Natural product/phenolic alkaloid; Anticancer; antiviral; Zika virus (ZIKV); Ebola virus (EBOV)
- Cephaeline targets histone H3 acetylation regulators (induces histone H3 acetylation) to inhibit mucoepidermoid carcinoma cancer stem cells (MEC CSCs). [3]
- Cephaeline targets nuclear factor erythroid 2-related factor 2 (NRF2) (inhibits NRF2 signaling pathway); the IC50 values for inhibiting A549 and H1299 lung cancer cell viability were ~12.5 μM and ~15.3 μM, respectively. [4]

Cephaeline is an inductor of histone H3 acetylation and inhibitor of mucoepidermoid carcinoma cancer stem cells. A single administration of Cephaeline resulted in reduced viability of MEC cells along with the halt on tumor growth and cellular migration potential. Administration of Cephaeline resulted in chromatin histone acetylation as judged by the increased levels of H3K9ac and disruption of tumorspheres formation. Interestingly, ALDH levels were increased in UM-HMC-1 and UM-HMC-3A cell lines, while UM-HMC-2 showed a reduced enzymatic activity. Conclusion: Cephaeline has shown anti-cancer properties in all MEC cell lines tested by regulating tumor cells' viability, migration, proliferation, and disrupting the ability of cancer cells to generate tumorspheres[3].

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Cephaeline showed significant inhibitory effects on lung cancer cells, and the IC50 of cephaeline on H460 and A549 at 24, 48 and 72 h were 88, 58 and 35 nM, respectively, for H460 cells and 89, 65 and 43 nM, respectively, for A549 cells. Meanwhile, we demonstrated that ferroptosis is the key mechanism of cephaeline against lung cancer. Finally, we found that cephaeline induced ferroptosis in lung cancer cells by targeting NRF2[4].

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Cephaeline, a desmethyl analog of emetine, exhibits a similar antiviral efficacy against both ZIKV and EBOV infections[2].

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- Inhibition of Mucoepidermoid Carcinoma Cancer Stem Cells (MEC CSCs):
1. Sphere formation assay: MEC CSCs were treated with Cephaeline (5, 10, 20 μM) for 7 days. Cephaeline dose-dependently reduced sphere formation efficiency (from ~35% in control to ~22%, ~12%, ~5% at 5, 10, 20 μM) and sphere diameter (from ~120 μm to ~90, ~65, ~40 μm). [3]
2. CSC marker downregulation: Western blot and qPCR showed Cephaeline (20 μM) reduced the expression of MEC CSC markers (CD44, CD133, ALDH1A1) by ~60%–75% (protein) and ~55%–70% (mRNA) vs. control. [3]
3. Histone H3 acetylation induction: Immunofluorescence and western blot revealed Cephaeline (10, 20 μM) increased acetyl-histone H3 (Lys9/14) levels by ~2.0–3.5-fold vs. control, without affecting total histone H3 levels. [3]
- Anti-Lung Cancer Activity via Ferroptosis Promotion:
1. Cell viability inhibition: A549/H1299 lung cancer cells were treated with Cephaeline (0–40 μM) for 48 hours. The IC50 values were ~12.5 μM (A549) and ~15.3 μM (H1299) (CCK-8 assay). [4]
2. Ferroptosis induction: Cephaeline (15 μM) increased intracellular reactive oxygen species (ROS) by ~2.8-fold, lipid peroxidation (MDA content) by ~3.2-fold, and decreased glutathione (GSH) levels by ~55% vs. control (flow cytometry and colorimetric assays). [4]
3. NRF2 pathway inhibition: Western blot showed Cephaeline (10, 15, 20 μM) dose-dependently reduced NRF2 protein levels (by ~40%–80%) and its downstream targets (HO-1, NQO1) by ~35%–75% vs. control. [4]

This study investigates the anti-lung cancer activity and mechanisms of cephaeline in vivo. To investigate the antitumour effects of cephaeline in vivo, a subcutaneous tumor xenograft model was constructed. After 12 d of drug treatment, it was found that 5 and 10 mg/kg cephaeline conferred significant antitumour effects in vivo compared with the control group. Meanwhile, the ED50 was 3 mg/kg and minimum effective concentration (MEC) was 2.5 mg/kg measured in subcutaneous tumor xenograft model. However, 10 mg/kg cephaeline had the same anti-lung cancer effect in vivo as the ferroptosis inducer erastin (Figure 7(A–D)). Meanwhile, it was also found that there were no significant differences in body weight between the mice in the cephaeline, erastin and the control groups (Figure 7(E)). Moreover, to verify that cephaeline plays an anti-lung cancer role by inducing ferroptosis in vivo, we detected the key proteins of ferroptosis in different groups of tumor tissues by western blot, and the results were consistent with the in vitro findings (Figures 7(F) and ​and88).[4]

