Variation of Saponins in Sanguisorba officinalis L. before and after Processing (Paozhi) and Its Effects on Colon Cancer Cells In Vitro

Abstract

The incidence of colon cancer is increasing year over year, seriously affecting human health and quality of life in recent years. However, traditional Chinese medicine (TCM) has been utilized for the treatment of colon cancer. S. officinalis Saponins (S-Saponins), the potential compound of TCM, displays multiple biological activities in colon cancer treatment. In our study, ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) combined with multivariate statistical analysis were performed to analyze and identify raw and processed saponins. Then, MTT and cell migration assays were used to preliminarily explore the effects of saponins in vitro on colon cancer cells. The results showed that 29 differential saponins compounds under Paozhi were identified by UHPLC-MS/MS. Moreover, in vitro validation showed that Sprocessed better inhibited the proliferation and migration of colon cancer cells than Sraw. This study provides a basis for the determination of the chemical fundamentals of the efficacy changes during Paozhi through inferring the changes in saponin components and its possible transformation mechanisms before and after processing S. officinalis. Meanwhile, it also provides new insights into potential bioactive ingredients for the treatment of colon cancer.

Keywords: UHPLC-MS/MS; activity study; content determination; potential ingredients; qualitative analysis; traditional Chinese medicine.

Figure 1

Negative-ion chromatogram of Sanguisorba officinalis L. before (Sraw) and after (Sprocessed) processing.

Figure 2

Positive-ion chromatogram of Sanguisorba officinalis L. before (Sraw) and after (Sprocessed) processing.

Figure 3

Mass spectrum and structure of disappeared components in Sanguisorba officinalis L. after processing. (A): 3β, l9α-dihydroxyursin-12-en-28-acid-β-D-glucose ester; (B): 2α,3α,19α-trihydroxyurs-12-en-28-acid-β-D-glucopyranosyl ester or isomer; (C): 2α,3α,19α-trihydroxyurs-12-en-28-oic-acid.

Figure 4

Mass spectrum and structure of new components in Sanguisorba officinalis L. after processing. (A): 2α,3,19-Trihydroxyurs-12-en-28-acid-β-D-glucopyranosyl ester or isomer; (B): 2α,3,19-Trihydroxyurs-12-en-28-acid-β-D-glucopyranosyl ester or isomer; (C): 3,11-dioxo-19α-hydroxy-urs-12-en-28-oic acid; (D): 3β-O-α-L-arabinopyranosylusr-12,18-dien-28-acid; (E): 2α,3β-dihydroxy-28-norurs-12,17,19(20),21-tetraen-23-oic acid; (F): ursolic acid; (G): 3,11-dioxo-19α-hydroxy-urs-12-en-28-acid; (H): 3-oxo-12-en-28-ursolic acid or isomer; (I): 3-oxo-12-en-28-ursolic acid or isomer.

Figure 5

Content change of chemical components in Sanguisorba officinalis L. before and after processing.

Figure 6

Cluster heat map of Sanguisorba officinalis L. before and after processing.

Figure 7

The possible transformation process in Sanguisorba officinalis L. under processing.

Figure 8

Multivariate statistical analysis score graph. (A): PCA scores of raw and processed Sanguisorba officinalis L. (2D, 3D); (B): The OPLS-DA model and VIP values of differential components in Sanguisorba officinalis L. before and after processing.

Figure 9

Sraw and Sprocessed inhibited the proliferation of human colon cancer cells. (A): Inhibition of the proliferation of HCT 116 colon cancer cells by different concentrations of Sraw and Sprocessed; (B): Inhibition of the proliferation of RKO colon cancer cells by different concentrations of Sraw and Sprocessed. Data are expressed as mean ± SEM (n = 6); “*” indicates the difference between one group and the control group, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0.0 μg/mL.

Figure 10

Wound healing assays in HCT116 cells showing inhibition by Sraw and Sprocessed (scratch analysis). (A): 0 μg/mL; (B): 80 μg/mL; (C): 160 μg/mL; (D): 320 μg/mL.

Figure 11

Wound healing assays in RKO cells showing inhibition by Sraw and Sprocessed (scratch analysis). (A): 0 μg/mL; (B): 80 μg/mL; (C): 160 μg/mL; (D): 320 μg/mL.