Content Determination Of Five Compounds in Semen Rehmanniae And Establishment Of Its HPLC Fingerprint

Table 3 Results of recovery test (n=6)

2.4 Sample determination

The six batches of Rehmannia collected were prepared according to the method under "2.2.2" and sampled and determined according to the chromatographic conditions under "2.1". The results of the content determination are shown in Table 4. The results show that among the five components, the content of rehmannia glycoside D is the highest, ranging from 0.460% to 0.730%; the content of leonuri glycoside is second, ranging from 0.100% to 0.270%, and the content of the two components in samples from different production areas is not significantly different. In sample SX1, except for echinacoside, the contents of the other four components are significantly higher than those of other batches of samples; the contents of echinacoside, cistancheside A, and verbascoside in Rehmannia glutinosa from different production areas are quite different and irregular.

CISTANCHE HERB WITH GOOD QUALITY CONSISTENCY

2.5 Establishment and similarity evaluation of HPLC fingerprint of Rehmannia glutinosa

The chromatograms and data of the multi-component content determination in Rehmannia glutinosa were imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software, and the chromatogram of the SX1 test sample was selected as the reference spectrum. The time window width was set to 0.10 min, and the median method was used to generate superimposed spectra and reference spectra. A total of 23 common peaks were identified (see Figure 3). The similarity between the fingerprints of the Rehmannia glutinosa sample and the reference was 0.904~0.950, indicating that the samples of Rehmannia glutinosa from different production areas and between raw Rehmannia glutinosa and cooked Rehmannia glutinosa were similar and had good quality consistency.

Table 4 Content determination results of 5 components in Semen Rehmanniae/%

13. rehmannioside D; 17. leonuride; 19. echinacoside; 20. castanoside A; 21. acteoside

Figure 3 Overlaying HPLC chromatograms of 6 batches of Semen Rehmanniae (SX1-SX4, HN1, HN2) and reference (R)

3 Discussion

3.1 Selection of chromatographic conditions

Four mobile phase systems, acetonitrile-water, acetonitrile-0.1% formic acid solution, methanol-water, and methanol-0.1% formic acid solution, were investigated in this study. The results showed that the five components had good separation effects in the acetonitrile system; compared with the acetonitrile-water mobile phase system, the peaks of rehmannia glutinosa and motherwort glycosides in the acetonitrile-0.1% formic acid solution mobile phase were narrower and had good peak symmetry. At the same time, due to the different ultraviolet absorption of the five components, the content of the components varies greatly. In order to more accurately determine the content of each component and to ensure that the absorption intensity of the five components is evenly distributed in the same chromatogram, the ultraviolet absorption of rehmannia glycoside D, motherwort glycoside, echinacoside, cistancheside A, and verbascoside was analyzed, and variable wavelength determination was selected, that is, 0-24 min, the first separated rehmannia glycoside was determined at 203 nm, and 24-40 min, the separated echinacoside, cistancheside A, and verbascoside were determined at 330 nm. In addition, this study also investigated the extraction effects of methanol, 70% methanol solution, 50% methanol solution, and 50% acetonitrile solution as extraction solvents. The results showed that 70% methanol solution as the extraction solvent can better dissolve the components to be tested, and the extracted sample has fewer impurities. Therefore, this study selected 70% methanol as the extraction solvent, acetonitrile-0.1% formic acid solution as the mobile phase system, and used variable wavelength to determine the contents of the five components in Rehmannia.

3.2 Analysis of sample determination results

From the determination results, it can be seen that after steaming, except for a slight increase in the content of echinacoside, the contents of the other four components were significantly reduced, especially the content of leonuriside and cistancheside A; the content of rehmannia glycoside D and cistancheside A in Rehmannia glutinosa from the same production area was slightly different, and the content of leonuriside, echinacoside and verbascoside was significantly different; the content of echinacoside in Rehmannia glutinosa from Henan production area was significantly higher than that of the samples from Shanxi production area, and the content of the other four components was slightly different. At the same time, according to the HPLC fingerprint established in this study, the similarity between the 6 batches of Rehmannia samples and the control fingerprint was 0.904~0.950, indicating that the main chemical components of the 6 batches of Rehmannia samples before and after processing were highly consistent.

