Schisandrone targets α-hemolysin (Hla) expression in Staphylococcus aureus. It down-regulates the transcription of hla, agrA, and RNAIII genes [2].

Schisandrone did not affect the growth of S. aureus USA300; the minimum inhibitory concentration (MIC) was 512 µg/mL. Growth curves showed that concentrations up to 32 µg/mL did not influence bacterial growth [2].
Schisandrone significantly decreased the hemolytic activity of S. aureus USA300 in a dose-dependent manner. At 4 µg/mL, hemolytic activity decreased to 89.11±1.31%; at 32 µg/mL, hemolytic activity decreased to 1.79±0.51%. Similar inhibition was observed for S. aureus Newman (hemolytic activity decreased to 40.52±1.03% at 32 µg/mL) and SA1B3G (decreased to 11.50±0.62% at 32 µg/mL) [2].
Schisandrone did not directly neutralize Hla-induced hemolysis in a neutralization assay (no significant effect) [2].
Schisandrone did not affect Hla heptamer formation (oligomerization assay showed no change in heptamer band with increasing concentrations up to 64 µg/mL) [2].
Cellular thermal shift assay (CETSA) showed no significant difference in Hla protein amount between Schisandrone-treated group and DMSO group with increasing temperature, indicating no direct binding to Hla [2].
Western blot analysis showed that Schisandrone decreased Hla protein expression in S. aureus USA300 supernatant in a dose-dependent manner (4-32 µg/mL) [2].
RT-qPCR showed that Schisandrone significantly down-regulated the transcription levels of hla, RNAIII, and agrA in a dose-dependent manner (P<0.001) [2].
Schisandrone (0-32 µg/mL) showed no apparent cytotoxicity to A549 cells; cell viability was close to DMSO-treated cells (MTT assay) [2].
Schisandrone protected A549 cells from S. aureus-induced injury. Fluorescence microscopy (live/dead staining) showed that increasing concentrations of Schisandrone (4-32 µg/mL) reduced red fluorescence (dead cells). LDH release assay confirmed dose-dependent protection (exact values not provided) [2].
Checkerboard synergy assay showed that Schisandrone combined with ceftiofur reduced the MIC of ceftiofur from 32 µg/mL to 2 µg/mL; FICI value was 0.125, indicating synergistic effect. No significant synergy was observed with ceftriaxone, cefoxitin, cefotaxime acid, or oxacillin [2].

In a murine pneumonia model (C57BL/6J mice infected intranasally with S. aureus USA300), Schisandrone treatment (40 mg/kg, subcutaneous injection every 12 h) improved survival rate. 72 h post-infection (2×10^8 CFU), survival rate in infection group was 10%, in Schisandrone-treated group was 30%, and in Schisandrone + ceftiofur combination group was 60% (30% higher than Schisandrone alone) [2].
Bacterial burden in lungs: untreated infected mice had 8.59±0.77 log10 CFU/g; Schisandrone (40 mg/kg) reduced to 6.46±0.65 log10 CFU/g; combination with ceftiofur reduced to 2.98±0.60 log10 CFU/g [2].
Histopathological examination (H&E staining) showed that Schisandrone treatment reduced inflammatory cell infiltration and alveolar damage compared to untreated infected mice; the combination group showed significantly reduced inflammatory cells and relatively intact alveolar structure [2].

Oligomerization assay: Purified Hla protein (20 µg) was incubated with 5 mM deoxycholate in PBS buffer with or without Schisandrone (0-64 µg/mL) for 25 min at 22°C. After incubation, the sample was mixed with 5× protein loading buffer without β-mercaptoethanol and incubated for 10 min at 55°C. The effect of Schisandrone on Hla oligomerization was observed by SDS-PAGE using 8% gel. Heptamer formation did not change significantly with increasing Schisandrone concentrations [2].
Cellular thermal shift assay (CETSA): Hla protein was expressed in E. coli BL21 containing pET28a-hla. After ultrasonic lysis and centrifugation, Schisandrone (32 µg/mL) or DMSO was added to the protein supernatant and incubated in the dark at 37°C for 1 h. Samples were centrifuged at 18,000 ×g for 20 min at low temperature. The supernatant was heated in a PCR machine at specified temperatures (25.0, 48.0, 52.0, 53.7, 56.2, and 58.0°C) for 5 min, then immediately cooled on ice for 4 min. After mixing with 5× loading buffer and boiling for 5 min, samples were analyzed by 12% SDS-PAGE. No significant difference in Hla amount was observed between Schisandrone-treated and DMSO groups [2].
Neutralization assay: Supernatant of S. aureus USA300 was incubated with various concentrations of Schisandrone for 20 min at 37°C, then mixed with defibrinated rabbit red blood cells and further incubated for 1 h at 37°C. Hemolytic activity was measured. Schisandrone did not directly neutralize Hla-induced hemolysis [2].

