An in vivo and in vitro assessment of the anti-breast cancer activity of crude extract and fractions from Prunella vulgaris L

Abstract

Prunella vulgaris L.(P. vulgaris) is a perennial herb belonging to the Labiate family and widely distributed in China, Japan, Korea and Europe. Medical monographs and previous studies have shown that P. vulgaris has significant anti-breast cancer activity, and its use in breast treatment has a long history. However, systematically reports about the material basis and mechanism of P. vulgaris on anti-breast cancer activity are limited. In the present study, we first screened the best active fraction from the crude extract (PVE) and ethanol eluted fractions of P. vulgaris by using MDA-MB-231, MCF-7, 4T1 cell models in vitro and a 4T1-BALB/c transplanted tumour mouse breast cancer model in vivo. Furthermore, the anti-breast cancer mechanism of the best active fraction was investigated. The results demonstrated that PVE and ethanol fractions exhibited anti-breast cancer activity, especially with the 50% ethanol eluted fraction (PV50), which effectively regulated the 4T1 cell cycle, inhibited tumour cell proliferation, and promoted cancer cell apoptosis. In case of in vivo assays, PV50 inhibited tumour growth and lung metastasis, as well as inducing cell apoptosis by promoting damage of nuclear DNA and increasing expression of cleaved caspase-3. In addition, the chemical compositions of PV50 were analyzed by HPLC and UPLC-MS/MS, which were identified as flavonoids, moderately polar triterpenes, and a small amount of phenolic acid. The PV50 could be applied as natural sources against breast cancer in the pharmaceutical industry. These findings provide a basis for understanding the mechanism of the anti-breast cancer activity of P. vulgaris.

Keywords: Apoptosis; Breast cancer; Caspase; Herb; Prunella vulgaris L..

Figure 1

(A, C, E) The effects of PVE and fractions on the viability of MDA-MB-231, MCF-7 and 4T1 cells. (B, D, F) The intersections of each curve with the 50% dashed line correspond to the drug concentration (IC50) that yielded a survival rate of 50% (N = 3, x^– ± s; ∗ and ∗∗ represent p < 0.05 and p < 0.01, respectively, compared to the control group).

Figure 2

(A) Micro-CT scan of mouse tumours. (B) Visual diagram of isolated tumours in each group (n = 6–10). (C) Tumour weight (n = 6). (D) Tumour volume (n = 6). ∗p < 0.05, ∗∗p < 0.01 vs TC.

Figure 3

(A) Hematoxylin and eosin-stained sections of tumours treated with Tam and vary fractions (200×). (B) Hematoxylin-eosin staining of breast tissue in normal mice (200×). (C) Body weight of each group. (D) Spleen/weight ratio of each group. ∗p < 0.05, ∗∗p < 0.01 vs TC (n = 8–10). (E) Hematoxylin-eosin staining of spleen (200×). (F) Hematoxylin-eosin staining of lung (200×).

Figure 4

(A) The effects of different doses and different times on the proliferation activity of 4T1 cells (∗ and ∗∗ represent p < 0.05 and p < 0.01, respectively, compared to the control group). (B) The drug concentration with a survival rate of 50% (IC50) in 4T1 cells under different incubation times. (C) The effects of PV50 on 4T1 cell colony formation (∗ and ∗∗ represent p < 0.05 and p < 0.01, respectively, compared to the control group).

Figure 5

(A) Comparison of the morphological characteristics of 4T1 cell apoptosis induced by PV50 (Hoechst 33342 staining, 200×). (B) The effects on the apoptosis of PV50 (μg·mL⁻¹) on 4T1 cells. (C, D) The effects of PV50 on cell cycle in 4T1 cells (∗ and ∗∗ represent p < 0.05, p < 0.01, respectively, compared to the control group). (E, F) The effects of PV50 on scratch healing.

Figure 6

(A) Micro-CT scan of mouse tumours. (B) Visual diagram of isolated tumours in each group. (C) Tumour volume. ∗p < 0.05, ∗∗p < 0.01 vs TC (n = 8–10). (D) Tumour weight. ∗p < 0.05, ∗∗p < 0.01 vs TC (n = 8–10). (E) Effects of drugs on tumour pathological structure in tumour-bearing mice.

Figure 7

(A) The body weight of each group. (B) The ratio of spleen/body weight. Different marks within treatments indicate significant differences at ^#p < 0.05 and ^##p < 0.01 compared to the TC group (n = 10). (C) The ratio of heart/body weight. (D) The ratio of liver/body weight. (E) The ratio of kidney/body weight. (F) The ratio of lung/body weight. Different marks within treatments indicate significant differences at ∗p < 0.05 and ∗∗p < 0.01 compared to the NC group (n = 10). (G) Histopathological sections of mouse organs in each group (200×).

Figure 8

(A) The effects of drugs on tumour cell apoptosis. Green spots represent apoptotic bodies, and blue spots represent cell nuclei (400×). (B) The effects of drugs on the expression of cleaved caspase-3 in tumour tissue (400×). (C) Cleaved caspase 3 and corresponding frequencies of TUNEL positive cells expressed as AI. Different marks within treatments indicate significant differences at ∗p < 0.05 and ∗∗p < 0.01 compared to the TC group (n = 3). (D) Weight change trends in 10 acutely toxic experimental mice.