In vitro activity: Andrographolide was found to suppress the expression of inducible nitric oxide synthase in a concentration-dependent manner. In the andrographolide-treated group, a reduction of Akt, c-Jun N-terminal kinase and p65 phosphorylation was observed. Andrographolide also caused a decrease in Bcl-2/NF-κb expression and a dose-dependent increase in the expression of Cleaved-Caspase3/Bax protein.

Kinase Assay: In vitro osteoclastogenesis assays are preformed to examine the effects of Andrographolide on osteoclast differentiation. Bone marrow macrophages (BMM) cells are prepared. Briefly, cells extracted from the femur and tibiae of a 6-week-old C57/BL6 mouse are incubated in complete cell culture media and 30 ng/mL M-CSF in a T-75 cm2 flask for proliferation. When changing the medium, the cells are washed in order to deplete residual stromal cells. After reaching 90% confluence, cells are washed with PBS three times and trypsinized for 30 min to harvest BMMs. Cells adhering to the bottom of the dish are classified as BMMs; these BMMs are plated in 96-well plates at a density of 8×103 cells per well in triplicate and incubated in a humidified incubator containing 5% CO2 at 37°C for 24 h. The cells are then treated with various concentrations of Andrographolide (0, 2.5, 5, or 10 μM) plus M-CSF (30 ng/mL) and RANKL (50 ng/mL). After 5 days, cells are fixed and stained for tartrate-resistant acid phosphatase (TRAP) activity. TRAP-positive multinucleated cells with more than five nuclei are counted as osteoclasts

Cell Assay: Effects of Andrographolide on cell proliferation are determined with a CCK-8. BMMs are plated in 96-well plates at a density of 3×103 cells per well in triplicate. Twenty-four hours later, the cells are treated with increasing concentrations of Andrographolide (0, 2.5, 5, 10 or 20 μM) for 2 days. Next, 10 μL CCK-8 is added to each well, and the plates are then incubated at 37°C for an additional 2 h. The optical density (OD) is then measured with an ELX800 absorbance microplate reader at a wavelength of 450 nm (650 nm reference). The cell viability is calculated

Treatment with Andrographolide (5 or 30 mg/kg) reduces the extent of bone loss induced by LPS. Moreover, Andrographolide slightly increases the BMD and cortex thickness compared to LPS treatment. Histological examination confirms the protective effects of Andrographolide on LPS-induced bone loss. LPS injection leads to inflammatory bone erosion and increased numbers of TRAP-positive osteoclasts.

Rats were dosed with andrographolide intragastrically for 5 consecutive days in a hepatoprotection study. Results indicated that andrographolide could up-regulate glutamate cysteine ligase catalytic and modifier subunits, heme oxygenase-1, superoxide dismutase-1, glutathione S-transferase protein and mRNA expression in the heart, liver, and kidney.

Dis Esophagus. 2016 Jan;29(1):54-61; Br J Pharmacol. 2014 Feb;171(3):663-75.; Toxicol Appl Pharmacol. 2014 Oct 1;280(1):1-9.