Tissue-Specific Metabolite Profiling on the Different Parts of Bolting and Unbolting Peucedanum praeruptorum Dunn (Qianhu) by Laser Microdissection Combined with UPLC-Q/TOF⁻MS and HPLC⁻DAD

Abstract

Background: Qianhu is a traditional Chinese medicine. It is thought that Qianhu roots will harden after bolting and not be suitable for medicinal purposes. Bolting Qianhu and unbolting Qianhu are referred to as "Xiong Qianhu" and "Ci Qianhu," respectively. In this study, the properties, microscopic and chemical characteristics of Ci Qianhu and Xiong Qianhu roots were compared using fluorescence microscopy, laser microdissection coupled with ultra-high-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry, and high-performance liquid chromatography with diode-array detection.

Results: Microscopy results showed that the area of secondary xylem in the root increased after bolting, with the cork and secretory canals showing strong fluorescence intensity. A total of 34 peaks, mostly pyranocoumarins, were identified in the tissues of Ci Qianhu and Xiong Qianhu. The secretory canals contained the highest variability of coumarins, whereas the secondary xylem contained the least coumarins. Moreover, seven coumarins, especially the pyran- coumarin, decreased after bolting. Generally, both before and after bolting, coumarin level was the highest in the bark, followed by the middle part, and the lowest in the inner part.

Conclusion: Thus, it was indicated that the area of secondary xylem increased after bolting, however the coumarin variant and content decreased in the secondary xylem of Qianhu. The result shows that the quality of Qianhu decreases after bolting, which supports the viewpoint that Xiong Qianhu is not suitable for medicinal use.

Keywords: HPLC–DAD; LMD; Peucedanum praeruptorum Dunn; UPLC-Q/TOF–MS; bolting; microstructure.

Figure 1

Macroscopic and microscopic morphology of Qianhu. (A: Sample1; B: Sample2) (A1) the Raw plants of Ci Qianhu;(A2) the medicinal materials of Ci Qianhu; (A3) the cross-section stained with phloroglucinol and concentrated hydrochloric acid of Ci Qianhu; (A4) observed under a normal light microscope of Ci Qianhu; (A5) observed under polarized light of Ci Qianhu; (B1) the Raw plants of Xiong Qianhu; (B2) the medicinal materials of Xiong Qianhu; (B3) the cross-section stained with phloroglucinol and concentrated hydrochloric acid of Xiong Qianhu; (B4) observed under a normal light microscope of Xiong Qianhu; (B5) observed under polarized light of Xiong Qianhu. CK, cork; Ph, phloem; SC, secretory canals; Cam, cambium; XV, xylem vessels; XR, xylem ray.

Figure 2

Microscopic characteristics of different parts of Qianhu. (A: Sample 8; B: Sample 4) (A1) observed under normal light mode of Ci Qianhu; (A2) observed under fluorescence mode of Ci oianhu (excitation wavelength: mixed 450–490 nm and 460–500 nm); (A3) observed under fluorescence mode of Ci Qianhu (equipped with triple band filter RGB); (B1) observed under normal light mode of Xiong Qianhu. (B2) observed under fluorescence mode of Xiong Qianhu (excitation wavelength: mixed 450–490 nm and 460–500 nm); (B3) observed under fluorescence mode of Xiong Qianhu (equipped with triple band filter RGB).

Figure 3

LC–MS base peak chromatograms of microdissected tissues from Ci Qianhu (Sample 8). The peak numbers referred to Table 1. SC1, secretory canals 1; SC2, secretory canals 2; SC3, secretory canals 3; SC4, secretory canals 4; Cam, cambium; Ck, cork; Ph1, secondary phloem1; Ph2, secondary phloem2; Ph3, secondary phloem3; Xr, xylem ray; Xvp, xylem vessels and parenchyma cells.

Figure 4

LC–MS base peak chromatograms of microdissected tissues from Xiong Qianhu (Sample 9). The peak numbers referred to Table 1. SC1, secretory canals 1; SC2, secretory canals 2; Cam, cambium; CK, cork; Ph, secondary phloem; XR, xylem ray; Xvp, xylem vessels and parenchyma cells.

Figure 5

Peaks identification: (A) Liquid chromatogram of reference substance, (B) Liquid chromatogram of sample. (1) peucedanol; (2) xanthotoxin; (3) bergapten; (4) imperatorin; (5) praeruptorin A; (6) praeruptorin B; (7) praeruptorin E.