HIF-1α; NLRP3 inflammasome (including NLRP3, ASC, caspase-1) [4]

Agnuside (100 μM, 12–20 h) can decrease the expression of iNOS, COX-2, and IL-8 proteins in RAW264.7 and HT-29 cells stimulated in LPS (1 μg/mL/100 ng/mL); Agnuside (0.1-2500 ng/mL, 20–96 h) can then, in a time- and dose-dependent manner, promote angiogenesis by promoting cell proliferation (EC50= 1.376 µg/mL) in HUVEC [3]. Agnuside (3 μM, 4 h) dramatically decreased LPS (10 μg/ml)-stimulated fibroblast-like synoviocytes (FLS) expression of caspase-1, ASC, NLRP3, HIF-1α, IL-1β, and IL. Inhibiting the factor at -18 levels [4].

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In LPS-treated rat fibroblast-like synoviocytes (FLSs), AGN (3 μM) for 24 h significantly reduced caspase-1 activity increased by LPS challenge [4].

AGN (3 μM) prevented LPS-induced upregulation of HIF-1α mRNA and protein levels in FLSs [4].

AGN reversed LPS-promoted mRNA expression of caspase-1, ASC, and NLRP3, and also decreased protein levels of pro-caspase-1 and cleaved caspase-1 p10 [4].

AGN significantly lowered the content of proinflammatory factors IL-1β and IL-18 in the supernatant of LPS-treated FLSs [4].

In LPS-treated FLSs, AGN (3 μM, 24 h) downregulated both mRNA and protein expressions of fibrotic markers TGF-β, TIMP1, and VEGF [4].

Agnuside (6.25 mg/kg; oral dose; single dose) reduces allergic mediator levels in Balb/C mice in a dose-dependent manner and is anti-allergic mediator [1]. Agnuside (6.25 mg/kg; oral model; single dose) inhibits allergy and autophagy in the Balb/C mouse model [1]. It can reduce synovitis and fibrosis in knee osteoarthritis (KOA) [4].

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In MIA-induced KOA model rats, oral AGN at 6.25 mg/kg for 21 days effectively relieved local hypoxia in synovial tissue as shown by pimonidazole staining and HIF-1α immunohistochemistry, with decreased percentage of HIF-1α-positive areas and reduced HIF-1α mRNA levels [4].

AGN treatment decreased both mRNA and protein levels of pro-caspase-1, caspase-1 p10, ASC, and NLRP3 in synovial tissues of KOA rats [4].

Serum levels of IL-1β and IL-18 were significantly downregulated in AGN-treated KOA rats compared to KOA group [4].

HE staining showed that AGN-treated KOA rats had orderly arranged synovial lining cells, loose connective tissue, and less inflammatory cell infiltration, with lower synovitis score according to Krenn's criteria [4].

Anatomical observation and Sirius Red staining revealed markedly decreased collagen deposition in AGN-treated KOA rats [4].

Immunohistochemical staining of type I collagen showed significantly lower percentage of collagen I-positive areas in AGN group [4].

AGN treatment significantly decreased mRNA and protein expressions of fibrotic markers TGF-β, TIMP1, and VEGF in synovial tissues of KOA rats [4].

Molecular docking was performed using AutoDockTools to understand the interaction mechanism between lead molecules and the VEGFR2 binding site. A grid-box surrounding all residues within 10 Å of the ATP binding site was defined. Glu885 was subjected to sidechain flexibility to account for receptor flexibility. A Lamarckian genetic algorithm was applied for 50 runs with a population size of 50. The binding, electrostatic, van der Waals, hydrogen bond, desolvation, and total internal energies were computed using AutoDock’s empirical binding free energy function. Agnuside was docked into the VEGFR2 active site, showing a binding energy of -11.28 kcal/mol. Its binding is characterized by a hydrophobic network with Leu889 and Val899 sidechains, hydrogen bond acceptor interactions with Lys868 and Asp1046 via its C=O linker, and hydrogen bond donor interaction with Glu885 via a hydroxyl group. [3]
- An additional angiogenesis assay was performed as a competitive ligand experiment to assess if agnuside actively competes with a highly selective VEGFR2 inhibitor (ZM 323881 HCl, IC50 < 2 nM) for VEGFR2 binding site occupancy. HUVECs were pre-treated with 0.1 ng/mL agnuside for 30 minutes to allow it to occupy the target receptor's active site, followed by the addition of 1 nM ZM 323881 HCl. [3]

Primary rat fibroblast-like synoviocytes (FLSs) were obtained from normal rats. Synovial tissues were washed with PBS, minced into 2-3 mm² pieces, digested in 0.1% collagenase type II for 30 min, filtered through a cell strainer, and centrifuged at 1500 rpm for 4 min. Cells were cultured in DMEM with 10% FBS and antibiotics. Passages 3-6 were used [4].

