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Apr 13, 2016 - ABSTRACT: Astragalus hoantchy, a widely cultivated medicinal plant species in traditional Chinese and Mongolian medicine, has been ofte...
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Allelochemicals from the Rhizosphere Soil of Cultivated Astragalus hoantchy Kai Guo,†,‡ Xiaofeng He,† Zhiqiang Yan,† Xiuzhuang Li,† Xia Ren,†,‡ Le Pan,†,‡ and Bo Qin*,† †

Key Laboratory of Chemistry of Northwestern Plant Resources of CAS 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 ‡ University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Astragalus hoantchy, a widely cultivated medicinal plant species in traditional Chinese and Mongolian medicine, has been often hampered by replant failure during cultivation, like many other herbs of the genus Astragalus. Root aqueous extracts of Astragalus herbs were reported to exhibit allelopathic activity against other plants and autotoxic activity on their own seedlings, but the allelochemicals released by Astragalus plants have not been specified so far. Ten compounds were isolated from the rhizosphere soil extract of cultivated A. hoantchy and elucidated by spectroscopic analysis. Compounds 1−6 observably showed allelopathic activity against Lactuca sativa seedlings and autotoxic activity against A. hoantchy seedlings. The isolated compounds were further confirmed and quantified by high-performance liquid chromatography (HPLC) in the rhizosphere soil, with a total concentration of 9.78 μg/g (dry weight). These results specify and verify the allelochemicals released by cultivated A. hoantchy into the soil environment, which may provide new insights into the allelopathic mechanisms of this medicinal plant and probably assist in clarifying the replant problems of Astragalus plants. KEYWORDS: Astragalus hoantchy, allelochemicals, rhizosphere soil, replant failure, autotoxicity

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INTRODUCTION Astragalus hoantchy, a perennial plant of the genus Astragalus, is cultivated in large areas in north and northwest China. Its roots are used as traditional Chinese medicine (the substitute of prescribed Radix Astragali) and Mongolian medicine, for general debility, chronic illnesses, increased overall vitality, and adjunct cancer therapy.1,2 In commercial cultivation, Astragalus plants have often been hampered by replant failure due to limited land resources and traditional monocropping habits, which leads to a serious reduction in both medicinal yield and quality.3,4 The problem of replant failure is mainly caused by factors including soil nutrient element depletion, plant diseases, and autotoxicity.5,6 Autotoxicity is an intraspecific allelopathy when mature plants inhibit the growth of seedlings of their own species through the release of autotoxic compounds into the environment.7,8 It has been recognized as the major cause of replant failure and reported in a number of plant species like crops and medicinal plants.9,10 Allelochemicals (including autotoxic compounds) may be introduced into soils through foliar leaching, root exudation, residue decomposition, and incorporation of organic debris. Therefore, the rhizosphere soil surrounding the plant roots contains these compounds in the greatest abundance.11,12 Crude extracts of different tissues from Astragalus plants have been reported to inhibit seed germination, radical elongation, and seedling growth of themselves, as well as many other plants.3,4,13,14 However, it remains obscure which compounds are responsible for the phytotoxic effects of Astragalus plants. To clarify chemically mediated allelopathy (including autotoxicity) in cultivated Astragalus plants, identification of allelochemicals and evaluation of their phytotoxic effects are urgently required. © XXXX American Chemical Society

Accordingly, in this study the isolation, identification, quantification, and biological evaluation of allelochemicals from the rhizosphere soil of cultivated A. hoantchy were undertaken. Our results demonstrated that different kinds of secondary metabolites were released and endowed with phytotoxic activities by cultivated A. hoantchy in the soil environment, which may likely play an important role in clarifying the replant problems of Astragalus plants. To the best of our knowledge, this is the first report for the genus Astragalus on allelochemicals and their phytotoxic activities in rhizosphere soil.

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MATERIALS AND METHODS

General Experimental Instruments and Procedures. 1H and 13 C NMR spectra were performed on a Bruker AM-400BB instrument (Bruker, Karlsruhe, Germany) with tetramethylsilane (TMS) as internal standard, operating at 400 and 100 MHz, respectively. Column chromatography (CC) was carried out on silica gel (200−300 mesh, type 60; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and Sephadex LH-20 (25−100 μm, Pharmacia Fine Chemical Co., Ltd., Berlin, Germany). Standard and preparative thin-layer chromatography (TLC and PTLC) were performed on silica gel (GF254, 10−40 μm; Qingdao Haiyang Chemical Co., Ltd.). Spots were detected on TLC under UV light or by heating after spraying with 5% H2SO4 in C2H5OH (v/v). High-Performance Liquid Chromatographic Analysis. HPLC analysis was carried out on a Waters 1525 binary HPLC pump (Waters, Milford, MA) with a Waters 2998 photodiode array detector, and the column used was a 250 mm × 4.6 mm, 5 μm, Phecda Rp C18 (Jiangsu Received: December 24, 2015 Revised: February 29, 2016 Accepted: April 13, 2016

