P-gp
Hypophyllanthin targets the NF-κB signaling pathway (by suppressing IKKα/β, IκBα, and NF-κBp65 phosphorylation, and IκBα degradation). [1]
Hypophyllanthin targets MAPK signaling pathway: it suppresses the phosphorylation of JNK, ERK, and p38. [1]
Hypophyllanthin targets the PI3K-Akt signaling pathway by suppressing LPS-induced Akt phosphorylation. [1]
Hypophyllanthin targets TLR4 and MyD88 expression. [1]
Hypophyllanthin inhibits P-glycoprotein (P-gp) function directly, likely through reversible binding to substrate binding sites, but does not affect multidrug resistance protein 2 (MRP2) activity. [2]

By preventing NF-κB, MAPK, and Akt from being activated, lutein inhibits the production of the COX-2, TNF-α, and IL-1β genes in U937 macrophages [1].

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In LPS-induced U937 macrophages, Hypophyllanthin (1.5 to 24 μM) showed no cytotoxic effect for 24 h. It inhibited TNF-α and IL-1β production with IC50 values of 12.18 μM and 9.39 μM, respectively. [1]
In the same model, Hypophyllanthin suppressed PGE2 production and COX-2 expression at both protein and gene levels in a concentration-dependent manner. [1]
Hypophyllanthin (2 h pretreatment) inhibited LPS-induced phosphorylation of IKKα/β, IκBα, and NF-κBp65, as well as IκBα degradation, in a concentration-dependent manner. [1]
Hypophyllanthin suppressed LPS-induced phosphorylation of JNK, ERK, and p38 MAPKs, and also suppressed Akt phosphorylation. [1]
Hypophyllanthin downregulated LPS-induced TLR4 and MyD88 protein expression in a concentration-dependent manner. [1]
In Caco-2 cells (21 days post-seeding), Hypophyllanthin (1 to 100 μM) increased intracellular accumulation of calcein (a P-gp substrate) in a concentration-dependent manner, with a 3.2-fold increase at 100 μM, indicating inhibition of P-gp function. It did not affect CDCF accumulation (MRP2 substrate). [2]
The inhibitory effect of Hypophyllanthin on P-gp was reversible: when cells were pre-treated with the lignan for 30 min followed by washout before adding calcein-AM, no inhibition was observed. In the co-treatment condition (lignan and substrate added together without pre-incubation), the inhibitory effect was present but with high variation. [2]
Prolonged exposure (up to 7 days) of Caco-2 cells to Hypophyllanthin (at non-cytotoxic concentrations) did not affect P-gp function. [2]

U937 cells were differentiated with 200 nM phorbol 12-myristate 13-acetate (PMA) for 24 h to obtain macrophage-like phenotype. Differentiated cells were seeded at 5×10^5 cells/mL in 96-well plates, treated with various concentrations of Hypophyllanthin (1.5, 3, 6, 12, 24 μM) for 24 h, then 10% v/v of Alamar blue reagent was added and incubated for 4 h. Cell viability was measured at 570 nm with reference at 600 nm. [1]
For ELISA, differentiated U937 cells were plated in 24-well plates, pretreated with or without Hypophyllanthin (1.5–24 μM) and dexamethasone (0.001–10 μM) for 2 h, then stimulated with 1 μg/mL LPS for 24 h. Supernatants were collected and TNF-α and IL-1β levels were measured using ELISA kits. [1]
For PGE2 assay, U937 macrophages were pretreated with Hypophyllanthin (1.5–24 μM) for 2 h followed by LPS stimulation for 24 h. Supernatant was collected and PGE2 concentration was quantified using a PGE2 assay kit with a standard curve. [1]
For qRT-PCR, total RNA was extracted from U937 macrophages using an RNA extraction kit. cDNA synthesis was performed, and relative quantification of mRNA (COX-2, TNF-α, IL-1β, GAPDH as housekeeping) was carried out using real-time PCR with SYBR Green master mix. The 2^(-ΔΔCt) method was used for analysis. [1]
For Western blot, U937 macrophages were pretreated with Hypophyllanthin (or inhibitors) for 2 h, then stimulated with LPS for indicated periods. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was quantified, and 20 μg of protein were separated by 10% SDS-PAGE, transferred to PVDF membrane, blocked with 5% non-fat milk, incubated with primary antibodies (COX-2, p-p38, p38, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, p-Akt, IκBα, p-IκBα, p-IKKα/β, p-NF-κBp65, TLR4, MyD88, β-actin), then HRP-conjugated secondary antibody, and detected by chemiluminescence. Band intensity was quantified with ImageJ. [1]
Caco-2 cells (passages 50-78) were seeded at 1.3×10^4 cells/cm² in 24-well plates and used at 21 days post-seeding. For P-gp and MRP2 activity assays, cells were washed with HBSS, then pre-incubated with Hypophyllanthin (or verapamil or indometacin) for 30 min at 37°C. Then substrate (0.4 μM calcein-AM for P-gp or 5 μM CDCFDA for MRP2) was added and co-incubated for another 30 min. Cells were washed with ice-cold PBS, lysed with 0.1% Triton X-100, and fluorescence (ex 485 nm, em 535 nm) was measured. Protein was quantified with Bradford reagent. [2]
For reversibility studies, three conditions were used: (1) standard pre/co-treatment: pre-incubation with Hypophyllanthin for 30 min then co-incubation with substrate for 30 min; (2) pre-treatment: cells exposed to lignan for 30 min, then washed, and substrate added for 30 min without lignan; (3) co-treatment: lignan and substrate added simultaneously for 30 min without pre-incubation. [2]
For prolonged exposure, Caco-2 cells (14 days after seeding) were treated with Hypophyllanthin for 2 or 7 days. On assay day, medium was replaced with fresh medium without lignan for 3 h before the calcein-AM uptake assay. [2]

