3-butylidenephthalide is a gamma-lactone that is phthalide substituted by a butylidene group at position 3. Isolated from Ligusticum porteri, it exhibits hypoglycemic activity.

Toxicity and Repellency of 3-Butylidenephthalide

At NCNPR, we are running a screening program and continuously screening natural products for their toxicity and repellency against mosquitoes and imported fire ants. The ethanolic extract of Ligusticum porteri J.M.Coult. & Rose (Apiaceae) roots, a herbaceous flowering plant native to the Rocky Mountains, showed strong repellency against mosquitoes. Two isomers and a mixture, were identified as the compounds responsible for the repellency of this extract against mosquitoes. (E/Z)-3-butylidenephthalide also showed increased repellency in combination with carotol against mosquitoes. The repellency and toxicity of (E/Z)-3-butylidenephthalide against imported fire ants have not been documented. Based on mosquito repellency data against mosquitoes, we tested (E/Z)-3-butylidenephthalide to determine its toxicity and repellency against imported fire ants. This study reports the toxicity and repellency of (E/Z)-3-butylidenephthalide against imported fire ants. Many natural products have been reported as repellents against insects including mosquitoes and imported fire ants. Ethanolic extract of roots of Ligusticum porteri showed high repellency against mosquitoes. .[1]

The reduced removal of the treated sand in digging bioassays indicated that the treatment has suppressed the digging ability of the fire ants. Based on the amount of sand removed as a criterion of repellency, in this study, (E/Z)-3-butylidenephthalide showed significantly higher repellency than DEET. DEET failed at the doses of 62.5 to 15.6 μg/g in different species of fire ants whereas (E/Z)-3-butylidenephthalide showed repellency up to the dosages of 0.6, 0.15, and 4.98 μg/g in red, black, and hybrid imported fire ants, respectively, suggesting that repellency varied among fire ant species and hybrids as a function of dose. Ali et al. reported the repellency of (Z)-3-butylidenephthalide, (E)-3-butylidenephthalide, and (E/Z)-3-butylidenephthalide against mosquitoes. Chen  reported the repellency of different phthalates in imported fire ants and reported the highest activity in methyl phthalate and diethyl phthalate. However, this is the first report on the repellency of (E/Z)-3-butylidenephthalide against imported fire ant species.The repellency causes disorientation which blocks the workers from effectively digging through treated mounds while the toxicity kills them. The ability of (E/Z)-3-butylidenephthalide to negatively affect the digging behavior of workers makes it a suitable candidate for treating the potting soil mixtures to prevent the entry of fire ants in the potted soil used to transport nursery plant materials. Since (E/Z)-3-butylidenephthalide was isolated from L. porteri root extract which has been used traditionally to treat many diseases in human beings, the compound may be potentially safe to use around human activity centers.

3-Butylidenephthalide in Targeting the Microenvironment

Butylidenephthalide (BP) is abundant in Chinese medicinal herbs such as Angelica sinensis , and Ligusticum chuanxiong . Studies have reported that phthalide compounds perform an antiproliferative function in HSCs through the inhibition of platelet-derived growth factor (PDGF), but in the in vivo data on fibrosis, this has not yet been reported. In our previous studies, 3-Butylidenephthalide was examined for antihepatocellular carcinoma activity by inducing Nur77 (also known as NR4A1) expression, which led to caspase-3-dependent apoptosis and triggered an antiplatelet effect through PDGF reduction. BP also downregulates EMT-related genes such as Snail (Snail/SNAI1) and Slug (Slug/SNAI2). Although EMT and tumor migration are the characteristics of activated HSCs that cause liver fibrosis, these considerations prompted us to study the effects of 3-Butylidenephthalide on liver fibrosis.[2]

EMT inducers like TGF-β and BMP-7 can be targeted for therapy; hence, we considered 3-Butylidenephthalide may reduce EMT, leading to the improvement in liver fibrosis. In our time-dependent study, the protein expression of BMP-7 was upregulated at an early stage (1 h after BP treatment), in contrast to TGF-β, which decreased at the latter stage (12 h) of BP treatment. Also, the RNA expression of bmp7 was induced at 0.5 to 1 h as shown. These data reminds us that 3-Butylidenephthalide induced BMP-7 can regulate the expression of TGF-β; thereby, reducing liver fibrosis. To identify the relationship between BMP-7 and TGF-β, we knocked down the expression of BMP-7 by using siRNA and treated cells with BP for 24 h. We observed that the downregulated expression of TGF-β was partially recovered in BMP-7 knockdown cells, leading to an increase in EMT, which has been reported to promote liver fibrosis. Furthermore, to test whether BP reduces fibrosis in a BMP-7 deficient liver, an in vivo study of a zebrafish liver fibrosis model was conducted by administering TAA treatment. We observed that 3-Butylidenephthalide did not reduce fibrosis in BMP-7 silenced fish, suggesting that BMP-7 could be the upstream regulator of TGF-β and EMT genes. These results connect correlate? with previous studies on liver and renal fibrosis, suggesting that BMP-7 is one of the possible mechanisms in reversing liver fibrosis by counteracting TGF-β and EMT.

In summary, we demonstrated that 3-Butylidenephthalide reduced EMT by regulating BMP-7 in order to ameliorate liver fibrosis. The effect of MET was triggered by BMP-7 and inhibited by TGF-β, and thus prevented the activation of HSC and α-SMA expression. In addition, 3-Butylidenephthalide induced liver regeneration and enabled the recovery of liver function. Third, BP induced MMP-9 expression and fibrinolysis in the liver and reduced previously accumulated collagen. These data suggest that BP has the potential to effectively treat liver fibrosis and target the liver microenvironment for its antifibrotic effects.

References

[1]Ali A, Shah FM, Khan IA. Toxicity and Repellency of (E/Z)-3-Butylidenephthalide: A Natural Compound Isolated from Ligusticum porteri ;Root Extract Evaluated Against Imported Fire Ants (Hymenoptera: Formicidae). Insects. 2024 Oct 23;15(11):828. doi: 10.3390/insects15110828. PMID: 39590427; PMCID: PMC11594892.

[2]Chuang HM, Su HL, Li C, Lin SZ, Yen SY, Huang MH, Ho LI, Chiou TW, Harn HJ. The Role of Butylidenephthalide in Targeting the Microenvironment Which Contributes to Liver Fibrosis Amelioration. Front Pharmacol. 2016 Apr 27;7:112. doi: 10.3389/fphar.2016.00112. PMID: 27199755; PMCID: PMC4847481.