In HepG2 cells, peiminine (2–6 μg/mL, 24 h) can decrease procaspase-3, procaspase-8, and procaspase-9 expression while raising caspase-3, 8, and 9 protein levels [1]. HepG2, Hela, SW480, and MCF-7 cells are clearly cytotoxically affected by periminine (2–6 μg/mL, 24-72 h) [1]. HepG2 cells are induced to arrest in the G2/M phase by peiminine (2–6 μg/mL, 24 h) [1].

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Peiminine exhibited significant cytotoxicity against Hela, HepG2, SW480, and MCF-7 cancer cell lines in vitro, with IC50 values at 24 hours of 4.89, 4.58, 5.07, and 5.12 µg/mL, respectively. [1]
In HepG2 cells, Peiminine inhibited cell viability in a time- and dose-dependent manner, with IC50 values of 4.58 µg/mL (24 h), 4.05 µg/mL (48 h), and 3.79 µg/mL (72 h). Treatment caused cytoplasmic membrane damage and reduced cell population. [1]
Peiminine induced apoptosis in HepG2 cells, as evidenced by DAPI staining showing chromatin condensation and apoptotic bodies, and by agarose gel electrophoresis showing dose-dependent DNA fragmentation. [1]
Flow cytometry analysis using Annexin V-FITC/PI staining confirmed Peiminine-induced apoptosis. After 24h treatment with 0, 2, 4, and 6 µg/mL, the early apoptotic cell percentages were 8.28%, 10.32%, 15.23%, and 19.36%, and late apoptotic cell percentages were 3.36%, 5.08%, 3.55%, and 5.51%, respectively. [1]
Peiminine treatment arrested the cell cycle at the G2/M phase in HepG2 cells. The percentage of cells in G2/M phase increased from 17.32% (control) to 39.99% with increasing drug concentration, while the percentages in G1 and S phases decreased. [1]
Peiminine treatment significantly decreased the mitochondrial membrane potential (ΔΨm) in HepG2 cells in a concentration-dependent manner, as measured by Rhodamine 123 staining and flow cytometry. [1]
Peiminine (4 µg/mL) increased caspase-3 activity in HepG2 cells. This activation was inhibited by pre-treatment with the caspase-3 specific inhibitor z-DEVD-fmk (50 µM), which also prevented the associated chromatin condensation and DNA fragmentation. [1]
A PathScan intracellular signaling array showed that Peiminine treatment up-regulated the expression of pro-apoptotic proteins Bax, cleaved PARP, and active caspase-3, -8, -9, while down-regulating the expression of the anti-apoptotic protein Bcl-2 and Chk2 in HepG2 cells. [1]
Western blot analysis confirmed that Peiminine treatment (2-6 µg/mL for 24h) decreased the protein levels of procaspase-3, -8, -9, Bcl-2, and full-length PARP, while increasing the levels of cleaved caspase-3, -8, -9, Bax, and cleaved PARP in HepG2 cells. The expression of p53 was also increased in a dose-dependent manner. [1]

Fritillaria (3 mg/kg, intraperitoneal injection, single dosage) can lessen intestinal tissue damage and the inflammatory symptoms of the animal model of thermally induced ductitis [2]. For six weeks, intraperitoneal fritillaria (10 mg/kg) given once every two days inhibits adipogenesis and bone loss brought on by OVX [3]. In an animal model of DNCB-induced allergic dermatitis, peiminine (1–5 mg/kg, given to the back skin once daily for 16 days) suppresses serum IL-6 and TNF-α [4]. Beminin, administered intraperitoneally once daily for four weeks at a dose of two to five mg/kg, protects against myocardial infarction damage and fibrosis [5]. In a lipopolysaccharide-induced acute lung damage model, belimine (1–5 mg/kg, i.p., single dose) may potentially reduce the response to injury and pulmonary edema [6].

