UPLC-Q-XActive Orbitrap high-resolution mass spectrometry quantitative analysis of the content of 9 components in Ligustrum lucidum from different origins and before and after processing
Ligustrum Lucidum Ait. is a dried and mature fruit of Ligustrum Lucidum Ait, a plant in the Oleaceae family. It has a sweet, bitter, and cool taste and enters the liver and kidney meridians. It has the effects of nourishing the liver and kidneys, improving vision, and blackening hair; It can be used for liver and kidney yin deficiency, dizziness and tinnitus, soreness and weakness of the waist and knees, premature graying of hair, unclear vision, internal heat quenching, bone steaming and hot flashes, etc. Fructus Ligustri Lucidi contains a variety of active ingredients, such as triterpenoids, iridoids, phenylethanol, flavonoids, volatile oils, sugars, amino acids, sterols, etc. It has a variety of pharmacological effects, such as anti diabetes, anti-tumor, liver protection, antioxidant, anti-inflammatory, hypoglycemic, lipid-lowering, immune regulation, etc. It is mainly used in clinical treatment of hyperlipidemia, osteoporosis, colon cancer, lung cancer, hepatitis and other diseases. The main active ingredients in Ligustrum lucidum are iridoid glycosides, but the efficacy of Ligustrum lucidum is not determined by the content of individual components. The components such as phenylethanol, flavonoids, and iridoids in Ligustrum lucidum have also been used for quality evaluation due to their antioxidant and anti osteoporosis activities, but they are still not perfect. Only by analyzing the content of compound groups related to the efficacy of Ligustrum lucidum can the quality of Ligustrum lucidum medicinal materials be truly evaluated. Therefore, this study established a method for simultaneously determining the content of multiple active ingredients in Ligustrum lucidum, including phenylethanol (salidroside, echinacoside), flavonoids (quercetin, apigenin, rutin, verbascoside, verbascoside), and cyclohexene ether terpenes (oleuropein, astragaloside), to evaluate the quality of Ligustrum lucidum.

The processing method of Ligustrum lucidum first appeared in the Song Dynasty’s book “Su Yang”. The traditional processed products of Ligustrum lucidum are still used today, including raw and alcoholic products. In modern times, purified products, steamed products, salt products, vinegar products, etc. are also added. Raw and alcoholic products are commonly used in clinical practice. Ligustrum lucidum raw products clear the liver and clear the eyes, nourish yin and moisten dryness, and the nourishing effect on the liver and kidneys is significantly enhanced after alcohol production. Research has shown that the enhanced liver and kidney tonifying effects of Ligustrum lucidum wine are closely related to the hydrolysis and transformation of iridoid glycosides in Ligustrum lucidum during processing. After processing, the content of iridoid glycosides and phenylethanolic glycosides in Ligustrum lucidum decreases, while the content of salidroside and phenylethanolic glycosides increases. However, these studies are relatively scattered and lack systematicity, which is insufficient to scientifically evaluate the changes in chemical composition before and after the processing of Fructus Zhenzi. In view of this, this study used UPLC-Q Exactive-Orbitrap high-resolution mass spectrometry to establish a simultaneous determination of the contents of salidroside, rutin, verbascoside, ligustilide, oleuropein, luteolin, apigenin, quercetin, and pineal acid in Ligustrum lucidum. The distribution pattern of chemical components in Ligustrum lucidum from different regions was analyzed, and the changes in chemical component content before and after processing were examined, in order to provide theoretical basis for standardizing the quality standards of Ligustrum lucidum decoction pieces and processed products.

