Ligusticum chuanxiong Hort as a medicinal and edible plant foods: Antioxidant, anti-aging and neuroprotective properties in Caenorhabditis elegans

Abstract

Ligusticum chuanxiong Hort. (CX) is a medicinal and edible plant including a variety of active substances, which may be an available resource for the treatment of related diseases. To expand the medicinal uses of CX, this study aims to explore the antioxidant, anti-aging and neuroprotective effects of the Ligusticum chuanxiong leaves (CXL) and rhizome (CXR) extracts. We first characterize CX phytochemical spectrum by LC-MS as well as antioxidant capacity. Acute toxicity, anti-oxidative stress capacity, lifespan and healthspan was evaluated in C elegans N2. Neuroprotective effect was evaluated in vitro and in vivo (C elegans CL4176 and CL2355). In this study, we detected 74 and 78 compounds from CXR and CXL, respectively, including phthalides, alkaloids, organic acids, terpenes, polyphenols and others. Furthermore, we found that CXs not only protect against oxidative stress, but also prolong the lifespan, alleviate lipofuscin, malondialdehyde (MDA) and reactive oxygen species (ROS) accumulation, and improve movement level, antioxidant enzyme activity in C elegans N₂. However, only CXR reduced the β-amyloid peptide (Aβ)-induced paralysis phenotype in CL4176s and alleviated chemosensory behavior dysfunction in CL2355s. In addition, CXR treatment reduced the production of Aβ and ROS, enhanced SOD activity in CL4176s. The possible mechanism of anti-aging of CXL and CXR is to promote the expression of related antioxidant pathway genes, increase the activity of antioxidant enzymes, and reduce the accumulation of ROS, which is dependent on DAF-16 and HSF-1 (only in CXR). CXR was able to activate antioxidase-related (sod-3 and sod-5) and heat shock protein genes (hsp-16.1 and hsp-70) expression, consequently ameliorating proteotoxicity related to Aβ aggregation. In summary, these findings demonstrate the antioxidant, anti-aging and neuroprotective (only in CXR) activities of the CX, which provide an important pharmacological basis for developing functional foods and drugs to relieve the symptoms of aging and AD. However, the material basis of neuroprotective activity and antiaging effects need to be elucidated, and the relationship between these activities should also be clarified in future studies.

Keywords: Caenorhabditis elegans; Ligusticum chuanxiong; antiaging activity; antioxidant activity; neuroprotective activity.

FIGURE 1

LC-MS profile of CXR and CXL. (A) LC-MS profile of CXR and CXL in negative ionization modes. (B) LC-MS profile of CXR and CXL in positive ionization modes.

FIGURE 2

Antioxidant activities of CXL and CXR with six gradient concentrations (0.0, 0.2, 0.4, 0.6, 0.8, 1.0, and 3.0 mg/ml). (A) ABTS radical scavenging activity. (B) DPPH radical scavenging activity. (C)Reducing power. Vc was used as the positive control.

FIGURE 3

Effect of CXL and CXR on stress resistance in C. elegans. (A) Survival curve of the N₂ strain under H₂O₂-induced oxidative stress. (B) Survival curve of the N₂ strain under paraquat-induced oxidative stress. (C) Survival curve of the N₂ strain under heat shock stress. Res was used as the positive control. Three independent biological replicates were performed. Significant differences were determined by a log-rank test, with *p < 0.05; **p < 0.01 and ***p < 0.001.

FIGURE 4

Effect of CXL and CXR on the antioxidant defense system in C. elegans. (A) The accumulation of ROS. (B) The content of MDA. (C,D) The activity of GSH-PX and SOD. Bars with different letters indicated statistical significance (p < 0.05).

