Article pubs.acs.org/JAFC
Allelochemicals in the Rhizosphere Soil of Euphorbia himalayensis Quan Liu,†,‡ Dengxue Lu,§ Hui Jin,† Zhiqiang Yan,† Xiuzhuang Li,† Xiaoyan Yang,† Hongru Guo,† and Bo Qin*,† †
Key Laboratory of Chemistry of Northwestern Plant Resources of the Chinese Academy of Sciences (CAS) and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, Gansu 730000, People’s Republic of China ‡ State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, Gansu 730020, People’s Republic of China § Institute of Biology, Gansu Academy of Sciences, Lanzhou, Gansu 730000, People’s Republic of China ABSTRACT: Weed infestation has been known to cause considerable reductions in crop yields, thereby hindering sustainable agriculture. Many plants in genus Euphorbia affect neighboring plants and other organisms by releasing chemicals into the environment. In view of the serious threat of weeds to agriculture, the allelochemicals of Euphorbia himalayensis and their allelopathic effects were investigated. The extract of root exudates from rhizosphere soil exhibited allelopathic activities against crops (wheat, rape, and lettuce) and grasses (Poa annua, Festuca rubra, and red clover). Bioassay-guided fractionation and isolation from the root extract of E. himalayensis led to the characterization of two ellagic acid derivatives and a jatrophane diterpene, which observably showed phytotoxic activities against lettuce, Festuca arundinacea, and F. rubra. They were further confirmed by ultra-performance liquid chromatography−tandem mass spectrometry to have concentrations of 3.6, 3.8, and 8.99 nmol/g in the rhizospere soil, respectively. Bioassay indicated that the combination of the allelochemicals could be selective plant growth regulator in agriculture. KEYWORDS: Euphorbia himalayensis, root exudates, ellagic acid derivatives, esulone A, allelopathy
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INTRODUCTION Weeds pose an important biological constraint to crop productivity. Many weeds release allelochemicals to interfere with the germination and growth of crops growing around them.1 It has long been suspected that an allelopathic mechanism should play an important role in the interference of weed with the crop plants. The genus Euphorbia is one of the largest and most widely distributed in the spurge family (Euphorbiaceae), in which most species are found in temperate regions. The plants of the genus are herbs, shrubs, and cactus-like, growing in nearly all climate types, and are often characterized by the presence of a milky latex.2 Many plants in the genus, such as leafy spurge, are reported to affect adjacent plants through allelochemicals released into the environment.3,4 A yield reduction of 4−85% has been reported in field crops with different Euphorbia species and distinct occurrence densities. The plants in this genus decrease herbage production by 10−100% in pasture and rangelands.5 Euphorbia species have herbicidal and insecticidal properties, but there is a need to establish their commercial utility potential as environmentally friendly plant protection measures. Euphorbia himalayensis Boiss. commonly grows in nonirrigated farmlands and infests cultivated fields in the Tibetan Plateau. The plant is also toxic to livestock and poses a serious threat to crop and livestock production. In light of the ecological benefits of E. himalayensis in farmland, the investigation of chemical constituents with phytotoxic activities was processed. Using a bioassay-directed approach, three active compounds were isolated and elucidated, which were responsible for obvious phytotoxic activities. Furthermore, these allelochemicals were © XXXX American Chemical Society
identified from the rhizosphere soil by high-performance liquid chromatography (HPLC) and ultraperformance liquid chromatography−mass spectrometry (UPLC−MS). To the best of our knowledge, this is the first report of this plant on the phytochemical investigation and allelopathic activities. These results would be useful to explore how E. himalayensis becomes a successful competitor against other plants in nature. In this study, a rapid and highly sensitive method was developed with the use of ultraperformance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS) to determine the levels of allelochemicals in the rhizospere soil.