Analysis of the content determination results of Ligustrum lucidum from different origins. This study established an LC-MS method to determine the content of 9 components in Ligustrum lucidum from different origins. The 2020 edition of the Chinese Pharmacopoeia stipulates that the limit of ligustilide should not be less than 0.70%. Among the 22 batches of Ligustrum lucidum medicinal materials tested from different origins, the content of ligustilide in Ligustrum lucidum medicinal materials from three origins (Xinxiang, Henan, Baoding, Hebei, and Huixian, Henan) was lower than the limit of the 2020 edition of the Chinese Pharmacopoeia, accounting for 13.64%. The reasons for this need further research. In different production areas, the content of salidroside and paeoniflorin is relatively high in Zhengzhou, Henan, Dangyang, Hubei, Guangyuan, Sichuan, Yangling, Shaanxi, Nanyang, Henan, Changsha, Hunan, and Changde, Hunan, with concentrations ranging from 0.9862 to 1.5440 mg/g and 12.8500 to 18.1066 mg/g, respectively. After processing Ligustrum lucidum, the content of Ligustrum lucidum glycosides significantly increased, all of which were higher than the content limit of the 2020 edition of the Chinese Pharmacopoeia. Using SIMCA14.1 software, principal component analysis was conducted on Ligustrum lucidum from different origins and processed Ligustrum lucidum from different origins. Ligustrum lucidum from different origins and processed Ligustrum lucidum from different origins can be divided into two categories: Category I includes S2 S4、S6、S8、S13、S14、S15；Z2、Z4、26、Z8、Z13、Z14、Z15； Class II includes S1 S3、S5、S7、S9、S10、S11、S12、S16~S22；Z1、Z3、Z5、Z7、Z9、Z10、Z11、Z12、Z16~Z22。 According to the VIP plot in partial least squares discriminant analysis, it can be seen that the contribution of ligustilide, verbascoside, and salidroside to the division of production areas is significant. The analysis results indicate that there are significant differences in the quality of Ligustrum lucidum medicinal materials from different production areas, which may be related to differences in the growth environment of the medicinal materials. Simply measuring the content of one or two components cannot effectively reflect the quality of the medicinal materials. It is necessary to simultaneously measure multiple components of Ligustrum lucidum in order to comprehensively reflect its intrinsic quality.

Analysis of the content determination results of Ligustrum lucidum before and after processing. There are various methods for processing Ligustrum lucidum, among which the most commonly used method is alcohol fermentation, which includes alcohol stewing and alcohol steaming. This study used the wine stewing method to preliminarily explore the changes in component content of Ligustrum lucidum during the processing. After stewing Ligustrum lucidum with alcohol, the content of salidroside, astragaloside, and oleuropein in Ligustrum lucidum increased significantly. Perhaps due to changes in the organizational structure or physicochemical properties of the components after alcohol production, it is beneficial for the infiltration, dissolution, displacement, diffusion and other dissolution processes of the components, resulting in a solubilizing effect and improving the dissolution rate of effective ingredients. The specific influencing factors need further investigation. Osmanthus glycosides, luteolin, and rutin have made significant contributions to the classification of Ligustrum lucidum before and after processing. Studies have shown that after processing Ligustrum lucidum, specifically Ligustrum lucidum glycosides are hydrolyzed into salidroside, and the content of salidroside in processed Ligustrum lucidum is significantly increased. This study established a method for determining the content of nine components, including salidroside, rutin, verbascoside, rutinoside, oleuropein, luteolin, apigenin, quercetin, and pineal glycoside, in Ligustrum lucidum from different origins and before and after processing using ultra-high performance liquid chromatography-mass spectrometry. Compared with UV detection, this research method has strong separation ability, high sensitivity, reliable qualitative analysis results, and low detection limit. The significant differences in the quality of Ligustrum lucidum medicinal materials from different origins may be related to factors such as region, climate, soil, growth period, and harvesting time. Specifically, Ligustrum lucidum glycoside, verbascoside, and salidroside contribute significantly to the division of origins. After the production of Ligustrum lucidum liquor, the content of salidroside, ligustilide, and oleuropein significantly increased, which may be due to the increased dissolution rate of active ingredients in the liquor production. The specific reasons need to be further investigated. This experimental method is simple, reliable, and highly sensitive, providing a reference for the improvement and enhancement of quality standards for raw and processed products of Ligustrum lucidum.