Comparative study on UPLC characteristic spectra and multi index component contents of different specifications of Qingpi medicinal materials
Qingpi was first recorded in the book “Pearl Sac”. Li Shizhen said, “Qingju peel was useless in ancient times, but it was not until the Song Dynasty that medical experts began to use it.” It was not until the Song Dynasty that immature and mature orange peel were compared and found to have slightly different medicinal effects, hence the distinction between Qingju peel and Huangju peel. Qingpi refers to the dried or unripe fruit skin of Citrus reticulata Blanco and its cultivated varieties in the Rutaceae family. There are two different specifications for Qingpi used in medicine: collecting young fruits that fall from the ground between May and June, drying them in the sun, and commonly known as “Geqingpi”; Harvesting immature fruits from July to August, longitudinally cutting them into four petals from the skin to the base, removing all the petals, and drying them in the sun, commonly known as “Four Flower Green Skin”. Green skin is warm in nature, with a bitter and pungent taste. Return to the liver, gallbladder, and stomach meridians. It has the effects of soothing the liver and breaking down qi, reducing accumulation and resolving stagnation. It is used for chest and rib pain, hernia pain, breast fetish, breast abscess, food accumulation and qi stagnation, and abdominal distension and pain. The main components of Qingpi are volatile oils, flavonoids, amino acids, etc. Modern pharmacological studies have shown that Qingpi has the effects of regulating the gastrointestinal tract, promoting bile flow, anti arrhythmic effects, expectorant effects, and treating mastitis. The two specifications of green skin have slightly different functional indications. One type of green skin mainly focuses on breaking qi stagnation, while the other type focuses on regulating liver and regulating qi.
In addition, the two also have slight differences in their composition. The 2020 edition of the Chinese Pharmacopoeia has made different regulations on the characteristics and identification of different specifications of green peel, and the content determination items only have limit requirements for hesperidin. The research results of Li et al. showed that the content of hesperidin and sinomenine in Geqingpi was higher than that in Sihuaqingpi, while the content of N-methyl amine and volatile oil was lower. The trend of high and low content of the four components in two specifications from different regions was basically the same. Zhao et al. used GC-MS to analyze the volatile oils of Eucommia ulmoides and Eucommia ulmoides, and found that nine compounds, including geraniol, nerolidol, and α – isoterpinene, were unique to Eucommia ulmoides. The main components, limonene and linalool, were more than twice that of Eucommia ulmoides, while the content of other components varied greatly. Yi et al. also pointed out that there are certain differences in the composition of Geqingpi and Sihuaqingpi by using gas chromatography and liquid chromatography to study their components. The above studies all indicate that there are differences in the chemical composition between Geqingpi and Sihuaqingpi.
This study established ultra high performance liquid chromatography (UPLC) characteristic spectra of Qingpi and Sihua Qingpi, combined with chemometric methods, to compare the differences between the two specifications of Qingpi and compare the content of five effective ingredients in the two specifications of Qingpi medicinal materials, providing reference for improving the quantity standards of Qingpi in the two specifications.

 

Preparation of test solution. This study used single factor analysis to evaluate the effects of extraction solvents (methanol, 70% methanol, 50% methanol, ethanol, 70% ethanol, 50% ethanol), extraction methods (ultrasonic extraction, heating reflux), and extraction times (15, 30, 45 minutes) on the characteristic spectra and content determination of each component, with the chromatographic peak “total peak area/sample size” and the contents of sinomenin, hesperidin, naringin, and hesperidin as evaluation indicators. The results showed that heating reflux with 70% methanol for 30 minutes had the best effect.
