Natural product; HSF1
Azadiradione has been reported to inhibit NF-κB signaling and may have anti-inflammatory activity. It may also interact with other cellular targets involved in inflammation, cell proliferation, and apoptosis. As a limonoid, azadiradione may exert its effects through modulation of various signaling pathways. However, the specific molecular targets of azadiradione have not been fully characterized.

Azadiradione exhibits antimycobacterial and anti-inflammatory activities. It has a role as a plant metabolite, an antimycobacterial drug and an anti-inflammatory agent. It is a limonoid, a tetracyclic triterpenoid, an acetate ester, a cyclic terpene ketone and a member of furans.
Azadiradione has been reported to exhibit anti-inflammatory and anticancer activities in vitro. It may inhibit NF-κB activation and reduce the production of pro-inflammatory cytokines. The compound has also been shown to induce apoptosis in certain cancer cell lines. However, specific IC50 values and detailed in vitro activity data are limited.

This study was carried out to evaluate the anti-nociceptive and anti-inflammatory activities of carbon tetrachloride extract (CTCE) of Azadirachta indica fruit skin and its isolated constituent azadiradione at two different dose levels (50 and 100 mg kg⁻¹ body weight). Anti-nociceptive screening by writhing test and hot-plate technique supported both peripheral and central mechanisms, respectively. Anti-inflammatory activity was observed using carrageenan-induced paw oedema model. The results concluded that the animals treated with 100 mg kg⁻¹ dose of CTCE and azadiradione exhibited significant anti-nociceptive and anti-inflammatory activities. This study had rationalised the ethnomedicinal use of the plant for wound, burns and injury by tribal people.[1]

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This study demonstrated that partial rescue of the defective protein quality control in HD model mouse by azadiradione (a bioactive limonoids found in the seed of Azadirachta indica) could potentially improve the disease pathology. Prolonged treatment of azadiradione to HD mice significantly improved the progressive deterioration in body weight, motor functioning along with extension of lifespan. Azadiradione-treated HD mice brain also exhibited considerable decrease in mutant huntingtin aggregates load and improvement of striatal pathology in comparison with age-matched saline-treated HD controls. Biochemical analysis further revealed upregulation and activation of not only HSF1 (master regulator of protein folding) but also Ube3a (an ubiquitin ligase involved in the clearance of mutant huntingtin) in azadiradione-treated mice. Our results indicate that azadiradione-mediated enhanced folding and clearance of mutant huntingtin might underlie improved disease pathology in HD mice and suggests that it could be a potential therapeutic molecule to delay the progression of HD[2].
In vivo, azadiradione has been studied for its anti-inflammatory and anticancer activities in animal models. It may reduce inflammation and inhibit tumor growth. However, detailed in vivo efficacy data are limited. The compound is a natural product and has been used in traditional medicine, but its therapeutic potential requires further investigation.

NF-κB inhibition assays are performed using reporter cell lines or by measuring NF-κB DNA binding activity. Cells are treated with azadiradione and stimulated with TNF-α or other NF-κB activators. NF-κB activity is measured using luciferase reporter assays or by EMSA. Cytokine levels (TNF-α, IL-6, IL-1β) are measured by ELISA. Cell viability is assessed using MTT assays.

Cellular assays for azadiradione employ various cancer cell lines or inflammatory cell types. Cells are treated with varying concentrations of azadiradione for 24-72 hours. Cell viability is assessed using MTT or CellTiter-Glo assays. NF-κB activation is assessed by Western blot for IκBα degradation and p65 phosphorylation. Cytokine production is measured by ELISA. Apoptosis is evaluated by Annexin V/PI staining.

Transgenic mouse line for HD (strain B6CBA-Tg (HDexon1) 62Gpb/3 J) was procured from The Jackson Laboratory and bred in the animal house facility of National Brain Research Centre. This HD transgenic mouse line (commonly referred as R6/2 line) expresses Exon1 region of huntingtin with 120 CAG repeats and exhibits very fast progressive neurological phenotype [44]. Animals had free access to pelleted diet and water ad libitum. All experiments were conducted according to the strict guideline proposed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forestry, Government of India and were approved by the Institutional Animal Ethics Committee of the National Brain Research Centre (Protocol number NBRC/IAEC/2017/120). After proper genotyping, female HD mice along with their wild type littermates were used for the present study. Azadiradione was dissolved in DMSO, diluted in saline (containing 5% DMSO) and then given intraperitoneal injection (dose 10 mg/kg body weight) to wild-type and HD mice at their age of 55 days. Each mouse was received total 15 doses at 1-day interval. In some pilot experiments, wild-type mice were treated azadiradione at a dose of 1 and 10 mg/kg body weight every day for 10 days. Control mice received with same volume of saline containing 5% DMSO.[2]
In vivo studies with azadiradione are typically performed in rodent models of inflammation or cancer. The compound is administered via oral or intraperitoneal routes at various doses. Inflammatory markers are measured in serum or tissues. Tumor growth is monitored in xenograft models. Tissue samples are collected for histology and molecular analysis. However, detailed published in vivo protocols are limited.