Hemolysis assay: Overnight cultures of S. aureus USA300 or Newman were inoculated 1:100 into TSB medium and grown to OD600=0.3 at 37°C. Different concentrations of Schisandrone (4-32 µg/mL) were added and cultured until OD600=2.5. Bacterial supernatant was harvested and mixed with PBS and defibrinated rabbit blood (100 µL supernatant + 875 µL PBS + 25 µL blood). After incubation at 37°C for 1 h and centrifugation (5000 rpm, 4°C, 3 min), hemoglobin release was measured at 543 nm. Schisandrone decreased hemolytic activity in a dose-dependent manner [2].
Western blot: S. aureus USA300 was cultured with different concentrations of Schisandrone (4-32 µg/mL) until OD600=2.5. Equal amounts of bacterial culture supernatants were subjected to 12% SDS-PAGE, transferred to PVDF membranes, blocked with 5% skimmed milk, incubated with anti-Hla rabbit polyclonal antibody (1:2,000) for 2 h, then with HRP-labeled goat anti-rabbit IgG (1:8,000) for 1 h. Immunoblots were visualized by ECL. Hla protein expression decreased dose-dependently [2].
RT-qPCR: S. aureus USA300 was grown to OD600=0.3, treated with Schisandrone (0-32 µg/mL) for 12 h at 37°C with shaking (220 rpm). Total RNA was extracted using Trizol, reverse transcribed to cDNA. 16S rRNA was used as endogenous control. The expression of hla, agrA, and RNAIII genes was analyzed by real-time PCR with cycling parameters: 95°C for 30 s, 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 30 s. Relative fold changes were calculated using the 2−ΔΔCt method. Transcription levels of hla, RNAIII, and agrA were significantly down-regulated (P<0.001) [2].
MTT cytotoxicity assay: A549 cells were seeded at 5×10^3 cells/well in 96-well plates and incubated for 24 h. Schisandrone (4-32 µg/mL) was added and incubated for 24 h at 37°C in 5% CO2. Then 10 µL MTT solution (5 mg/mL) was added for 4 h. Crystals were dissolved in 100 µL DMSO, and absorbance measured at 490 nm. Cell viability was close to DMSO-treated controls, indicating no apparent cytotoxicity [2].
Live/dead and LDH cytotoxicity assay: A549 cells (5×10^4 cells/well) were seeded in 24-well plates for 24 h. S. aureus USA300 (10^6 CFU/mL) was centrifuged, supernatant discarded, and resuspended in 1640 medium without FBS and antibiotics. Then 100 µL bacterial culture and 400 µL 1640 medium containing different concentrations of Schisandrone (0-32 µg/mL) were added to cells. After 5 h incubation at 37°C, supernatant was collected. Live/dead staining was observed by fluorescence microscopy (Calcein/PI). LDH release was measured at 490 nm. Schisandrone protected A549 cells from S. aureus-induced injury in a dose-dependent manner [2].
Checkerboard synergy assay: Two-fold serial dilutions of antibiotics (ceftriaxone, ceftiofur, cefoxitin, cefotaxime acid, oxacillin) were combined with different concentrations of Schisandrone in 96-well plates. S. aureus USA300 (1×10^5 CFU/mL) was added and incubated at 35°C for 16 h. Absorbance at 600 nm was measured. The fractional inhibitory concentration index (FICI) was calculated: FICI = FIC_A + FIC_B = C_A/MIC_A + C_B/MIC_B. Synergistic effect (FICI=0.125) was observed with ceftiofur [2].

