The study indicates that periplogenin induces necroptotic cell death. The compound's mechanism involves the necroptosis pathway, requiring the involvement of receptor interaction protein kinase 1 (RIPK1) and mixed lineage kinase domain-like protein (MLKL). This is evidenced by the ability of the RIPK1 inhibitor Necrostatin-1 (Nec-1) and the MLKL inhibitor Necrosulfonamide (NSA) to rescue cells from cell death. The study also implicates reactive oxygen species (ROS) as key mediators of this process. [1]

- Periplogenin significantly inhibited the viability of cultured human HaCaT keratinocytes (a psoriasis-relevant in vitro model) in a dose-dependent manner. The IC50 value for HaCaT cells was 1.56 µg/ml. It was less cytotoxic toward Hs-68 human fibroblast cells (IC10 > 10 µg/ml). [1]
- Periplogenin did not induce biochemical features of apoptosis, such as nuclear condensation, chromatin fragmentation, or cleaved caspase-3 expression, as shown by DAPI staining, TUNEL assay, and western blotting. The general caspase inhibitor z-VAD-fmk did not attenuate periplogenin-induced cell death. [1]
- Periplogenin did not induce autophagy, as the levels of Beclin-1 and LC3-II did not increase following treatment, even in the presence of the autophagosome-lysosome fusion inhibitor chloroquine (CQ). Autophagy inhibitors (3-MA, LY294002, CQ) could not decrease periplogenin-induced cell death. [1]
- Periplogenin caused propidium iodide (PI) to permeate into cells and increased lactate dehydrogenase (LDH) release, indicating loss of plasma membrane integrity. Flow cytometry analysis showed an increase in the percentage of primary necrotic cells (FITC negative, PI positive). [1]
- Transmission electron microscopy analysis of periplogenin-treated HaCaT cells revealed morphological characteristics of necrosis, such as cell swelling, organelle swelling, and plasma membrane disruption, without the apoptotic features seen in H2O2-challenged cells. [1]
- The necroptosis inhibitors Nec-1 (an inhibitor of RIPK1) and NSA (an inhibitor of MLKL) were able to significantly rescue cell viability loss and inhibit necrosis in periplogenin-treated HaCaT cells, confirming the involvement of necroptosis. [1]
- Periplogenin induced a dose- and time-dependent accumulation of reactive oxygen species (ROS). Treatment with 2 µg/ml periplogenin increased ROS levels by 3.14-fold (12h) and 6.23-fold (24h). The antioxidant N-acetyl-cysteine (NAC) reduced ROS production and significantly reversed cell death caused by periplogenin, indicating that the necroptotic cell death is mediated by oxidative stress. Nec-1 could also significantly inhibit the ROS levels caused by periplogenin. [1]

- In a TPA (12-O-tetradecanoylphorbol 13-acetate)-induced psoriasis-like mouse model (epidermal hyperplasia), topical administration of periplogenin (2 µg per site) for 11 consecutive days ameliorated skin lesions. It significantly reduced ear thickness and ear weight compared to the TPA-treated control group. Histological analysis via H&E staining showed that periplogenin attenuated epidermal hyperplasia and leukocyte infiltration. [1]
- In an IMQ (imiquimod)-induced psoriasis-like mouse model (skin inflammation), topical administration of periplogenin (2 µg per site) for 11 consecutive days ameliorated the course of the disease, reducing scaling, erythema, ear thickness, and ear weight. Histological analysis confirmed that periplogenin prevented IMQ-induced epidermal hyperplasia and increased leukocyte infiltration. The reference compound dexamethasone (200 mg per site) also ameliorated these conditions. [1]

- Compound Screening and Cell Viability (MTT assay): HaCaT cells were plated in triplicate in 96-well plates (1.5×10^4 cells/well). After 12 hours, they were treated with 5 µg/ml of compounds or DMSO in 3% serum. After 44 hours, 20 µl of MTT (5 mg/ml) was added for 4 hours. The formazan precipitate was dissolved in 100 µl DMSO and absorbance measured at 570 nm. For viability assays, cells were treated with periplogenin for 44 hours at indicated concentrations. Cell viability was calculated as (OD experimental / OD control) × 100%. [1]
- Real-Time Cell Analysis (RTCA): HaCaT cells were seeded at 1.5×10^4 cells/well on an E-Plate. After 12 hours, they were treated with DMSO or periplogenin (0.5, 1, and 2 µg/ml). Dynamic cell index (CI) values, which integrate living cell number and size, were monitored at 15-minute intervals for 38 hours. A decreased CI indicated reduced cell viability. [1]
- Nuclear Staining (DAPI and TUNEL): HaCaT cells treated with periplogenin (2 µg/ml), ADM (2 µg/ml), or DMSO for 12-24 hours were fixed with 4% paraformaldehyde, stained with DAPI (0.5 µg/ml), and analyzed by fluorescence microscopy. For TUNEL, fixed and permeabilized cells were stained with TUNEL reagent (TMR red) for 60 minutes, followed by DAPI staining, and analyzed under a fluorescence microscope. [1]
- Flow Cytometry (Annexin V/PI): After exposure to periplogenin (2 µg/ml) for 12 or 24 hours, cells were harvested, washed, and stained using an annexin V-FITC Apoptosis Detection Kit. Flow cytometry discriminated viable cells (FITC-/PI-), early apoptotic (FITC+/PI-), late apoptotic/secondary necrotic (FITC+/PI+), and primary necrotic cells (FITC-/PI+). [1]
- Western Blotting: After treatment with periplogenin or DMSO for different durations, cells were harvested and proteins extracted. Western blots were performed to analyze the expression of cleaved caspase-3, Beclin-1, and LC3, with GAPDH as a loading control. [1]
- Propidium Iodide (PI) Uptake Assay: Cells were treated with 2 µg/ml periplogenin for 48 hours, then incubated with PI (4 µg/mL) for 15 minutes in the dark. After washing, cells were observed under a fluorescence microscope to assess cell membrane integrity. [1]
- Lactate Dehydrogenase (LDH) Assay: HaCaT cells were seeded in 96-well plates and incubated with various concentrations of periplogenin for 24 or 48 hours. LDH released into the supernatant was detected using an LDH assay kit. Absorbance was measured at 450 nm. Cells treated with 1% Triton-X-100 served as a control for maximal LDH release (100%). [1]
- Electron Microscopy Analysis: After treatment with DMSO, H2O2, or periplogenin for different durations, HaCaT cells were harvested, processed, and examined by electron microscopy to visualize morphological changes. [1]
- Reactive Oxygen Species (ROS) Assay: Cells were treated with different doses of periplogenin for indicated times. After treatment, they were harvested and incubated with DCFH-DA (10 µmol/L) in serum-free medium for 30 minutes at 37°C. Cells were then washed, resuspended, and analyzed using a flow cytometer. [1]