In summary, the content determination method and HPLC fingerprint established in this study are accurate, reliable, and have good specificity. They can be used to characterize the changes in the components and the degree of processing of Rehmannia glutinosa before and after processing, and provide technical support for the quality control of Rehmannia glutinosa. However, this study has the disadvantages of small sample batches and incomplete coverage of production areas, which requires further analysis and verification.

References

[1] Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China: Volume 1 [S]. Beijing: China Medical Science and Technology Press, 2020: 129-130.

[2] Xu Jun, Fu Zhetong. Research on the historical evolution of the names of processed Rehmannia glutinosa and the study of its efficacy [J]. Chinese Patent Medicine, 2017, 39 (9): 1913-1916.

[3] Zhu Jue, Zhu Xiangmei, Shi Yuhe, et al. Research progress on Rehmannia glutinosa and prediction analysis of its quality markers [J]. Chinese Patent Medicine, 2022, 45 (2): 1273-1281.

[4] Chen Jinpeng, Zhang Kexia, Liu Yi, et al. Research progress on the chemical composition and pharmacological effects of Rehmannia glutinosa [J]. Chinese Herbal Medicine, 2022, 52 (6): 1772-1784.

[5] Li Hongwei, Meng Xiangle. Research progress on chemical components and pharmacological effects of Rehmannia glutinosa［J］.

Drug Evaluation Research, 2015, 38(2): 218-228.

[6] Zhang Boyong, Jiang Zhenzuo, Wang Yuefei, et al. Analysis of chemical components of fresh Rehmannia glutinosa, raw Rehmannia glutinosa and cooked Rehmannia glutinosa by UPLC/ESI-Q-TOF MS［J］. Chinese Patent Medicine, 2016, 38(5): 1104-1108.

[7] Zhang Lixian, Wei Yue, Li Feifei, et al. Fingerprint pattern recognition and multi-component content determination of different processed Rehmannia glutinosa products［J］. Hubei Agricultural Sciences, 2023, 62(9): 135-141.

[8] Chen Jinpeng, Zhang Kexia, Liu Yi, et al. Research progress on chemical components and pharmacological effects of Rehmannia glutinosa［J］. Chinese Herbal Medicine, 2021, 52(6): 1772-1784.

[9] Tao Yi, Jiang Yanhui, Tang Kejian, et al. Comparative study of chemical components of Rehmannia glutinosa before and after processing by UHPLC-Q-TOF/MS［J］. New Chinese Medicines and Clinical Pharmacology, 2016, 27(1): 102-106.

［10］ Xu Zhuo, Dai Xinxin, Su Shulan, et al. Dynamic accumulation of nucleoside and amino acid components in Rehmannia glutinosa based on UPLC-TQ-MS［J］. Chinese Herbal Medicine, 2021, 52(23): 7323-7330.

［11］ Li Yujiao. Study on the processing technology and mechanism of "nine steaming and nine exposure" of Rehmannia glutinosa［D］. Jinan: Shandong University, 2023.

［12］ Wang Yue, Zhou Qiao, Zhang Xuelan, et al. HPLC Simultaneous determination of the contents of eight components in Rehmannia glutinosa and its different processed products by UPLC fingerprint analysis and multi-index component content determination [J]. Chinese Medicinal Materials, 2021, 44 (7): 64-68.

[13] Guo Xiaokai, Xue Shujuan, Chen Suiqing. Quality evaluation of Rehmannia glutinosa based on UPLC fingerprint and multi-index component content determination [J]. Chinese Medicinal Materials, 2021, 44 (4): 917-923.