A

DOI: 10.1021/acs.jafc.5b06093 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Chromatographic Data for 1−6 and Their Concentrations in Rhizosphere Soil of Cultivated A. hoantchy compd

regression equation

R2

detection wavelength (nm)

retention time (min)

concn (μg/g)

1 2 3 4 5 6

y = 52870x + 27067 y = 9553x + 49667 y = 354.06x + 31.5 y = 454.32x + 1674.4 y = 193.84x + 506.13 y = 66.09x + 977.71

0.9999 0.9994 0.9994 0.9987 0.9988 0.9989

210 240 210 210 210 210

21.81 158.48 56.11 98.18 40.33 34.93

0.21 1.28 1.56 1.22 1.18 4.33

Figure 1. Phytotoxic effects of crude methanol extract from rhizosphere soil against L. sativa and A. hoantchy seedlings at concentrations of 0, 50, 100, 200, and 400 μg/mL. Values are presented as percentage of the mean compared with control. Means significantly lower than DMSO controls are indicated with one asterisk (*) (Dunnett’s one-sided t-test; p < 0.05) or two asterisks (**) (p < 0.01). Error bars are one standard error of the mean and N = 5.

Figure 2. Structures of 1−10 isolated from rhizosphere soil of A. hoantchy. B

DOI: 10.1021/acs.jafc.5b06093 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Allelopathic effects of 1−10 on (A) root length and (B) stem length of L. sativa seedlings at concentrations of 0, 25, 50, 100, and 200 μg/mL. Values are presented as percentage of the mean compared to control. Means significantly lower than DMSO controls are indicated with one asterisk (*) (Dunnett’s one-sided t-test; p < 0.05) or two asterisks (**) (p < 0.01). Error bars are one standard error of the mean and N = 5. gently collected in five different areas from the same farm in Anyang, Gansu Province, China, in November 2014. After removal of fibrous roots and root contaminants, the collected samples were dried in the dark at room temperature and crushed to pass through a 0.356 mm screen sieve. The sieved rhizosphere soil samples were blended well and divided into two parts. The first group of samples (7.0 kg) were extracted ultrasonically with methanol three times (30 min each), and the filtrate was concentrated in vacuum to near dryness on a rotary evaporator, yielding a dry residue (2.0 g) for isolation of allelochemicals. Another group of samples (100 g) were treated to obtain dry residue in the same way as the first one, and then the dry residue was dissolved in methanol and passed through a 0.45 μm nylon membrane filter prior to HPLC analysis for quantification of allelochemicals. Isolation of Allelochemicals from Rhizosphere Soil. Dry residue (2.0 g) was subjected to silica gel CC with step gradient elution with CHCl3/EtOAc (100:0, 60:1, 30:1, 10:1, 5:1, 2:1, and 0:1 v/v) as the mobile phase and finally was washed with MeOH to give eight fractions (Fr.1−Fr.8). Fr.3 was fractionated on silica gel CC with petroleum ether (PE)/EtOAc (10:1) as the mobile phase and submitted to PTLC to

Hanbang Co., Ltd., Jiangsu, China). Acetonitrile solvent for HPLC analysis was HPLC gradient grade (Spectrum Chemical Mfg. Corp., Gardena, CA). Compounds were monitored at 210 and 240 nm, respectively, and UV spectra were recorded between 209.8 and 400 nm. The mobile phase, which was composed of acetonitrile (A) and water (B), was programmed as follows: 0−5 min, 10% A; 5−25 min, 10%−30% A; 25−30 min, 30%−45% A; 30−80 min, 45%−55% A; 80−90 min, 55%−65% A; 90−120 min, 65%−85% A; 120−130 min, 85%−100% A; 130−160 min, 100% A. The flow rate was 0.8 mL/min and the injection volume was 20 μL at a column temperature of 35 °C. Plant Material. Plants and seeds of cultivated A. hoantchy were collected from an A. hoantchy farm in Anyang, Gansu Province, China, in October 2014 and identified by Professor Huanyang Qi from Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (CAS). The voucher specimen (Ah-P-01 and Ah-S-01) was deposited in Lanzhou Institute of Chemical Physics, CAS. Seeds of L. sativa were purchased from a seed company (Sichuan Zhongdu Co., Ltd., Sichuan, China). Sample Preparation Procedures for Rhizosphere Soil. Soil samples about 15 cm deep surrounding the roots of A. hoantchy were C

