Natural product; p50; NF-κB ; Estrogen - related receptor α (ERRα) [1]

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].

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Andrographolide can suppress RANKL - induced osteoclastogenesis. It interferes with the interaction between ERRα and co - activator PGC - 1β, inhibits the transcriptional activity of ERRα, thereby down - regulating the expression of mitochondrial glutaminase, disrupting the metabolic adaptation during osteoclast differentiation, and inhibiting the formation of mature osteoclasts. This was verified by immunoprecipitation and dual - luciferase reporter assays [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].

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Andrographolide can prevent inflammatory bone loss in vivo. In the ovariectomized female rat model and high - fat diet - induced male rat model, it can effectively inhibit bone resorption and significantly improve bone mass loss, which is related to its mechanism of targeting ERRα to regulate the metabolic adaptation of osteoclastogenesis [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].

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Culture bone marrow - derived macrophages (BMMs) and pre - osteoclast cell line RAW264.7 cells. Add Andrographolide at different concentrations to the culture medium, and stimulate the cells with RANKL at the same time. Then, use TRAP staining to detect the formation of osteoclasts, and use RT - qPCR to detect the expression of osteoclast - related genes, such as Acp5, Ctsk, and Atp6v0d2. In addition, immunoprecipitation is used to detect the interaction between ERRα and PGC - 1β, and the dual - luciferase reporter system is used to detect the transcriptional activity of ERRα [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.

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Ovariectomized female rats and high - fat diet - induced male rats were used as animal models. Andrographolide was dissolved in dimethyl sulfoxide (DMSO) and then diluted with normal saline. The rats were injected intraperitoneally with Andrographolide at a dose of 50 mg/kg per day. After a certain period of time, the bone mass of the rats was evaluated by micro - CT, and the bone resorption situation was observed by histological staining [1]

[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 latanoid diterpenoid compound isolated from the leaves and roots of Andrographis paniculata, possessing anti-HIV, anti-inflammatory, and antitumor activities. It functions as a metabolite, anti-inflammatory drug, anti-HIV agent, and antitumor agent. It is a γ-lactone, primary alcohol, secondary alcohol, latanoid diterpenoid, and carbon-bicyclic compound. Andrographolide (HMPL-004) is a plant product extracted from a naturally growing Chinese herb. This herb has a long history of use in Traditional Chinese Medicine for treating upper respiratory tract infections and other inflammatory and infectious diseases. Andrographolide has been reported to exist in Andrographis paniculata, Citronella cirrhosa, and Ginkgo biloba, with relevant data reported. Andrographolide is a latanoid diterpenoid compound produced by Andrographis paniculata with broad therapeutic uses, including anti-inflammatory, antiplatelet aggregation, and potential antitumor properties. Due to the diverse therapeutic activities of andrographolide, several hypotheses have been proposed regarding its mechanism of action. The anti-inflammatory effect of this compound appears to be related to the inhibition of macrophage production of nitric oxide (NO). This drug may activate the NO/cyclic guanosine monophosphate (cGMP) pathway and inhibit the phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC) and PI3K/AKT-MAPK signaling pathways in activated platelets, thereby inhibiting platelet aggregation. In activated platelets, these three signaling pathways are all downstream of collagen-binding-mediated integrin activation and affect the binding of fibrinogen to its receptor. Furthermore, andrographolide may exert its anticancer activity by inducing cell cycle arrest at the G0/G1 phase and stimulating lymphocyte proliferation and activation. These processes may lead to reduced tumor cell proliferation and enhanced immunocytotoxicity.

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Drug Indications
It has been studied for the treatment of ulcerative colitis.

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Mechanism of Action
HMPL-004 acts on multiple cellular targets in inflammatory signaling pathways, thereby inhibiting the expression of inflammatory cytokines, including TNF-α, IL-1β, and IL-6. Cellular experiments showed that HMPL-004 inhibited the production of TNF-α and IL-1β. HMPL-004 also inhibited NF-κB activation. NF-κB is a transcription factor that regulates multiple genes that play crucial roles in host defense and inflammation. The mechanism of action of HMPL-004 was further confirmed in an IBD animal model. Treatment of IBD rats with HMPL-004 significantly reduced plasma cytokine concentrations, including TNF-α and IL-1β.

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Background and Objectives: Osteoclasts play a crucial role in diseases such as osteoporosis, rheumatoid arthritis, and bone metastases. Therefore, finding natural compounds that can inhibit osteoclast formation and/or function is of great significance for the treatment of osteoclast-related diseases. This study investigated the effects of andrographolide (AP, a diterpenoid lactone isolated from the traditional Chinese medicine and Indian medicinal plant Andrographis paniculata) on osteoclastogenesis and LPS-induced osteolysis. Experimental Methods: The effects of AP on osteoclast differentiation and bone resorption were detected in vitro. Western blot and RT-PCR were used to investigate its potential molecular mechanisms. This study evaluated the osteoprotective activity of AP in vivo in a mouse osteolysis model. Main Results: In vitro experiments showed that AP concentration-dependently inhibited RANKL-mediated osteoclast differentiation and bone resorption, and reduced the expression of osteoclast-specific markers, including tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K. Further molecular analysis revealed that AP attenuated the RANKL-induced NF-κB signaling pathway by inhibiting phosphorylation of TGF-β-activated protein kinase 1, inhibiting IκBα phosphorylation and degradation, and thus preventing nuclear translocation of the NF-κB p65 subunit. AP also inhibited the ERK/MAPK signaling pathway but did not affect the p38 or JNK signaling pathways. Conclusions and Significance: AP inhibits RANKL-induced osteoclastogenesis in vitro by attenuating the NF-κB and ERK/MAPK signaling pathways, thereby preventing bone loss in vivo. These data suggest that AP is a promising natural compound for the treatment of osteoclast-associated bone diseases. [1] Andrographolide is a diterpenoid compound known for its anti-inflammatory effects. It can be isolated from various plants of the Andrographis genus, commonly known as "Andrographis". This purified compound has been tested for anti-inflammatory effects under various stress conditions, such as ischemia, fever, arthritis, hepatotoxicity or neurotoxicity, carcinogenesis and oxidative stress. In addition to its anti-inflammatory effects, andrographolide also has immunomodulatory effects, effectively enhancing cytotoxic T cells, natural killer (NK) cells, phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC). All these properties of andrographolide lay the foundation for its use in inhibiting viral replication and viral pathogenesis. This article explores the antiviral properties of andrographolide in various viral infections, aiming to develop a new, highly effective antiviral drug with multiple effects. [2] Osteoporosis is a systemic metabolic bone disease that is closely related to abnormal osteoclast activity. ERRα is an important regulator of osteoclast differentiation and a key transcription factor affecting mitochondrial energy metabolism. Andrographolide, as a potential antagonist of ERRα, provides a new direction for the development of drugs for the prevention and treatment of osteoporosis. [1]

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.)