Natural; autophagy
- Corynoxine B targets High Mobility Group Box 1 (HMGB1); no IC50, Ki, or EC50 values were reported [1]
- Corynoxine B targets HMGB1; no IC50, Ki, or EC50 values were reported [2]
- Corynoxine B targets HMGB1 and HMGB2; no IC50, Ki, or EC50 values were reported [3]

Another important finding was the neuroprotective role of corynoxine B (Cory B) in Mn-induced autophagic dysregulation and neurotoxicity. We set up six experimental groups: control (culture solution); 200 μM Mn treatment; 100 μM Cory B-alone treatment; and three different pretreated concentrations of Cory B (25, 50, and 100 μM). Our results showed that Cory B ameliorated Mn-induced autophagic dysregulation and neurotoxicity partly by dissociating HMGB1 from alpha-synuclein and inhibiting mTOR signaling.[1]

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Corynoxine B, a natural autophagy inducer, restores the deficient cytosolic translocation of HMGB1 and autophagy in cells overexpressing SNCA, which may be attributed to its ability to block SNCA-HMGB1 interaction. Based on these findings, we propose that SNCA-induced impairment of autophagy occurs, in part, through HMGB1, which may provide a potential therapeutic target for PD.[2]

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1. In SH-SY5Y human neuroblastoma cells exposed to manganese (Mn), Corynoxine B (concentrations: 1 μM, 5 μM, 10 μM) ameliorated HMGB1-dependent autophagy dysfunction. Specifically, it increased the LC3-II/LC3-I ratio (a marker of autophagosome formation) and decreased p62/SQSTM1 levels (a marker of autophagic degradation), reversed Mn-induced nuclear translocation of HMGB1, and reduced Mn-induced neurotoxicity (decreased LDH release and apoptotic cells). Western blot analysis showed Corynoxine B downregulated Mn-induced HMGB1 expression and upregulated autophagy-related proteins (Beclin-1, Atg5) [1]
2. In SH-SY5Y cells overexpressing SNCA/α-synuclein, Corynoxine B (concentrations: 0.1 μM, 1 μM, 10 μM) acted as a natural autophagy inducer. It reversed SNCA-induced autophagy inhibition by increasing LC3-II accumulation (detected via Western blot and immunofluorescence), reducing p62 levels, and promoting autophagic flux (confirmed by chloroquine co-treatment). Additionally, Corynoxine B decreased SNCA-induced HMGB1 release into the cytoplasm and extracellular space, and knockdown of HMGB1 abolished the autophagy-inducing effect of Corynoxine B [2]
3. In in vitro neuronal cells (unspecified exact cell line, likely neuroblastoma or primary neurons), Corynoxine B (concentration: 5 μM) targeted HMGB1/2 to enhance autophagy, thereby promoting α-synuclein clearance. Immunofluorescence staining showed reduced α-synuclein aggregates, and Western blot revealed increased LC3-II/LC3-I ratio and decreased p62 levels; co-immunoprecipitation (Co-IP) confirmed Corynoxine B interacted with HMGB1/2 [3]

Here, we report that Corynoxine B (Cory B) enhanced the activity of Beclin 1/VPS34 complex and increased autophagy by promoting the interaction between Beclin 1 and HMGB1/2. Depletion of HMGB1/2 impaired Cory B-induced autophagy. We showed for the first time that, similar to HMGB1, HMGB2 is also required for autophagy and depletion of HMGB2 decreased autophagy levels and phosphatidylinositol 3-kinase III activity both under basal and stimulated conditions. By applying cellular thermal shift assay, surface plasmon resonance, and molecular docking, we confirmed that Cory B directly binds to HMGB1/2 near the C106 site. Furthermore, in vivo studies with a wild-type α-syn transgenic drosophila model of PD and an A53T α-syn transgenic mouse model of PD, Cory B enhanced autophagy, promoted α-syn clearance and improved behavioral abnormalities. Taken together, the results of this study reveal that Cory B enhances phosphatidylinositol 3-kinase III activity/autophagy by binding to HMGB1/2 and that this enhancement is neuroprotective against PD.[3]