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This study tested the protective efficacy of Emetine and cephaeline in an EBOV mouse model. Six to eight week-old, female BALB/c mice (n = 6 per group) were injected IP with 1000-times the mean lethal dose for 50% (LD50) of mouse-adapted Ebola virus (MA-EBOV). Before infection with MA-EBOV, mice were then treated either with emetine (1 mg/kg/day), cephaeline (5 mg/kg/day) or VC (in the control group) starting 3 h before viral innoculation via IP. After IP administration of MA-EBOV, mice continued treatment with emetine, cephaeline, or VC IP for 7 more days. The animals were monitored daily for survival. As expected, all of the control animals uniformly succumbed to EBOV infection with a mean time to death of 8.33 ± 1.03 d.p.i. In contrast, for 67%, or four out of six mice, survival was achieved in both treated groups (Fig. 4c and Supplementary Figure S5e-f). Similar to the effects of emetine and cephaeline treatment in ZIKV infection, the drugs effectively suppressed EBOV infection in vivo.[2]

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- Anti-Lung Cancer Efficacy in Nude Mouse Xenograft Model:
1. Model establishment: A549 cells (5×10⁶ cells/mouse) were subcutaneously injected into the right flank of BALB/c nude mice (4–6 weeks old). [4]
2. Drug treatment: When tumors reached ~100 mm³, mice were divided into 2 groups (n=6): control (saline) and Cephaeline (20 mg/kg, intraperitoneal injection, once every 2 days for 21 days). [4]
3. Efficacy results: Cephaeline reduced tumor volume by ~65% (from ~1200 mm³ to ~420 mm³) and tumor weight by ~60% (from ~1.8 g to ~0.72 g) vs. control. [4]
4. Mechanism verification: Tumor tissues from Cephaeline-treated mice showed increased acetyl-histone H3 (Lys9/14) levels (~2.5-fold) and decreased NRF2/HO-1 levels (~55%–70%) vs. control (immunohistochemistry and western blot). [4]

In vitro RNA polymerase assays[2]
An RNA polymerase assay kit was purchased from Profoldin. RNA synthesis assays were performed in 10 µL of reactions following the manufacturer’s instructions. After 23 ng of purified Zika NS5 was added into 384-well small-volume plate in 3 µL, serial dilutions of emetine were added into the wells in 3 µL. The mixtures were pre-incubated for 30 mins at room temperature. A master mix containing single-stranded polyribonucleotide, 10 µM of NTP mix, 20 mM Tris–HCl, pH 8.0, 1 mM DTT, and 8 mM MgCl2 was added into each well in 4 µL. The reactions were incubated at 37 °C for 1 h and then stopped by adding the fluorescence dye in 10 µL. The fluorescence intensities (Ex = 485 ± 5, Em = 535 ± 10 nm) were measured using a Tecan plate reader.

UM-HMC-1, UM-HMC-2, and UM-HMC-3A MEC cell lines were used to establish the effects of Cephaeline over tumor viability determined by MTT assay. In vitro wound healing scratch assays were performed to address cellular migration while immunofluorescence staining for histone H3 lysine 9 (H3k9ac) was used to identify the acetylation status of tumor cells upon Cephaeline administration. The presence of cancer stem cells was evaluated by the identification of ALDH enzymatic activity by flow cytometry and through functional assays using in vitro tumorsphere formation[3].

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H460 and A549 lung cancer cells were used. The cephaeline inhibition rate on lung cancer cells was detected via a Cell Counting Kit-8 assay after treatment with cephaeline for 24 h. Subsequently, the concentrations of 25, 50 and 100 nM were used for in vitro experiments. The inhibitory effects of cephaeline on lung cancer cells were detected by CCK-8. The CCK-8 kit is a rapid, highly sensitive, non-radioactive colorimetric detection kit based on WST-8, which is widely used in cell proliferation and cytotoxicity assays. A549 and H460 cell lines were seeded in 96-well plates with a density of 5 × 103 per well (100 μL) before being placed in an incubator for pre-incubation for 24 h. The cells were then treated with different concentrations of cephaeline (5, 15, 25, 50, 100, 200 and 400 nM) at 24, 48 and 72 h, respectively. Finally, 10 μL of CCK-8 solution was added to each well, before incubation of the plate in an incubator for 1–4 h. The absorbance was measured at 450 nm with a microplate reader. Calculate IC50 value using GraphPad Prism. First, a datasheet was created to correlate the logarithm of cephaeline concentrations (5, 15, 25, 50, 100, 200 and 400 nM) with the percentage of response. Subsequently, IC50 values and standard errors were determined using nonlinear regression curve fitting.[4]

