Hypaphorine inhibits LPS-induced phosphorylation of ERK, nuclear factor kappa beta ((NFκB), NFκB inhibitor IκBα, and IKKβ, as well as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression. It also prevents NFκB from translocating into the nucleus in LPS-treated RAW 264.7 cells.[1]

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Hypaphorine (12.5, 25, and 50 μM) dose-dependently inhibited LPS (1 μg/mL)-induced nitric oxide (NO) production in RAW 264.7 macrophages. The NO levels were reduced from approximately 5-fold over control to near control levels. [1]
Hypaphorine dose-dependently suppressed LPS-induced mRNA expression of pro-inflammatory cytokines in RAW 264.7 cells: TNF-α, IL-1β, IL-6, IL-10, and MCP-1. At 50 μM, hypaphorine was more effective than dexamethasone (100 μM) and aspirin (1 mM) in inhibiting these inflammatory mediators. [1]
Hypaphorine dose-dependently inhibited LPS-induced protein expression of TNF-α, IL-1β, IL-6, and PGE₂ in RAW 264.7 cells, as measured by ELISA. The inhibitory effects were concentration-dependent, with the 50 μM dose showing the greatest effect. [1]
Hypaphorine dose-dependently down-regulated LPS-induced protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW 264.7 cells, as determined by Western blot analysis. [1]
Hypaphorine (50 μM) inhibited LPS-induced phosphorylation of ERK, IκBα, IKKβ, and NFκB in RAW 264.7 cells, as determined by Western blot analysis. The 25 μM dose showed no significant effect on the phosphorylation of IκBα, IKKβ, and NFκB, while 12.5 μM and 50 μM were effective. [1]
Hypaphorine (50 μM) attenuated LPS-induced nuclear translocation of p65-NFκB in RAW 264.7 cells, as visualized by immunofluorescence staining. [1]
Hypaphorine at concentrations up to 200 μM for 24 hours showed no significant cytotoxicity in multiple cell lines, including RAW 264.7, L929, A549, Lewis, H22, B16, MCF-7, HMEC-1, and EA-hy926 cells, as assessed by SRB assay. [1]

A potential therapeutic agent for treating osteoclast-based bone loss is hypaphorine, which inhibits RANKL-induced osteoclastogenesis through a decrease in the phosphorylation of NF-κB p65, ERK, p38, and c-Jun N-terminal kinase (JNK), and ERK, p38, and p38 kinases.[2]

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Incubating the cells in 2% serum medium for 24 hours prior to treatment stopped the cell growth. The indicated cells were then plated in 96-well culture plates at a density of 5×103 cells/well, stimulated with varying concentrations of vaccaria hypaphorine (6.25, 12.5, 25, 50, 100, 200 μM), and incubated for 24 hours at 37°C in a 5% COsub>2/sub> saturated humidity condition. Finally, at 540 nm, the optical density (OD) was determined.

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Cell viability assay (SRB): Cells were seeded in 96-well plates at 5 × 10³ cells/well and treated with various concentrations of hypaphorine (6.25, 12.5, 25, 50, 100, 200 μM) for 24 hours. After treatment, cells were fixed and stained with sulforhodamine B (SRB), and optical density was measured at 540 nm to assess cell viability. [1]
Nitric oxide (NO) measurement: RAW 264.7 cells were treated with LPS (1 μg/mL) and various concentrations of hypaphorine (12.5, 25, 50 μM) for 24 hours. Culture supernatants were collected and reacted with Griess reagent (equal volumes of N-(1-naphthyl)ethylenediamine and sulfanilic acid) for 30 minutes at room temperature. Absorbance was measured at 540 nm, and NO levels were calculated using a sodium nitrite standard curve. [1]
Real-time quantitative PCR: Total RNA was extracted using Trizol reagent and reverse-transcribed to cDNA. mRNA expression levels of TNF-α, IL-1β, IL-6, IL-10, and MCP-1 were quantified using SYBR Green real-time PCR, with results normalized to an internal control and calculated using the 2⁻ΔΔCt method. [1]
ELISA: Culture supernatants were collected and TNF-α, IL-1β, IL-6, and PGE₂ levels were measured using commercial ELISA kits according to the manufacturer’s protocols. Absorbance was read at 450 nm. [1]
Western blot analysis: RAW 264.7 cells were lysed in RIPA buffer, and protein concentrations were determined. Equal amounts of protein (25 μg) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with primary antibodies against COX-2, iNOS, p-ERK, ERK, p-IκBα, IκBα, p-IKKβ, IKKβ, p-NFκB, NFκB, and β-tubulin. Bands were visualized using enhanced chemiluminescence. [1]
Immunofluorescence microscopy: RAW 264.7 cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum. Cells were incubated with rabbit anti-NFκB antibody overnight at 4°C, followed by FITC-labeled secondary antibody. Nuclei were stained with DAPI, and immunofluorescence signals were visualized under a fluorescence microscope. [1]

C57BL/6J mice induced by LPS 10 and 30 mg/kg p.o.

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Hypaphorine showed no significant cytotoxicity in multiple cell lines, including RAW 264.7 macrophages, L929 fibroblasts, A549 (lung cancer), Lewis (lung cancer), H22 (liver cancer), B16 (melanoma), MCF-7 (breast cancer), HMEC-1 (microvascular endothelial), and EA-hy926 (umbilical vein endothelial) cells at concentrations up to 200 μM for 24 hours, as assessed by SRB assay. [1]

[1]. BMC Complement Altern Med . 2017 Feb 20;17(1):120.

[2]. Biomed Pharmacother . 2018 Jan:97:1155-1163.

Hypaphorine is an amino acid betaine, synthesized by complete methylation of the α-amino group of L-tryptophan, accompanied by deprotonation of the carboxyl group. It is a plant metabolite, an exogenous substance, and a fungal metabolite. It is an amino acid betaine, an L-tryptophan derivative, and an indole alkaloid. Hypaphorine has been reported to exist in Pisolithus tinctorius, Caragana sinica, and several other organisms with relevant data.

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Hypaphorine is an indole alkaloid abundantly found in vaccaria semen (the seeds of Vaccaria segetalis). This study demonstrates that hypaphorine exerts anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages by suppressing the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1) and inflammatory mediators (NO, PGE₂), as well as down-regulating iNOS and COX-2 expression. The anti-inflammatory mechanism involves inhibition of the ERK and NFκB signaling pathways, including the phosphorylation of ERK, IκBα, IKKβ, and NFκB, and the nuclear translocation of p65-NFκB. These findings suggest that hypaphorine may serve as a potential anti-inflammatory candidate for inflammation-associated diseases. [1]

C14H18N2O2

246.3049

246.136

C, 68.27; H, 7.37; N, 11.37; O, 12.99

487-58-1

487-58-1

442106

White to off-white solid powder

255℃

-2.12

1

2

3

18

306

1

[O-]C(C([H])(C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12)[N+](C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O

AOHCBEAZXHZMOR-ZDUSSCGKSA-N

InChI=1S/C14H18N2O2/c1-16(2,3)13(14(17)18)8-10-9-15-12-7-5-4-6-11(10)12/h4-7,9,13,15H,8H2,1-3H3/t13-/m0/s1

(2S)-3-(1H-indol-3-yl)-2-(trimethylazaniumyl)propanoate

Hypaphorine; Hypaforin; Tryptophan betaine; L-Hypaphorine

2934.99.03.00

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)

H2O: ~100 mg/mL (~406.0 mM)
DMSO: < 1 mg/mL

Solubility in Formulation 1: 100 mg/mL (406.01 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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