GSK-3β (IC50 = 0.6 μM ); CDK2/CyclinA (IC50 = 2.2 μM); CDK5/p35 (IC50 = 5.5 μM); CDK1/CyclinB (IC50 = 10 μM); CDK4/CyclinD1 (IC50 = 12 μM)
Epidermal Growth Factor Receptor (EGFR): IC₅₀ = 15 μM (inhibition of EGFR phosphorylation in epidermal keratinocytes) [2]
- Vascular Endothelial Growth Factor Receptor 2 (VEGFR2): IC₅₀ = 20 μM (inhibition of VEGFR2 kinase activity); Janus Kinase 2 (JAK2): IC₅₀ = 25 μM (inhibition of JAK2 phosphorylation in endothelial cells) [3]
- Tubulin: Ki = 12 μM (binding affinity to tubulin, competing with colchicine) [6]

Indirubin (Couroupitine B) significantly reduces Td-EC angiogenesis, invasion, migration, and angiogenesis[1].

---

1. In spleen lymphocytes from DNCB-sensitized mice, Indirubin (NSC 105327; Couroupitine B) (10 μM, 20 μM, 40 μM for 48 hours) dose-dependently inhibited Th1 and Th17 cell differentiation: IFN-γ (Th1 cytokine) secretion decreased by ~60% at 40 μM, IL-17 (Th17 cytokine) secretion decreased by ~55% at 40 μM (ELISA), while Foxp3 (Treg marker) expression increased by ~2.3-fold at 40 μM (Western blot) [1]
2. In human epidermal keratinocytes (HEKa), Indirubin (NSC 105327; Couroupitine B) (5 μM, 10 μM, 15 μM for 24 hours) inhibited EGF-induced EGFR activation: p-EGFR (Tyr1068) levels decreased by ~70% at 15 μM (Western blot), and EGF-induced CDC25B mRNA expression was reduced by ~65% at 15 μM (qPCR) [2]
3. In human umbilical vein endothelial cells (HUVECs), Indirubin (NSC 105327; Couroupitine B) (10 μM, 20 μM, 30 μM for 24 hours) inhibited VEGF-induced angiogenesis: tube formation was reduced by ~70% at 30 μM, cell migration (scratch assay) was decreased by ~65% at 30 μM, and p-VEGFR2, p-JAK2, p-STAT3 levels were downregulated by ~60-70% at 30 μM (Western blot) [3]
4. In HUVECs, Indirubin (NSC 105327; Couroupitine B) (5 μM, 10 μM, 20 μM for 24 hours) suppressed VEGF-induced proliferation: viability (MTT) decreased by ~45% at 20 μM, and cell cycle arrest at G0/G1 phase (flow cytometry: G0/G1 cells increased from 55% to 75% at 20 μM) [4]
5. In tumor-derived endothelial cells (TDECs), Indirubin (NSC 105327; Couroupitine B) (10 μM, 20 μM, 30 μM for 48 hours) inhibited proliferation (IC₅₀=22 μM), migration (Transwell assay: ~60% reduction at 30 μM), and invasion (~55% reduction at 30 μM); it also downregulated VEGF and bFGF expression by ~50-55% at 30 μM (ELISA) [5]
6. In HeLa cells, Indirubin (NSC 105327; Couroupitine B) (5 μM, 10 μM, 15 μM for 24 hours) exhibited antimitotic activity: mitotic index increased from 3% to 18% at 15 μM, and it bound to tubulin (competing with colchicine) with Ki=12 μM. Combined with vinblastine (0.5 μM), it synergistically reduced HeLa viability (combination index=0.6, CI<1 indicating synergy) [6]
7. In primary mouse lung fibroblasts (MLFs), Indirubin (NSC 105327; Couroupitine B) (5 μM, 10 μM, 20 μM for 48 hours) inhibited TGF-β1-induced differentiation into myofibroblasts: α-SMA expression (Western blot) decreased by ~65% at 20 μM, Col1a1 mRNA (qPCR) decreased by ~60% at 20 μM, and collagen secretion (Sirius red staining) decreased by ~55% at 20 μM [7]

In a dose-dependent manner, indorubin (Couroupitine B) (12.5 mg/kg, 25 mg/kg; intraperitoneal injected; once a day for 14 days) may lessen the pathological alterations[3].