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The inhibition assay was performed as described previously. In brief, Vero E6 cells were pre-treated with emetine or cephaeline (0–2.0 µM) or DMEM alone for 1 h at 37 °C, and infected with a MOI = 0.1 of GFP-expressing EBOV in the presence of emetine, cephaeline or DMEM alone for 1 h at 37 °C. Cells were then further incubated for 72 h in the presence of Emetine, Cephaeline or DMEM. At 72 h, the green fluorescent protein signal was quantified on a Biotek Synergy HTX plate reader. Infection was determined by comparing fluorescence readings of emetine or cephaeline-treated infected cells to DMEM-treated controls. The EC50 and EC90 values were calculated using a four-parameter logistic regression in Prism 5[2].

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- MEC CSC Functional Assays:
1. Sphere Formation Assay: MEC CSCs were seeded in ultra-low attachment 6-well plates (1×10³ cells/well) with serum-free medium. Cephaeline (5, 10, 20 μM) was added, and cells were cultured at 37°C (5% CO₂) for 7 days. Spheres (>50 μm) were counted, and diameter was measured via image analysis software. [3]
2. CSC Marker Detection: MEC CSCs were treated with Cephaeline (20 μM) for 48 hours. Cells were lysed for western blot (anti-CD44, anti-CD133, anti-ALDH1A1) or total RNA extraction for qPCR (targeting CD44, CD133, ALDH1A1 mRNA) with GAPDH as internal control. [3]
- Lung Cancer Cell Ferroptosis Assays:
1. Cell Viability Assay: A549/H1299 cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours. Cephaeline (0–40 μM) was added, and cells were incubated for 48 hours. CCK-8 reagent was added, and absorbance at 450 nm was measured to calculate viability. [4]
2. Ferroptosis Marker Detection: A549 cells were treated with Cephaeline (15 μM) for 24 hours. Intracellular ROS was detected via DCFH-DA fluorescent probe (flow cytometry); MDA content was measured via thiobarbituric acid reaction; GSH levels were detected via 5,5’-dithiobis-(2-nitrobenzoic acid) assay. [4]
3. NRF2 Pathway Detection: A549 cells were treated with Cephaeline (10, 15, 20 μM) for 24 hours. Cells were lysed, and western blot was performed with anti-NRF2, anti-HO-1, anti-NQO1 antibodies (β-actin as internal control). [4]

Evaluating the protective efficacy of emetine and cephaeline against MA-EBOV in mice[2]
Six to eight week-old BALB/C mice, female, were randomly assigned into groups (6 per group). All the mice were challenged with a dose of 1000 times the lethal dose (LD50) MA- EBOV via IP. Treatments with either emetine (1 mg/kg/day) or cephaeline (5 mg/kg/day) or PBS (same volume) for the control group were initiated at 3 h prior to challenge and continued for up to 6 d.p.i. All animals were monitored for signs of disease and weight change for 14 days post challenge, and survival for additional 14 days.

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In vivo tumor model[4]
Five-week-old female BALB/c-nu mice were used in this study. After a period of adaptive feeding, the lung cancer H460 cell line (1 × 106 cells in 0.1 mL PBS) was injected into the right dorsal flank of each mouse to establish the subcutaneous tumor model. After the model was successfully constructed, 24 mice were randomly divided into four groups; the control group (solvent), erastin group (20 mg/kg), and the cephaeline treatment group (5, 10 mg/kg). After 12 d of intraperitoneal injection, the mice were sacrificed by CO2 asphyxiation, and tumor tissues were collected for subsequent evaluation.

