In SH-SY5Y-APP695 cells, ligtroside upregulates the mRNA expression of SIRT1, CREB1, complex I, and GPx1 [1].

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In Vitro: Ligstroside (0.05 μM, 24 h) significantly increased basal ATP levels in SH-SY5Y-APP695 cells compared to solvent control [1].
Ligstroside (0.05 μM, 24 h) significantly elevated endogenous respiration, complex I respiration, coupled complex I+II respiration, leak I respiration (after oligomycin), and the electron transfer system (ETS) capacity in SH-SY5Y-APP695 cells as measured by high-resolution respirometry [1].
Ligstroside (0.05 μM, 24 h) significantly increased citrate synthase (CS) activity, a marker of mitochondrial content, in SH-SY5Y-APP695 cells [1].
Ligstroside (0.05 μM, 24 h) significantly increased mRNA expression of SIRT1 (175.2% of control, p<0.05), CREB1 (137.2% of control, p<0.05), GPx1 (163.0% of control, p<0.05), and complex I (KI, 177.2% of control, p<0.05) in SH-SY5Y-APP695 cells, while significantly decreasing mRNA expression of CAT (66.6% of control, p<0.05) and SOD2 (50.2% of control, p<0.05) as determined by qRT-PCR. Numerical increases were observed for PGC-1α, NRF1, and TFAM [1].
Ligstroside (0.05 μM, 24 h) did not alter Aβ1-40 levels in SH-SY5Y-APP695 cells as measured by ELISA [1].
Ligstroside protected SH-SY5Y-APP695 cells against rotenone (25 μM)-induced ATP decrease (based on Supplementary Table 2, with first significant effects at 0.05 μM) [1].

In models of early Alzheimer disease and brain aging, ligstroside (50 mg/kg; 6 months of dietary supplementation) prevents mitochondrial dysfunction [1].
In Vivo: Female NMRI mice (aged 12 months) were fed a diet supplemented with Ligstroside at 50 mg/kg diet (equivalent to 6.25 mg/kg body weight) for 6 months. Ligstroside treatment significantly increased the number of spontaneous alternations in the Y-maze test (15.29 ± 1.24) compared to aged control mice (11.2 ± 0.96), indicating improved spatial working memory [1].
Ligstroside significantly increased basal ATP levels in dissociated brain cells of aged mice (1.28 ± 0.15 mmol/mg protein) compared to aged control animals (0.82 ± 0.05 mmol/mg protein) and young control animals (1.18 ± 0.01 mmol/mg protein) [1].
Ligstroside significantly extended lifespan in aged NMRI mice: survival rate was 85% in ligstroside-fed mice compared to 67% in aged control mice (p<0.01, log-rank test) [1].

Cell Assay: Human neuroblastoma SH-SY5Y cells stably transfected with human wild-type APP695 (SH-SY5Y-APP695) were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum, 0.3 mg/ml hygromycin, 60 units/ml penicillin, 60 μg/ml streptomycin, MEM Non-Essential Amino Acids, and 1 mM sodium pyruvate at 37°C in 5% CO2. Cells were passaged every 3-4 days and used at 80-90% confluence [1].
For ATP measurement, cells were incubated with Ligstroside (0.05 μM) for 24 h. ATP levels were determined using a bioluminescence assay based on light production from ATP and luciferin in the presence of luciferase, following a published protocol. Rotenone (25 μM) was used to induce mitochondrial dysfunction [1].
For mitochondrial respiration analysis, cells (10⁶ cells/ml) in mitochondrial respiration medium were permeabilized with digitonin (10 μg/10⁶ cells). Oxygen consumption was monitored at 37°C using an Oxygraph-2k with DatLab software. A substrate-uncoupler-inhibitor titration protocol was applied: complex I respiration (glutamate 10 mM, malate 2 mM, ADP 2 mM), coupled complex I+II respiration (succinate 10 mM), leak respiration (oligomycin 2 μg/ml), electron transfer system capacity (FCCP titration), complex II non-coupled respiration (rotenone), residual oxygen consumption (antimycin A 2.5 μM), and complex IV activity (TMPD 0.5 mM, ascorbate 2 mM) corrected for autoxidation (sodium azide ≥100 mM) [1].
For citrate synthase activity, cell suspensions were frozen in liquid nitrogen and stored at -80°C. CS activity was measured spectrophotometrically according to a previously published protocol [1].
For Aβ1-40 detection, cells were lysed after 24 h treatment with Ligstroside (0.05 μM) and protein concentration was normalized. A specific solid-phase sandwich ELISA was performed according to the manufacturer's instructions [1].
For qRT-PCR, total RNA was isolated from 500,000 cells using an RNeasy Mini Kit with RNA Protect stabilization. RNA was quantified by absorbance at 260/280 nm and 260/230 nm. Residual genomic DNA was removed with a TURBO DNA-free kit. cDNA was synthesized from 1 μg RNA using an iScript cDNA Synthesis Kit. qRT-PCR was conducted on a CfX 96 Connect system using SYBR Green assay with optimized primer concentrations (Table 1). Cycling conditions: initial denaturation 95°C for 3 min, then 45 cycles of 95°C for 10 s, 58°C for 30 s, and 72°C for 29 s. Gene expression was analyzed using the 2-ΔΔCq method normalized to GAPDH, beta-Actin, and PGK1. Melt curve analysis and agarose gel confirmation were performed [1].

