In MC3T3-E1 cells, neoeriocitrin promotes osteogenic differentiation possibly via the MAPK/ERK1/2 pathway, as it partially rescues the inhibition of cell differentiation induced by PD98059 (an ERK1/2 inhibitor) [1]
Acetylcholinesterase (AChE): IC50 = 80.97 μmol/L. [2]

In MC3T3-E1 preosteoblast cells, neoeriocitrin significantly promoted cell proliferation in a dose-dependent manner at concentrations of 2, 4, 8, 10, and 20 μg/ml after 3 days of treatment, with the best effect observed at 2 μg/ml (MTT assay). Compared to naringin at the same concentration, neoeriocitrin exhibited significantly higher proliferative activity (P<0.01). [1]
Alkaline phosphatase (ALP) activity, an early marker of osteogenic differentiation, was significantly increased by neoeriocitrin in a dose-dependent manner (2-20 μg/ml) after 5 days of treatment. At 2 μg/ml, neoeriocitrin enhanced ALP activity significantly better than naringin (P=0.043). [1]
Real-time PCR analysis showed that after 4 days (for Runx2 and COL1) or 8 days (for OCN) of treatment with 2 μg/ml neoeriocitrin, the mRNA expression levels of Runx2, type I collagen (COL1), and osteocalcin (OCN) were up-regulated. Compared to naringin, neoeriocitrin enhanced Runx2 expression by 56%, COL1 by 37%, and OCN by 14% higher. [1]
In the presence of PD98059 (50 μmol/l), an ERK1/2 inhibitor, ALP activity decreased by 30% at day 5, and the expression of COL1 (day 4) and OCN (day 8) was significantly reduced. Treatment with 2 μg/ml neoeriocitrin significantly rescued ALP activity and the expression of these osteogenic markers. [1]
In the AChE inhibition assay, neoeriocitrin showed dose-dependent inhibitory activity against AChE with an IC50 of 80.97 μmol/L. [2]
In PC12 cells, pretreatment with neoeriocitrin (100 μg/mL) for 1 hour followed by treatment with Aβ(25-35) (10 μM) for 48 hours significantly increased cell viability to 67.66% (compared to Aβ(25-35)-only treatment, which reduced viability to 15.12%, P<0.001). [2]

For alkaline phosphatase (ALP) activity measurement, MC3T3-E1 cells were plated in 24-well plates and allowed to reach 80% confluence. The culture medium was replaced with osteogenic supplements (OS) containing ascorbic acid and β-glycerophosphate. Various concentrations (2-20 μg/ml) of neoeriocitrin were added and cells were cultured for 5 days. The cell monolayer was washed with ice-cold PBS and lysed with RIPA buffer. The lysate was centrifuged, and the supernatant was used for ALP activity measurement using a commercial alkaline phosphatase activity kit. Protein concentration was determined using a protein assay kit. [1]
For acetylcholinesterase (AChE) inhibition assay, a microplate reader method was used. The assay mixture (220 μl) contained sample solution (20 μl), phosphate-buffered saline (80 μl), dithiobis(nitrobenzoic acid) (40 μl), and AChE solution (20 μl). The microplate was incubated at 37°C for 10 min, then acetylthiocholine iodide (40 μl) was added and incubated for another 10 min at 37°C. SDS solution (1%, 60 μl) was added to stop the reaction. Absorbance was measured at 405 nm. Inhibition percentage was calculated. For IC50 determination, seven different concentrations of each inhibitor were assayed in triplicate. [2]
For ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) screening of AChE inhibitors, incubation mixtures were prepared by mixing sample solution (5 mg/ml, 5 μl) and AChE (10 μl) in ammonium acetate buffer (185 μl, containing 20 mM ammonium acetate and 10% isopropanol, pH 6.8). The mixture was incubated at 37°C for 30 min, then injected into an ultrafiltration chamber and centrifuged at 13000×g for 10 min at 25°C. The filter was washed three times with buffer by centrifugation. Bound ligands were released by adding methanol (200 μl) and centrifugation at 13000×g for 10 min (repeated three times). The ultrafiltrate was evaporated under vacuum and the residue was redissolved in 50% methanol for LC analysis. Control experiments were performed using denatured enzyme. The degree of binding (DB) was calculated as (Ab - Ac)/Aa × 100, where Aa is peak area of compound without enzyme, Ab and Ac are peak areas with active and denatured AChE, respectively. [2]

