clones EU2, EU5, and EU8, and contains terpene volatiles such as tetrahydrogeraniol, ethyllinalool, naringenin, and hemlock. It has pharmacological properties such as anti-inflammatory, antibacterial, and anti-tumor effects, which have certain guiding significance for the further development of medicinal value of Eucommia ulmoides bark.

Supervised methods are used to explore completely unknown data features, classify raw data information based on sample characteristics, group target data with similar features into homologous classes, and use corresponding visualization techniques to visually express them. Currently, common unsupervised methods include cluster analysis and principal component analysis. This article uses UPLC measurement data for clustering, dividing 8 clones of Eucommia ulmoides into 3 groups. Principal component analysis extracts 3 principal components, among which the cyclohexene ether terpenoid substances geniposide, geniposide, and aucubin contribute the most in the first dimension. Furthermore, the PLS-DA model was used to analyze the differences in volatile components of Eucommia ulmoides bark among different clones, and 22 volatile substances were selected as differential volatile components, mainly ester volatile components. The cyclohexene ether terpenes such as aucubin and geniposide are the main secondary metabolites in the bark of Eucommia ulmoides, and their synthetic precursors are monoterpenes such as vanillin. Therefore, the next step is to consider using GC-MS technology for targeted metabolomic analysis of terpene volatiles in Eucommia ulmoides bark. In addition, Eucommia ulmoides exhibits the same bark characteristics in its early stages, with a bluish gray color, no cracking, significant skin pores, and no identifiable morphological features. Fourier transform infrared spectroscopy technology can be applied to quickly distinguish Eucommia ulmoides bark from different clones in the leaf forest mode in the later stage.