Hypophyllanthin at concentrations from 1.5 to 24 μM showed no cytotoxic effect on differentiated U937 cells for 24 h as determined by Alamar blue assay. [1]
In Caco-2 cells, the highest concentrations of Hypophyllanthin used in each experiment were non-cytotoxic as determined by trypan blue extrusion assay, and treatment had no effects on cell morphology or cell attachment. [2]
Prolonged exposure (up to 7 days) to Hypophyllanthin at non-cytotoxic concentrations did not affect P-gp function. [2]

[1]. Anti-Inflammatory Effects of Hypophyllanthin and Niranthin Through Downregulation of NF-κB/MAPKs/PI3K-Akt Signaling Pathways. Inflammation. 2018 Jun;41(3):984-995.

[2]. Phyllanthin and hypophyllanthin inhibit function of P-gp but not MRP2 in Caco-2 cells. J Pharm Pharmacol. 2013 Feb;65(2):292-9.

It has been reported that Phyllanthus urinaria and Phyllanthus niruri contain hypobenzalin, and relevant data are available. See also: Phyllanthus amarus (top part).

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Hypophyllanthin is a major lignan in Phyllanthus species. Its anti-inflammatory mechanism involves downregulation of NF-κB, MAPKs (JNK, ERK, p38), and PI3K-Akt signaling pathways, as well as suppression of TLR4 and MyD88 expression, leading to reduced production of pro-inflammatory mediators (TNF-α, IL-1β, PGE2) and COX-2. [1]
Hypophyllanthin directly inhibits P-gp activity likely via reversible competitive binding to the substrate binding sites, without affecting MRP2 activity. It does not alter P-gp function after prolonged exposure for up to 7 days. [2]

C24H30O7

430.4908

430.199

33676-00-5

182140

White to off-white solid

1.158

511.1±50.0 °C at 760 mmHg

129-130℃

200.8±30.0 °C

0.0±1.3 mmHg at 25°C

1.536

3.77

0

7

8

31

560

3

COC[C@H]1CC2=CC(=C3C(=C2[C@@H](C4=CC(=C(C=C4)OC)OC)[C@@H]1COC)OCO3)OC

LBJCUHLNHSKZBW-XGHQBKJUSA-N

InChI=1S/C24H30O7/c1-25-11-16-8-15-10-20(29-5)23-24(31-13-30-23)22(15)21(17(16)12-26-2)14-6-7-18(27-3)19(9-14)28-4/h6-7,9-10,16-17,21H,8,11-13H2,1-5H3/t16-,17-,21+/m0/s1

(7R,8R,9S)-9-(3,4-dimethoxyphenyl)-4-methoxy-7,8-bis(methoxymethyl)-6,7,8,9-tetrahydrobenzo[g][1,3]benzodioxole

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 : ~33.33 mg/mL (~77.42 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.81 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (5.81 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.)