To mimic the inflammatory environment of KOA and activate NLRP3 inflammasome, FLSs were stimulated with LPS (10 μg/ml) in DMEM for 6 h as the KOA group. The LPS+AGN group was treated with AGN (3 μM) for 24 h after LPS challenge [4].

Caspase-1 activity was measured using a caspase-1 activity assay kit. After treatment, FLSs were harvested, and absorbance was measured at 405 nm using a microplate reader. Relative changes of caspase-1 activity were calculated compared to normal group [4].

Western blotting: FLSs were mixed with RIPA lysate and ground for 10-15 min. Protein levels were quantified by BCA assay. Samples were electrophoresed in SDS-PAGE, transferred to PVDF membranes, blocked with 5% nonfat dry milk for 2 h, incubated with primary antibody (1:1000) overnight at 4°C, then secondary antibody for 2 h. Bands were visualized by ECL method, and gray values were quantified with actin as internal control [4].

Quantitative real-time PCR: Total RNA was extracted with TRIzol, reverse transcribed using PrimeScript RT reagent Kit. qPCR was performed using SYBR-Green PCR on an ABI PRISM 7300. mRNA levels were normalized to GAPDH and calculated by 2^(-ΔΔCT) method [4].

ELISA: IL-1β and IL-18 levels in culture media were determined using commercial rat ELISA kits according to manufacturer's instructions [4].

Animal/Disease Models: Balb/C female mouse model [1]
Doses: 30 mg/kg, 60 mg/kg
Route of Administration: po (oral gavage) (Bao); single dose;
Experimental Results: LC3B expression diminished, Beclin1/p62 expression increased ( LC3B and Beclin1/p62 are autophagy markers). Reduces IgE and IL-4/IL-10 levels in a dose-dependent manner. (IgE and IL-4/IL-10 are allergic inflammatory mediators) Animal/Disease Models: KAO rat model [4]
Doses: 6.25 mg sodium iodoacetate (MIA): 1 mg
Route of Administration: po (oral gavage); Single dose
Experimental Results: Reduce the degree of local hypoxia in the synovial tissue of rats and Dramatically reduce the levels of pro-fibrotic substances in the synovial tissue. Inhibits HIF-1α accumulation and NLRP3 inflammasome activation.

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Twenty-four 2-month-old SD male rats (weight 210-250 g) were randomly assigned to three groups: normal, KOA, and KOA+AGN (n=8 each). On Day 1, KOA model was constructed by intra-articular injection of 1 mg monosodium iodoacetate (MIA) into both knees. After 14 days (Day 14), drug administration began [4].

AGN (6.25 mg) was prepared as suspension in 0.5% w/v sodium carboxymethyl cellulose in 10 ml sterilized physiologic saline. The KOA+AGN group received an oral dose (1 ml/100 g body weight/day) by oral gavage for 21 days (until Day 35). The other two groups received equal volume of sodium carboxymethyl cellulose solution [4].

On Day 35, all rats were anesthetized with Nembutal, then abdominal aortic serum and synovial tissues were collected [4].

For pimonidazole staining to investigate synovium tissue hypoxia, rats were injected with pimonidazole HCl at 60 mg/kg for 45 min prior to sacrifice [4].

Synovial tissues were fixed in 10% neutral formalin, embedded in paraffin, and cut into slices for HE staining (scored according to Krenn's criteria), Sirius Red staining, and immunohistochemistry [4].

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Twenty-four 2-month-old SD male rats (weight 210-250 g) were randomly assigned to three groups: normal, KOA, and KOA+AGN (n=8 each). On Day 1, KOA model was constructed by intra-articular injection of 1 mg monosodium iodoacetate (MIA) into both knees. After 14 days (Day 14), drug administration began [4].