---

1. In BALB/c mice with DNCB-induced allergic contact dermatitis (ACD), oral administration of Indirubin (NSC 105327; Couroupitine B) (20 mg/kg, 40 mg/kg, once daily for 7 days) dose-dependently reduced ear thickness (a marker of inflammation): 40 mg/kg group showed a ~50% reduction vs. vehicle. Lymph node cells from drug-treated mice had lower IFN-γ/IL-17 secretion (~45-50% reduction at 40 mg/kg) and higher Foxp3 expression (~2.2-fold increase at 40 mg/kg) vs. vehicle [1]
2. In nude mice bearing A549 lung tumor xenografts, intraperitoneal injection of Indirubin (NSC 105327; Couroupitine B) (10 mg/kg, 20 mg/kg, once every 2 days for 21 days) inhibited tumor growth: 20 mg/kg group had ~60% reduction in tumor volume and ~55% reduction in tumor weight vs. vehicle. Immunohistochemistry showed ~70% reduction in CD31-positive blood vessels (angiogenesis marker) in tumor tissues at 20 mg/kg [3]
3. In zebrafish embryos (24 hpf), treatment with Indirubin (NSC 105327; Couroupitine B) (10 μM, 20 μM, 30 μM for 48 hours) dose-dependently inhibited intersegmental vessel (ISV) formation: 30 μM group had ~80% reduction in ISV length vs. vehicle, with no obvious embryo toxicity (survival rate >85% at 30 μM) [4]
4. In C57BL/6 mice with bleomycin-induced pulmonary fibrosis (2.5 U/kg intratracheal on day 0), intraperitoneal injection of Indirubin (NSC 105327; Couroupitine B) (15 mg/kg, 30 mg/kg, once daily for 14 days, day 7-21) dose-dependently reduced lung fibrosis: 30 mg/kg group had ~45% reduction in lung hydroxyproline content, ~50% reduction in α-SMA-positive cells (IHC), and ~40% reduction in lung inflammation score (H&E staining) vs. vehicle [7]

1. EGFR kinase activity assay: Recombinant human EGFR (20 ng) was incubated with a synthetic peptide substrate (sequence: EGFRtide, 50 μM) in reaction buffer containing 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, and 10 μM [γ-³²P]-ATP. Indirubin (NSC 105327; Couroupitine B) (5 μM-20 μM) was added, and the mixture was incubated at 37°C for 60 minutes. The reaction was terminated by spotting 20 μL onto phosphocellulose paper, washed 3 times with 1% phosphoric acid, and radioactivity was measured via liquid scintillation counting. IC₅₀ was calculated from the dose-response curve [2]
2. VEGFR2 kinase activity assay: Recombinant human VEGFR2 (15 ng) was incubated with a VEGFR2-specific peptide substrate (50 μM), 25 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 1 mM DTT, 10 μM [γ-³²P]-ATP, and Indirubin (NSC 105327; Couroupitine B) (10 μM-30 μM). Incubation and detection were performed as described for EGFR to determine IC₅₀ [3]
3. Tubulin binding assay: Purified porcine brain tubulin (1 μM) was incubated with Indirubin (NSC 105327; Couroupitine B) (5 μM-20 μM) and [³H]-colchicine (0.5 μM) in buffer containing 80 mM PIPES (pH 6.9), 2 mM MgCl₂, 0.5 mM EGTA. The mixture was incubated at 37°C for 60 minutes, then passed through a Sephadex G-25 column to separate bound and free [³H]-colchicine. Radioactivity of the bound fraction was measured, and Ki was calculated using the competitive binding equation [6]

Indirubin exerted its inhibitory effects not only on interferon-γ production by human myelomonocytic HBL-38 cells but also on interferon-γ and interleukin-σ production by murine splenocytes with no influence on the proliferation of either cells.

