Natural product; p50; NF-κB

Andrographolide (AP) reduces the expression of osteoclast-specific markers and inhibits RANKL-mediated osteoclast differentiation and bone resorption in vitro. Andrographolide reduces inflammation by preventing TNF-induced NF-B activation through covalent modification of reduced Cys62 of p50, without affecting I-B degradation or p50/p65 nuclear translocation. Without affecting p38 or JNK signaling, andrographolide also blocks the ERK/MAPK signaling pathway. RAW 264.7 cells' differentiation into osteoclasts is inhibited by andrographolide in a concentration-dependent manner. In both BMMs and RAW 264.7 cells, andrographolide inhibits osteoclast formation in a concentration-dependent manner without obviously having cytotoxic effects. Treatment with andrographolide significantly reduces the area of bone resorption. After 2.5 μM Andrographolide treatment, only about 30% of the bone resorption seen in the control group is accomplished. After receiving treatment with 10 μM Andrographolide, osteoclastic bone resorption is almost entirely inhibited[1].

Treatment with andrographolide (5 or 30 mg/kg) lessens the severity of bone loss brought on by LPS. In addition, andrographolide slightly improves BMD and cortex thickness in comparison to LPS treatment. The protective effects of andrographolide against bone loss caused by LPS are confirmed by histological analysis. Increased TRAP-positive osteoclast numbers and inflammatory bone erosion are caused by LPS injection[1].

In vitro osteoclastogenesis assays are preformed to examine the effects of Andrographolide on osteoclast differentiation. BMM cells, or bone marrow macrophages, are created. In a T-75 cm2 flask with complete cell culture media and 30 ng/mL M-CSF, cells taken from the femur and tibiae of a 6-week-old C57/BL6 mouse are incubated for proliferation. The cells are washed when the medium is changed to remove any stromal cells that may still be present. Cells are three times PBS-washed and then trypsinized for 30 minutes to harvest BMMs once they have reached 90% confluence. BMMs are cells that stick to the bottom of the dish. BMMs are plated in 96-well plates at a density of 8×103 cells per well in triplicate and incubated for 24 hours at 37 °C in a humidified incubator containing 5% CO2. Then, different concentrations of andrographolide (0, 2.5, 5, or 10 μM) are added to the cells along with M-CSF (30 ng/mL) and RANKL (50 ng/mL). Cells are fixed and stained to check for tartrate-resistant acid phosphatase (TRAP) activity after five days. Osteoclasts are multinucleated cells that are positive for TRAP and have five or more nuclei[1].

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. After twenty-four hours, the cells are exposed to increasing doses of andrographolide (0, 2.5, 5, 10, or 20 μM) for two days. The plates are then incubated at 37°C for an additional 2 hours after the addition of 10 μL of CCK-8 to each well. The optical density (OD) is then determined using an ELX800 absorbance microplate reader at a wavelength of 450 nm (650 nm reference). Calculations are made to determine cell viability[1].

Mice: Four groups of seven C57BL/6 mice each are created using 8-week-old mice. One day prior to the injection of LPS (5 g/g body weight), mice are administered i.p. injections of andrographolide (5 or 30 mg/kg body weight) or PBS as a control. Over the course of eight days, either andrographolide or PBS is intraperitoneally injected. On days one and four, LPS is administered intraperitoneally. All mice are euthanized 8 days after receiving the initial LPS injection, and their left femurs are all scanned using a high-resolution micro-CT with a 9-m resolution.

[1]. Andrographolide suppresses RANKL-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo. Br J Pharmacol. 2014 Feb;171(3):663-75.

[2]. Broad-spectrum antiviral properties of andrographolide. Arch Virol. 2017 Mar;162(3):611-623.

Andrographolide is a labdane diterpenoid isolated from the leaves and roots of Andrographis paniculata that exhibits anti-HIV, anti-inflammatory and antineoplastic properties. It has a role as a metabolite, an anti-inflammatory drug, an anti-HIV agent and an antineoplastic agent. It is a gamma-lactone, a primary alcohol, a secondary alcohol, a labdane diterpenoid and a carbobicyclic compound.
Andrographolide (HMPL-004) is a botanical product extracted from a herb that occurs naturally in China. The herb has an extensive history of use in TCM for the treatment of upper respiratory tract infections and other inflammatory and infectious diseases.
Andrographolide has been reported in Andrographis paniculata, Cymbopogon schoenanthus, and Ginkgo biloba with data available.
Andrographolide is a labdane diterpenoid that is produced by the Andrographis paniculata plant, which has a broad range of therapeutic applications including anti-inflammatory and anti-platelet aggregation activities and potential antineoplastic properties. Since andrographolide has multiple therapeutic activities there are several proposed mechanisms of action for this agent. The anti-inflammatory effects of this agent appear to be related to the inhibition of nitric oxide (NO) production by macrophages. This agent may activate the NO/cyclic GMP pathway and inhibit both the phospholipase C gamma 2 (PLC gamma2)/protein kinase C (PKC) and PI3K/AKT-MAPK signaling pathways in activated platelets to inhibit platelet aggregation. In activated platelets, these three signaling pathways are downstream of integrin activation mediated by collagen binding and influence the association of fibrinogen with its receptors. Additionally, andrographolide may exert its anti-cancer activity through the induction of cell cycle arrest at G0/G1 phase and the stimulation of lymphocyte proliferation and activation. These processes could result in decreased proliferation of and increased immunocytotoxicity against tumor cells.

