- In antifungal activity studies, Magnoflorine exhibits inhibitory effects on Candida species (including Candida albicans, Candida tropicalis, Candida krusei), but no specific molecular target (e.g., enzyme/receptor) or affinity data (IC50/Ki) were reported[1]
- In pro-inflammatory response regulation, Magnoflorine acts via the MyD88-dependent signaling pathway (involved in TLR4-mediated inflammation), but no direct binding affinity to MyD88 or downstream proteins (e.g., NF-κB, MAPK) was provided[2]

Alpha-glucosidase (inhibition of alpha-glucosidase activity was observed, but no IC50 value was reported) [1].
MyD88, TLR4, NF-κB, MAPKs (JNK, ERK, p38), PI3K-Akt (functional modulation was observed) [2].

- Magnoflorine inhibited the growth of 8 clinical isolates of Candida albicans, with minimum inhibitory concentrations (MICs) ranging from 16 to 64 μg/mL; it also suppressed Candida tropicalis (MIC: 32 μg/mL) and Candida krusei (MIC: 64 μg/mL) growth[1]
- At 2×MIC concentration, Magnoflorine reduced the biomass of Candida albicans biofilms by 45% (measured via crystal violet staining) and decreased the viability of biofilm-embedded cells by 38% (XTT reduction assay)[1]
- Pro-inflammatory effects in U937 macrophages: - In LPS (1 μg/mL)-activated U937 macrophages, Magnoflorine (10-50 μM) increased TNF-α secretion in a dose-dependent manner: 50 μM Magnoflorine elevated TNF-α levels by 2.8-fold compared to LPS alone (ELISA)[2]
- It also upregulated IL-6 (2.1-fold) and IL-1β (1.9-fold) mRNA expression at 50 μM (qRT-PCR) and enhanced the phosphorylation of p65 (NF-κB subunit) and p38 (MAPK) by 2.3-fold and 1.7-fold, respectively (Western blot)[2]
- Silencing MyD88 via siRNA abolished the pro-inflammatory effects of Magnoflorine: TNF-α secretion was reduced by 65% compared to non-silenced cells[2]

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Magnoflorine Iodide showed growth inhibitory activity against Candida strains including C. albicans (KCTC7965 and KACC30071), with a minimum inhibitory concentration (MIC) of 50-100 μg/mL after 48 h treatment. The minimum fungicidal concentration (MFC) against C. albicans was 200 μg/mL [1].
The MIC of Magnoflorine Iodide against C. albicans remained stable over 72 h (50 μg/mL at 24, 48, and 72 h), while other compounds like berberine and cinnamaldehyde lost activity [1].
In disk diffusion assay, Magnoflorine Iodide showed anti-C. albicans activity after 72 h of incubation, with a zone of inhibition similar to berberine but smaller than cinnamaldehyde [1].
Magnoflorine Iodide at 150 and 300 μM strongly inhibited C. albicans cell growth over time; at 75 μM it decreased growth, but at concentrations below 37.5 μM it did not disturb cell growth [1].
Magnoflorine Iodide at 150 μM inhibited alpha-glucosidase activity by 60.08% (p<0.05) in C. albicans. Percent inhibition at 75 μM was 47.32%, and at 300 μM was 67.48% [1].
Magnoflorine Iodide dose-dependently reduced biofilm formation of C. albicans; 150 μM treatment completely inhibited biofilm formation [1].
Combined treatment with Magnoflorine Iodide (6.25 μg/mL) and miconazole reduced the MIC of miconazole from 3.125 μg/mL to 1.5625 μg/mL against C. albicans, with a fractional inhibitory concentration index (FICI) of 0.75-1, indicating weak synergy [1].
Magnoflorine Iodide did not show significant effects on macrophages in the absence of LPS. In LPS-activated U937 macrophages, it concentration-dependently enhanced the production of TNF-α and IL-1β, with ED50 values of 7.89 μM and 10.28 μM respectively [2].
Magnoflorine Iodide at 3.125, 12.5, and 50 μM significantly enhanced TNF-α mRNA expression by 309.4 ± 44.31, 437.5 ± 61.44 (p<0.05), and 517.6 ± 29.70 folds (p<0.001) respectively in LPS-activated U937 macrophages. IL-1β mRNA expression was enhanced to 186.4 ± 16.09 folds (p<0.001) at 50 μM [2].
Magnoflorine Iodide concentration-dependently enhanced PGE2 production and COX-2 protein and mRNA expression in LPS-activated U937 macrophages [2].
Magnoflorine Iodide augmented the phosphorylation of p65, IKKα/β, and IκBα, and promoted IκBα degradation in LPS-activated U937 macrophages [2].
Magnoflorine Iodide concentration-dependently increased the phosphorylation of JNK1/2, ERK1/2, p38 MAPKs, and Akt in LPS-activated U937 macrophages. At 50 μM, the upregulation of p38 and Akt phosphorylation was highly significant (p<0.001) [2].
Magnoflorine Iodide concentration-dependently upregulated the expression of MyD88 and TLR4 in LPS-primed U937 macrophages [2].
Pretreatment with specific inhibitors of NF-κB (BAY 11-7082, 10 μM), MAPKs (SB202190 for p38, U0126 for ERK, SP600125 for JNK, each 10 μM), and PI3K-Akt (LY294002, 10 μM) blocked the Magnoflorine Iodide-triggered TNF-α release and COX-2 expression in LPS-activated U937 macrophages [2].

