Antitumor and antioxidant effects of Clinacanthus nutans Lindau in 4 T1 tumor-bearing mice

Abstract

Background: Clinacanthus nutans Lindau (C. nutans) is a species of in Acanthaceae family and primarily used in South East Asian countries. C. nutans is well known as Sabah snake grass in Malaysia, and its leaves have diverse medicinal potential in conventional applications, including cancer treatments. On the basis of literature search, there is less conclusive evidence of the involvement of phytochemical constituents in breast cancer, in particular, animal tumor models. The current study aimed to determine the antitumor and antioxidant activities of C. nutans extract in 4 T1 tumor-bearing mice.

Methods: C. nutans leaves were subjected to methanol extraction and divided into two different concentrations, 200 mg/kg (low-dose) and 1000 mg/kg (high-dose). The antitumor effects of C. nutans extracts were assessed using bone marrow smearing, clonogenic, and splenocyte immunotype analyses. In addition, hematoxylin and eosin, tumor weight and tumor volume profiles also used to indicate apoptosis appearance. Serum cytokine levels were examined using ELISA assay. In addition, nitric oxide assay reflecting antioxidant activity was performed.

Results: From the results obtained, the methanol extract of C. nutans leaves at 200 mg/kg (P < 0.05) and 1000 mg/kg (P < 0.05) showed a significant decrease in nitric oxide (NO) and malondialdehyde (MDA) levels in the blood. On the other hand, C. nutans extract (1000 mg/kg) also showed a significant decrease in the number of mitotic cells, tumor weight, and tumor volume. No inflammatory and adverse reactions related to splenocytes activities were found in all treated groups of mice. Despite its promising results, the concentration of both C. nutans extracts have also reduced the number of colonies formed in the liver and lungs.

Conclusion: In conclusion, C. nutans extracts exert antitumor and antioxidant activities against 4 T1 mouse breast model with no adverse effect and inflammatory response at high dose of 1000 mg/kg, indicating an effective and complementary approach for cancer prevention and treatment.

Keywords: Antioxidant; Antitumor; Breast tumor; Clinacanthus nutans Lindau.

Fig. 1

The weight and volume of tumors from untreated and C. nutans–treated groups. a Weight of tumors was measured after being harvested on 28 days of post-treatment. b Volume of tumors was measured using a Vernier caliper. Each value represents the mean ± standard error of the mean. Significance is set at *P < 0.05

Fig. 2

Flow cytometry analysis of immune markers (CD4, CD8, CD3 and NK 1.1) on the splenocytes of the untreated mice, treated mice with low- and high-dose of methanol C. nutans extract. The percentage of the CD4/CD3 T-cell and CD8/CD3 T-cell population was increased significantly for both low and high-dose of treatment when compared to the untreated and control groups. The population of natural killer (NK) 1.1/CD3 cells was also increased in both low and high-dose of C. nutans treatment when compared to untreated and control groups

Fig. 3

Enzyme-linked immunosorbent assay analysis on the detection of the level of in IL-2 and IFN-γ in serum of the untreated group, treated mice with low and high-dose of C. nutans extract. The levels of expression for both IL-2 and IFN-γ have increased for both low and high C. nutans treatment when compared with the untreated group. Each value represents the mean ± standard error of the mean. Significance is set at *P < 0.05

Fig. 4

Level of nitric oxide assay from the untreated and treated groups (low-dose and high-dose of C. nutans). Each value represents the mean ± standard error of the mean. Significance is set at *P < 0.05. The level of NO decreased significantly in low-dose and high-dose of treatment compared to the untreated group

Fig. 5

Level of MDA from the untreated and treated groups (low-dose and high-dose of C. nutans). Each value represents the mean ± standard error of the mean. Significance is set at *P < 0.05. The level of MDA decreased significantly in low-dose and high-dose of treatment compared to the untreated group

Fig. 6

Histology analysis of the untreated, low-dose and high-dose of C. nutans. a Both tumor samples of the untreated and treated groups are stained with hematoxylin and eosin (H&E). (b) The number of mitotic cells decreased significantly in low-dose and high-dose of C. nutans treatment compared to the untreated group. Notes: a Circles represent cells undergoing mitosis. Magnification: 40X. Significance is set at *P < 0.05

Fig. 7

Clonogenic assay of mice organs. a Representative images of colonies formed in the lung and liver organs. b Bar chart of the total 4 T1 colonies formed from the mashed lung and liver harvested from the untreated, treated mice with low-dose and high-dose of C. nutans treatment after 10 days of incubation. Notes: a Lung, dilution factor: 10³; liver, dilution factor: 10³. b Each value represents mean ± standard error of the mean; *P < 0.05

Fig. 8

Bone marrow cells stained with Giemsa viewed under a phase-contrast microscope. Notes: Circles indicate the presence of abnormal cells base on the different morphology. Magnification: 40X