Murine lung infection model (pneumonia): C57BL/6J mice (~22 g) were randomly divided into five groups (n=10 per group): S. aureus USA300 infection group, Schisandrone treatment group, ceftiofur group, antibiotic combination group, and uninfected group. For survival studies, mice were infected intranasally with S. aureus USA300 (2×10^8 CFU). Two hours after infection, mice in treatment groups were subcutaneously injected with 40 mg/kg Schisandrone or equal volume of PBS (containing 0.5% DMSO) administered every 12 h. Survival was monitored at 12 h, 24 h, 36 h, 48 h, 72 h, and survival curve was drawn (n=10) [2].
For bacterial load and histopathology: Mice (n=8 per group) were infected intranasally with S. aureus USA300 (1×10^8 CFU). After 24 h, mice were sacrificed. Left lung was taken and weighed, homogenized in sterile normal saline, and bacterial CFU were counted by serial dilution on BHI plates. Right lung was fixed with 10% formalin, embedded in paraffin, sectioned (6 µM thick), and stained with hematoxylin-eosin (H&E) for histopathological observation under optical microscope [2].

Schisandrone showed no apparent cytotoxicity to A549 cells at concentrations up to 32 µg/mL (MTT assay, cell viability close to DMSO-treated cells) [2].
No acute toxicity or adverse effects were reported in mice treated with 40 mg/kg Schisandrone (subcutaneous injection every 12 h) [2].

[1]. Schisandrone, a New 4-Aryltetralone Lignan fromSchisandra sphenanthera. Planta Medica, 1985, 51(03), 217–219.

[2]. An inhibitory effect of schisandrone on α-hemolysin expression to combat methicillin-resistant staphylococcus aureus infections. World J Microbiol Biotechnol. 2022 Nov 8;39(1):3.

There have been reports that schisandrin exists in Schisandra sphenanthera and Schisandra arisanensis, and relevant data are available for reference.

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Schisandrone (C21H24O5) has a molecular weight of 356, m.p. 176-177°C, [α]D17 -32.5° (c 0.12, CHCl3). Its structure was elucidated as (2S,3S,4R)-4-aryltetralone lignan. It was isolated from dried fruits of Schisandra sphenanthera (10.5 kg yielded 6.6 g of Schisandrone) [1].
Schisandrone has been reported to have anti-oxidant activity and a therapeutic effect on Alzheimer's disease, and protective effect on cortical neuron injury induced by ketamine in rats [2].
The anti-virulence mechanism of Schisandrone involves down-regulation of agrA transcription, which leads to reduced RNAIII and hla expression, thereby inhibiting Hla production and hemolytic activity without affecting bacterial growth or directly binding to Hla [2].
Schisandrone contains no pan-assay interference structure (PAINS), indicating strong activity and development potential [2].

C21H24O5

356.4123

356.162

98619-25-1

14078177

White to off-white solid

1.2±0.1 g/cm3

508.2±50.0 °C at 760 mmHg

180-181℃

176.1±23.6 °C

0.0±1.4 mmHg at 25°C

1.557

4.23

1

5

4

26

495

3

O=C1C2=C([H])C(=C(C([H])=C2[C@@]([H])(C2C([H])=C([H])C(=C(C=2[H])OC([H])([H])[H])OC([H])([H])[H])[C@]([H])(C([H])([H])[H])[C@]1([H])C([H])([H])[H])OC([H])([H])[H])O[H]

DRKPZVVNEGETTG-XAAFQQQXSA-N

InChI=1S/C21H24O5/c1-11-12(2)21(23)15-9-16(22)18(25-4)10-14(15)20(11)13-6-7-17(24-3)19(8-13)26-5/h6-12,20,22H,1-5H3/t11-,12+,20-/m1/s1

(2S,3S,4R)-4-(3,4-dimethoxyphenyl)-7-hydroxy-6-methoxy-2,3-dimethyl-3,4-dihydro-2H-naphthalen-1-one

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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