Animal/Disease Models: Female NMRI mice, 12 months old [1]
Doses: 50 mg/kg
Route of Administration: Received supplementary diet for 6 months (equivalent to 6.25 mg/kg body weight)
Experimental Results: demonstrated improvement in spatial working memory; Compared with aged control animals, aged mice had restored brain ATP levels and Dramatically extended their lifespan.

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Animal Protocol: Female NMRI mice (12 months old at start) were maintained on a 12 h light/dark cycle. Mice were randomized into groups (aged control, aged + oleocanthal, aged + ligstroside). Young control NMRI mice (3 months old) were purchased. Mice were fed a standardized C1000 diet. The treatment group received the same diet supplemented with Ligstroside at 50 mg/kg diet (equivalent to 6.25 mg/kg body weight) for 6 months. The feeding period for young control mice started 3 months later to ensure same endpoint. Behavioral testing was performed before starting point and at the end of feeding period. Mice were killed by cervical dislocation and decapitation; brains were quickly dissected on ice after removal of cerebellum, brain stem, and olfactory bulb. All experiments were approved by the regional authority (Regierungsprasidium Darmstadt; #V54 - 19 c 20/15 - FU/1062) [1].
For the one-trial Y-maze test, a custom-made Y-maze (polyvinyl chloride, arm length 36 cm, arm height 7 cm, arm width 5 cm, 120° angle between arms) was used. The mouse was placed into one arm and the sequence of entries was recorded for 5 min. Spontaneous alternation was calculated as (number of alternations / number of entries) / 2 [1].
For preparation of dissociated brain cells (DBCs), one hemisphere was used. DBCs were re-suspended in DMEM without supplements. For ATP measurement, DBCs were seeded in 50 μl aliquots into a 96-well plate and incubated for 3 h in a humidified incubator (5% CO2). Six wells were incubated with sodium nitroprusside (0.5 mM for ATP measurement) in DMEM. Protein content was determined using a protein assay kit [1].
For survival analysis, mice were fed for 6 months and survival rates were calculated [1].

[1]. Purified oleocanthal and ligstroside protect against mitochondrial dysfunction in models of early Alzheimer's disease and brain ageing. Exp Neurol. 2020;328:113248.

[2]. Chemical structures of constituents from the flowers of Osmanthus fragrans var. aurantiacus. J Nat Med. 2015;69(1):135-141.

Ligstroside is a sericocyclic ether glycoside, a methyl ester of 3,4-dihydro-2H-pyran-5-carboxylic acid, with hydroxyl, ethylene, and carboxymethyl groups substituted at positions 2, 3, and 4, respectively. The terminal hydroxyl group at position 2 is converted to β-D-glucoside, and the carboxylic acid moiety at the carboxymethyl substituent is converted to the corresponding 4-hydroxyphenethyl ester (2S,3E,4S stereoisomers). Ligstroside is an important phenolic compound in olive varieties, possessing plant metabolite and antitumor activity. It is a sericocyclic ether glycoside, methyl ester, diester, pyran compound, phenolic compound, and β-D-glucoside. It has been reported that Ligstroside is present in Fraxinus taiwanensis, Ligustrum obtusifolium, and other organisms with relevant data.

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Additional Info: Ligstroside is a secoiridoid polyphenol found in extra virgin olive oil (EVOO) during the early ripening period of olive fruits, and can also be isolated from privet leaves. It is not commercially available. Unlike oleocanthal, Ligstroside does not affect Aβ1-40 levels, suggesting its beneficial effects on mitochondrial function are independent of the amyloid-β pathway. Ligstroside upregulates SIRT1, CREB1, and GPx1 mRNA expression, indicating potential activation of mitochondrial biogenesis via the PGC-1α cascade. In vivo, Ligstroside improved spatial working memory, restored brain ATP levels, and extended lifespan in aged mice, highlighting its potential for preventing age-related cognitive decline and mitochondrial dysfunction [1].

C25H32O12

524.51438

524.189

35897-92-8

14136859

White to off-white solid powder

1.4±0.1 g/cm3

736.9±60.0 °C at 760 mmHg

245.1±26.4 °C

0.0±2.5 mmHg at 25°C

1.614

-0.31

5

12

11

37

834

7

C/C=C/1\[C@@H](C(=CO[C@H]1O[C@H]2[C@@H]([C@H]([C@@H]([C@H](O2)CO)O)O)O)C(=O)OC)CC(=O)OCCC3=CC=C(C=C3)O

GMQXOLRKJQWPNB-MVVLSVRYSA-N

InChI=1S/C25H32O12/c1-3-15-16(10-19(28)34-9-8-13-4-6-14(27)7-5-13)17(23(32)33-2)12-35-24(15)37-25-22(31)21(30)20(29)18(11-26)36-25/h3-7,12,16,18,20-22,24-27,29-31H,8-11H2,1-2H3/b15-3+/t16-,18+,20+,21-,22+,24-,25-/m0/s1

methyl (4S,5E,6S)-5-ethylidene-4-[2-[2-(4-hydroxyphenyl)ethoxy]-2-oxoethyl]-6-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4H-pyran-3-carboxylate

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)

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.)