Kinase experiment:

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℃ 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].

Cell experiment:

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℃ 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].

Animal experiment:

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.

IC50: 11.0 ± 0.28 μg/ml for α-glucosidase; 11.3 ± 0.29 μg/ml for α-amylase

Andrographolide is an irreversible antagonist of NF-κB. The nuclear factor NF-κB pathway has been considered as a prototypical proinflammatory signaling pathway, based on the role of NF-κB in the expression of proinflammatory genes including chemokines, cytokines and adhesion molecules.

In vitro: 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 [1].

In vivo: 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 [2].

Clinical trial: N/A

References:
[1] Wang ZM,Kang YH,Yang X,Wang JF,Zhang Q,Yang BX,Zhao KL,Xu LP,Yang LP,Ma JX,Huang GH,Cai J,Sun XC.  Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro. Dis Esophagus.2016 Jan;29(1):54-61.
[2] Chen HW,Huang CS,Li CC,Lin AH,Huang YJ,Wang TS,Yao HT,Lii CK.  Bioavailability of andrographolide and protection against carbon tetrachloride-induced oxidative damage in rats. Toxicol Appl Pharmacol.2014 Oct 1;280(1):1-9.