DOI: 10.1021/acs.jafc.5b06093 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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and isoastragaloside I (10),21 were isolated from the crude methanol extract of the rhizosphere soil sample, and their structures were characterized by spectroscopic analysis in comparison with literature data. Allelopathic Activities of Purified Compounds on L. sativa Seedlings. Compounds 1−10, isolated from the rhizosphere soil of cultivated A. hoantchy, were evaluated for their allelopathic activities on seedling growth of L. sativa. Results indicated that 1−6 inhibited the growth of tested seedlings at different degree and their inhibitory effects were dose-dependent, while 7−10 did not show obvious growth regulation effect at any concentration tested (Figure 3). The inhibitory activity of 1−6 against the root length of seedlings was much stronger than that against the stem length, which means these compounds decreased the seedling growth mainly by inhibiting the elongation of root.

yield 2 (3.5 mg). Fr.6 was separated on Sephadex LH-20 eluted with CHCl3/MeOH (1:1) to obtain three fractions (Fr.6-1−Fr.6-3). Fr.6-1 and Fr.6-2 were subjected to PTLC to give 7 (2 mg) and 1 (1.5 mg), respectively. Separation of Fr.6-3 on silica gel CC with CHCl3/EtOAc (10:1) as the mobile phase gave 8 (4 mg). Fr.8 was applied to Sephadex LH-20 washed with CHCl3/MeOH (1:1) to give two fractions (Fr.8-1 and Fr.8-2). Fr.8-1 was fractionated on silica gel CC with CHCl3/ MeOH (15:1) as the mobile phase to afford four fractions (Fr.8-1-1− Fr.8-1-4), and then Fr.8-1-3 and Fr.8-1-4 were purified by silica gel CC eluted with CHCl3/MeOH (10:1) to provide 9 (6 mg) and 10 (4 mg), respectively. Fractionation of Fr.8-2 on silica gel CC with CHCl3/ MeOH (20:1) as the mobile phase gave nine fractions (Fr.8-2-1−Fr.8-2-9). Fr.8-2-2 was fractionated on silica gel CC washed with CHCl3/Me2CO (2:1) to yield 3 (5 mg). Fr.8-2-4 and Fr.8-2-6 were submitted to PTLC to afford 4 (4 mg) and 5 (4.5 mg), respectively. Separation of Fr.8-2-9 on silica gel CC eluted with CHCl3/Me2CO (1.5:1) gave 6 (12 mg). Bioassays. Inhibitory activities on the growth of A. hoantchy and L. sativa seedlings were evaluated by the plate-culture method. Seeds of A. hoantchy were submerged for 20 min in concentrated sulfuric acid for skin breaking to improve the germination rate and then washed thoroughly with distilled water to remove the sulfuric acid. Seeds of A. hoantchy after skin breaking and those of L. sativa were soaked in 10% hypochlorite for 7 min, washed five times with distilled sterile water, and then germinated on filter paper in the dark at 25 °C for 72 and 48 h, respectively. Isolated pure compounds from the rhizosphere soil were prepared as a stock solution of 20 mg/mL after being dissolved in the requisite amount of dimethyl sulfoxide (DMSO) and the crude extract from the rhizosphere soil of A. hoantchy was prepared as 40 mg/mL. The stock solutions were successively diluted with distilled water to obtain concentrations of 200, 100, 50, and 25 μg/mL for pure compounds and 400, 200, 100, and 50 μg/mL for the crude extract. The same volume of DMSO was added to distilled water as control, and the final percentage of DMSO in water was less than 1%. After germination, uniformly growing seedlings were transferred to 6-well plates (VWR Scientific, Inc., Shanghai, China), and the processed solutions with a final volume of 1 mL were added to each of the wells (each concentration dealing with at least six seedlings). Then the seedlings were incubated in a constant-temperature humidity chamber in the dark at 25 °C (A. hoantchy for 72 h, L. sativa for 48 h). Root length and stem length were measured at the end of the experiments. 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. Identification and Quantification of Allelochemicals in Rhizosphere Soil. Identification of allelochemicals in rhizosphere soil of cultivated A. hoantchy was completed by comparing the retention times with standards of 1− 6. Quantification of these compounds was also undertaken, and standard curves were constructed by the linear regression method (results are shown in Table 1).