Azadiradione has molecular formula C28H34O5 and molecular weight 450.57. It has CAS number 26241-51-0. The compound is a naturally occurring limonoid isolated from neem (Azadirachta indica). It is soluble in DMSO and other organic solvents. The compound should be stored at -20°C for long-term stability. It is a research-use only compound.

Toxicological data for azadiradione are limited. As a naturally occurring compound from neem, it is generally considered to have low toxicity, but specific toxicological data are not extensively reported. The compound should be handled with appropriate laboratory safety precautions. Standard toxicological studies would be required for any therapeutic development.

[1]. Anti-nociceptive and anti-inflammatory activities of Azadirachta indica fruit skin extract and its isolated constituent azadiradione. Nat Prod Res. 2013;27(16):1463-7.

[2]. Azadiradione Restores Protein Quality Control and Ameliorates the Disease Pathogenesis in a Mouse Model of Huntington's Disease. Mol Neurobiol. 2018 Aug;55(8):6337-6346.

Azadirachtin is a tetracyclic triterpenoid compound with the structure 4,4,8-trimethylandrost-1,14-diene, substituted with oxo groups at positions 3 and 16, acetoxy groups at position 7, and furan-3-yl groups at position 17. It was isolated from neem (Azadirachta indica) and possesses anti-mycobacterial and anti-inflammatory activities. Azadirachtin can be used as a plant metabolite, an anti-mycobacterial drug, and an anti-inflammatory agent. It is a limonene compound, a tetracyclic triterpenoid compound, an acetate compound, a cyclic terpene ketone compound, and also belongs to the furan class of compounds. Azadirachtin has been reported to exist in Dysoxylum parasiticum, Quivisianthe papinae, and other organisms with relevant data.
Azadiradione is a naturally occurring limonoid compound isolated from the neem tree (Azadirachta indica) and other plants in the Meliaceae family. It has been studied for its anti-inflammatory and anticancer activities. Azadiradione may inhibit NF-κB signaling and reduce pro-inflammatory cytokine production. The compound is a research-use only reagent for studying inflammation and cancer biology.

C28H34O5

450.57

450.241

C, 74.64; H, 7.61; O, 17.75

26241-51-0

12308714

Typically exists as solid at room temperature

5.417

0

5

3

33

975

7

CC(OC1CC2C(C(C=C[C@]2(C)C2CCC3(C(C4C=COC=4)C(=O)C=C3[C@]12C)C)=O)(C)C)=O

KWAMDQVQFVBEAU-HMWIRDDCSA-N

InChI=1S/C28H34O5/c1-16(29)33-23-14-20-25(2,3)22(31)8-11-26(20,4)19-7-10-27(5)21(28(19,23)6)13-18(30)24(27)17-9-12-32-15-17/h8-9,11-13,15,19-20,23-24H,7,10,14H2,1-6H3/t19-,20+,23-,24-,26-,27-,28-/m1/s1

[(5R,7R,8R,9R,10R,13S,17R)-17-(furan-3-yl)-4,4,8,10,13-pentamethyl-3,16-dioxo-6,7,9,11,12,17-hexahydro-5H-cyclopenta[a]phenanthren-7-yl] acetate

Azadiradione; 26241-51-0; CHEBI:67280; Azadiradione (~90%); 24-Norchola-1,14,20,22-tetraene-3,16-dione, 7-(acetyloxy)-21,23-epoxy-4,4,8-trimethyl-, (5alpha,7alpha,13alpha,17alpha)-; [(5R,7R,8R,9R,10R,13S,17R)-17-(furan-3-yl)-4,4,8,10,13-pentamethyl-3,16-dioxo-6,7,9,11,12,17-hexahydro-5H-cyclopenta[a]phenanthren-7-yl] acetate; (5a,7a,13a,17a)-7-(Acetyloxy)-21,23-epoxy-4,4,8-trimethyl-24-norchola-1,14,20,22-tetraene-3,16-dione; CHEMBL1215754;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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