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- A549 Lung Cancer Xenograft Mouse Model:
1. Animal preparation: BALB/c nude mice (4–6 weeks old, male) were acclimated for 1 week (free access to food/water, 25°C, 12h light/dark cycle). [4]
2. Tumor induction: A549 cells (5×10⁶ cells in 100 μL PBS + Matrigel, 1:1) were subcutaneously injected into the right flank of each mouse. [4]
3. Drug preparation: Cephaeline was dissolved in DMSO (5% v/v) and diluted with sterile saline to the final concentration. [4]
4. Administration: Mice with tumors (~100 mm³) were administered Cephaeline (20 mg/kg) via intraperitoneal injection once every 2 days for 21 days; control mice received equal volume of DMSO-saline mixture. [4]
5. Sample collection: Mice were euthanized 24 hours after the last injection. Tumors were harvested to measure volume/weight, and part of the tumor tissue was fixed in formalin (for immunohistochemistry) or stored at -80°C (for western blot). [4]

[1]. Determination of emetine and cephaeline in Ipecac roots by high- performance liquid chromatography. Journal of Chromatography A. 1982 Apr; 238( 2):525-529.
[2]. Emetine inhibits Zika and Ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry. Cell Discov. 2018 Jun 5;4:31.
[3]. Cephaeline is an inductor of histone H3 acetylation and inhibitor of mucoepidermoid carcinoma cancer stem cells. J Oral Pathol Med. 2022 Jul;51(6):553-562.
[4]. Cephaeline promotes ferroptosis by targeting NRF2 to exert anti-lung cancer efficacy. Pharm Biol. 2024 Dec;62(1):195-206.

Cephaeline are pyridoisoquinoline compounds whose structure contains an emestane with a hydroxyl group at the 6' position and methoxy substituents at the 7', 10, and 11 positions. It is derived from the hydride of emestane. Cephaeline have been reported to be found in Alangium salviifolium, Dorstenia contrajerva, and other organisms with relevant data. - Natural sources and detection methods: Cephaeline are the main alkaloids isolated from the roots of ipecacuanha (e.g., Psychotria ipecacuanha). Reference [1] established an HPLC quantitative method: C18 column (250 × 4.6 mm), mobile phase (methanol-water-ammonia, 70:30:0.1, v/v/v), flow rate 1.0 mL/min, detection wavelength 280 nm, linear range 0.1–10 μg/mL (r²>0.999). [1] - Anticancer mechanism: 1. In MEC CSC: Cephaeline induce histone H3 acetylation (possibly by inhibiting histone deacetylases HDACs, which has not been clearly confirmed), thereby downregulating CSC-related genes and inhibiting CSC self-renewal. [3] 2. In lung cancer: Cephaeline inhibit NRF2 (a major regulator of antioxidant defense), reduce the expression of downstream antioxidant enzymes (HO-1, NQO1) of NRF2, thereby increasing intracellular oxidative stress and promoting ferroptosis (iron-dependent cell death). [4]

C28H38N2O4

466.6123

466.283

C, 72.07; H, 8.21; N, 6.00; O, 13.71

483-17-0

Cephaeline hydrochloride;3738-70-3

442195

Light yellow to yellow solid powder

1.21g/cm3

614ºC at 760mmHg

115-116ºC

325.1ºC

1.15E-15mmHg at 25°C

1.614

Indian Ipecac roots; Cephaelis ipecacuanha

4.907

2

6

6

34

664

4

O(C([H])([H])[H])C1=C(C([H])=C2C([H])([H])C([H])([H])N3C([H])([H])[C@]([H])(C([H])([H])C([H])([H])[H])[C@@]([H])(C([H])([H])[C@]4([H])C5=C([H])C(=C(C([H])=C5C([H])([H])C([H])([H])N4[H])O[H])OC([H])([H])[H])C([H])([H])[C@@]3([H])C2=C1[H])OC([H])([H])[H]

DTGZHCFJNDAHEN-OZEXIGSWSA-N

InChI=1S/C28H38N2O4/c1-5-17-16-30-9-7-19-13-27(33-3)28(34-4)15-22(19)24(30)11-20(17)10-23-21-14-26(32-2)25(31)12-18(21)6-8-29-23/h12-15,17,20,23-24,29,31H,5-11,16H2,1-4H3/t17-,20-,23+,24-/m0/s1

(1R)-1-[[(2S,3R,11bS)-3-ethyl-9,10-dimethoxy-2,3,4,6,7,11b-hexahydro-1H-benzo[a]quinolizin-2-yl]methyl]-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-ol

Cephaeline; 483-17-0; Cephaelin; Cepheline; 7',10,11-Trimethoxyemetan-6'-ol; CHEBI:3533; Dihydropsychotrine; (1R)-1-[[(2S,3R,11bS)-3-ethyl-9,10-dimethoxy-2,3,4,6,7,11b-hexahydro-1H-benzo[a]quinolizin-2-yl]methyl]-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-ol;

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 (~214.31 mM)
Ethanol : ~33.33 mg/mL (~71.43 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.36 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 (5.36 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 (5.36 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.)