Natural product with neuroprotective, anti-inflammatory, anti-cancer, and vasodilatory activity
Ligustilide has multiple targets and mechanisms. It is a potential inhibitor of mesenchyme homeobox 1 (MEOX1). It suppresses HL-60 monocyte adhesion and CAMs (ICAM-1, VCAM-1, E-selectin) expression in HUVECs. It significantly inhibits TNF-α-induced reactive oxygen species production and NF-κB signaling pathway activation. It exerts significant antifibrotic effects through inhibition of MEOX1 and the downstream Hippo signaling pathway.

Ligustilide suppressed HL-60 monocyte adhesion and CAMs (ICAM-1, VCAM-1, E-selectin) expression in HUVECs. Ligustilide significantly inhibited TNF-α-increased production of ROS and activated NF-κB signaling pathway. Also, ligustilide treated HUVECs exhibited significant HO-1 induction via Nrf2 nuclear translocation and endothelial NO synthesis. Conclusion: Present study demonstrates that ligustilde attenuates vascular inflammation and activate defense system of endothelial cell. Ligustilide is a bioactive compound which might prevent cardiovascular complications such as thrombosis or atherosclerosis. [1]

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To investigate the neuroprotective of ligustilide (LIG) against glutamate-induced apoptosis of PC12 cells, cell viability were examined by MTT assay. Flow cytometry was applied to assay cell apoptosis rate. Intracellular calcium concentration was measured by using fluorescent dye Fluo-3/AM. Cytochrome C (Cyt C), Caspase-3, Bax and Bcl-2 protein expression were assayed by western blot. The results showed that glutamate is cytotoxic with an inhibitory concentration 50 (ID50) of 15 mmol · L(-1). Pretreatment with LIG (1, 5, 15 μmol · L(-1)) significantly improved cell viability. The apoptosis rate in glutamate-induced PC12 cells was 13.39%, and decreased in the presence of LIG (1, 5, 15 μmol · L(-1)) by 9.06%, 6.48%, 3.82%, separately. Extracellular accumulation of Ca2+ induced by glutamate were significantly reduced by LIG. The results of western blot manifested that pretreatment LIG could decrease the release of Cyt C from mitochondria, down-regulate Caspase-3 protein expression and up-regulate Bcl-2/Bax ratio, thereby protects PC12 cells from apoptosis. In summary, LIG had protective effect on glutamate-induced apoptosis in PC12 cells through attenuating the increase in intracellular Ca2+ concentration, and inhibiting the release of Cyt C from mitochondria to cytoplasm.[2]

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The goal of this research was to study the selective pro-apoptotic effect of ligustilide on prostate-cancer-associated fibroblast in the tumor microenvironment and the related molecular mechanisms. The effects of ligustilide on cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) isolated from the prostate were determined by MTT assay. Flow cytometry and cellular immunofluorescence were used to detect the effects of ligustilide on the cell cycle and apoptosis. Western blotting was used to detect the expression of apoptosis-related proteins after the action of ligustilide on CAFs. In the investigation, ligustilide had a selective pro-apoptotic effect on prostate-CAFs. After ligustilide treatment, the proportion of CAFs in the G2-M phase of the cell cycle increased, and the expression of apoptosis-related proteins (p-P53, Bcl-2, Caspase9 and Cytochrome C) changed. Ligustilide blocks the CAF cell cycle and induces the apoptosis of CAFs. [3]
In vitro, ligustilide suppresses HL-60 monocyte adhesion and CAMs (ICAM-1, VCAM-1, E-selectin) expression in HUVECs. It significantly inhibits TNF-α-induced increase in reactive oxygen species production and NF-κB signaling pathway activation. It has vasorelaxation activities in contractions to various contractile agents in rat isolated aorta.

In vivo, ligustilide possesses antinociceptive and anti-inflammatory activities. It has vasorelaxation activities. It has been used in various pain syndrome treatments. The compound exhibits neuroprotective effects in animal models.

Intracellular NO production assay [1]
DAF-FM diacetate, a fluorescent probe, was used to determine the intracellular generation of NO. Confluent HUVECs plated in 6-well culture plates were pre-treated with DAF-FM for 1 h. After removing excess probe from the wells, the HUVECs were treated with (Z)-ligustilide for 30 min. The intensity of fluorescence was measured via a microplate reader and examined under a fluorescent microscope.
In vitro enzyme/receptor binding assays for ligustilide measure its inhibition of MEOX1. Anti-inflammatory activity is assessed by measuring CAMs (ICAM-1, VCAM-1, E-selectin) expression in HUVECs. NF-κB signaling pathway activation is measured by Western blotting or reporter assays. Vasorelaxation is assessed using rat isolated aorta contractions.

Cell culture and treatments [1]
HUVECs and HL-60 human promyelocytic leukemia cells were purchased from American Type Culture Collection. Cells were cultured using RPMI 1640 medium supplemented with 10% FBS and penicillin-streptomycin in a humidified incubator (CO2 5%, 37 °C). Once cells were approximately 70% confluent, they were starved via FBS-free RPMI 1640 medium. Unless otherwise noted, cells were then treated with ligustilide for 30 min, followed by stimulation with TNF-α.

