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^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
C57BL/6 mice (8 weeks old) are divided into four groups of seven mice each. Mice are injected i.p. with Andrographolide (5 or 30?mg/kg body weight) or PBS as a control 1 day before injection of LPS (5?μg/g body weight). Andrographolide or PBS is injected intraperitoneally every other day for 8 days. LPS is injected intraperitoneally on days one and four. All mice are killed 8 days after the initial LPS injection, and the left femurs of all animals are scanned with a high-resolution micro-CT at a resolution of 9?μm.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Zhai ZJ, et al. Andrographolide suppresses RANKL-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo. Br J Pharmacol. 2014 Feb;171(3):663-75.  [Content Brief]

  [2]. Gupta S, et al. Broad-spectrum antiviral properties of andrographolide. Arch Virol. 2017 Mar;162(3):611-623.  [Content Brief]