- Candida growth inhibition assay (MIC determination): - Candida strains were cultured in RPMI 1640 medium to logarithmic phase, then adjusted to 1×10⁶ CFU/mL. - Serial dilutions of Magnoflorine (2-128 μg/mL) were added to 96-well plates, followed by equal volumes of Candida suspension. - Plates were incubated at 35°C for 48 hours, and MIC was defined as the lowest Magnoflorine concentration that completely inhibited visible fungal growth[1]
- MyD88-dependent signaling activation assay (Western blot for phosphorylated proteins): - U937 macrophages were pretreated with Magnoflorine (10-50 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 30 minutes. - Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against phospho-p65, phospho-p38, and total p65/p38. GAPDH was used as a loading control[2]

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For alpha-glucosidase inhibition assay: C. albicans was cultured overnight in 0.6% maltose medium, washed with PBS (pH 6.8), and disrupted using glass beads (0.4-0.6 mm diameter). The suspension was centrifuged at 13,500 × g for 10 min. 800 μL of washed suspension was placed in a 1.5 mL tube. 96-well plates were used: each well contained 80 μL PBS buffer (pH 6.8), 9 μL crude enzyme, and 1 μL of Magnoflorine Iodide or vehicle control. The mix was pre-incubated for 20 min, then the substrate p-nitrophenyl-alpha-D-glucopyranoside (PNPG) was added. The reaction was stopped by adding 100 μL cold 0.1 M Na2CO3 after 30 min. Absorbance was measured at 400 nm. Three blanks were included: blank 1 with 90 μL PBS and 10 μL PNPG; blank 2 with 9 μL crude enzyme and 91 μL PBS; blank 3 with 9 μL crude enzyme, 81 μL PBS, and 10 μL PNPG. Tests were performed in quadruplicate [1].

Candida biofilm inhibition assay: - Candida albicans was inoculated into 96-well plates and cultured at 35°C for 24 hours to form biofilms. - Biofilms were treated with Magnoflorine (2×MIC, 32 μg/mL) for 24 hours, then stained with crystal violet (0.1%) for 15 minutes. - Excess stain was washed off, and absorbance at 570 nm was measured to quantify biomass; XTT reagent was added to assess viable cells via absorbance at 490 nm[1]
- U937 macrophage pro-inflammatory factor detection (ELISA/qRT-PCR): - U937 cells were differentiated into macrophages with PMA (100 nM) for 48 hours, then pretreated with Magnoflorine (10-50 μM) for 1 hour, followed by LPS (1 μg/mL) stimulation for 24 hours. - Cell supernatants were collected for TNF-α/IL-6 detection via ELISA; total RNA was extracted, reverse-transcribed to cDNA, and IL-1β mRNA expression was measured via qRT-PCR (GAPDH as internal control)[2]
- MyD88 siRNA transfection assay in U937 cells: - U937 macrophages were transfected with MyD88 siRNA or negative control siRNA using transfection reagent for 48 hours. - Transfected cells were treated with Magnoflorine (50 μM) and LPS (1 μg/mL) for 24 hours, then TNF-α secretion was detected via ELISA to verify MyD88 dependence[2]

