Exploring the quality differences between Perilla frutescens leaves and Cycas alba leaves based on pre column derivatization GC-MS technology
Perilla frutescens (L.) Britt. is an annual upright herbaceous plant of the genus Perilla in the family Lamiaceae. Throughout history, important herbal medicines have referred to those with leaves that are completely purple on both sides or have a green face and a purple back as “Su” or “Perilla”, while those with leaves that are completely green are called “Yan” or “Bai Su”. Both can be consumed as vegetables, but there are still some differences in their usage. Perilla frutescens is widely used in the field of natural food coloring agents due to its high content of natural pigments. Referring to the description of the characteristics of Perilla frutescens leaves in the Chinese Pharmacopoeia (2020 edition) as “two sides purple or green on the upper surface, purple on the lower surface”, it can be inferred that Perilla frutescens is a plant source of authentic Perilla frutescens medicinal materials. White Su can be used not only as a vegetable, but also as a food preservative. In the 1970s, the “Flora of China” referred to the opinion of foreign taxonomist Elmer Drew Merrill and merged “Perilla frutescens” and “White Su” into one species, calling the original variant P Frutescens var. Frutescens. In the 1980s, Chinese medicine scholars suggested naming “Perilla frutescens” as P. frutescens based on their research and differences in efficacy between Perilla frutescens and Paeonia lactiflora frutescens var. arguta， The name of ‘Bai Su’ is P frutescens var. frutescens， And Liu et al. found through field observation and cultivation experiments that after sowing, all perilla seeds were still perilla, while after sowing, all leaves of white perilla seeds were green on both sides, and no variation was found. It can be seen that there is still some controversy in the classification of Perilla frutescens plants, and relevant scholars need to continuously search for reliable and universally applicable features as the basis for the classification of Perilla frutescens and Cymbidium.
Perilla frutescens (L.) Britt. is a dried leaf (or with tender branches) of Perilla frutescens, which has the effects of relieving external coldness, promoting qi circulation, and regulating the stomach. It is commonly used to treat wind cold, cold, cough, nausea, vomiting during pregnancy, and fish and crab poisoning. Since the Flora of China combines Perilla frutescens and Perilla frutescens into one species, the source plants of Perilla frutescens leaves should also include Perilla frutescens. However, there are significant differences between Perilla frutescens leaves and Perilla frutescens leaves in terms of appearance, chemical composition, and other aspects. Li et al. collected different germplasm of Perilla frutescens from various parts of China, observed and recorded their morphological characteristics after planting, and determined their chemical types using GC-MS technology. The results showed that the leaves of Perilla frutescens were mostly two-sided green and PK (purple ketone) type, while the leaves of Perilla frutescens were mostly two-sided purple and PA (purple aldehyde) type. There were significant differences in leaf size, leaf flatness, leaf edge and other morphological characteristics between the two; Dai et al. compared the content of phenolic acids in Perilla frutescens leaves and Perilla frutescens leaves using HPLC technology, and found that the content of protocatechuic aldehyde, caffeic acid, ferulic acid, and rosmarinic acid in Perilla frutescens leaves was higher than that in Perilla frutescens leaves; Shangguan et al. used visible spectrophotometry to compare the differences in total flavonoid content between Perilla frutescens leaves and Perilla frutescens leaves. The results showed that the total flavonoid content in Perilla frutescens leaves was higher than that in Perilla frutescens leaves; Huang et al. compared and analyzed the content of rosmarinic acid in perilla leaves and white perilla leaves using HPLC technology, and found that the content of rosmarinic acid in perilla leaves was higher than that in white perilla leaves during the same harvesting period. At present, research on the differences in chemical composition between Perilla frutescens leaves and Perilla frutescens leaves mainly focuses on volatile oils, flavonoids, phenolic acids, and other components. There is still a lack of research on the differences in the content of amino acids, organic acids, sugars, and other components between the two. It is necessary to analyze the differences between Perilla frutescens leaves and Perilla frutescens leaves more systematically and comprehensively.
This study aims to establish a pre column derivatization GC-MS analysis method for perilla leaves and white perilla leaves, and determine the content of amino acids, organic acids, sugars, fatty acids, phenolic acids, and other components in both; And compare the differences in chemical composition between the two using multivariate statistical analysis methods such as principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), and screen for differential biomarkers; By systematically studying the differences in chemical composition between Perilla frutescens leaves and Perilla frutescens leaves, certain references will be provided for the botanical classification of Perilla frutescens and the application of different germplasm resources of Perilla frutescens.