For MTT proliferation assay, MC3T3-E1 cells were collected by trypsin treatment, suspended in medium, and plated at a density of 5×10^4 cells/ml into 96-well culture plates. After 24 h, the medium was replaced with media containing 5% FBS and osteogenic supplements (OS: 10 μg/ml ascorbic acid and 10 mM β-glycerophosphate). Various concentrations (2-20 μg/ml) of neoeriocitrin were added. After 72 h incubation, MTT solution (5 g/l in PBS) was added to each well (1:10 volume ratio) and incubated at 37°C for 4 h. DMSO was added to dissolve the dark blue crystals, and after 10 min at room temperature, absorbance was read at 492 nm using a microplate reader. [1]
For quantitative real-time PCR, MC3T3-E1 cells were cultured to 80% confluence, then induced with OS medium containing 2 μg/ml neoeriocitrin for 4 days (for Runx2 and COL1) or 8 days (for OCN). Total RNA was extracted using Trizol reagent, and single-strand cDNA synthesis was performed using a reverse transcription kit. Real-time quantitative PCR was performed using SYBR green kit. The reaction conditions were: initial step at 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s. Gene expression levels were normalized to GAPDH. Primers sequences for Runx2, COL1, OCN, and GAPDH were provided. [1]
For the PD98059 rescue experiment, MC3T3-E1 cells were pretreated with or without PD98059 (50 μmol/l) for 60 min after adding OS for 2 h, then stimulated with 2 μg/ml neoeriocitrin for 5 days (for ALP activity) or for 4 days (for COL1) and 8 days (for OCN) for real-time PCR. [1]
For PC12 cell culture and viability assay, rat PC12 cells were maintained in Dulbecco's modified Eagle medium supplemented with penicillin (100 units/ml), streptomycin (100 μg/ml), 5% fetal bovine serum, and 5% horse serum at 37°C in 5% CO2. To evaluate neuroprotective effect, PC12 cells were divided into groups: untreated control, 10 μM Aβ(25-35) alone, 10 μM Aβ(25-35) + 10 μM donepezil hydrochloride, and 10 μM Aβ(25-35) + neoeriocitrin (10, 25, 50, 100, 200 μg/ml). Cells were pretreated with compounds for 1 h, then treated with Aβ(25-35) for 48 h. Cell viability was measured by MTT assay: cells were transferred to 96-well plates at 2×10^4 cells/well, MTT (5 mg/ml, 10 μl) was added, incubated at 37°C for 2 h, then medium removed and DMSO (100 μl) added to dissolve formazan. Absorbance was measured at 340 nm. Viability was expressed as percentage of untreated control. [2]

In PC12 cells, no significant toxic effects were observed when cells were incubated with 100 μg/mL neoeriocitrin (cell viability was 67.66% of control, indicating no overt cytotoxicity). [2] No other toxicity data are reported.

[1]. Comparison of neoeriocitrin and naringin on proliferation and osteogenic differentiation in MC3T3-E1. Phytomedicine. 2011 Aug 15;18(11):985-9.

[2]. Extraction and Isolation of Acetylcholinesterase Inhibitors from Citrus limon Peel Using an in vitro Method. J Sep Sci. 2020 Jan 30.

Neohesperidin is a flavanone glycoside formed by the substitution of sennaol at the 7-position via a glycosidic bond with a 2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl residue. It is a plant metabolite. It is a neohesperidin, a disaccharide derivative, a trihydroxyflavanone, a flavanone glycoside, and also a member of the 4'-hydroxyflavanone class of compounds. Its function is related to sennaol. Neohesperidin has been reported to exist in Dendrobium (Pyrrosia serpens), Citrus hystrix, and several other organisms with relevant data.

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Osteoporosis is a major public health problem leading to fractures and deformities. Chinese herbal medicine, particularly Rhizoma Drynariae (Drynaria Rhizome), has been used for thousands of years to treat bone diseases. Naringin is a well-established effective component, but neoeriocitrin, also isolated from Drynaria Rhizome, shows better bone formation potential by increasing osteoblast number and accelerating differentiation. The enhancement of new bone formation is obtained by increasing the number of osteoblasts and/or accelerating osteoblast differentiation. [1]
Alzheimer's disease (AD) is associated with amyloid-beta (Aβ) peptide and acetylcholinesterase (AChE). Aβ induces cell apoptosis and increases AChE activity, while AChE further promotes Aβ aggregation, forming a more toxic complex. Neoeriocitrin was identified from Citrus limon peel as a potential multi-target anti-AD agent because it inhibits AChE and protects PC12 cells from Aβ(25-35)-induced injury. [2]

C27H32O15

596.5340

596.174

C, 54.36; H, 5.41; O, 40.23

13241-32-2

114627

White to off-white solid powder

1.73g/cm3

960.7ºC at 760 mmHg

277ºC

318.3ºC

0mmHg at 25°C

1.727

-0.9

9

15

6

42

924

11

O([C@@]1([H])[C@@]([H])([C@@]([H])([C@]([H])([C@]([H])(C([H])([H])[H])O1)O[H])O[H])O[H])[C@]1([H])C([H])(OC2=C([H])C(=C3C(C([H])([H])C([H])(C4C([H])=C([H])C(=C(C=4[H])O[H])O[H])OC3=C2[H])=O)O[H])O[C@@]([H])(C([H])([H])O[H])C([H])([C@@]1([H])O[H])O[H]

OBKKEZLIABHSGY-DOYQYKRZSA-N

InChI=1S/C27H32O15/c1-9-20(33)22(35)24(37)26(38-9)42-25-23(36)21(34)18(8-28)41-27(25)39-11-5-14(31)19-15(32)7-16(40-17(19)6-11)10-2-3-12(29)13(30)4-10/h2-6,9,16,18,20-31,33-37H,7-8H2,1H3/t9-,16-,18+,20-,21+,22+,23-,24+,25+,26-,27+/m0/s1

(2S)-7-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxy-2-(3,4-dihydroxyphenyl)-5-hydroxy-2,3-dihydrochromen-4-one

Neoeriocitrin; Eriodictyol 7-O-neohesperidoside

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 (~167.64 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.19 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 (4.19 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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 (Please use freshly prepared in vivo formulations for optimal results.)