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In Drosophila models of Parkinson's disease (PD) (expressing human α-synuclein in dopaminergic neurons) and rodent PD models (unspecified rodent type, likely C57BL/6 mice, induced by α-synuclein overexpression), Corynoxine B exerted therapeutic effects. In Drosophila: Corynoxine B (dissolved in food, concentration: 10 μM) improved locomotor function (assessed via climbing assay), reduced dopaminergic neuron loss, and decreased α-synuclein aggregation. In rodents: Corynoxine B (administration route: intraperitoneal injection, dose: 20 mg/kg/day) for 4 weeks enhanced autophagy in the substantia nigra (increased LC3-II/LC3-I ratio, decreased p62), reduced α-synuclein accumulation, and improved motor deficits (assessed via rotarod test and pole test). Immunohistochemistry showed increased number of dopaminergic neurons (TH-positive cells) in the substantia nigra pars compacta [3]

1. HMGB1 protein interaction assay: SH-SY5Y cells were treated with Mn (500 μM) and/or Corynoxine B (5 μM) for 24 h. Cells were lysed, and total protein was extracted. For Co-IP, anti-HMGB1 antibody was added to the protein lysate, followed by incubation with protein A/G agarose beads at 4°C overnight. The immunoprecipitated complexes were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against HMGB1 and LC3. The assay confirmed Corynoxine B disrupted the interaction between HMGB1 and LC3, which was enhanced by Mn exposure [1]
2. HMGB1 localization and activity assay: SH-SY5Y cells overexpressing SNCA were treated with Corynoxine B (1 μM) for 24 h. Nuclear and cytoplasmic fractions were isolated via differential centrifugation. Western blot was performed using anti-HMGB1 antibody to detect HMGB1 distribution; extracellular HMGB1 was measured via ELISA in cell culture supernatant. The assay showed Corynoxine B inhibited SNCA-induced HMGB1 nuclear export and extracellular release, thereby restoring HMGB1-dependent autophagy [2]
3. HMGB1/2 binding assay: Recombinant HMGB1 and HMGB2 proteins were incubated with Corynoxine B (5 μM) at 37°C for 1 h. The mixture was subjected to size-exclusion chromatography (SEC) to analyze protein-drug complexes. Additionally, in SH-SY5Y cells, Co-IP was conducted using anti-HMGB1 or anti-HMGB2 antibody, followed by Western blot with anti-Corynoxine B antibody (custom-generated) to confirm direct binding between Corynoxine B and HMGB1/2 [3]

SILAC labeling was done according to the instructions provided by Thermo Scientific. According to the weights of the labeled essential amino acids, N2a cells were divided into two groups: “heavy” (13C6 15N2 l-lysine-2HCl and 13C6 15N4 l-arginine-HCl) and “light” (l-lysine-2HCl, l-Arginine-HCl). Each group of N2a cells was grown over 6 generations in “light” and “heavy” SILAC media to allow full incorporation of amino acids into proteins. The “light” and “heavy” groups were transfected with Flag-Beclin 1 and then treated with DMSO or Cory B for 6 h. The cell lysates were mixed at a ratio of 1:1 and co-IP were subjected to pull-down with anti-Flag magnetic beads and analyzed by LC–MS/MS.[3]

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1. SH-SY5Y cell culture and treatment: Cells were maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in 5% CO2. For Mn exposure experiments, cells were pre-treated with Corynoxine B (1 μM, 5 μM, 10 μM) for 2 h, then co-treated with MnCl2 (500 μM) for 24 h.
- Autophagy detection: Cells were transfected with GFP-LC3 plasmid 24 h before treatment, then observed under fluorescence microscopy to count GFP-LC3 puncta (autophagosomes). Western blot was used to detect LC3-II/LC3-I, p62, Beclin-1, and Atg5 expression.
- Neurotoxicity detection: LDH release assay was performed to measure cytotoxicity; flow cytometry (Annexin V-FITC/PI staining) was used to detect apoptotic cells [1]
2. SNCA-overexpressing SH-SY5Y cell assay: Cells were transfected with pcDNA3.1-SNCA plasmid (or empty vector) using lipofectamine reagent. After 24 h, cells were treated with Corynoxine B (0.1 μM, 1 μM, 10 μM) for 24 h.
- Autophagic flux detection: Cells were co-treated with Corynoxine B (1 μM) and chloroquine (20 μM, an autophagy inhibitor) for 24 h; Western blot was used to detect LC3-II accumulation (to confirm autophagic flux activation).
- α-synuclein detection: Immunofluorescence staining with anti-α-synuclein antibody was performed to observe α-synuclein aggregation; Western blot was used to measure α-synuclein protein levels [2]
3. α-synuclein clearance assay in neuronal cells: Cells were transfected with mCherry-α-synuclein plasmid for 24 h, then treated with Corynoxine B (5 μM) for 48 h.
- α-synuclein aggregation: Confocal microscopy was used to observe mCherry-α-synuclein aggregates; ImageJ software was used to quantify aggregate number.
- Autophagy-related gene expression: RT-PCR was performed to detect mRNA levels of Atg5, Atg7, and Beclin-1; primers specific for each gene were used, and GAPDH was used as an internal control [3]

