Quality analysis of roses based on UPLC fingerprint and determination of two chemical components
Rose flower is the dried flower of Rosa chinensis Jacq., a plant in the Rosaceae family. It is also known as rose red, victorious snow, slender guest, fighting snow red, four season flower, precious moon flower, and bright snowflake, and is known as the queen of flowers. Rose flowers were first recorded in the Compendium of Materia Medica. They have a sweet taste, a warm nature, and belong to the liver meridian. They have the effects of promoting blood circulation, regulating menstruation, soothing the liver, and relieving depression. They can be used to treat menstrual disorders, dysmenorrhea, and amenorrhea; Blood stasis, swelling and pain, burns, injuries from falls, abscesses, and scrofula. It is planted nationwide, mainly in Yunnan, Hubei, Sichuan, and Gansu, and can be harvested throughout the year. The chemical composition of roses is rich and diverse, including flavonoids, tannins, phenolic acids, volatile oils, pigments, and glycoside compounds.
In recent years, research on roses has made certain progress. Wu et al. used UPLC-Q-Tof-MS technology to study the differences in non-volatile chemical components between roses and roses. The results showed that 43 compounds were detected in roses and 25 compounds were detected in roses. However, the main components detected in roses were phenolic glycosides, tannins, and phospholipids, and the only flavonoid component detected was kaempferol glucoside. According to literature research reports, flavonoids are the main active components of rose flowers. Shangguan et al. studied rose flowers at different harvesting times, measured the total flavonoid content of rose flowers at different harvesting periods, and evaluated their antioxidant activity based on their ability to scavenge DPPH and hydroxyl radicals. They explored the correlation between total flavonoid content and antioxidant activity, but did not study the total flavonoids of rose flowers from different origins. At present, there are many studies on roses, but there are few reports on the quality standards of roses. Modern pharmacological research has found that hyperoside and isoquercitrin have antioxidant stress and cell apoptosis, anti acute liver injury, anti-inflammatory, antidepressant, vascular protection and other effects, which are consistent with the effect of soothing the liver and relieving depression in roses. This indicates that hyperoside and isoquercitrin are one of the main active ingredients in roses. Under the content determination of rose flowers in the 2020 edition of the Chinese Pharmacopoeia, only the isocratic elution method is used to limit the total amount of hyperoside and isoquercitrin. Under this chromatographic condition, only the information of hyperoside and isoquercitrin is reflected, but it cannot fully reflect the quality of rose flowers. The information of other characteristic components such as flavonoids and phenolic acids cannot be comprehensively reflected. Fingerprint spectrum can comprehensively reflect the chemical components contained in traditional Chinese medicine, and has become an important means of comprehensive quality control of traditional Chinese medicine. Based on this, this experiment established a UPLC fingerprint method for rose flowers, and simultaneously measured the total amount of its flavonoid components, hyperoside and isoquercitrin, under the conditions of fingerprint, in order to achieve qualitative and quantitative control of rose flowers solely through fingerprint methods. Cluster analysis and principal component analysis were combined to evaluate rose flowers from different origins, laying the foundation for quality control research of rose flowers. In addition, this method is also applicable to the quality standards of roses in the same family and genus, and can be applied to the identification research of roses and roses.

 

Determination of preparation method and chromatographic conditions for the test sample. This experiment investigated the extraction solvent, extraction method, extraction time, and solid-liquid ratio. The chromatographic method was used to maximize the information content, achieve the best extraction efficiency, and ensure the stability of the method. Finally, 50% methanol was used as the extraction solvent, followed by 30 minutes of ultrasonic treatment and a solid-liquid ratio of 1.0g: 50mL as the sample preparation method. In terms of chromatographic conditions, different chromatographic columns, mobile phase systems, column temperatures, flow rates, and detection wavelengths were investigated. After comparison, it was found that selecting Agilent SB C18 as the chromatographic column, acetonitrile 0.2% phosphoric acid as the mobile phase, column temperature of 35 ℃, flow rate of 0.4mL/min, the peak shape and separation degree of the chromatographic peaks were good; The test solution was scanned at full wavelength under UV visible light at 190-400nm, and the results showed that the common peaks of the fingerprint spectrum at 254nm had high response values and good peak shapes. Moreover, the maximum absorption of hyperoside and isoquercitrin was observed at 254nm, so 254nm was selected as the detection wavelength.
Analysis of fingerprint spectrum and content determination results. This study used the “Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation Software” (2012 version) to conduct similarity analysis on the fingerprint spectra of 15 batches of rose flowers. A total of 30 common peaks were detected, and the similarity of the fingerprint spectra of the 15 batches of samples was greater than 0.95, indicating consistency in the chemical composition of rose flowers from different batches. A total of 10 characteristic peaks were identified through comparison with reference standards, of which 8 were flavonoids. Using cluster analysis and principal component analysis to analyze the fingerprint spectra of roses from different origins, when the clustering distance is 10, roses from different batches can be divided into 5 categories, indicating that roses from different origins have certain differences. PCA analysis shows that there are certain differences in the quality of roses from different regions. By measuring the content of hyperoside and isoquercitrin in 15 batches of rose flowers, the total content of hyperoside and isoquercitrin was 3.88-8.67mg/g, which was higher than the pharmacopoeia value of 0.38%. This indicates that all 15 batches of rose flowers are qualified medicinal materials, with the highest total content of hyperoside and isoquercitrin in Zhoukou City, Henan Province.
The established fingerprint method can be applied to the quality evaluation of roses. The fingerprint spectra of 15 batches of rose medicinal materials were analyzed using the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software” (2012 version), and a total of 22 common peaks were identified. The similarity of the fingerprint spectra of 15 batches of rose medicinal materials was greater than 0.95. Rose and rose are both members of the Rosaceae family and can be distinguished in terms of their morphological, microscopic, and physicochemical characteristics. However, there have been no reports on fingerprint identification. The UPLC fingerprint established in this study can be used for the identification of rose and rose. From the comparison of fingerprint spectra, it can be seen that the two have 15 common components, with rose having 10 exclusive components and rose having 5 exclusive components.
On the basis of establishing the UPLC fingerprint of rose flowers, this study identified multiple flavonoid components and evaluated them using chemical pattern recognition method; Establish a method for determining the content of hyperoside and isoquercitrin in roses, providing a basis for quality control of roses. This method is simple, accurate, and can provide reference for subsequent research on roses.