- TPA-Induced Epidermal Hyperplasia Model: The backs of female Balb/c mice (6-8 weeks old) were shaved. One day later, 20 µl of TPA solution (50 µg/ml in 1% DMSO/99% methanol) was applied to the skin surface of the back and on both ears for 8 days. After 8 days, mice were treated topically with periplogenin (2 µg), vehicle control (1% DMSO/99% methanol), or dexamethasone (200 mg per site) every day for 11 consecutive days. At the end, mice were killed, ears were collected and measured, and 1-cm^2 punch biopsies were taken from the treated dorsal skin. [1]
- IMQ-Induced Skin Inflammation Model: Mice received a daily topical dose of 60 mg of IMQ cream (5%) on both ears for 8 consecutive days (3.125 mg of active compound). Control mice were treated with vehicle (1% DMSO/99% methanol). After 8 days, mice were treated topically with periplogenin (2 µg), vehicle control, or dexamethasone (200 mg per site) every day for 11 consecutive days. Mice were then killed, and the ears were collected and measured. [1]

Pharmacokinetic studies have indicated that periplogenin is rapidly absorbed and eliminated from rat plasma after oral administration of Cortex Periplocae extract. [1]

Periplogenin was less cytotoxic toward normal Hs-68 human fibroblast cells (IC10 > 10 µg/ml) than toward HaCaT keratinocyte cells (IC50 = 1.56 µg/ml), suggesting a degree of selective cytotoxicity. In animal studies, topical administration of periplogenin was tolerated and ameliorated skin lesions without any described systemic toxicity or side effects in the TPA- and IMQ-induced psoriasis-like mouse models. [1]

[1]. Periplogenin induces necroptotic cell death through oxidative stress in HaCaT cells and ameliorates skin lesions in the TPA- and IMQ-induced psoriasis-like mouse models. Biochem Pharmacol. 2016 Apr 1;105:66-79.

[2]. Phellopterin isolated from Angelica dahurica reduces blood glucose level in diabetic mice. Heliyon. 2018 Mar; 4(3): e00577.

Periplogenin has been reported to be found in Periploca sepium, Streptocaulon juventas, and other organisms with available data.

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- Psoriasis is a common chronic inflammatory and hyperproliferative skin disorder affecting 2-3% of the world's population. It is characterized by epidermal hyperplasia with dysregulated keratinocyte differentiation, pronounced inflammatory cell infiltration, and increased vascularization. Hyperproliferation and abnormal differentiation of epidermal keratinocytes are essential in the pathophysiology of psoriasis. [1]
- Periplogenin is a cardiac aglycone from the root bark of Periploca sepium Bunge (Cortex Periplocae, known as Xiangjiapi in Chinese), which has been used traditionally to treat rheumatoid arthritis and strengthen tendons and bones. [1]
- This study is the first to demonstrate that periplogenin induces necroptotic cell death through oxidative stress in HaCaT cells and ameliorates skin lesions in psoriasis-like mouse models, making it a promising candidate for future anti-psoriatic drug research. [1]

C23H34O5

390.5131

390.241

C, 70.74; H, 8.78; O, 20.48

514-39-6

10574

White to off-white solid powder

245-248℃

2.719

3

5

1

28

733

8

O([H])[C@]12C([H])([H])C([H])([H])[C@]([H])(C3=C([H])C(=O)OC3([H])[H])[C@@]1(C([H])([H])[H])C([H])([H])C([H])([H])[C@]1([H])[C@@]3(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[C@]3(C([H])([H])C([H])([H])[C@@]21[H])O[H])O[H]

QJPCKAJTLHDNCS-FBAXFMHRSA-N

InChI=1S/C23H34O5/c1-20-7-3-15(24)12-22(20,26)9-5-18-17(20)4-8-21(2)16(6-10-23(18,21)27)14-11-19(25)28-13-14/h11,15-18,24,26-27H,3-10,12-13H2,1-2H3/t15-,16+,17-,18+,20+,21+,22-,23-/m0/s1

3-[(3S,5S,8R,9S,10R,13R,14S,17R)-3,5,14-trihydroxy-10,13-dimethyl-2,3,4,6,7,8,9,11,12,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl]-2H-furan-5-one

Desoxostrophanthidin; Periplogenin; UNII-B6808P7IY9

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 (~256.08 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.40 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 (6.40 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 (6.40 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.)