A caspase-3 activity assay was performed. HepG2 cells were treated with Peiminine alone (4 µg/mL) or in combination with the caspase-3 inhibitor z-DEVD-fmk (10 µmol/L). Caspase-3 activity in cell lysates was assessed using a colorimetric assay kit. The assay utilizes a substrate that is cleaved by active caspase-3 to release a chromophore (p-nitroaniline, pNA). The absorbance of the released pNA was measured at 405 nm using a spectrophotometer, and the caspase-3 activity was determined relative to controls. [1]

Cytotoxicity assay[1]
Cell Types: HepG2, Hela, SW480, MCF-7
Tested Concentrations: 2 μg/. mL, 4 μg/mL, 6 μg/mL, 8 μg/mL, 10 μg/mL, 12 μg/mL and 14 μg/mL
Incubation Duration: 24 hrs (hours), 48 hrs (hours), 72 hrs (hours)
Experimental Results: For HepG2, Hela, SW480 and The IC50 values of MCF-7 cells after 24 hrs (hours) of survival were 4.58, 4.89, 5.07 and 5.12 μg/mL respectively.

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Apoptosis analysis[1]
Cell Types: HepG2
Tested Concentrations: 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL
Incubation Duration: 24 h
Experimental Results: Dissociated chromosomes produce DNA in a dose-dependent manner fragment.

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Cell cycle analysis[1]
Cell Types: HepG2
Tested Concentrations: 2 μg/mL, 4 μg/mL, 6 μg/mL
Incubation Duration: 24 hrs (hours)
Experimental Results: G1 phase percentage diminished from 65.15% ± 0.78 to 49.55% ± 0.17 Increase in concentration . As the concentration increased, the percentage of G2/M phase increased from 17.32%±0.20 to 39.99%±0.47.

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Western Blot Analysis[1]
Cell Types: HepG2
Tested Concentrations: 2 μg/mL, 4 μg/mL, 6 μg/mL
Incubation Duration: 24 h
Experimental Results: diminished expression of procaspase-3, PARP

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Cell viability was assessed by MTT assay. Cells (Hela, HepG2, SW480, MCF-7) were seeded in 96-well plates and treated with various concentrations of Peiminine (0-14 µg/mL) for 24, 48, or 72 hours. MTT solution was added to each well and incubated for 4 hours. The formed formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm using a microplate reader. Cell viability percentage and IC50 values were calculated. [1]
Apoptosis was assessed by DAPI nuclear staining. HepG2 cells treated with Peiminine (4 µg/mL for 24h) were fixed, stained with DAPI solution, and observed under a fluorescence microscope for morphological changes characteristic of apoptosis, such as chromatin condensation and nuclear fragmentation. [1]
DNA fragmentation was assessed by agarose gel electrophoresis. HepG2 cells treated with Peiminine (0-6 µg/mL for 24h) were lysed, and genomic DNA was extracted using a phenol/chloroform/isoamyl alcohol mixture. After RNase A treatment, the DNA was separated on a 1.5% agarose gel and visualized to observe the DNA laddering pattern indicative of apoptosis. [1]
Quantitative apoptosis and cell cycle analysis were performed by flow cytometry. For apoptosis, cells treated with Peiminine were collected, stained with Annexin V-FITC and propidium iodide (PI) in binding buffer, and analyzed by flow cytometry to distinguish live, early apoptotic, late apoptotic, and necrotic cells. For cell cycle analysis, treated cells were fixed, stained with PI solution containing RNase A, and analyzed by flow cytometry to determine the distribution of cells in different phases (G0/G1, S, G2/M). [1]
Mitochondrial membrane potential (ΔΨm) was assessed using Rhodamine 123 staining. HepG2 cells treated with Peiminine were incubated with Rhodamine 123 dye, harvested, washed, and analyzed by flow cytometry. The fluorescence intensity reflects the ΔΨm, with a decrease indicating mitochondrial depolarization. [1]
Changes in intracellular signaling molecules were analyzed using a commercial antibody array kit. Protein lysates from control and Peiminine-treated HepG2 cells were applied to the array membrane coated with antibodies against various signaling proteins. After incubation and washing, the bound proteins were detected using chemiluminescence, and the signal intensity was quantified. [1]
Protein expression was analyzed by Western blotting. HepG2 cells treated with Peiminine were lysed, and total protein was quantified. Equal amounts of protein were separated by SDS-PAGE, transferred to a PVDF membrane, and blocked. The membrane was incubated overnight with primary antibodies against target proteins (e.g., Bcl-2, Bax, caspases, PARP, p53, β-actin), followed by incubation with HRP-conjugated secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence detection kit. [1]

Animal/Disease Models: Ulcerative colitis model [2]
Doses: 3mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Reduce inflammation, mucosal ulceration, involvement of all layers of the digestive system, and inflammatory cell infiltration. Reduces the levels of MPO and NO produced in rectal tissue. Splenocyte proliferation is diminished. Reduces production of f IL-1β, IL-6 and TNF-α cytokines. The expression levels of IL-1β, IL-6, TNF-α, iNOS, and COX2 genes were diminished.