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MATERIALS AND METHODS
General Experimental Instruments and Procedures. Electron ionization−mass spectrometry (EI−MS) was recorded on a VG ZABHS instrument (VG, East Sussex, U.K.), and electrospray ionization (ESI) ion-trap data were recorded on an Esquire 6000 instrument (Bruker Daltonics, Billerica, MA). 1H and 13C nuclear magnetic resonance (NMR) spectra with tetramethylsilane (TMS) as the internal standard were measured on a Varian Mercury-400BB instrument (Bruker, Karlsruhe, Germany) operating at 400 and 100 MHz, respectively. Silica gel (200−300 mesh) (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex LH-20 (25−100 mm) (Pharmacia Fine Chemical Co., Ltd., Berlin, Germany), and silica gel 60 RP18 (230−400 mesh) (Merck, Whitehouse Station, NJ) were used in column chromatography (CC). Thin-layer chromatography (TLC) was performed on precoated silica Received: April 28, 2014 Revised: July 27, 2014 Accepted: August 2, 2014
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CC on silica gel CC (2 × 75 cm), eluted with a gradient of petroleum ether/Me2CO [10:1 (1.5 L), 5:1 (0.8 L), 2:1 (0.6 L), and 0:1 (0.32 L)], and then repeated silica gel CC to produce compound 3 (9 mg). Fraction F was chromatographed on silica gel CC (2 × 75 cm), eluting with a CHCl3/CH3OH gradient [20:1 (0.5 L), 5:1 (1 L), 2:1 (1.6 L), and 0:1 (0.4 L)], then was passed through a Sephadex LH-20 column (1.1 × 60 cm, 30 g) (1:1 CHCl3/CH3OH), and repeated silica gel CC to produce compound 1 (16 mg) and compound 2 (6 mg). Compound 1. White amorphous powder. ESI−MS m/z: 461.13 [M − H]−. 1H NMR (400 MHz, DMSO) δ: 7.73 (s, 1H, H-5), 7.48 (s, 1H, H-5′), 4.09 (s, 3H, 3-OMe), 4.05 (s, 3H, 3′-OMe), 5.16 (d, 1H, J = 7.6 Hz, H-1″), 3.21−3.87 (m, 5H, H-2″, H-3″, H-4″, H-5″). 13C NMR (100 MHz, DMSO) δ: 114.0 (C-1), 141.4 (C-2), 141.7 (C-3), 151.1 (C-4), 111.7 (C-5), 112.6 (C-6), 158.3 (C-7), 110.9 (C-1′), 140.8 (C-2′), 140.0 (C-3′), 152.7 (C-4′), 111.7 (C-5′), 112.6 (C-6′), 158.2 (C-7′), 61.5 (3-OMe), 60.9 (3′-OMe), 101.7 (C-1″), 72.9 (C-2″), 76.0 (C-3″), 69.1 (C-4″), 65.7 (C-5″). Compound 2. White amorphous powder. ESI−MS m/z: 329.10 [M − H]−. 1H NMR (400 MHz, DMSO) δ: 7.48 (s, 2H, H-5, H-5′), 4.03 (s, 6H, 2× OMe). 13C NMR (100 MHz, DMSO) δ: 111.5 (C-1, C-1′), 141.1 (C-2, C-2′), 140.1 (C-3, C-3′), 152.1 (C-4, C-4′), 111.3 (C-5, C-5′), 112.0 (C-6, C-6′), 158.3 (C-7, C-7′), 60.8 (2× OMe). Compound 3. White amorphous powder. ESI−MS m/z: 689.25 [M − H]−. 