FIGURE 5

Effect of CXL and CXR on lifespan and healthspan of C. elegans. (A) Lifespan. (B) Representative pictures of lipofuscin in worms treated with CXL and CXR. (C) The content of lipofuscin was quantified according to ImageJ software. (D) The frequency of the body bends. (E) The frequency of the head swing. (F) The three levels of locomotivity were measured, A-free movement, B-movement after prodding, C-weak movement after prodding. Bars with different letters indicated statistical significance (p < 0.05). Res was used as the positive control.

FIGURE 6

The molecular mechanism of CXL and CXR in the anti-aging. (A) The expression level of age-related genes in C. elegans. (B) The survival curve of skn-1 mutant worms. (C) The survival curve of daf-16 mutant worms. (D) The survival curve of hsf-1 mutant worms. Bars with different letters indicated statistical significance (p < 0.05). Significant differences were determined by a log-rank test, with *p < 0.05; **p < 0.01 and ***p < 0.001.

FIGURE 7

Effect of CXL and CXR on the AChE inhibition activity. (A) AChE inhibition activity of the CXL and CXR in vitro. (B) AChE activity of the CXL and CXR in worms. HupA was used as positive control. Bars with different letters indicated statistical significance (p < 0.05).

FIGURE 8

Effect of CXL and CXR on Aβ-induced chemotactic dysfunction in C. elegans. (A) The schematic of chemotaxis assay. (B) The chemotaxis index after treatment of CL2122 worms with CXL or CXR. (C) The chemotaxis index after treatment of CL2355 worms with CXL or CXR. Bars with different letters indicated statistical significance (p < 0.05).

FIGURE 9

Effect of CXL and CXR on Aβ-induced paralysis phenotype in CL4176 C. elegans. (A) Paralysis curve shown non-paralyzed CL4176 worms after the CXL and CXR treatment. (B) Statistics of the number of Aβ deposits in nematodes from the different groups. (C) Thioflavin T staining images of Aβ deposition in CL4176 C. elegans that were maintained at a permissive temperature (16°C) were used as negative controls. (D) Thioflavin T staining images of Aβ deposition in CL4176 C. elegans after the increase of temperature. (E) Thioflavin T staining images of Aβ deposition in CL4176 C. elegans after treatment with CXR. (F) Res was used as a positive control. The Fluorescence images were observed by a fluorescent microscope at ×40 magnification. Aβ aggregates are remarked with white arrow. Bars with different letters indicated statistical significance (p < 0.05). Significant differences were determined by a log-rank test, with *p < 0.05; **p < 0.01, and ***p < 0.001.

FIGURE 10

Effect of CXR on oxidative stress and expression of related genes in CL4176 C. elegans. (A) The ROS was measured in worms from the different groups at 36 h after temperature uplift to 25°C using 2′, 7′-dichlorofluorescein diacetate. (B) The SOD activity in worms from the different groups. (C) The relative expression of related genes was calculated using the method of 2^−ΔΔCt and the gene act-1 was used as the internal reference. Bars with different letters indicated statistical significance (p < 0.05).

FIGURE 11

Effect of CXL and CXR on the body size and reproduction of (C) elegans. (A) Brood size. (B) Representative picture of body length of (C) elegans. (C) Body length was quantitated by image J. Bars with different letters indicated statistical significance (p < 0.05).

FIGURE 12

Hypothetical model of the mode of action of CXR and CXL on anti-aging and CXR on neuroprotection. The green circle represents cytomembrane. After entering the cytomembrane, CXL and CXR likely down-regulates ROS level via up-regulating DAF-16 or HSF-1 (only in CXR), which in turn likely activates antioxidant oxidase-related and heat shock protein genes expression, respectively, consequently improving the lifespan. CXR alleviates ROS and Aβ accumulation likely via activating DAF-16 or HSF-1 that promote downstream expression of related genes, and reduction in ROS level inhibits aggregation of Aβ, consequently displaying neuroprotective potential. Truncated lines indicate a decrease in the process. Lines with arrow indicate a increase in the process. Red: a possible mechanism of CXs on anti-aging. Blue: a possible mechanism of CXR on neuroprotection.