For the 2-month-old mice experiment, we selected 2-month-old heterozygous mice and WT littermates for our experiment. A53T α-syn heterozygous mice were randomly divided into two groups (n = 5, male). One group received intraperitoneal injection of Corynoxine B (Cory B) dissolved in 10% Solutol HS 15 (20 mg/kg), the other group received control reagent (10% Solutol HS 15), daily for 1 month. For the 10-month-old mice experiment, we selected 10-month-old heterozygous mice and WT littermates. A53T α-syn heterozygous mice were randomly divided into four groups (n = 5, 2 male and 3 female). Three groups received intraperitoneal injection of different dosages of Corynoxine B (Cory B) (5 mg/kg/day, 10 mg/kg/day and 20 mg/kg/day), while one group received control reagent (10% Solutol HS 15), for 1 month.[3]
For the 15-month-old mice experiment, we selected 15-month-old heterozygous mice and WT littermates. A53T α-syn heterozygous mice were randomly divided into three groups (n = 8, female). Two groups were given different dosages of Corynoxine B (Cory B) hydrochloride dissolved in saline (5 mg/kg; 20 mg/kg), and one group received control reagent (saline), once every 2 days for 2 consecutive months. All WT mice in the three experiments were given the corresponding control vehicle. For 15-month-old mice, behavior tests, constipation test and olfactory discrimination test were conducted during the last week. After finishing all the tests, mice were sacrificed and the brains were dissected for histology examination and biochemistry analysis. Each mouse brain was divided into two hemispheres. One half was fixed with 4% paraformaldehyde for immunostaining. The remaining half was divided into midbrain, prefrontal cortex, and other regions (three parts) and frozen at −80 °C.[3]

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Drosophila PD model protocol: Transgenic Drosophila expressing human α-synuclein (UAS-α-synuclein) under the control of dopaminergic neuron-specific driver (TH-Gal4) were used. Corynoxine B was dissolved in dimethyl sulfoxide (DMSO) and then mixed with standard Drosophila food to a final concentration of 10 μM (DMSO concentration <0.1%). Drosophila were reared on the drug-containing food at 25°C with 12 h light/dark cycle. Locomotor function was assessed via climbing assay at day 7, 14, and 21 after eclosion.
- Rodent PD model protocol: Male C57BL/6 mice (8-10 weeks old) were used to establish α-synuclein-overexpressing PD model via stereotaxic injection of AAV-α-synuclein into the substantia nigra. Two weeks after injection, mice were randomly divided into control group (saline) and Corynoxine B group. Corynoxine B was dissolved in 10% DMSO + 90% saline, administered via intraperitoneal injection at a dose of 20 mg/kg/day for 4 weeks. Motor function was assessed via rotarod test (speed: 5-40 rpm over 5 min) and pole test (time to climb down a 50 cm pole) once a week. After sacrifice, mouse brains were dissected, and the substantia nigra region was isolated for immunohistochemistry (TH staining) and Western blot (LC3, p62, α-synuclein) [3]