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Drug Indication
Investigated for use/treatment in ulcerative colitis.

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Mechanism of Action
HMPL-004 acts on multiple cellular targets in the inflammatory signal transduction pathways resulting in suppressed inflammation cytokine expression including TNF-α, IL-1β and IL-6. HMPL-004 was demonstrated to inhibit TNF-α and IL-1β production in cell-based assays. HMPL-004 is also able to inhibit NF-kB activation. NF-kB is a family of transcriptional factors that regulate a wide spectrum of genes critically involved in host defence and inflammation. The mechanism of action of HMPL-004 was further supported in laboratory IBD animal models. Treatment of IBD rats with HMPL-004 caused a significant drop in plasma cytokine concentrations, including TNF-α and IL-1β.

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Background and purpose: Osteoclasts play a pivotal role in diseases such as osteoporosis, rheumatoid arthritis and tumour bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. Here, we examined changes in osteoclastogenesis and LPS-induced osteolysis in response to andrographolide (AP), a diterpenoid lactone isolated from the traditional Chinese and Indian medicinal plant Andrographis paniculata. Experimental approach: Effects of AP on osteoclast differentiation and bone resorption were measured in vitro. Western blots and RT-PCR techniques were used to examine the underlying molecular mechanisms. The bone protective activity of AP in vivo was assessed in a mouse model of osteolysis. Key results: AP concentration-dependently suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro and reduced the expression of osteoclast-specific markers, including tartrate-resistant acid phosphatase, calcitonin receptors and cathepsin K. Further molecular analysis revealed that AP impaired RANKL-induced NF-κB signalling by inhibiting the phosphorylation of TGF-β-activated kinase 1, suppressing the phosphorylation and degradation of IκBα, and subsequently preventing the nuclear translocation of the NF-κB p65 subunit. AP also inhibited the ERK/MAPK signalling pathway without affecting p38 or JNK signalling. Conclusions and implications: AP suppressed RANKL-induced osteoclastogenesis through attenuating NF-κB and ERK/MAPK signalling pathways in vitro, thus preventing bone loss in vivo. These data indicated that AP is a promising natural compound for the treatment of osteoclast-related bone diseases. [1]

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Andrographolide, a diterpenoid, is known for its anti-inflammatory effects. It can be isolated from various plants of the genus Andrographis, commonly known as 'creat'. This purified compound has been tested for its anti-inflammatory effects in various stressful conditions, such as ischemia, pyrogenesis, arthritis, hepatic or neural toxicity, carcinoma, and oxidative stress, Apart from its anti-inflammatory effects, andrographolide also exhibits immunomodulatory effects by effectively enhancing cytotoxic T cells, natural killer (NK) cells, phagocytosis, and antibody-dependent cell-mediated cytotoxicity (ADCC). All these properties of andrographolide form the foundation for the use of this miraculous compound to restrain virus replication and virus-induced pathogenesis. The present article covers antiviral properties of andrographolide in variety of viral infections, with the hope of developing of a new highly potent antiviral drug with multiple effects. [2]

C20H30O5

350.45

350.209

C, 68.54; H, 8.63; O, 22.83

5508-58-7

5318517

White to off-white solid powder

1.2±0.1 g/cm3

557.3±50.0 °C at 760 mmHg

229-232ºC

195.5±23.6 °C

0.0±3.4 mmHg at 25°C

1.568

1.62

3

5

3

25

597

6

O([H])[C@]1([H])C([H])([H])C([H])([H])[C@@]2(C([H])([H])[H])[C@]([H])(C([H])([H])/C(/[H])=C3/C(=O)OC([H])([H])[C@@]/3([H])O[H])C(=C([H])[H])C([H])([H])C([H])([H])[C@]2([H])[C@]1(C([H])([H])[H])C([H])([H])O[H]

BOJKULTULYSRAS-OTESTREVSA-N

InChI=1S/C20H30O5/c1-12-4-7-16-19(2,9-8-17(23)20(16,3)11-21)14(12)6-5-13-15(22)10-25-18(13)24/h5,14-17,21-23H,1,4,6-11H2,2-3H3/b13-5+/t14-,15-,16+,17-,19+,20+/m1/s1

(3E,4S)-3-[2-[(1R,4aS,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylidene-3,4,4a,6,7,8-hexahydro-1H-naphthalen-1-yl]ethylidene]-4-hydroxyoxolan-2-one

Andrographolide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.13 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.13 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.13 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)