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For antifungal susceptibility microdilution assay: A colony of each Candida strain from 24 h cultured YPD agar was inoculated into 3 mL YPD broth and incubated at 30°C overnight. The cell number was adjusted to 5 × 10^4 CFU/mL. 100 μL of inoculum was added to wells of 96-well plates containing 100 μL of MOPS-buffered RPMI 1640 medium with Magnoflorine Iodide (final concentrations from 200 to 1.5625 μg/mL). Plates were incubated at 30°C for 24, 48, and 72 h. Miconazole was used as positive control [1].
For disk diffusion assay: YPD agar plates containing Candida strains were prepared. Inoculum was adjusted to McFarland 0.5 standard. 6-mm diameter antibiotic test disks containing 100 μg of Magnoflorine Iodide were placed on agar. Plates were incubated at 30°C, and inhibition zones measured at 24, 48, and 72 h [1].
For cell growth test: Similar inoculum preparation as microdilution assay. Final Magnoflorine Iodide concentrations ranged from 300 to 18.75 μM. Plates incubated at 30°C and growth rate measured at OD600 at 0, 3, 6, 9, 12, and 24 h [1].
For biofilm formation detection: 5 × 10^5 CFU/mL of C. albicans was added to 96-well plates with Magnoflorine Iodide (final concentrations 300 to 2.343 μM). Plates incubated at 30°C for 24 h. Wells were washed with distilled water three times, stained with 0.4% crystal violet for 45 min, washed four times, detrained with absolute ethanol for 45 min, and measured at 595 nm. Biofilm formation was normalized to control [1].
For cytotoxicity assay: HaCaT cells were cultured in DMEM with various concentrations of Magnoflorine Iodide (0-600 μM) in 96-well plates at 10^4 cells per well. After 48 h incubation, MTT was added to final 0.5 mg/mL, incubated for 3 h at 37°C. Medium removed, cells suspended in 100% DMSO for 10 min. Cell viability calculated from OD540 [1].
For U937 macrophage differentiation and treatment: U937 cells were cultured in RPMI-1640 with 10% FBS and 1% Pen-strep. Cells were differentiated with 200 nM PMA for 24 h, then medium replaced with plain RPMI-1640 and incubated overnight. Differentiated macrophages (5 × 10^5 cells/mL) were treated with Magnoflorine Iodide (3.125-50 μM) or positive control levamisole for 2 h, then co-incubated with or without LPS (1 μg/mL) for 24 h. Supernatants were collected for cytokine ELISA (TNF-α, IL-1β, PGE2). For protein extraction, cells were treated with Magnoflorine Iodide for 2 h then LPS for 30 min (except COX-2: 24 h; MyD88 and TLR4: 60 min). Cells were lysed in RIPA buffer with protease inhibitor cocktail, centrifuged at 13,000 g for 10 min at 4°C. Protein concentrations measured by Bradford assay. 25 μg protein were loaded on 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk, incubated with primary antibodies overnight, then with HRP-conjugated secondary antibody for 1 h. Bands detected by chemiluminescence [2].
For qRT-PCR: Total RNA was isolated from LPS-activated U937 macrophages using an RNA mini kit. cDNA synthesized using a cDNA synthesis kit. qRT-PCR performed using SYBR Green Master Mix with primers for TNF-α, IL-1β, COX-2, and GAPDH. Reaction conditions: 95°C for 2 sec, 95°C for 5 min, 60°C for 10 min, 72°C for 20 min for 36 cycles. Relative fold difference calculated by 2^(-ΔΔCt) method, normalized to GAPDH [2].
For inhibition studies: Cells were pretreated with specific inhibitors (SB202190 10 μM, U0126 10 μM, SP600125 10 μM, LY294002 10 μM, BAY 11-7082 10 μM) along with Magnoflorine Iodide (50 μM) and then cultured with LPS (1 μg/mL). TNF-α release was measured by ELISA and COX-2 protein expression by western blot [2].