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RESULTS AND DISCUSSION Isolation and Identification of Allelochemicals from Rhizosphere Soil of Cultivated A. hoantchy. Results showed that the crude methanol extract of the rhizosphere soil sample significantly inhibited growth of A. hoantchy and L. sativa seedlings at high concentrations (≥200 μg/mL) (Figure 1), indicating that there are potential autotoxic and allelopathic compounds that influence the growth of Astragalus plants themselves and other plants. The phytotoxic effect of the crude extract was dose-dependent, which was in accordance with previous studies.13,14 Ten compounds (Figure 2), including 4-hydroxybenzaldehyde (1),15 stigmast-4-en-3-one (2),16 cycloastragenol (3),17 5α,6β-dihydroxydaucosterol (4),18 3-(O-xylopyranosyl)cycloastragenol (5),17 astragaloside IV (6),17 methylnissolin (7),19 (22E)-5α,8α-epidioxy-6,22-dien-3β-ol (8),20 astragaloside I (9),21

Figure 4. Typical pictures of effects of 1−6 on growth length of (A) L. sativa seedlings and (B) A. hoantchy seedlings at concentrations of 0, 25, 50, 100, and 200 μg/mL. D

DOI: 10.1021/acs.jafc.5b06093 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 5. Autotoxic effects of 1−10 on (A) root length and (B) stem length of A. hoantchy seedlings at concentrations of 0, 25, 50, 100, and 200 μg/mL, respectively. Values are presented as percentage of the mean compared to control. Means significantly lower than DMSO controls are indicated with one asterisk (*) (Dunnett’s one-sided t-test; p < 0.05) or two asterisks (**) (p < 0.01). Error bars are one standard error of the mean, and N = 5.

When the treated concentration attained 50 μg/mL, 1−4 significantly inhibited the root length of L. sativa seedlings, whereas 5 and 6 showed significant inhibitory effect at higher concentrations (≥100 μg/mL), and 4 was much more effective at higher concentrations (≥100 μg/mL) than the other compounds. A typical picture of effects of 1−6 on growth length of L. sativa seedlings is shown in Figure 4. Autotoxic Activities of Purified Compounds on A. hoantchy Seedlings. Results indicated that 1−6 had remarkable autotoxic effects against A. hoantchy seedlings whereas 7−10 did not show obvious growth regulation effect at any treated concentration, which is in accordance with the results of L. sativa seedlings (Figure 5). It could also be found that these compounds mainly inhibited the seedling growth of A. hoantchy by decreasing the elongation of root through comparing root length with stem length, and the inhibitory effects did not show a sensible difference among 1−6. When A. hoantchy seedlings were treated with 1−6 at 100 or 200 μg/mL, their root

and stem growth were significantly inhibited, which indicated that these compounds might be released by the plants into the surrounding soil environment and endowed with autotoxic activity against themselves. The typical picture of effects of 1−6 on growth length of A. hoantchy seedlings is shown in Figure 4. Identification and Quantification of Allelochemicals in Rhizosphere Soil. To further identify and quantify allelochemicals in the soil environment of cultivated A. hoantchy, HPLC was carried out on the crude methanol extract of the second group of soil sample and the isolated pure compounds 1−6. By comparing the retention times of crude extract with those of pure compounds, the existence of allelochemicals in the rhizosphere soil was further confirmed (Figure 6). Standard curves were obtained by the linear regression method; peak areas at 210 nm (1, 3−6) or 240 nm (2) were plotted to calculate the concentrations; and finally, the concentrations of 1−6 in rhizosphere soil were determined (Table 1). E

DOI: 10.1021/acs.jafc.5b06093 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 6. HPLC analysis of crude methanol extract of rhizosphere soil from cultivated A. hoantchy at (A, B) 210 nm and (C) 240 nm, as well as purified 1−6 at (a, b) 210 nm and (c) 240 nm under the same conditions with LC retention times of 21.81, 158.48, 56.11, 98.18, 40.33, and 34.93 min, respectively.