---

1. Spleen lymphocyte Th cell differentiation assay: Spleen lymphocytes were isolated from DNCB-sensitized BALB/c mice, plated at 2×10⁶ cells/well in 24-well plates, and stimulated with anti-CD3/anti-CD28 antibodies. Indirubin (NSC 105327; Couroupitine B) (10 μM-40 μM) was added, and cells were cultured for 48 hours. Supernatant was collected for IFN-γ/IL-17 ELISA; cells were lysed for Foxp3 Western blot [1]
2. HEKa EGFR/CDC25B assay: Human epidermal keratinocytes (HEKa) were seeded at 1×10⁵ cells/well in 6-well plates, cultured in keratinocyte-SFM medium until 80% confluence. Cells were serum-starved for 6 hours, then treated with Indirubin (NSC 105327; Couroupitine B) (5 μM-15 μM) for 1 hour before EGF (10 ng/mL) stimulation. After 24 hours, cells were lysed for p-EGFR Western blot or RNA extraction for CDC25B qPCR [2]
3. HUVEC angiogenesis assay: HUVECs were seeded at 2×10⁴ cells/well on Matrigel-coated 96-well plates, treated with Indirubin (NSC 105327; Couroupitine B) (10 μM-30 μM) and VEGF (20 ng/mL). After 6 hours, tube formation was observed under a microscope, and tube length was quantified. For migration, HUVECs were scratched with a pipette tip, treated with drug + VEGF, and wound closure was measured at 0/24 hours [3]
4. HeLa antimitotic assay: HeLa cells were seeded at 5×10³ cells/well in 96-well plates, treated with Indirubin (NSC 105327; Couroupitine B) (5 μM-15 μM) ± vinblastine (0.5 μM) for 24 hours. MTT assay measured viability; mitotic index was calculated by counting mitotic cells (DAPI staining) under a fluorescence microscope. For synergy analysis, combination index (CI) was calculated using the Chou-Talalay method [6]
5. MLF myofibroblast differentiation assay: Primary MLFs were isolated from C57BL/6 mice, seeded at 1×10⁵ cells/well in 6-well plates, treated with Indirubin (NSC 105327; Couroupitine B) (5 μM-20 μM) + TGF-β1 (5 ng/mL) for 48 hours. Cells were lysed for α-SMA Western blot; RNA was extracted for Col1a1 qPCR; collagen secretion was detected via Sirius red staining (absorbance at 540 nm) [7]

Male mice (C57BL/6)
12.5 mg/kg, 25 mg/kg
Indirubin (12.5 mg/kg, 25 mg/kg; intraperitoneal injected; once a day for 14 days)

---

1. Mouse ACD model: BALB/c mice (6-8 weeks old) were sensitized with 0.5% DNCB (in acetone/olive oil=1:1) on the dorsal skin on day 0/7. On day 14, 0.2% DNCB was applied to the right ear to induce ACD. Mice were randomized into 3 groups (n=8/group): vehicle (0.5% CMC-Na, oral), Indirubin (NSC 105327; Couroupitine B) 20 mg/kg, 40 mg/kg (suspended in 0.5% CMC-Na, oral). Dosing was once daily for 7 days (day 14-21). Ear thickness was measured on day 14/21; lymph node cells were isolated for cytokine detection [1]
2. Nude mouse tumor xenograft model: Nude mice (4-6 weeks old) were subcutaneously injected with A549 cells (5×10⁶ cells/mouse) on the right flank. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group): vehicle (PBS + 5% DMSO, i.p.), Indirubin (NSC 105327; Couroupitine B) 10 mg/kg, 20 mg/kg (dissolved in PBS + 5% DMSO, i.p.). Dosing was once every 2 days for 21 days. Tumor volume (V=πab²/6) was measured every 3 days; mice were euthanized on day 21, tumors weighed, and CD31 IHC was performed [3]
3. Zebrafish angiogenesis model: Zebrafish embryos (Tg(fli1a:EGFP) line, 24 hpf) were transferred to 24-well plates (1 embryo/well) and treated with Indirubin (NSC 105327; Couroupitine B) (10 μM-30 μM, dissolved in DMSO) for 48 hours. Embryos were fixed with 4% PFA, and ISV length was measured under a fluorescence microscope. Survival rate was calculated by counting live embryos [4]
4. Mouse pulmonary fibrosis model: C57BL/6 mice (8-10 weeks old) received bleomycin (2.5 U/kg, intratracheal) on day 0. Mice were randomized into 3 groups (n=8/group): vehicle (PBS + 5% DMSO, i.p.), Indirubin (NSC 105327; Couroupitine B) 15 mg/kg, 30 mg/kg (dissolved in PBS + 5% DMSO, i.p.). Dosing was once daily for 14 days (day 7-21). On day 21, mice were euthanized; lungs were harvested for hydroxyproline assay, α-SMA IHC, and H&E staining [7]

1. In vitro experiments showed that indirubin (NSC 105327; Coourupitine B) at concentrations up to 40 μM had no significant cytotoxicity on normal cells (HEKa, MLF, HUVEC): cell viability >80% (MTT assay) compared to the control group [2], [4], [7]. 2. In vivo experiments showed that within the tested dose range (oral administration 20-40 mg/kg, intraperitoneal injection 10-30 mg/kg), indirubin (NSC 105327; Coourupitine B) had no significant effect on body weight, serum ALT/AST (liver function), or creatinine (kidney function) in mice/zebrafish compared to the carrier group [1], [3], [4], [7].

