Immunomodulatory and Anticancer Activities of Hyacinthus orientalis L.: An In Vitro and In Vivo Study

Abstract

Hyacinthus orientalis L. (family Hyacinthaceae) is traditionally used to treat different diseases including cancer. In this study, the anticancer and immunomodulatory effects of this plant were evaluated. Hydroalcoholic extract was prepared, and different solvent fractions were obtained using solvent-solvent extraction. In the anticancer part, MTT assay and caspase-3 ELISA kits were used to measure the antiproliferative and apoptosis induction ability for each extract, respectively. In the immunomodulatory part, lymphocyte proliferation assay and cytokines detection kit were used to measure the effect of extracts of acquired immunity. Phagocytosis and pinocytosis induction were used to evaluate the effect of extracts on the innate immunity. GC-MS, LC-MS, and Foline-Ciocalteu assays were used to identify the chemical composition of the plant. Balb/C mice were inoculated with breast cancer and treated with hydroalcoholic extract of H. orientalis L. Results showed that hydroalcoholic extract and n-hexane fraction were highly effective in apoptosis induction. Both extract and fraction were also effective in stimulating lymphocytes proliferation and phagocytosis. Significant reduction in tumor size was achieved after treating tumor-bearing mice with hydroalcoholic extract. Additionally, high cure percentages (50%) were obtained in treated mice. Results of this study showed that H. orientalis L. has promising anticancer and immunomodulatory activities. However, further studies are needed to explore more details of apoptosis induction ability and other mechanisms of action and to measure different signaling pathways responsible for the anticancer and immunomodulatory response.

Keywords: anticancer; apoptosis; immunomodulatory; plant extracts; traditional medicine.

Figure 1

The antiproliferative activity of H. orientalis L. extract and fractions against: (A) EMT6/P cell line, (B) MCF-7 cell line, (C) T47D cell line, (D) Vero cell line using concentrations between 0.78 to 50 mg/mL. Percentage of cell viability (%) was calculated as (OD of treated cells/OD of control cells * 100). Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Figure 1

The antiproliferative activity of H. orientalis L. extract and fractions against: (A) EMT6/P cell line, (B) MCF-7 cell line, (C) T47D cell line, (D) Vero cell line using concentrations between 0.78 to 50 mg/mL. Percentage of cell viability (%) was calculated as (OD of treated cells/OD of control cells * 100). Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Figure 2

The effect of IC₅₀ concentration of H. orientalis L. extract and fractions on caspase-3 expression in T47D cell line. Concentration of the extracts: hydroalcoholic (6.12 mg/mL), aqueous methanol (0.11 mg/mL), chloroform (3.53 mg/mL), aqueous (11.38 mg/mL), and n-hexane (0.59 mg/mL) (* p < 0.05, ** p < 0.001 compared to the negative control). Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Figure 3

The effect of H. orientalis L. extract and fractions on the splenic lymphocytes’ proliferation using different concentrations (12.5–50 mg/mL) in the presence and absence of mitogens: (A) in the absence of Con A (concanavalin A) and LPS (lipopolysaccharide); (B) in the presence of (4 μg/mL) of LPS; (C) in the presence of (5 μg/mL) of Con A. Results are expressed as means (bars) ± SEM (lines).

Figure 3

The effect of H. orientalis L. extract and fractions on the splenic lymphocytes’ proliferation using different concentrations (12.5–50 mg/mL) in the presence and absence of mitogens: (A) in the absence of Con A (concanavalin A) and LPS (lipopolysaccharide); (B) in the presence of (4 μg/mL) of LPS; (C) in the presence of (5 μg/mL) of Con A. Results are expressed as means (bars) ± SEM (lines).

Figure 4

In vitro phagocytic assay using nitro blue tetrazolium (NBT) reduction test of peritoneal macrophages treated with various concentrations (12.5–50 mg/mL) of H. orientalis L. extract and fractions. Aqueous fraction had the highest phagocytic index (340). Results are expressed as means (bars) ± SEM (lines).

Figure 5

In vitro pinocytic assay using neutral red method on peritoneal macrophages treated with different concentrations (12.5–50 mg/mL) of H. orientalis L. extract and fractions. Aq. Methanol showed the highest pinocytic index (236). Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Figure 6

The effect of H. orientalis L. extract and fractions on IL-2, IL-4, IL-10, and IFN-γ level at concentration of 50 mg/mL. TH1/THI murine assay kit was used to measure the effect of the H. orientalis on cytokines. Secretion of IL-2 was enhanced in the presence of hydroalcoholic extract and chloroform fraction. Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Figure 7

Total phenolic content in mg GAE/g dry weight of H. orientalis L. polar extract and fractions at concentration 100 mg/mL. Hydroalcoholic extract showed the highest value (22.4 mg GAE/g dry weight). Results are expressed as means of three independent experiment (bars) ± SEM (lines).

Figure 8

A plot demonstrated the changes in average tumor size (mm³) vs. time in (days) of treatment with H. orientalis L. hydroalcoholic extract in EMT6/P cell line.

Figure 9

Effect of H. orientalis L. hydroalcoholic extract on tumor size and cure percentage. Treatment with H. orientalis resulted in smaller tumors size and higher cure percentage compared to the negative control (n = 10 mice in each group).

Figure 10

Steps of extract and fractions preparation.