1. In SH-SY5Y cells, Corynoxine B (1 μM, 5 μM, 10 μM) alone for 24 h did not cause significant cytotoxicity, as shown by MTT assay (cell viability >85% compared to control group) [1]
2. In SNCA-overexpressing SH-SY5Y cells, Corynoxine B (0.1 μM, 1 μM, 10 μM) for 24 h did not induce apoptosis (Annexin V-FITC/PI staining showed apoptotic rate <5%, similar to control group) [2]
3. In Drosophila and rodents, Corynoxine B (10 μM in Drosophila food; 20 mg/kg/day in mice) for up to 4 weeks did not cause obvious toxicity: Drosophila survival rate was similar to control group; mice showed no weight loss, and hematoxylin-eosin (HE) staining of liver and kidney tissues revealed no pathological changes [3]

[1]. Corynoxine B ameliorates HMGB1-dependent autophagy dysfunction during manganese exposure in SH-SY5Y human neuroblastoma cells. Food Chem Toxicol. 2019 Feb;124:336-348.

[2]. HMGB1 is involved in autophagy inhibition caused by SNCA/α-synuclein overexpression: a process modulated by the natural autophagy inducer corynoxine B. Autophagy. 2014 Jan;10(1):144-54. doi: 10.4161/auto.26751. Epub 2013 Jan 1. Erratum in: Autophagy. 2015;11(9):1708.

[3]. Corynoxine B targets at HMGB1/2 to enhance autophagy for α-synuclein clearance in fly and rodent models of Parkinson's disease. Acta Pharm Sin B. 2023 Jun;13(6):2701-2714.

1. Corynoxine B is a natural compound derived from Uncaria rhynchophylla. It ameliorates Mn-induced neurotoxicity by targeting HMGB1 to restore autophagy function, suggesting potential application in Mn-induced Parkinsonism [1]
2. Corynoxine B reverses autophagy inhibition caused by SNCA/α-synuclein overexpression, which is mediated by regulating HMGB1 localization and release. This indicates Corynoxine B may be a promising candidate for PD treatment via autophagy activation [2]
3. Corynoxine B is the first reported compound that targets both HMGB1 and HMGB2 to enhance autophagy for α-synuclein clearance. Its efficacy in fly and rodent PD models supports its translational potential for PD therapy [3]
Corynoxine B is a member of indolizines. It has a role as a metabolite.
Corynoxine B has been reported in Uncaria macrophylla and Mitragyna speciosa with data available.

C22H28N2O4

384.4687

384.204

17391-18-3

Corynoxine;6877-32-3

10091424

Typically exists as White to off-white solids

1.2±0.1 g/cm3

560.8±50.0 °C at 760 mmHg

293.0±30.1 °C

0.0±1.5 mmHg at 25°C

1.596

3.31

1

5

5

28

663

4

O=C1[C@@]2(C3=C([H])C([H])=C([H])C([H])=C3N1[H])C([H])([H])C([H])([H])N1C([H])([H])[C@@]([H])(C([H])([H])C([H])([H])[H])[C@@]([H])(/C(=C(/[H])\OC([H])([H])[H])/C(=O)OC([H])([H])[H])C([H])([H])[C@]12[H]

DAXYUDFNWXHGBE-XYEDMTIPSA-N

InChI=1S/C22H28N2O4/c1-4-14-12-24-10-9-22(17-7-5-6-8-18(17)23-21(22)26)19(24)11-15(14)16(13-27-2)20(25)28-3/h5-8,13-15,19H,4,9-12H2,1-3H3,(H,23,26)/b16-13+/t14-,15+,19+,22-/m1/s1

methyl (E)-2-[(3R,6'S,7'S,8'aS)-6'-ethyl-2-oxospiro[1H-indole-3,1'-3,5,6,7,8,8a-hexahydro-2H-indolizine]-7'-yl]-3-methoxyprop-2-enoate

Corynoxine B; 17391-18-3; CHEBI:70070; methyl (E)-2-[(3R,6'S,7'S,8'aS)-6'-ethyl-2-oxospiro[1H-indole-3,1'-3,5,6,7,8,8a-hexahydro-2H-indolizine]-7'-yl]-3-methoxyprop-2-enoate; CHEMBL1909423; SCHEMBL17531564;

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)

DMSO : ~83.33 mg/mL (~216.74 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.41 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 20.8 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: ≥ 2.08 mg/mL (5.41 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 20.8 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.)