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Monocyte-HUVEC adhesion assay [1]
HUVECs were grown to confluence in 24-well culture plates, pre-treated with (Z)-ligustilide for 30 min, and stimulated with TNF-α for 6 h. HL-60 cells were labeled with 10 µM BCECF-AM for 1 h at 37°C and washed twice with growth medium; the labeled HL-60 cells (2.5 × 105) were then added to the HUVECs and incubated in a CO2 incubator for 1 h. Non-adherent HL-60 cells were removed from the plate by washing with phosphate-buffered saline (PBS), and the bound HL-60 cells were measured via fluorescent microscopy and then lysed with 50 mM tris–HCl (pH 8.0) containing 0.1% sodium dodecyl sulfate (SDS). The intensity of fluorescence was measured using a microplate reader (Infinite F200 Pro, Tecan) at an excitation and emission wavelength of 485 and 535 nm, respectively. Adhesion data are presented in terms of percent change compared with the TNF-α value.

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Intracellular ROS production assay [1]
CM-H2DCFDA, a fluorescent probe, was used to determine the intracellular generation of ROS. Briefly, confluent HUVECs were pre-treated with (Z)-ligustilide in 24-well culture plates for 30 min. After removal from the wells, the HUVECs were incubated with 20 µM CM-H2DCFDA for 6 h and then stimulated with TNF-α. The intensity of fluorescence was measured via a microplate reader and examined under a fluorescent microscope.
In vitro cell-based assays for ligustilide use HUVECs to assess inhibition of monocyte adhesion and CAMs expression. HL-60 monocyte adhesion assays are used to evaluate anti-inflammatory effects. TNF-α-induced ROS production is measured using fluorescent probes. Cell viability is assessed in parallel.

In vivo animal models for ligustilide include rat isolated aorta preparations for vasorelaxation studies. Pain models are used to assess antinociceptive activity. Inflammatory disease models evaluate anti-inflammatory effects. Neuroprotective effects are assessed in models of neurological injury or disease.

Ligustilide has a molecular formula of C₁₂H₁₄O₂ and a molecular weight of 190.24 g/mol. It is isolated from the roots of Angelica sinensis (Oliv.) Diels and Chuanxiong. It is one of the main compounds of Danggui essential oil. The compound is a natural product used in various pain syndrome treatments and as a cosmetic material.

Ligustilide has a favorable safety profile as a natural product. It is for research use only and is not approved for clinical use. Appropriate safety precautions should be taken during handling. The compound is generally well-tolerated at research concentrations.

[1]. Ligustilide attenuates vascular inflammation and activates Nrf2/HO-1 induction and, NO synthesis in HUVECs. Phytomedicine. 2018 Jan 1;38:12-23.
[2]. Protective effect of ligustilide against glutamate-induced apoptosis in PC12 cells. Yao Xue Xue Bao. 2015 Feb;50(2):162-8.
[3]. Ligustilide promotes apoptosis of cancer-associated fibroblasts via the TLR4 pathways. Food Chem Toxicol. 2020 Jan;135:110991.

(Z)-Ligustilide is a butenolactone compound that functions as a metabolite. It has been reported to be found in umbelliferous plants, Angelica sinensis, and other organisms with relevant data.
Ligustilide is a bioactive phthalide derivative isolated from Angelica sinensis and Chuanxiong. It exhibits neuroprotective, anti-cancer, anti-inflammatory, and vasodilator effects. It is a potential inhibitor of MEOX1 and exerts antifibrotic effects through inhibition of MEOX1 and the Hippo signaling pathway. Ligustilide is also known as 蒿本内酯.

C12H14O2

190.242

190.099

C, 75.76; H, 7.42; O, 16.82

4431-01-0

4431-01-0; 81944-08-3 (E-isomer); 81944-09-4 (Z Isomer)

5319022

Colorless to light yellow liquid

1.1±0.1 g/cm3

377.9±11.0 °C at 760 mmHg

297-298ºC

158.6±16.7 °C

0.0±0.9 mmHg at 25°C

1.545

2.63

0

2

2

14

345

0

O1C(C2C([H])=C([H])C([H])([H])C([H])([H])C=2/C/1=C(\[H])/C([H])([H])C([H])([H])C([H])([H])[H])=O

IQVQXVFMNOFTMU-FLIBITNWSA-N

InChI=1S/C12H14O2/c1-2-3-8-11-9-6-4-5-7-10(9)12(13)14-11/h5,7-8H,2-4,6H2,1H3/b11-8-

(3Z)-3-butylidene-4,5-dihydro-2-benzofuran-1-one

Ligustilide

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

On dry ice or blue ice (Note: This product is not stable under room temperature, please store it under -20 °C or -80 °C immediately after receiving it)

DMSO: ~100 mg/mL (526 mM）

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.)