1H NMR (400 MHz, DMSO) δ: 2.47 (m, 1H, H-1a), 2.93 (m, 1H, H-1b), 5.43 (dd, 1H, J = 4.2 and 1.0 Hz, H-3), 3.75 (dd, 1H, J = 4.2 and 9.0 Hz, H-4), 6.03 (d, 1H, J = 9.0 Hz, H-5), 6.08 (s, 1H, H-7), 4.81 (d, 1H, J = 9.2 Hz, H-8), 6.12 (d, 1H, J = 16.0 Hz, H-11), 5.88 (dd, 1H, J = 16.0 and 9.0 Hz, H-12), 4.34 (m, 1H, H-13), 1.31 (s, 3H, H-16), 5.75 (brs, 1H, H-17a), 6.12 (brs, 1H, H-17b), 1.34 (s, 3H, H-18), 1.26 (s, 3H, H-19), 1.41 (d, 3H, J = 6.0 Hz, H-20), 2.03 (s, 3H, 3-OAc), 2.08 (s, 3H, 15-OAc), 7.03−7.35 (m, 6H, 2× OBz), 7.52− 7.81 (m, 4H, 2× OBz), 2.34 (s, 1H, 2-OH), 3.30 (d, 1H, J = 9.2 Hz, 8-OH). 13C NMR (100 MHz, DMSO) δ: 50.6 (C-1), 79.0 (C-2), 80.6 (C-3), 46.6 (C-4), 65.6 (C-5), 136.8 (C-6), 72.6 (C-7), 72.8 (C-8), 211.5 (C-9), 48.4 (C-10), 134.3 (C-11), 134.8 (C-12), 42.0 (C-13), 205.1 (C-14), 90.8 (C-15), 25.1 (C-16), 124.0 (C-17), 23.0 (C-18), 20.0 (C-19), 23.5 (C-20), 169.7 [3-OAc(CO)], 20.5 [3-OAc(Me)], 169.8 [15-OAc(CO)], 21.9 [15-OAc(Me)], 165.0 [5-OBz(CO)], 128.5 (1′,5-OBz), 129.6 (2′,6′,5-OBz), 127.9 (3′,5′,5-OBz), 133.1 (4′,5-OBz), 166.0 [7-OBz(CO)], 128.8 (1″,7-OBz), 129.7 (2″,6″,7OBz), 128.0 (3″,5″,7-OBz), 133.1 (4″,7-OBz). HPLC Analysis. HPLC analysis was measured on an Agilent apparatus equipped with a diode array detector (DAD) and a reversephase C18 column with 5 μm particle sizes. UV spectra were recorded at 200−400 nm, and the compounds were monitored at 254 and 275 nm. The mobile phase was composed of (A) 0.1% aqueous acetic acid and (B) methanol/acetonitrile (1:2, v/v) for analysis of compounds 1 and 2 and programmed as follows: 0−5 min, 15% B; 5−10 min, from 15 to 40% B; 10−20 min, from 40 to 55% B; 20−35 min, from 55 to 70% B; and 35−42 min, 70% B. The flow rate was 0.6 mL/min with an injection volume of 20 μL at the temperature of 30 °C. To analyze compound 3 in the root exudates, the other mobile phase was composed of (A) 0.1% aqueous acetic acid and (B) methanol. The gradient schedule was as follows: 0−5 min, 40% B; 5−10 min, from 40 to 55% B; 10−20 min, from 55 to 60% B; 20−25 min, 60% B; 25−40 min, from 60 to 70% B; 40−45 min, from 70 to 90% B; and 45−50 min, from 90 to 100% B. The method for analysis of compounds 1−3 in the extract of roots at the concentration of 0.1 mg/mL was the same as that with the compounds 1 and 2. The analytical HPLC methods described above were validated by compounds 1 and 3. Mean recovery rates (accuracy) were >98% with a relative standard deviation of 328), (C) compound 1 (461 > 313), (D) compound 2 (329 > 314), (E) compound 2 (329 > 299), and (F) UPLC chromatogram with a photodiode array detector at 254 nm.