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Animal/Disease Models: Ovariectomized (OVX) rat model [3]
Doses: 10 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Reduce bone loss caused by surgical castration. Improved the expression of COL1A1 and β-catenin and diminished the increased expression of PPAR-γ in trabecular bone.

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Animal/Disease Models: allergic dermatitis model [4]
Doses: 1 mg/kg, 5 mg/kg
Route of Administration: Apply to the back skin
Experimental Results: Reduce bone loss caused by surgical castration. Improved the expression of COL1A1 and β-catenin and r

[1]. The effects and mechanism of peiminine-induced apoptosis in human hepatocellular carcinoma HepG2 cells . PLoS One, 2019, 14(1): e0201864.

[2]. Anti-inflammatory activity of peiminine in acetic acid-induced ulcerative colitis model . Inflammopharmacology, 2023: 1-9.

[3]. Peiminine regulates bone-fat balance by canonical Wnt/β-catenin pathway in an ovariectomized rat mode . Phytotherapy Research, 2023.

[4]. Effect of peiminine on DNCB-induced atopic dermatitis by inhibiting inflammatory cytokine expression in vivo and in vitro . International immunopharmacology, 2018, 56: 135-142.

[5]. Peiminine inhibits myocardial injury and fibrosis after myocardial infarction in rats by regulating mitogen-activated protein kinase pathway . The Korean Journal of Physiology & Pharmacology: Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 2022, 26(2): 87-94.

[6]. Peiminine attenuates acute lung injury induced by LPS through inhibiting lipid rafts formation . Inflammation, 2020, 43: 1110-1119.

[7]. Peiminine ameliorates bleomycin-induced acute lung injury in rats. Mol Med Rep. 2013 Apr;7(4):1103-10.

Imperialine alkaloid is an alkaloid.
It has been reported that fritillarine exists in Fritillaria ebeiensis, Fritillaria monantha and other organisms with relevant data.

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Fritillarine is a natural compound extracted from the bulbs of Fritillaria thunbergii and Bolbostemma paniculatum and used in traditional Chinese medicine. [1]
This study concluded that fritigrine induces apoptosis in human liver cancer HepG2 cells through exogenous (death receptor) and endogenous (mitochondrial) apoptosis pathways. This is mediated by upregulation of pro-apoptotic proteins (Bax, p53, cleaved caspase-3, -8, -9, cleaved PARP) and downregulation of anti-apoptotic protein Bcl-2, ultimately leading to caspase-3 activation, loss of mitochondrial membrane potential and cell cycle arrest in the G2/M phase. [1]
This study cites previous research showing that fritillary alkaloids can also inhibit the proliferation of colorectal cancer cells by inducing apoptosis and autophagy. [1]

C27H43NO3

429.6352

429.324

18059-10-4

167691

White to off-white solid powder

1.2±0.1 g/cm3

567.1±50.0 °C at 760 mmHg

212-213ºC

296.8±30.1 °C

0.0±3.5 mmHg at 25°C

1.585

3.9

2

4

0

31

755

12

C[C@H]1CC[C@H]2[C@@]([C@H]3CC[C@@H]4[C@H]([C@@H]3CN2C1)C[C@H]5[C@H]4CC(=O)[C@@H]6[C@@]5(CC[C@@H](C6)O)C)(C)O

IQDIERHFZVCNRZ-YUYPDVIUSA-N

InChI=1S/C27H43NO3/c1-15-4-7-25-27(3,31)21-6-5-17-18(20(21)14-28(25)13-15)11-22-19(17)12-24(30)23-10-16(29)8-9-26(22,23)2/h15-23,25,29,31H,4-14H2,1-3H3/t15-,16-,17+,18+,19-,20-,21-,22-,23+,25-,26+,27-/m0/s1

(1R,2S,6S,9S,10S,11S,14S,15S,18S,20S,23R,24S)-10,20-dihydroxy-6,10,23-trimethyl-4-azahexacyclo[12.11.0.02,11.04,9.015,24.018,23]pentacosan-17-one

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 : ≥ 100 mg/mL (~232.75 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.82 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 (5.82 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 (5.82 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.)