 

 

The current classification dispute between Perilla frutescens and Perilla frutescens has not been resolved. The Latin name of “Flora of China” combines Perilla frutescens and Perilla frutescens into one species, but the Chinese Pharmacopoeia explicitly states that the leaves are single-sided purple or double-sided purple, indicating that the medicinal Perilla frutescens varieties specified in the Pharmacopoeia do not include Perilla frutescens. For many years, research on the differences between perilla leaves and white perilla leaves has mainly focused on secondary metabolites. Currently, more than 200 chemicals have been isolated and identified in perilla leaves, mainly volatile oils (perilla aldehyde, perilla ketone, etc.), phenolic acids (rosmarinic acid, caffeic acid, etc.), and flavonoids (luteolin, apigenin, etc.). These components make significant contributions to the quality of perilla leaves. In fact, in addition to the secondary metabolites that have been extensively studied by previous researchers, there are also some highly polar primary metabolites in perilla leaves, such as sugars, amino acids, organic acids, etc., which also make important contributions to the quality of perilla leaves. Part of the reason for the limited research on these primary metabolites is the lack of comprehensive analytical methods. In order to compare the differences in the content of these components between perilla leaves and white perilla leaves, pre column derivatization GC-MS technology was used in this study. This technology modifies the target substance into a similar compound using chemical methods before GC-MS analysis of the sample, thereby reducing the polarity and boiling point of the target compound, enhancing the volatility and thermal stability of the substance, and making it more convenient for qualitative and quantitative analysis of non-volatile substances. The pre column derivatization method for perilla leaf samples established by our research institute has mild conditions, simple operation, good sample stability, and accurate results. It can be used as a rapid and high-throughput analysis and determination method for amino acids, sugars, fatty acids, phenolic acids, and other components in perilla leaves.

Perilla leaves are a traditional medicinal and edible plant. The qualitative and quantitative analysis results of various components in Perilla frutescens leaves and Cymbidium bungeanum leaves in this study showed that both contain six essential amino acids for human body (L-valine, L-leucine, L-isoleucine, L-threonine, L-phenylalanine, L-lysine), and their average content in Perilla frutescens leaves is higher than that in Cymbidium bungeanum leaves, indicating that in terms of amino acid level, the nutritional value of Perilla frutescens leaves is better than that of Cymbidium bungeanum leaves. At the same time, it was found that both perilla leaves and white perilla leaves contain high levels of rosmarinic acid. Rosmarinic acid is a naturally occurring phenolic acid with various activities such as anti-inflammatory, anticancer, antibacterial, antioxidant, and immunosuppressive. Comparative results show that the content of rosmarinic acid in perilla leaves is significantly higher than that in white perilla leaves, indicating that perilla leaves are a better source of medicinal plants. The content of tartaric acid, L-alanine, L-glycine, L-glutamic acid, palmitic acid, oleic acid, and other components in white perilla leaves is higher than that in perilla leaves, indicating certain edible and medicinal value. The above results indicate that there are significant differences in the primary metabolites of Perilla frutescens leaves and Perilla frutescens leaves. Separating Perilla frutescens from Perilla frutescens leaves in botanical classification is beneficial for the proper purification of Perilla frutescens medicinal materials. The above results are of great significance for the quality control of perilla leaves and the screening of high-quality perilla germplasm.