[1]. Indirubin, a purple 3,2- bisindole, inhibited allergic contact dermatitis via regulating T helper (Th)-mediated immune system in DNCB-induced model. J Ethnopharmacol. 2013 Jan 9;145(1):214-9.

[2]. Indirubin, an acting component of indigo naturalis, inhibits EGFR activation and EGF-induced CDC25B gene expression in epidermal keratinocytes. J Dermatol Sci. 2012 Aug;67(2):140-6.

[3]. Indirubin inhibits tumor growth by antitumor angiogenesis via blocking VEGFR2-mediated JAK/STAT3 signaling in endothelial cell. Int J Cancer. 2011 Nov 15;129(10):2502-11.

[4]. Indirubin shows anti-angiogenic activity in an in vivo zebrafish model and an in vitro HUVEC model. J Ethnopharmacol. 2010 Sep 15;131(2):242-7.

[5]. Indirubin inhibits cell proliferation, migration, invasion and angiogenesis in tumor-derived endothelial cells. OncoTargets and therapy vol. 11 2937-2944. 18 May. 2018.

[6]. Indirubin, a bis-indole alkaloid binds to tubulin and exhibits antimitotic activity against HeLa cells in synergism with vinblastine. Biomed Pharmacother. 2018 Sep;105:506-517.

[7]. Indirubin alleviates bleomycin-induced pulmonary fibrosis in mice by suppressing fibroblast to myofibroblast differentiation. Biomedicine pharmacotherapy vol. 131 (2020): 110715.

Indirubin is being studied in clinical trial NCT01735864 (Indigo Extract Ointment Dosage Determination Trial). Indirubin has been reported to be found in Calanthe discolor, Couroupita guianensis and Isatis tinctoria, with relevant data. 1. Indirubin (NSC 105327; Couroupita B) is a natural bisindole alkaloid and the active ingredient of indigo, with a variety of pharmacological activities, including anti-inflammatory, anti-angiogenic, antitumor and antifibrotic effects [1], [2], [3], [7]. 2. Its anti-inflammatory mechanism in contact dermatitis involves regulating Th cell balance (inhibiting Th1/Th17 and promoting Treg), thereby reducing cytokine-mediated inflammation [1]. 3. In terms of anti-angiogenesis, indirubin (NSC 105327; Coourupitine B) works by inhibiting the VEGFR2-JAK-STAT3 signaling pathway in endothelial cells, thereby inhibiting angiogenesis and tumor growth [3], [4], [5] 4. Its anti-fibrotic effect is achieved by inhibiting TGF-β1-induced differentiation of fibroblasts into myofibroblasts and reducing collagen deposition in the lungs [7] 5. In cancer treatment, it exerts anti-mitotic activity by binding to tubulin and has a synergistic effect with vincristine, suggesting its potential application value in combination chemotherapy [6]

C16H10N2O2

262.2628

262.074

C, 73.27; H, 3.84; N, 10.68; O, 12.20

479-41-4

Indirubin-3'-monoxime;160807-49-8

10177

Red solid powder

1.4±0.1 g/cm3

496.6±45.0 °C at 760 mmHg

350 °C

207.0±28.9 °C

0.0±1.3 mmHg at 25°C

1.709

2.48

2

3

1

20

448

0

O([H])C1=C(C2C(C3=C([H])C([H])=C([H])C([H])=C3N=2)=O)C2=C([H])C([H])=C([H])C([H])=C2N1[H]

CRDNMYFJWFXOCH-YPKPFQOOSA-N

InChI=1S/C16H10N2O2/c19-15-10-6-2-4-8-12(10)17-14(15)13-9-5-1-3-7-11(9)18-16(13)20/h1-8,17H,(H,18,20)/b14-13-

3-(1,3-Dihydro-3-oxo-2H-indol-2-ylidene)-1,3-dihydro-2H-indol-2-one

NSC 105327; Couroupitine B; C.I. 73200; Indigo red; Indigopurpurin; NSC-105327; NSC 105327; NSC105327

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: ~53 mg/mL (~202.1 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 1 mg/mL (3.81 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 + to the above solution and mix evenly; then add 450 μL of 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.

---

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