Article pubs.acs.org/JAFC
Phytotoxic Allelochemicals from Roots and Root Exudates of Trifolium pratense Quan Liu,† Rui Xu,† Zhiqiang Yan,† Hui Jin,† Haiyan Cui,† Liqin Lu,† Denghong Zhang,† and Bo Qin*,†,‡ †
Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China ‡ State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, People’s Republic of China ABSTRACT: Trifolium pratense, a widespread legume forage plant, is reported to exhibit phytotoxic activity on other plants, but the active metabolites have not been clarified so far. A bioassay-guided fractionation of the root extracts led to the isolation of five isoflavonoids, which were elucidated by spectroscopic analysis. All of the purified compounds observably showed phytotoxic activities against Arabidopsis thaliana. Moreover, the inhibitory effects were concentration-dependent. The furan ring linked at C4 and C-2′ positions by an oxygen atom and a 1,3-dioxolane at C-4′ and C-5′ positions are considered to be critical factors for the phytotoxic activity. The concentrations of (6aR,11aR)-maackiain and (6aR,11aR)-trifolirhizin, concluded to be allelochemicals from soil around plants of T. pratense, were determined by HPLC and LC-MS to be 4.12 and 2.37 μg/g, respectively. These allelochemicals, which showed remarkable activities against the weed Poa annua may play an important role in assisting the widespread occurrence of T. pratense in nature. KEYWORDS: Trifolium pratense, allelopathy, isoflavonoids, phytotoxicity
■
INTRODUCTION Many legume plants biosynthesize a range of isoflavones and play an important role in traditional diets throughout the world.1 Consumption of legumes has been linked to reduced risk of diabetes and obesity, coronary heart disease, colon cancer, prostate cancer, and gastrointestinal disorders.2 The functions of isoflavones as well as their roles in human nutrition and pharmacology offer several potential applications in ecology and agriculture.3,4 Trifolium pratense L., also known as red clover, belonging to the family Leguminosae (Fabaceae), is an important forage plant that grows widely in many parts of the world. The plant is frequently used as an alternative medicine for hormone replacement therapy (HRT) to treat menopausal disorders.5 It is also a health product for humans commonly used for the treatment of eczema and psoriasis6,7 and relief of menopausal symptoms in clinic.8 Many isoflavones that have been found in the plant have multiple pharmacological and biological activities.9 Previous research on red clover indicated that it significantly delayed the seed germination and inhibited the growth of other plants, which assists its widespread distribution in nature.10−13 One important factor of its highly competitive nature and broad ecological adaptability was the allelopathic effect, which is due to the phytotoxic activities of secondary metabolites produced by the plant on neighboring plants. The existence of allelopathy as an ecological phenomenon has been well documented over the past few decades in both natural and agricultural ecosystems.14,15 Plants could affect neighboring plants and other organisms by releasing chemicals into the environment. Once allelopathic plant species release allelochemicals from their roots to the soil, a series of interactions between allelochemicals with soil abiotic and biotic factors can take place. © XXXX American Chemical Society
In light of the wide occurrence of ecological benefits of T. pratense, we decided to investigate the chemical constituents with phytotoxic activities. In this study, using a bioactivityguided approach, the phytochemical investigation led to the isolation of five active compounds that are responsible for the observed plant growth inhibitory activities on the model plant Arabidopsis thaliana. This study investigated the phytotoxic activities and structure−activity relationship of these compounds on seedling growth of A. thaliana. Furthermore, the allelochemicals were identified from the soil (soil rhizosphere plus surrounding soil) where the plant had grown by means of HPLC and UV spectroscopy, which were further confirmed by LC-MS. To our knowledge, this is the first report on the occurrence, structure, and content of phytotoxins and allelochemicals of this globally distributed plant.
■
MATERIALS AND METHODS
General Experimental Procedures. 1H and 13C NMR spectra were performed on a Bruker AM-400BB instrument (Bruker, Karlsruhe, Germany) with TMS as internal standard, operating at 400 and 100 MHz, respectively. EI-MS was measured with a VG ZABHS instrument (VG, East Sussex, UK), and ESI-ION trap was measured with an Esquire 6000 instrument (Bruker Daltonics, Billerica, MA, USA). Column chromatography (CC) used Sephadex LH-20 (25−100 mm) (Pharmacia Fine Chemical Co., Ltd., Berlin, Germany), silica gel 60 RP-18 (230−400 mesh) (Merck, Rahway, NJ, USA), and silica gel (200−300 mesh) (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). TLC was performed on precoated silica gel 60 F254 plates (Qingdao Haiyang Chemical Co., Ltd.), and spots Received: January 18, 2013 Revised: May 23, 2013 Accepted: June 5, 2013
A
dx.doi.org/10.1021/jf401241e | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Figure 1. Structures of compounds (1−5) isolated from the roots of T. pratense. for the culture of plants in laboratory) at 22 °C in an incubator with a 16/8 h day/night photoperiod. Seven-day-old seedlings were transferred into 990 μL of liquid 1/2 MS medium supplemented with 1.5% sucrose in 24-well plates (diameter = 1.6 cm and height = 1.5 cm) (VWR Scientific Inc., USA). The plants were treated with different concentrations (50, 100, 200, and 400 μg/mL for crude extract and fractions prepared with different solvents; 25, 50, 100, 200, and 400 μg/mL for compounds 1−5) of the test materials, with four replicates per treatment. Samples to be tested were dissolved in DMSO, and 10 μL was applied to each well containing the seedlings (10 μL of DMSO was added as control). Then the plates were sealed with parafilm and incubated in a rotary shaker at 95−105 rpm with a photoperiod of 16 h light/8 h dark at 22 °C for 7 days. After treatment, the fresh weights of each plant were measured, and the percentages of the fresh weights of the plants were determined by reference to the fresh weights of control. The phytotoxic bioassays of allelochemicals against P. annua were the same as for A. thaliana. Seven-day-old seedlings of P. annua were monitored for 7 days, and the fresh weights were measured at the end of the experiment. The results were analyzed by ANOVA (p < 0.05), and IC50 values were calculated by probit analysis based on percent of either radicle growth or fresh weight inhibition. Sample Preparation Procedures of the Soil. Seeds of T. pratense were sown into the soil and were grown for 1 year. The soil where T. pratense had grown was 1.08 kg (height = 8.5 cm; volume = 1.71 × 10−3 m3) and was extracted ultrasonically with MeOH three times (3 L of MeOH and 30 min each). The filtrate was concentrated in vacuum to near dryness on a rotary evaporator. Then the extract was dissolved in 100 mL of methanol to obtain the final solution. This solution was filtered through a 0.45 μm filter membrane prior to HPLC analysis. HPLC and LC-MS Analysis. Analytical HPLC was performed using an Agilent apparatus equipped with a diode array detector (DAD) and a reverse-phase C18 column (5 μm particle size, 4.6 mm × 25 cm). Compounds were monitored at 254 and 275 nm, and UV spectra were recorded between 200 and 400 nm. The mobile phase, which was composed of water (A) and methanol (B), was programmed as follows: 0−5 min, B 40%; 5−10 min, B from 40 to 70%; 10−20 min, B 70%; 20−35 min, B from 70 to 85%; 35−40 min, B from 85 to 100%. The flow rate with an injection volume of 20 μL was 0.6 mL/min at the column temperature of 30 °C. The compound (6aR,11aR)-trifolirhizin was used to validate the analytical HPLC method described above. Mean recovery rates (accuracy) for (6aR,11aR)-trifolirhizin were >98% with a relative standard deviation of
