Rhinacanthus nasutus extracts prevent glutamate and amyloid-β neurotoxicity in HT-22 mouse hippocampal cells: possible active compounds include lupeol, stigmasterol and β-sitosterol

Abstract

The Herb Rhinacanthus nasutus (L.) Kurz, which is native to Thailand and Southeast Asia, has become known for its antioxidant properties. Neuronal loss in a number of diseases including Alzheimer's disease is thought to result, in part, from oxidative stress. Glutamate causes cell death in the mouse hippocampal cell line, HT-22, by unbalancing redox homeostasis, brought about by a reduction in glutathione levels, and amyloid-β has been shown to induce reactive oxygen species (ROS) production. Here in, we show that ethanol extracts of R. nasutus leaf and root are capable of dose dependently attenuating the neuron cell death caused by both glutamate and amyloid-β treatment. We used free radical scavenging assays to measure the extracts antioxidant activities and as well as quantifying phenolic, flavonoid and sterol content. Molecules found in R. nasutus, lupeol, stigmasterol and β-sitosterol are protective against glutamate toxicity.

Keywords: Alzheimer’s disease; amyloid-β; glutamate; herbal medicine; neuron degeneration; oxidative stress.

Figure 1

The protective effect of Rhinacanthus nasutus ethanol extract against 5 mM glutamate. Phase contrast micrographs of HT-22 cells. (A) Control cells; (B) Glutamate 5 mM; (C) Glutamate 5 mM + R. nasutus root ethanol extract 10 μg; (D) R. nasutus root ethanol extract 10 μg.

Figure 1

The protective effect of Rhinacanthus nasutus ethanol extract against 5 mM glutamate. Phase contrast micrographs of HT-22 cells. (A) Control cells; (B) Glutamate 5 mM; (C) Glutamate 5 mM + R. nasutus root ethanol extract 10 μg; (D) R. nasutus root ethanol extract 10 μg.

Figure 2

Quantification of HT-22 cell survival. (A) Trypan blue exclusion assay; (B) Lactate dehydrogenase release assay; (C) MTT assay. ** ANOVA dunnett’s post hoc test p < 0.01.

Figure 3

The protective effect of Hexane extract of R. nasutus root against 5 mM glutamate. Phase contrast micrographs of HT-22 cells. (A) Control cells; (B) Glutamate 5 mM; (C) Glutamate 5 mM + R. nasutus root hexane extract 10 μg·mL⁻¹; (D) R. nasutus root hexane extract 10 μg·mL⁻¹.

Figure 4

Quantification of HT-22 cell survival. (A) Trypan blue exclusion assay; (B) Lactate dehydrogenase release assay. * ANOVA Dunnett’s post hoc test p < 0.05; ** ANOVA Dunnett’s post hoc test p < 0.01.

Figure 5

Protective effect of R. nasutus leaf ethanol extract against 5 mM glutamate. Phase contrast micrographs of HT-22 cells. (A) Control cells; (B) Glutamate 5 mM; (C) Glutamate 5 mM + R. nasutus leaf ethanol extract 10 μg·mL⁻¹; (D) R. nasutus leaf ethanol extract 10 μg·mL⁻¹.

Figure 6

Quantification of HT-22 cell survival. (A) Trypan blue exclusion assay; (B) Lactate dehydrogenase release assay. * ANOVA Dunnett’s post hoc test p < 0.05; ** ANOVA Dunnett’s post hoc test p < 0.01.

Figure 7

Hexane extract of R. nasutus provides no protection against 5 mM glutamate toxicity (Trypan blue exclusion).

Figure 8

The effect of R. nasutus extracts on the production of reactive oxygen species (ROS) after exposure to 5 mM Glutamate. (A) The effect of the ethanol extract of R. nasutus root on the ROS production in response to 5 mM Glutamate; (B) The effect of the ethanol extract of R. nasutus leaf on the ROS production in response to 5 mM Glutamate; (C) The effect of the hexane extract of R. nasutus root on the ROS production in response to 5 mM Glutamate; (D) The effect of the hexane extract of R. nasutus leaf on the ROS production in response to 5 mM Glutamate. * ANOVA p < 0.05; *** ANOVA p < 0.001.

Figure 9

Protective effect of R. nasutus extracts against 2 μM amyloid-β_25–35 toxicity. (A) Ethanol extract of R. nasutus root; (B) Hexane extract of R. nasutus root; (C) Ethanol extract of R. nasutus leaf; (D) Hexane extract of R. nausutsleaf. * ANOVA Dunnett’s post hoc test p < 0.05; ** ANOVA Dunnett’s post hoc test p < 0.01.

Figure 10

Structures of compounds found in R. nasutus, which were quantified using HPLC.

Figure 11

Chromatographs of R. nasutus extracts. (A) Ethanol extract of R. nasutus root (50 mg·mL⁻¹); (B) Hexane extract of R. nasutus root (50 mg·mL⁻¹); (C) Ethanol extract of R. nasutus leaf (50 mg·mL⁻¹); (D) Hexane extract of R. nasutus leaf (50 mg·mL⁻¹). Peaks: 1. Lupeol, 2. Stigmasterol, 3. β-sitosterol.

Figure 11

Chromatographs of R. nasutus extracts. (A) Ethanol extract of R. nasutus root (50 mg·mL⁻¹); (B) Hexane extract of R. nasutus root (50 mg·mL⁻¹); (C) Ethanol extract of R. nasutus leaf (50 mg·mL⁻¹); (D) Hexane extract of R. nasutus leaf (50 mg·mL⁻¹). Peaks: 1. Lupeol, 2. Stigmasterol, 3. β-sitosterol.

Figure 12

Protection of HT-22 cells against glutamate toxicity by (A) lupeol, (B) stigmasterol, (C) β-sitosterol. Measured using trypan blue exclusion assay. * ANOVA Dunnett’s post hoc test p < 0.05; ** ANOVA Dunnett’s post hoc test p < 0.01.

Figure 13

(A) β-Amyrin structure (B) β-amyrin protection against glutamate toxicity in HT-22 cells. ** ANOVA Dunnett’s post hoc test p < 0.01.