Although it is now known that the genus Astragalus contains numerous phenols, flavonoids, polysaccharides, terpenoids, and triterpene saponins with various biological effects, such as

improving immune function and anti-inflammatory, antioxidation, and antibacterial activity,19,22 the phytotoxic effects of these secondary metabolites in genus Astragalus have not been F

DOI: 10.1021/acs.jafc.5b06093 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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allelochemicals released into the soil have not been identified until now. In this study, 1−6, which were isolated from the crude rhizosphere soil extract of cultivated A. hoantchy and quantified in the rhizosphere soil by HPLC, showed observabley phytotoxic activities against L. sativa and the plant itself. These results may help to reveal the allelopathy mechanism of genus Astragalus and probably provide guidance for controlling the replant failure of cultivated Astragalus plants. On the basis of the present study, further research should be emphasized on the allelopathy mechanism of these verified allelochemicals as well as the discovery of probable degradation measures (such as organic fertilizers or microbes with decomposition ability) toward these allelochemicals.

reported so far. All the allelochemicals (1−6) isolated from the rhizosphere soil of A. hoantchy in this study had been found in the aerial parts or roots of genus Astragalus,15−20 and this means that these low-molecular-weight compounds are probably released from living or decomposed plant tissues of A. hoantchy and endowed with phytotoxic activities by the plant during its growth. Allelochemicals can be grouped into three main chemical classes: terpenoids, N-containing compounds, and phenolic compounds.23 Phenols are ubiquitous in plant materials and have been demonstrated to inhibit germination and growth of various plants.24 Compound 1 belongs to phenols and has been reported to have phytotoxic effects in some plants.25 Steroids, including steroid saponins, are common plant chemicals with several potent bioactivities such as antifungal and antiviral activities.26 A few studies have reported the potential phytotoxic activity of steroids; for instance, 7α-hydroxysitosterol,27 7-oxositosterol,27 (22E)-6α-hydroxystigmata-4,22-dien-3-one,28 spinasterol,29 spinasterone,29 and spinasterol glucopyranosyl29 were found to exhibit phytotoxic effects against some plants. In this study, two steroids, stigmast-4-en-3-one (2) and 5α,6β-dihydroxydaucosterol (4), were detected to have phytotoxic effects for the first time, and their inhibitory activity is inferred to be associated with the presence of an α,β-unsaturated carbonyl moiety in 2 and a glycosyl group at the C-3 position in 4, in accordance with previous reports.27−29 Compounds 3, 5, 6, 9, and 10 are typical secondary metabolites in the plants of Astragalus genus with a cycloartane-type skeleton, and 3 is the genuine sapogenin of the other four compounds. Among them, 3 and 6 have been well-documented to possess potent bioactivities as the main active constituents that contribute to the beneficial effects of Radix Astragali;30,31 nevertheless, none of them has been reported to have phytotoxic activity. In this study, we found that 3, 5, and 6 showed significant allelopathic and autotoxic effects at different concentrations while 9 and 10 did not. The inhibitory effect of 6 was a little more severe than that of 5 on root length of L. sativa and A. hoantchy, from which it might be assumed that the number of glucose residues influenced the phytotoxic activities of triterpene saponins. In addition, by comparing the chemical structures and inhibitory effects of 9 and 10 with those of 5 and 6, it is clear that the number and location of substituted acetyls may also affect the phytotoxic activities of triterpene saponins. Although a few studies have reported the plant growth regulation effects of triterpene saponins,32−34 our work undoubtedly is a new supplement toward this content. During the soil sample collection process, a large amount of fibrous roots were noticed in the soil. It is likely that the allelochemicals found in soils are derived partly from root exudates or from plant tissue degradation. Once released into the soil and accumulated, these compounds may play a major role in mediating the phytotoxic effects that interfere with seedling growth of A. hoantchy and other plants. Compound 6 possessed the highest concentration of 4.33 μg/g (dry weight), and the total content of the six bioactive compounds reached up to 9.78 μg/g in the rhizosphere soil (Table 1). Moreover, considering the coexistence and interaction of allelochemicals in soils, a potentially much stronger phytotoxic effect is expected as these compounds (or possibly the inactive compounds) act additively or synergistically in natural settings.23,35 It has been considered that the homogeneity of both allelochemicals and chemical compositions accumulated in the soil environment is the major reason for the allelopathy (including autotoxicity) of genus Astragalus,36 but the specific

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b06093. 1 H and 13C NMR data for 1−10 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone +86-931-4968371; fax +86-931-8277088; e-mail [email protected]. Funding

This work was supported by the National Natural Science Foundation of China (31570354, 21302195, and 31300290), Agricultural Biotechnology Research and Development Program of Gansu Province (GNSW-2015-25), Cooperation Program to Gansu Province of Lanzhou Branch of the Chinese Academy of Sciences, 135 key cultivation program of the Chinese Academy of Sciences, and the Province-academy Cooperation Program of Henan Province of China (102106000021). Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.jafc.5b06093 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.5b06093 J. Agric. Food Chem. XXXX, XXX, XXX−XXX