AUTOTOXIC COMPOUNDS FROM FRESH ALFALFA LEAF EXTRACTS: IDENTIFICATION AND BIOLOGICAL ACTIVITY

Size: px

Start display at page:

Download "AUTOTOXIC COMPOUNDS FROM FRESH ALFALFA LEAF EXTRACTS: IDENTIFICATION AND BIOLOGICAL ACTIVITY"

Fay Barker
5 years ago
Views:

1 Journal of Chemical Ecology, Vol. 26, No. 1, 2000 AUTOTOXIC COMPOUNDS FROM FRESH ALFALFA LEAF EXTRACTS: IDENTIFICATION AND BIOLOGICAL ACTIVITY ILL-MIN CHUNG, 1, * DAVID SEIGLER, 2 DARRELL A. MILLER, 3 and SUK-HUN KYUNG 4 1 Department of Crop Science, KonKuK University Seoul, South Korea, Department of Plant Biology, University of Illinois Urbana-Champaign, Illinois Department of Crop Science, University of Illinois Urbana-Champaign, Illinois Department of Applied Biology and Chemistry, Konkuk University Seoul, South Korea, (Received January 28, 1999; accepted September 15, 1999) Abstract Many investigators have attempted to identify the allelochemicals in alfalfa (Medicago sativa), that cause autotoxicity. The autotoxic compounds from fresh alfalfa leaves were separated and quantified, and their biological activity was determined. Chemical separation procedures involved an 80% methanol extract of fresh alfalfa leaves, treatment with activated charcoal, microcrystalline cellulose thin-layer chromatography (MCTLC), and finally separation by Sephadex LH-20 column chromatography. The various fractions were examined further by high-performance liquid chromatography (HPLC). Preliminary identification by HPLC analysis resulted in peaks with retention times close to those of chlorogenic (m/ z 354) and salicylic acid (m/ z 138) standards, and these compounds were confirmed with GC-MS. Several other peaks remain unidentified. Chlorogenic acid occurs in relatively large amounts (0.39 mg/ g) in alfalfa aqueous extracts as compared to salicylic acid (0.03 mg/ g), and bioassays suggest that chlorogenic acid is involved in alfalfa autotoxicity. Key Words Alfalfa extract, autotoxicity, bioassay, chlorogenic acid, salicylic acid, HPLC, GC-MS. * To whom correspondence should be addressed / 00/ $18.00/ Plenum Publishing Corporation

2 316 CHUNG, SEIGLER, MILLER, AND KYUNG INTRODUCTION Secondary plant metabolites and their degradation products are important in all agroecosystems. The following groups of compounds, among others, are known to produce toxic effects: terpenoids and steroids, phenols, coumarins, flavonoids, tannins, alkaloids, and cyanogenic glycosides (Rice, 1984). Phenolic compounds have been intensively studied with regard to their phytotoxicity (Putnam, 1985). During the past 20 years, many workers have reported autotoxicity in alfalfa (Nielsen et al., 1960; Guenzi et al., 1964; McElgunn and Heinrichs, 1970; Klein and Miller, 1980; Bohnenblust, 1983; Kehr et al., 1983; Goplen and Webster, 1969; Miller, 1983; Hall and Henderlong, 1989; Dornbos et al., 1990; Hedge and Miller, 1992). Various toxic compounds potentially involved in autotoxicity are localized in the seed coat, fresh alfalfa leaves, stems, crowns, dry hay, old roots, and soil residues (Miller, 1983). The effects of autotoxicity often are expressed as a reduction of alfalfa yield and difficulty in reestablishing plants in fields due to low seed germination and poor seedling growth. Chung and Miller (1995a) found that alfalfa aqueous extracts inhibit alfalfa seed germination and seedling growth, and that autotoxicity is concentration dependent and due to water-soluble toxic substances. Autotoxic or allelopathic compounds may serve as natural herbicides by inhibiting germination and seedling growth of certain weed species, but an understanding of the chemical structure of the compounds causing low germination and poor establishment is necessary for resolution of the complicated dynamics of alfalfa autotoxicity. Waller et al. (1993) showed that growth and development of cheatgrass (Bromus secalinus L.), barnyard grass [Echinochloa crusgalli (L.) Beauv.], pigweed (Amaranthus retroflexus L.), dandelion (Taraxacum officinale Weber), and coffeeweed [Daubentonia punicea (Cav.) D. C.] were inhibited by alfalfa root saponins. Chung and Miller (1995b) found that alfalfa residue reduced weed growth and development due to water-soluble allelochemicals present in the residue. Few investigations have identified specific compounds responsible for autotoxic effects. Saponins were dismissed as autotoxicity agents in alfalfa (Miller, 1983). When the isoflavonoid, medicarpin, was provided exogeneously to alfalfa seed, germination percentage was reduced by 59% (Miller et al., 1988). Caffeic, chlorogenic, isochlorogenic, p-coumaric, p-hydroxybenzoic, and ferulic acids are released from alfalfa root exudates and residues (Abdul-Rahman and Habib, 1989). Hall and Henderlong (1989) reported that alfalfa contains autotoxic compounds that were characterized as phenolic compounds. Dornbos et al. (1990) isolated medicarpin, 4-methoxymedicarpin, sativan, and 5- methoxysativan from alfalfa. Medicarpin inhibited alfalfa and velvetleaf (Abutilom theophrastic Medic.) germination and seedling growth. Medicarpin may

