Evaluation of the quality of pomegranate leaves from different regions based on fingerprint spectra and chemical stoichiometry
Pomegranate leaves are the dried leaves of the plant Psidium guajava Linn. in the family Rosaceae, mainly distributed in Guangdong, Guangxi, Sichuan, Yunnan, Fujian, and Taiwan. They have the effects of drying dampness and strengthening the spleen, clearing heat and detoxifying, astringent intestines and stopping diarrhea, and anti-inflammatory and hemostatic effects. Modern research has shown that the main active ingredients in pomegranate leaves include flavonoids (guaiacol, isoquercitrin, hyperoside, naringin, naringin, naringenin, quercetin) and triterpenoids, which have hypoglycemic, antioxidant, and antibacterial effects.
At present, pomegranate leaves have not been included in the 2020 edition of the Chinese Pharmacopoeia, but are included in the Guangxi Chinese Herbal Medicine Standards (1990 edition), Guangdong Province Chinese Herbal Medicine Standards Volume 1 (2004 edition), Hunan Province Chinese Herbal Medicine Standards (2009 edition), Hunan Province Chinese Herbal Medicine Processing Standards (2010 edition), Guangxi Zhuang Medicine Quality Standards (Volume 2, 2011 edition), Fujian Province Chinese Herbal Medicine Processing Standards (2012 edition), and Sichuan Province Chinese Herbal Medicine Processing Standards (2015 edition), and are local standard medicinal materials. The above standards only qualitatively identified quercetin in pomegranate leaves through thin-layer chromatography. The research and analysis methods on the quality of pomegranate leaf medicinal materials mainly focus on the content of major components and multi-component quantification, which is difficult to comprehensively reflect the quality of medicinal materials. Meanwhile, as an important economic fruit tree, guava is abundant in resources, but its origin varies, making it difficult to control the quality of guava leaves. Overall, the quality standards and evaluation research of pomegranate leaves are not yet perfect and urgently need to be addressed.
Chinese medicine fingerprint is a comprehensive and quantifiable identification method for evaluating the authenticity, quality, and stability of Chinese medicinal materials and semi-finished Chinese medicine preparations. It is now widely used to evaluate the quality of Chinese medicinal materials. The research group established a multi evaluation method for the flavonoid components in pomegranate leaves in the early stage, providing a certain foundation for the quality control of pomegranate leaves. This study takes pomegranate leaves as the research object and collects 31 batches of pomegranate leaf samples from different origins for fingerprint analysis. Similarity evaluation, cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis are used to study the correlation between pomegranate leaf quality and origin, providing a reference for the quality evaluation and quality standard research of pomegranate leaves.

Referring to the previous research of the research group, the extraction method was determined to be 60% ethanol ultrasonic extraction for 30 minutes, with a flow system of acetonitrile-0.2% phosphoric acid water, a Zorbax SE-C18 column (250mm x 4.6mm, 5um), a Du temperature of 35 ℃, and an injection volume of 10uL. At the same time, the effects of wavelengths (230, 254, 360nm) and different ratios of gradient elution in the flow system on the HPLC fingerprint of pomegranate leaves were investigated. As a result, under the chromatographic conditions determined in this experiment, the pomegranate leaf sample solution had the highest number of peaks, and the HPC baseline had a stable peak shape and good separation.

This experiment collected guava leaf medicinal materials with regional representatives, including Guangdong, Guangxi, Fujian, Hainan, and Sichuan, ensuring the universality of guava leaf fingerprint spectra. Using similarity evaluation to analyze the HPLC fingerprint spectra of pomegranate leaves, it was found that there were certain differences in the similarity of the HPC fingerprint spectra of 31 batches of pomegranate leaves, and the quality of pomegranate leaves from different regions was affected by geographical factors.

The results of cluster analysis and principal component analysis were consistent, and it was found that the 31 batches of pomegranate leaf samples were mainly divided into two categories, with Guangxi, Guangdong, Fujian, and Hainan clustered together as one category, and Sichuan and Yunnan clustered together as one category. According to the geographical distribution map and administrative divisions of China, Sichuan and Yunnan are located on the second tier (with an average elevation of 1000-2000m) of the boundary between the Greater Khingan Range, Taihang Mountains, Wushan Mountains, and Xuefeng Mountains, belonging to the southwestern region; Guangxi, Guangdong, Fujian, and Hainan are located on the third tier (with an average elevation of less than 500m) bounded by the Greater Khingan Range, Taihang Mountains, Wushan, and Xuefeng Mountains, and belong to coastal areas. Using orthogonal partial least squares discriminant analysis, seven main components causing differences in pomegranate leaves between these two types of production areas were identified, and five components were identified: tannic acid, hyperoside, isoquercitrin, naringin, and guava glycoside. Through quantitative analysis of tannic acid, hyperoside, isoquercetin renoside, and guava glycoside in guava leaf samples, it was found that the content of the five components in coastal areas (Guangdong Guangxi, Fujian Hainan) was lower than that in southwestern areas (Sichuan, Yunnan), and there was a significant difference (see Figure 7). In summary, there are significant regional differences in the secondary metabolites of pomegranate leaves, indicating that the quality of pomegranate leaves is closely related to their growth environment and may be affected by local climate conditions and environmental factors. Further investigation and research are needed to determine the specific influencing factors.

This study focuses on pomegranate leaves and uses high-performance liquid chromatography to establish a chemical fingerprint of pomegranate leaf medicinal materials. The content of flower acid, golden peach bark, and quinolones in pomegranate leaf medicinal materials was determined, and 31 batches of pomegranate leaf medicinal materials from different regions were analyzed using a chemical calculation method. It was found that the quality of pomegranate leaf medicinal materials is affected by regional factors. The established method is simple and accurate, providing a method and data reference for the quality control and establishment of quality standards for pomegranate leaf medicinal materials.