NF-κB p65 [1]

Wnt/β-catenin (active β-catenin) [1]

HIF-1α [2]

VEGFA [2]

PHD-2 [2]

In dose-dependent fashion, spencezhenide (0.2-5.0 μg/mL) dramatically lowers CoCl2-induced VEGFA mRNA expression in ARPE-19 cells [2].

---

specnuzehneide did not induce significant cytotoxicity in rat chondrocytes at concentrations of 0–200 μM after 24 h and 48 h incubation (CCK‑8 assay). It did not affect the expression of collagen II, sox9, MMP‑3, or MMP‑9 under normal conditions. [1]

In IL‑1β‑stimulated (5 ng/ml) rat chondrocytes, specnuzehneide (10–200 μM) down‑regulated mRNA expression of MMP‑3, MMP‑9, IL‑6, iNOS, and COX‑2 after 24 h; the mRNA levels of collagen II and sox9 were not significantly improved. Protein levels (Western blot) after 48 h: COX‑2 down‑regulated by all concentrations; MMP‑3, MMP‑9, IL‑6, iNOS significantly decreased at 200 μM; collagen II and sox9 protein significantly increased only at 200 μM. [1]

In LPS‑induced RAW264.7 cells (10 ng/ml LPS, 24 h), specnuzehneide (50–200 μM) significantly down‑regulated both mRNA and protein levels of iNOS and COX‑2. [1]

specnuzehneide (50–200 μM, 6 h) inhibited IL‑1β‑induced (5 ng/ml) increase of NF‑κB p65 (total and phosphorylated) and β‑catenin (total and active non‑phospho) in rat chondrocytes, as shown by Western blot. No inhibition of MAPK pathway (ERK, JNK, p38) was observed. [1]

Luciferase reporter gene assay: specnuzehneide (50–200 μM, 1 h pre‑treatment) decreased IL‑1β‑induced (5 ng/ml, 6 h for NF‑κB‑Luc; 12 h for TCF‑LEF RE) transcriptional activity of NF‑κB and Wnt/β‑catenin pathways. [1]

Immunofluorescence microscopy: specnuzehneide (200 μM, 1 h pre‑treatment) reduced IL‑1β‑induced nuclear translocation of NF‑κB p65 (30 min stimulation) and β‑catenin (6 h stimulation). [1]

Western blot of nuclear/cytoplasmic fractions: specnuzehneide (200 μM, 6 h) reduced nuclear protein levels of NF‑κB p65 and β‑catenin without affecting cytoplasmic levels. [1]

In ARPE‑19 cells under hypoxia‑mimicking condition (150 μM CoCl₂), specnuzehneide (0.2, 1.0, 5.0 μg/ml) significantly decreased VEGFA secretion (ELISA) after 48 h. [2]

qRT‑PCR (24 h): specnuzehneide (0.2–5.0 μg/ml) down‑regulated mRNA expression of VEGFA and PHD‑2 in CoCl₂‑treated ARPE‑19 cells, but did not significantly affect HIF‑1α mRNA. [2]

Western blot (24 h): specnuzehneide (0.2–5.0 μg/ml) significantly decreased protein levels of HIF‑1α and PHD‑2 in CoCl₂‑treated ARPE‑19 cells. [2]

In a rat osteoarthritis model (ACL and MM transection), intra‑articular injection of specnuzehneide (200 μM, 0.14 mg/kg) every 7 days for 8 weeks significantly reduced cartilage degeneration. The mean OARSI grade was 2.95 in the SPN group vs. 4.6 in the OA group. Immunohistochemistry showed decreased MMP‑3 and COX‑2 levels in the SPN group. [1]

μCT analysis of femoral subchondral bone: specnuzehneide treatment significantly increased connectivity density (Conn.D) compared to OA group, but did not significantly improve BV/TV or Tb.N. [1]

In an oxygen‑induced retinopathy (OIR) rat model (80% O₂ from postnatal day 7 to 12, then returned to room air), intraperitoneal administration of specnuzehneide (5.0 and 10.0 mg/kg) on PD12 significantly reduced retinal neovascular areas (ADPase staining) and neovascular nuclei/vessels (H&E staining) in a dose‑dependent manner. [2]