Establishment and analysis of feature maps. The results of the feature map study showed that there were 7 common peaks in the UPLC feature maps of both Geqingpi and Sihuaqingpi. Five characteristic components of Geqingpi and Sihuaqingpi were identified through reference standards, namely sinomenin, naringin, hesperidin, naringin, and hesperidin. We investigated different aqueous phases (0.1% phosphoric acid and 0.1% formic acid), two different types of chromatographic columns Waters ACQUITY HSS T3 (100mm × 2.1mm, 1.8 μ m), YMC Triart C18 (100mm × 2.1mm, 1.9 μ m), and compared the effects of different detection wavelengths (275nm and 284nm), column temperatures (38, 40, 42 ℃), and flow rates (0.28, 0.30, 0.32mL/min) on the chromatographic peaks of Qingpi characteristic spectra. The results showed that using Waters ACQUITY HSS T3 (100mm × 2.1 mm, 1.8 μ m) chromatographic column with 0.1% formic acid as the aqueous phase, under the chromatographic conditions of 275nm detection wavelength, column temperature of 40 ℃, and flow rate of 0.30mL/min, the chromatographic peaks were obtained. The separation degree is good, the baseline is stable, the number of chromatographic peaks is high, and the signal is correspondingly high.
The difference in chemical composition between Geqingpi and Sihuaqingpi. The UPLC characteristic spectra of different specifications of Qingpi medicinal materials established in this study identified 7 common peaks in both Geqingpi and Sihuaqingpi, of which 5 characteristic peaks were identified. Peak 1 was determined to be sinomenine; Peak 3 is Yunxiang pomelo peel glycoside; Peak 4 is hesperidin; Peak 6 is Chenpi extract; Peak 7 is orange peel extract. In this study, HCA, PCA, and OPLS-DA were used to classify different specifications of green peel medicinal materials into two categories; Further analysis reveals that the chemical components represented by peaks 1, 4, and 2 are the differential components between these two specifications of Qingpi medicinal herbs. Therefore, these three components can be used as entry points for further analysis to distinguish between these two specifications of Qingpi medicinal herbs.
Flavonoids are the main active ingredients in Qingpi. The content determination results of five known flavonoids in Geqingpi and Sihuaqingpi showed that the content of sinomenin and hesperidin in Geqingpi was higher than that in Sihuaqingpi, while the content of naringin and hesperidin in Geqingpi was lower than that in Sihuaqingpi. Overall, the content of naringin in the two specifications of Qingpi Yunxiangyupi was relatively similar. Independent sample t-test analysis showed that the differences in sinomenin, hesperidin, and naringin were statistically significant. This result is consistent with the research findings of Li et al. In addition to the flavonoids studied in this experiment, volatile oil is also one of the effective components of Qingpi medicinal herbs. Zhao’s research on the volatile oils of Geqingpi and Sihuaqingpi showed that nine compounds, including geraniol, nerolidol, and α – isoterpinene, are unique to Geqingpi. The main components, limonene and linalool, are more than twice that of Sihuaqingpi. In clinical application, the functional indications of Four Flower Green Skin are milder than those of Geqing Skin, which may be related to the lower content of active ingredients such as hesperidin and sinomenin, as well as volatile oil in Four Flower Green Skin compared to Geqing Skin. According to relevant literature, Geqingpi is a young fruit collected from May to June; Four flowered green skin is harvested from immature fruits in July to August, and the difference between the two lies in the harvesting time and maturity stage. From the above results, it can be seen that different growth and development stages can affect the accumulation of active ingredients in Qingpi. However, during the reproductive and developmental period, the active ingredients in Qingpi do not simply show an increasing or decreasing trend, but rather some increase and some decrease: Chenpi extract and hesperidin show an increasing trend; Simulin, hesperidin, and naringin showed a decreasing trend. Whether the differences in these contents directly or indirectly lead to different efficacy focuses of different specifications of Qingpi requires further pharmacological experiments to prove. Based on the difference in the content of the five active ingredients in two different specifications of Qingpi, combined with the different emphasis of the two effects, we can better implement the traditional Chinese medicine syndrome differentiation and treatment, and improve the efficiency of treatment if the prescription is separately labeled for use in clinical applications. Meanwhile, if the current pharmacopoeia standards can establish separate quality standards for Geqingpi and Sihuaqingpi, it can be more conducive to the quality control of Qingpi medicinal materials.