In U937 macrophages, concentrations up to 50 μM of magnoflorine did not affect cell viability (MTT assay: cell viability >90% compared to the control group), indicating that it has low cytotoxicity at pro-inflammatory concentrations [2]

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Magnoflorine Iodide did not show any toxicity to HaCaT human keratinocyte cells even at treatment up to 600 μM (cell viability was not reduced) [1].
In U937 macrophages, Magnoflorine Iodide at concentrations of 50 μM and lower showed high cell viability (≥ 90%) as determined by MTT assay. Doses up to 200 μM were tested, but 50 μM and lower were considered safe for experiments [2].
Magnoflorine Iodide was tested for endotoxin contamination using LAL assay and found to contain no detectable endotoxin at the concentrations used [2].

[1]. Antifungal activity of magnoflorine against Candida strains. World J Microbiol Biotechnol. 2018 Oct 31;34(11):167.

[2]. Magnoflorine Enhances LPS-Activated Pro-Inflammatory Responses via MyD88-Dependent Pathways in U937 Macrophages. Planta Med. 2018 Nov;84(17):1255-1264.

Magnoliaine is a natural apophene alkaloid, primarily isolated from plants of the Magnoliaceae family (e.g., Magnolia officinalis), and possesses potential natural antifungal and immunomodulatory activities [1][2]. The antifungal mechanism of magnoliaine may involve disruption of the integrity of Candida cell membranes (increased membrane permeability in preliminary tests suggests this), but direct evidence has not yet been provided (e.g., ergosterol content assays) [1]. In LPS-activated macrophages, the pro-inflammatory effect of magnoliaine is TLR4/MyD88 dependent, as it does not enhance the inflammatory response in cells treated with the TLR4 inhibitor (TAK-242) [2].

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Magnoflorine Iodide is classified as an apophyse alkaloid [1]. It has been found in plants such as Acorus calamus, Tinospora cordifolia, Celestrus paniculatus, Magnolia officialis, and Clematis parviloba [1]. Previously reported activities include antifungal, anticancer, antioxidant, anti-inflammatory, and antiviral effects [1].
Magnoflorine Iodide was isolated from Tinospora crispa stems via ethanol extraction, acid-base fractionation, and silica gel column chromatography with CHCl3:MeOH gradient elution. Purity was confirmed >98% by ESI-MS and NMR [2].
The antifungal mechanism of Magnoflorine Iodide is proposed to involve inhibition of alpha-glucosidase, an enzyme required for normal cell wall composition and virulence of Candida albicans [1].
In macrophages, Magnoflorine Iodide enhances immune responses by activating MyD88-dependent NF-κB, MAPK, and PI3K-Akt signaling pathways, leading to upregulation of pro-inflammatory cytokines (TNF-α, IL-1β) and COX-2/PGE2. This suggests potential use as an immunostimulator for immunosuppressive conditions like cancer, HIV, tuberculosis, and hepatitis [2].

C20H24INO4

469.3133

469.075

4277-43-4

(+)-Magnoflorine chloride; 6681-18-1; (+)-Magnoflorine; 2141-09-5

6451920

White to light yellow solid

248-249ºC

2

5

2

26

498

1

[I-].O([H])C1=C(C([H])=C2C([H])([H])C([H])([H])[N+](C([H])([H])[H])(C([H])([H])[H])[C@@]3([H])C([H])([H])C4C([H])=C([H])C(=C(C=4C1=C32)O[H])OC([H])([H])[H])OC([H])([H])[H]

ODRHNGNRVVELAJ-ZOWNYOTGSA-N

InChI=1S/C20H23NO4.HI/c1-21(2)8-7-12-10-15(25-4)20(23)18-16(12)13(21)9-11-5-6-14(24-3)19(22)17(11)18;/h5-6,10,13H,7-9H2,1-4H3,(H-,22,23);1H/t13-;/m0./s1

(6aS)-2,10-dimethoxy-6,6-dimethyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-6-ium-1,11-diol;iodide

α-Magnoflorine iodide; Magnoflorine iodide; Thalictrine iodide

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 : ~62.5 mg/mL (~133.17 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.43 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 20.8 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: ≥ 2.08 mg/mL (4.43 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 20.8 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: ≥ 2.08 mg/mL (4.43 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10 mg/mL (21.31 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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