Chondrocyte viability: Rat chondrocytes (5000/well in 96‑well plates) were incubated with specnuzehneide (0, 10, 50, 100, 200 μM) for 24 h and 48 h, then CCK‑8 reagent (10 μl) added for 4 h, and OD measured at 450 nm. [1]

mRNA expression in chondrocytes: Cells were pre‑treated with specnuzehneide (0–200 μM) for 1 h, then stimulated with IL‑1β (5 ng/ml) for 24 h. Total RNA extracted with TRIzol, cDNA synthesized, real‑time PCR performed with SYBR Green. Primers for MMP‑3, MMP‑9, IL‑6, iNOS, COX‑2, collagen II, sox9, GAPDH. [1]

Western blot in chondrocytes: Cells pre‑treated with specnuzehneide (0–200 μM) for 1 h, then with IL‑1β (5 ng/ml) for 48 h. Whole cell lysates prepared with RIPA buffer containing protease/phosphatase inhibitors. Proteins separated by 10% SDS‑PAGE, transferred to PVDF, blocked with 5% BSA, incubated with primary antibodies (anti‑MMP‑3, MMP‑9, IL‑6, iNOS, COX‑2, collagen II, sox9, β‑catenin, active β‑catenin, NF‑κB p65, phospho‑NF‑κB p65, GAPDH, β‑actin, TBP), then HRP‑conjugated secondary antibodies, developed with ECL. [1]

NF‑κB and Wnt/β‑catenin signaling: Chondrocytes treated with specnuzehneide (0–200 μM) for 6 h with/without IL‑1β (5 ng/ml). For MAPK: pre‑treated 2 h then IL‑1β 30 min. Western blot as above. [1]

Immunofluorescence: Chondrocytes on coverslips fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X‑100, incubated with primary antibodies (NF‑κB p65 or β‑catenin) overnight at 4 °C, then fluorochrome‑conjugated secondary antibody for 2 h, mounted with DAPI‑containing medium. [1]

Luciferase reporter assay: Chondrocytes stably transfected with NF‑κB‑Luc or TCF‑LEF RE construct. Transfected cells pre‑treated with specnuzehneide (0–200 μM) for 1 h, then stimulated with IL‑1β (5 ng/ml) for 6 h (NF‑κB) or 12 h (TCF‑LEF). Luciferase activity measured. [1]

ARPE‑19 cell culture and VEGFA secretion: ARPE‑19 cells seeded in 96‑well plates (8×10³/well) in DMEM/F12 with 10% FBS. After 24 h, medium replaced with FBS‑free medium. Cells treated with specnuzehneide (0.2, 1.0, 5.0 μg/ml) or YC‑1 (1.0 μg/ml). 24 h later, CoCl₂ (150 μM) added to mimic hypoxia. Conditioned medium collected after another 48 h, centrifuged, supernatants stored at −80 °C. VEGFA measured by ELISA. [2]

RNA isolation and qRT‑PCR in ARPE‑19 cells: Cells seeded in 6‑well plates (1×10⁵/well), treated as above for 24 h, harvested, total RNA extracted with TRIzol, cDNA synthesized, qRT‑PCR with primers for VEGFA, HIF‑1α, PHD‑2, β‑actin. [2]

Western blot in ARPE‑19 cells: Cells seeded in culture bottles (2×10⁵/bottle), treated for 24 h, lysed in RIPA buffer, protein quantitated by BCA, separated on 7.5% SDS‑PAGE, transferred to nitrocellulose, blocked with 5% skim milk, incubated with anti‑HIF‑1α (1:2000), anti‑PHD‑2 (1:1500), or anti‑β‑actin (1:800), then HRP‑labeled secondary antibodies, developed with ECL. [2]

Rat osteoarthritis model: Sprague‑Dawley rats (200‑250 g, 6 weeks old) underwent surgical transaction of anterior cruciate ligament (ACL) and medial meniscus (MM) of the knee joint under pentobarbital anesthesia (40 mg/kg). One week post‑surgery, rats received intra‑articular injection of specnuzehneide solution (200 μM, 0.14 mg/kg) in 200 μl volume every 7 days for 8 weeks. Control OA group received equal volume of vehicle. Sham group received sham surgery. Rats sacrificed after 8 weeks, knees fixed in 4% paraformaldehyde. [1]

