Fosane sesquiterpenes with phytotoxic activity in Ligustrum chuanxiong
Phytoallelopathy refers to the release of chemicals from one plant to the environment, which directly or indirectly have harmful or beneficial effects on another plant. Releasing allelopathic substances is a survival strategy evolved by species in special habitats to gain ecological advantages. It may affect the growth of other plants and the succession of plant communities in the same habitat, and even affect the distribution pattern and biodiversity of plant populations in the region. The development of eco-friendly natural herbicides using allelopathic substances as lead compounds is currently a research hotspot, among which sesquiterpenes have great potential.
Various plants of the Ligularia genus in the Asteraceae family, such as L. virgaurea, L. macrophylla, and L. sagitta, exhibit significant allelopathic activity. Sesquiterpenes are considered the main allelopathic active components in these plants and often exhibit phytotoxic activity. The plant L. cymbulifera, belonging to this genus, is widely distributed in the Hengduan Mountains and densely grows on alpine meadows at 2000-4000m in the Shangri La area of Yunnan Province. It is a dominant species in local alpine pastures. The main secondary metabolites of Ligularia angustifolia are sesquiterpenes, carbon reducing sesquiterpenes, bisabolane sesquiterpenes, and pyrrolizidinge alkaloids. In the early stage, the research team isolated and detected multiple sesquiterpenes with plant toxic activity from its roots and rhizosphere soil. The experimental results suggest that the sesquiterpenes of the taxane class are likely to be synthesized and released into the soil around the rhizosphere by Ligustrum chuanxiong, as potential allelopathic substances that synergistically interfere with the germination and growth of other plant seeds, thereby helping Ligustrum chuanxiong gain ecological advantages in the Hengduan Mountains. In order to search for more potential allelopathic substances and further study their mechanisms of action, and to screen lead compounds for the development of eco-friendly natural herbicides, this study continues to investigate the phytotoxic sesquiterpenes of the phorane class in Ligularia baicalensis.

This article isolated 13 sesquiterpenes from the 95% methanol fraction of Ligustrum chuanxiong root, including compound 1, which is a new eremophilane-12,8-lactam type sesquiterpene alkaloid; Compounds 2-5 are eremophilane-12,8-olides, a type of sesquiterpenes belonging to the class of Foshanes; Compounds 6-10 are eremophilane-14,6-olides type sesquiterpenes of the Foshane class; Compounds 11, 12, and 13 are furanemophilane type furanene sesquiterpenes (see Figure 1). The plant toxicity activity of the above compounds was investigated, and the results showed that compounds 1-13 had no inhibitory activity on Arabidopsis seed germination, but all exhibited certain inhibitory activity on Arabidopsis seed root growth, and some compounds could advance seed germination time by 1-2 days compared to the blank control group at low concentrations. This result is consistent with the literature: sesquiterpenes have stronger activity in inhibiting root elongation than in inhibiting seed germination, and plant toxic compounds with allelopathic potential often exhibit a promoting effect on seed germination at low concentrations. Recent studies have shown that plants have evolved a new strategy called “suicide germination” or “honeypot strategy” in combating parasitic plants: allelopathic substances released by host plants, such as Strigolactones (SLs), can induce and stimulate seed germination of parasitic plants, such as Striga spp. and Orobanche spp. If grown in an environment without a host plant, these germinated seeds will accelerate their death due to lack of nutrient supply, thereby reducing the reserve of parasitic plant seeds in the soil. In the face of complex environmental pressures, allelopathic substances often play multiple ecological roles. The results of this experiment suggest that in addition to directly inhibiting the germination and growth of other plant seeds, the sesquiterpenes in Ligustrum chuanxiong may also adopt a strategy similar to the “suicidal germination” mentioned above, which is to induce and promote the germination of other plant seeds, and then induce the death of root tip cells in some way, inhibiting their nutrient absorption and accelerating their death, thereby reducing the consumption of nutrients in the surrounding soil by these plants. At present, there are no similar research reports, and this speculation needs further verification through experiments.

Comparing the effects of compounds 3, 5, and 13 with other compounds on promoting early seed germination, the results suggest that the formation of lactone rings at the 6-position and 15th position substituents can affect the promotion effect; If there are 3 side chains and 10 hydroxyl groups substituted, it may have an inhibitory effect; The presence of a 6-digit side chain may have a promoting effect. On the other hand, the inhibitory activity of compounds 1-13 on Arabidopsis root growth is related to compound concentration and structure (see Figure 4). Based on the results of previous experiments, the inhibitory activity of sesquiterpenes from Ligustrum chuanxiong on Arabidopsis root growth follows the following pattern: furanermophiliane type sesquiterpenes have better plant toxicity, and furanermophiliane type>eremophiliane-14,6-olides type>eremophiliane-12,8-oolides type>modified eremophiliane type. Comparing the inhibitory activities of compounds 11, 12, and 13 on Arabidopsis root growth, the EC50 of compound 13 was 44.15 ± 5.21 μ g/mL, while the EC50 of compounds 11 and 12 were 2.30 ± 2.92 and 75.62 ± 1.33 μ g/mL, respectively. This suggests that the activity is better when the 6-position and 15th position substituents of furanermophilane type diterpenes do not form lactone rings. Comparing the inhibitory activities of compounds 6-10 on Arabidopsis root growth, compounds 6 and 9 showed similar activity, indicating that the double bond on the A ring of eremophilane-14,6-olides type sesquiterpenes has little effect on plant toxicity. Comparing the inhibitory activities of compounds 2-5 on Arabidopsis root growth, there is not much difference in activity between compounds 2 and 5, indicating that the 6th and 15th substituents of eremophilane-12,8-olides sesquiterpenes form lactone rings, and the presence of substituents at the 3rd position has little effect on activity. Comparing the inhibitory activities of compounds 1 and 3, 3, 5 and 4, 6 and 7, 9 and 10 on Arabidopsis root growth, it was found that the plant toxicity was enhanced when there was a hydroxyl substitution at position 10, which is consistent with previous research results. Compound 13 exhibited the best plant toxicity activity in this experiment. From the above structure-activity analysis, it can be concluded that furanermophilane type sesquiterpenes containing 10 hydroxyl groups and 6 side chain substitutions have the potential to serve as eco-friendly natural herbicide lead compounds. Therefore, the next step will be to search for more compounds of this type from Ligustrum chuanxiong and screen for lead compounds for the development of eco-friendly natural herbicides.