peaks of compounds 1−3 were enhanced obviously after the injection of the standards into soil samples and no new peaks appeared. The allelochemicals in the soil matrix were further identified by means of mass spectra, because they were the key to research of allelopathic effects. UPLC−MS/MS and HPLC− DAD were successfully used in this study to determine the existence of the allelochemicals in the rhizosphere soil of E. himalayensis. Accurate MS and MS/MS data confirmed the composition of each of the daughter ions. Fragmentation of the protonated molecular ion of compound 1 ([M − H]− m/z 461) in negative-ion ESI mode produces a number of daughter ions, notably m/z 446 (loss of CH3), m/z 328 (loss of C4H6O5), and m/z 313 (loss of xylopyranosyl) (Figure 7). Although compounds 1 and 2 have the same nucleus, the protonated molecular ion [M − H]− m/z 329 of compound 2 produces corresponding daughter ions of m/z 314 (loss of CH3) and m/z 299 (loss of two CH3). The most dominant daughter ion observed at m/z 121 was chosen as the transition (m/z 689 → 121) for verification of compound 3 using a multiple reaction monitoring (MRM) program on UPLC−MS/MS. The detection of three compounds in the UPLC−MS/MS chromatogram of the rhizosphere soil is shown in Figure 8. The standard curves were obtained by use of the linear regression method, and peak areas at 275 nm were plotted to calculate concentrations; thus, the concentrations of compounds 1, 2, and 3 in the soil were determined as 3.6, 3.8, and 8.99 nmol/g of soil, respectively. It is well-known that plants usually synthesize secondary metabolites used to adapt to the environment and enable their survival and well-being. The compounds of ellagic acid type, which belonged to compounds 1 and 2, were reported to exhibited varied activities, such as inhibitors of auxin transport.9
Allelochemicals 1 and 2 possessed the same skeleton and maybe mutual reverse in nature. The replacement of the xylose group at the C-4 position would improve the water solubility and enlarge the application range of compound 2, although the activity of compound 2 was slightly weaker than that of compound 1. Compound 3 belongs to jatrophane diterpenes usually isolated from Euphorbiaceae plants and exhibits cytotoxic, antitumor, insect deterrent, and antimicrobial activities in vitro.10 The phytotoxic compounds isolated from roots were also present in the root exudates, suggesting that they were allelochemicals of E. himalayensis. These two types of compounds might be leached into the soil and accumulated in the rhizosphere, resulting in a competitive superiority for E. himalayensis with its neighboring plants in the natural plant ecosystem. It is also interesting to note that compounds 1−3 have been reported to present in roots of leafy spurge, an invasive species in the same genus.7,11 Of particular interest is the observation that compounds 1 and 2 were also found in the root exudates of leafy spurge. It is known that the skeletal structures of secondary metabolites from the same genera are usually the same or similar.12 E. himalayensis, a toxic weed to livestock and humans in the Tibetan Plateau, exhibits allelopathic effects on crops, but the phytotoxic activities of the purified metabolites have not been reported thus far. In this study, compounds 1−3 isolated from the root extract of E. himalayensis showed observably phytotoxic activities. These compounds were also determined as allelochemicals of the plant released into the soil environment. The bioassay showed that the extract of the root exudates from the rhizosphere soil of E. himalayensis had significant allelopathic effects on crops and grasses. Our findings suggest that E. himalayensis is able to exert an effect on other plants through the release of allelochemicals. Production of these F
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chemicals can increase the fitness of E. himalayensis and provide the plant a superiority to become a successful competitor in the natural plant ecosystem. The bioassay results showed that the extract of the root exudates had an inhibitory effect at a lower concentration but promoted the growth of wheat seedlings once the test concentration was increased to 200 μg/mL, suggesting that the combination of the allelochemicals could be explored as a plant growth inhibitor for weed control and promoter for the growth and development of Gramineae crops, such as wheat in agriculture.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-931-4968372. Fax: +86-931-8277088. E-mail:
[email protected]. Funding
This work was funded by the National Natural Science Foundation of China (21102154 and 31070386), the Associate Scholar Program for Talents Cultivation Plan of “Western Light” of CAS, and the Basic Research Program of Lanzhou Institute of Chemical Physics, CAS (080423SYR1). Notes
The authors declare no competing financial interest.
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REFERENCES
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