AGN (6.25 mg) was prepared as suspension in 0.5% w/v sodium carboxymethyl cellulose in 10 ml sterilized physiologic saline. The KOA+AGN group received an oral dose (1 ml/100 g body weight/day) by oral gavage for 21 days (until Day 35). The other two groups received equal volume of sodium carboxymethyl cellulose solution [4].

On Day 35, all rats were anesthetized with Nembutal, then abdominal aortic serum and synovial tissues were collected [4].

For pimonidazole staining to investigate synovium tissue hypoxia, rats were injected with pimonidazole HCl at 60 mg/kg for 45 min prior to sacrifice [4].

Synovial tissues were fixed in 10% neutral formalin, embedded in paraffin, and cut into slices for HE staining (scored according to Krenn's criteria), Sirius Red staining, and immunohistochemistry [4].

Agnuside is described as a non-toxic, natural small molecule extract of Vitex agnus-castus. [3]

[1]. Agnuside mitigates OVA-LPS induced perturbed lung homeostasis via modulating inflammatory, autophagy, apoptosis-fibrosis response and myeloid lineages in mice model of allergic asthma. Int Immunopharmacol. 2022 May;106:108579.

[2]. Iridoid derivatives from Vitex rotundifolia L. f. with their anti-inflammatory activity. Phytochemistry. 2023 Jun;210:113649.

[3]. Pillarisetti P, Myers KA. Identification and characterization of agnuside, a natural proangiogenic small molecule. Eur J Med Chem. 2018 Dec 5;160:193-206.

[4]. Agnuside Alleviates Synovitis and Fibrosis in Knee Osteoarthritis through the Inhibition of HIF-1α and NLRP3 Inflammasome. Mediators Inflamm. 2021 Mar 16;2021:5534614.

Agnuside is a benzoic acid ester formed by the condensation of the carboxyl group of 4-hydroxybenzoic acid with the primary hydroxyl group of aucubin. It is an iridoid glycoside found in various Vitex species, including Vitex agnus-castus. Agnuside has multiple functions, including as a plant metabolite, anti-inflammatory agent, an angiogenic agent, and a cyclooxygenase 2 inhibitor. It is a terpene glycoside, iridoid monoterpene, benzoic acid ester, phenolic compound, β-D-glucoside, cyclopentapran, and monosaccharide derivative. Its function is related to aucubin. Agnuside has been reported in Vitex negundo, Castilleja tenuiflora, and several other organisms with relevant data. See also: Vitex fruit (partial); Vitex negundo leaf.

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AGN (agnuside) is a nontoxic, natural small molecule isolated from the extract of Vitex negundo L. (Verbenaceae), belonging to iridoid glycosides [4].

AGN has been demonstrated to have antioxidation, anti-inflammatory, analgesia, and many other properties [4].

This study is the first to reveal that HIF-1α and NLRP3 inflammasomes are effective intervention targets for AGN [4].

AGN alleviates synovitis and fibrosis in experimental knee osteoarthritis (KOA) through inhibition of HIF-1α accumulation and NLRP3 inflammasome activation, suggesting its potential value in treating KOA in humans [4].

C22H26O11

466.4352

466.147

11027-63-7

442416

White to yellow solid powder

1.6±0.1 g/cm3

785.5±60.0 °C at 760 mmHg

134-136ºC

273.5±26.4 °C

0.0±2.9 mmHg at 25°C

1.681

-1.24

6

11

7

33

747

9

O(C1([H])C([H])(C([H])(C([H])(C([H])(C([H])([H])O[H])O1)O[H])O[H])O[H])C1([H])C2([H])C(C([H])([H])OC(C3C([H])=C([H])C(=C([H])C=3[H])O[H])=O)=C([H])C([H])(C2([H])C([H])=C([H])O1)O[H]

GLACGTLACKLUJX-QNAXTHAFSA-N

InChI=1S/C22H26O11/c23-8-15-17(26)18(27)19(28)22(32-15)33-21-16-11(7-14(25)13(16)5-6-30-21)9-31-20(29)10-1-3-12(24)4-2-10/h1-7,13-19,21-28H,8-9H2/t13-,14+,15+,16+,17+,18-,19+,21-,22-/m0/s1

[(1S,4aR,5S,7aS)-5-hydroxy-1-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-7-yl]methyl 4-hydroxybenzoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~100 mg/mL (~214.39 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.36 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 (5.36 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 (5.36 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.)