Oxygen‑induced retinopathy (OIR) rat model: Sprague‑Dawley rat pups with nursing mothers were placed in 80% oxygen atmosphere from postnatal day (PD) 7 to PD 12 (5 days). On PD 12, returned to normoxia and intraperitoneally (i.p.) administered with specnuzehneide (5.0 or 10.0 mg/kg) in 0.5 ml normal saline. Positive control received conbercept (1.0 mg/kg), negative control received normal saline. On PD 17, pups sacrificed, eyes enucleated, fixed in 4% paraformaldehyde for 2 h. Retinal flat mounts prepared and stained with ADPase. For histology, paraffin sections (6 mm) stained with H&E. [2]

specnuzehneide showed no significant cytotoxicity in rat chondrocytes at concentrations up to 200 μM after 24 h and 48 h incubation (CCK‑8 assay). [1]

[1]. Specnuezhenide Decreases Interleukin-1β-Induced Inflammation in Rat Chondrocytes and Reduces Joint Destruction in Osteoarthritic Rats.Front Pharmacol. 2018 Jun 28;9:700.

[2]. Inhibition of Hypoxia-Induced Retinal Angiogenesis by Specnuezhenide, an Effective Constituent of Ligustrum lucidum Ait., through Suppression of the HIF-1α/VEGF Signaling Pathway.Molecules. 2016 Dec 21;21(12). pii: E1756.

specnuzehneide is an iridoid glycoside isolated from Ligustrum lucidum fruits. It has been used as a quality control marker for this herb and its preparations. [2]

The compound exhibits anti‑inflammatory effects in osteoarthritis by inhibiting NF‑κB and Wnt/β‑catenin signaling pathways, reducing cartilage matrix‑degrading enzymes (MMP‑3, MMP‑9, etc.) and increasing chondrocyte‑specific genes (collagen II, sox9). It also reduces joint destruction and subchondral bone degeneration in OA rats. [1]

In diabetic retinopathy models, specnuzehneide inhibits hypoxia‑induced retinal neovascularization via suppression of HIF‑1α/VEGF signaling pathway, reducing VEGFA secretion from retinal pigment epithelial cells. [2]

Potential therapeutic applications: osteoarthritis and diabetic retinopathy. No FDA warnings or clinical trial data reported. [1][2]

C31H42O17

686.6550

686.242

39011-92-2

11146840

White to off-white solid

1.5±0.1 g/cm3

893.8±65.0 °C at 760 mmHg

282.0±27.8 °C

0.0±0.3 mmHg at 25°C

1.636

-0.31

8

17

14

48

1120

12

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])O[H])O[H])O[C@@]1([H])/C(=C(/[H])\C([H])([H])[H])/C([H])(C(C(=O)OC([H])([H])[H])=C([H])O1)C([H])([H])C(=O)OC([H])([H])[C@]1([H])[C@]([H])([C@@]([H])([C@]([H])([C@]([H])(OC([H])([H])C([H])([H])C2C([H])=C([H])C(=C([H])C=2[H])O[H])O1)O[H])O[H])O[H]

STKUCSFEBXPTAY-DTYPFZMBSA-N

InChI=1S/C31H42O17/c1-3-16-17(18(28(41)42-2)12-45-29(16)48-31-27(40)24(37)22(35)19(11-32)46-31)10-21(34)44-13-20-23(36)25(38)26(39)30(47-20)43-9-8-14-4-6-15(33)7-5-14/h3-7,12,17,19-20,22-27,29-33,35-40H,8-11,13H2,1-2H3/b16-3+/t17-,19+,20+,22+,23+,24-,25-,26+,27+,29-,30+,31-/m0/s1

methyl (4S,5E,6S)-5-ethylidene-4-[2-oxo-2-[[(2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-[2-(4-hydroxyphenyl)ethoxy]oxan-2-yl]methoxy]ethyl]-6-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4H-pyran-3-carboxylate

Specnuezhenide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~250 mg/mL (~364.08 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (9.10 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 62.5 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.

---

Solubility in Formulation 2: ≥ 6.25 mg/mL (9.10 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 62.5 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.

---

View More

Solubility in Formulation 3: ≥ 6.25 mg/mL (9.10 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 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

---

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