Sesquiterpenoids from the Inflorescence of Ambrosia artemisiifolia

The successful invasion of Ambrosia artemisiifolia is largely due to allelopathy. As an invasive alien plant, A. artemisiifolia has spread rapidly in Asia and Europe. Studies have shown that sesquiterpenoids play an important role in plant allelopathy. However, it is unclear whether the inflorescence of A. artemisiifolia also contains allelopathic components. In this paper, our phytochemical research focuses on the inflorescence of A. artemisiifolia. Twenty sesquiterpenoids, including four new ones (1–4) were isolated through successive chromatographic columns and identified by spectroscopic methods. At a concentration of 200 μg/mL, all the compounds tested were evaluated for their allelopathic activities on seedling growth of wheat. Our results indicate that nine compounds inhibited both the root and shoot growth of seedlings. Compounds 14, 15, 17, and 20 significantly inhibited root length, which was more than 50% shorter than the control. This study identified the chemical profile of the sesquiterpenoids occurring in the inflorescence of A. artemisiifolia. The bioactivity screening results provide further understanding of the chemical basis of allelopathy in A. artemisiifolia.


Introduction
Ambrosia artemisiifolia L. (Asteraceae), which is native to North America, is an invasive weed widely distributed throughout temperate regions of the world [1]. This invasive plant has the potential to destroy native ecosystems and reduce agricultural yields [2]. Allelopathy describes the chemicals exuded from roots, rhizomes, leaves, stems, and/or litter of an individual and the resultant beneficial or deleterious effects on other organisms and is an important factor in the successful invasion of alien plants [3]. Meanwhile, because allelopathic components generally have strong phytotoxic activities against many weeds, the allelochemicals have the potential to develop new herbicides, which have the advantages of safety, easy degradation, and no resistance in weed control [4]. Previous studies reported that different solvent extracts of A. artemisiifolia had significant inhibitory effects on crop growth and seed germination, weeds, and other plants [5,6]. Studies have shown that sesquiterpenoids are the main chemical components of Ambrosia and play an important role in plant allelopathy [7][8][9][10]. Every year between July and August, clusters of columnar inflorescence from A. artemisiifolia produce large amounts of pollen causing a series of allergic reactions and affecting human health [11]. However, it is unclear whether the inflorescence of A. artemisiifolia also contains allelopathic components. Therefore, our phytochemical research focuses primarily on the inflorescence of A. artemisiifolia, from which we obtained twenty sesquiterpenoids, including four new ones (1)(2)(3)(4). Moreover, all the compounds were evaluated for their allelopathic activities on the seedling growth of wheat. from which we obtained twenty sesquiterpenoids, including four new ones (1−4). Moreover, all the compounds were evaluated for their allelopathic activities on the seedling growth of wheat.

Results and Discussions
Air-dried and powdered inflorescence of A. artemisiifolia was extracted with petroleum ether and then further extracted with ethanol. A total of 20 sesquiterpenoids ( Figure  1), including four new ones (1−4), were isolated from the petroleum ether extract and ethanol extracts using chromatographic methods.

Allelopathy Activity
Compounds 1-20 were evaluated for their allelopathic activities on the seedling growth of wheat (T. Aestivum) at a concentration of 200 µg/mL. Our results show that fourteen compounds (1-4, 5, 9-12, 14-15, 17, and 19-20) inhibited the root growth of the tested seedlings to varying degrees. Compounds 14, 15, 17, and 20 significantly inhibited root length, which was more than 50% shorter than that of the control ( Figure 6). In contrast, the inhibitory effect of these active compounds on shoot growth was reasonably weak, with relative shoot length being no less than 60% of the control (Figure 7). It is notable that compound 14, a pseudoguaiane-type sesquiterpene, showed the most potent allelopathic activity both on root and shoot length. Comparing the structure-activity relationships of compounds 11-14 showed that 14 had an acetonide group on the adjacent hydroxyl of C-3/C-4, which may be related to a high allelopathic effect. Previous research has demonstrated that parthenolides, germacrene-type sesquiterpenes, have strong allelopathic inhibitory activity [29]. The structural analogue of 17 also showed good inhibitory activity, but compounds 18 and 19 with an additional hydroxyl at C-14 and C-9, respectively, exhibited very weak allelopathic activities.    Sesquiterpenoids, which play an important role in plant allelopathy, are abunda many invasive plants and can significantly inhibit the growth of neighboring plants [ On the other hand, allelopathic components generally have strong phytotoxic acti against many weeds [30,31]. Our findings reveal the chemical profile of the sesquit noids occurring in the inflorescence of A. artemisiifolia, and our bioassay results are u in understanding the chemical basis of allelopathy in A. artemisiifolia. Sesquiterpenoids, which play an important role in plant allelopathy, are abundant in many invasive plants and can significantly inhibit the growth of neighboring plants [9,10]. On the other hand, allelopathic components generally have strong phytotoxic activities against many weeds [30,31]. Our findings reveal the chemical profile of the sesquiterpenoids occurring in the inflorescence of A. artemisiifolia, and our bioassay results are useful in understanding the chemical basis of allelopathy in A. artemisiifolia.

Plant Material
The inflorescence of A. artemisiifolia was collected from Cangwu County, Guangxi province, China, in August 2019, which was identified by Prof. Dai-gui Zhang (Key laboratory of Plant Resources Conservation and Utilization, Jishou University). A voucher specimen (zdg20190801) was deposited in Hunan Agricultural University. Seeds of Triticum aestivum L. were purchased from a seed company (Jiangsu Dingsheng Seed Co., Zhenjiang, China).

Extraction and Isolation
The air-dried and powdered inflorescence of A. artemisiifolia (9.0 kg) was extracted with petroleum ether (10 L × 4) and then extracted with ethanol (95%, 10 L × 4). The ethanol extract was suspended in water and partitioned with EtOAc and n-butanol successively, as described in our previous research [32].

Bioassays
Inhibitory activities on the growth of T. aestivum seedlings were evaluated using the plate culture method [33]. To enhance the germination rate, T. aestivum seeds were submerged in a 0.3% potassium permanganate solution for 15 min before being thoroughly rinsed with distilled water. After skin breaking, T. aestivum seeds were germinated on filter paper in the dark at 25 • C for 24 h. After being dissolved in acetone or DMSO, each compound (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) was prepared as a stock solution of 2 mg/mL. The stock solutions were diluted with distilled water (containing 1% Tween 80) to obtain concentrations of 200 µg/mL. The same volume of acetone or DMSO was added to distilled water (containing 1% Tween 80) as a control. Following germination, uniformly growing seedlings (10 seeds) were transferred to 9 cm diameter Petri dish lined with filter paper. Each dish was treated with 7 mL of the prepared corresponding concentration of test solution (or control solution). Each treatment was replicated 3 times. The seedlings were then incubated in a constant temperature humidity chamber in the dark at 25 • C for 72 h. At the end of the experiment, root and stem lengths were measured.

Statistical Analysis
All data were subjected to analysis of variance by use of SPSS 18.0. Significant differences in seedling growth between treatment and control were calculated by one-way analysis of variance (ANOVA). Relative length (percent) was determined by the formula [treated length)/control length] × 100.  Data Availability Statement: All data included in this study are available upon request by contact with the corresponding author.