3 AUTOTOXIC COMPOUND FROM ALFALFA LEAF 317 control the growth of plants surrounding an alfalfa plant, inhibit alfalfa seedling establishment, and control some weeds. The objectives of this study were to separate, identify, and quantify compounds from fresh alfalfa leaves that are responsible for alfalfa autotoxicity and to determine their phytotoxicity to alfalfa germination and seedling growth. METHODS AND MATERIALS Extraction of Leaves. Fresh alfalfa leaves (cv. WL-320) at the vegetative stage of growth were collected from the Agronomy and Plant Pathology South Farm, University of Illinois. The extraction procedure was based on the method of Mabry (1970). Fresh leaves were extracted with 80% methanol in a Waring blender for 10 min. The extract was filtered through four layers of cheesecloth and filter paper (Whatman No. 1). Extracts were concentrated under vacuum (30 C) to about one third the original volume or until most of the methanol had been removed. The remaining aqueous solution was partitioned with chloroform to remove nonpolar compounds, and the chloroform extraction was repeated three times. The aqueous extracts were combined and concentrated under vacuum and freeze dried. The chloroform washes are designated as extracts in the following discussion. Approximately 50 g of dried materials were obtained from 1 kg fresh leaves. Separation and Purification Procedures. Extract (3 g) was dissolved in distilled water and filtered with activated charcoal to determine whether activated charcoal altered the toxicity of fresh alfalfa extract. Preliminary germination bioassays indicated that chemicals of interest were still present (data not shown), and charcoal-treated samples were evaporated under vacuum and freeze dried. Most of the original active material was recovered (98%). The solid material was redissolved in distilled water (10 ml) and chromatographed on preparative microcrystalline cellulose TLC plates (0.1 mm, Whatman). Plates were developed with the lower phase of chloroform methanol water (5 : 6 : 4) and air-dried. Each chromatographic plate was divided into 2-cm bands, which were scraped from the plate, and biological compounds were desorbed with 50% methanol. The resulting suspensions were filtered through filter paper (Whatman No. 42) and evaporated under vacuum. Material from each band was resuspended with acetone and methanol. Solvents were evaporated under nitrogen and the residual material freeze dried to remove traces of water. The remaining solids from each band were weighed and dissolved in 50% methanol (10 ml). The solution from each band was divided into two parts (5 ml each). One sample was used for bioassay as described below in the bioassay test to determine the presence of bioactive compounds, while the remaining sample was refrozen and stored in a freezer (5 C). Sephadex LH-20 Column Chromatography. Column chromatography was used to purify the extractable compounds from bioassay-active bands 5 9

4 318 CHUNG, SEIGLER, MILLER, AND KYUNG obtained from microcrystalline cellulose TLC plates. These bands were combined, dissolved in methanol and water (1 : 1), and chromatographed on a Sephadex LH-20 column. Fractions (100 ml) were collected and concentrated under vacuum. Concentrated samples were evaporated under nitrogen and freeze-dried. Solid materials obtained were redissolved in distilled water (10 ml). Each fraction (10 ml) was divided into two parts. One fraction was used for bioassays to determine autotoxic activity, and the remaining fraction was stored in a freezer ( 13 C). Chemicals and Extracts. Chemical standards used for HPLC analysis were chlorogenic acid, p-coumaric acid, and salicyclic acid from Aldrich Chemical Co. and gallic acid, quercetin, and rutin from Sigma Chemical Co. All chemicals were purchased as high purity standards. Solvents were HPLC spectral grade, and all solvents and distilled water were degassed before use. All solvent ratios were based on a volume basis. HPLC Instrumentation. Extracts for HPLC analysis were collected from the Sephadex LH-20 column chromatography. A Beckman HPLC system with two 110A solvent metering pumps, a Beckman 25-cm C 18 Ultrasphere ODS column, and a Micromeritics 725 Autoinjector with a 20-ml sample loop was employed. Mobile phase A consisted of 98% water and 2% glacial acetic acid in M ammonium acetate, and mobile phase B was 68% water, 25% methanol, 5% butanol, and 2% glacial acetic acid in M ammonium acetate. Both extracts and standard compounds were used in the following gradient system: (1) from 0.0 to 1.0 min, isocratic with 10% B; (2) from 1.0 to 21.0 min, a linear gradient from 10 to 25% B; (3) from 21.0 to 36.0 min, a linear gradient from 25 to 45% B; (4) from min, a linear gradient from 45 to 100% B; (5) from 50.0 to min, the flow was increased to 1.20 ml/ min: (6) from min, a linear gradient from 100 to 10% B; (7) from 92.0 to min, the flow was decreased to 1.00 ml/ min; and (8) at 99.0 min, the sample loop was rinsed and gradient repeated. The UV detector was set to 254 nm. Analyses of extracts and standard compounds were based on the method of Banwart et al. (1985). Standard compounds were chromatographed alone and as mixtures. Retention times for the standard compounds and the major peaks in the extract were recorded. GC-MS Instrumentation. GC-MS analyses were performed according to the method of Porter et al. (1985). The GC contained a fused silica capillary column (Ultra-2, 50 m 0.25 mm ID 0.33 mm). The carrier was helium and the flow rate was 30 m/ min. The temperature gradient was from 50 C to 200 C at 25 C/ min. The collected eluant was frozen and placed in a freeze-dryer to remove the methanol water solvent. The remaining dried material was redissolved in methanol, and a portion was evaporated to dryness under nitrogen. The dried material was analyzed by direct probe electron impact with the GC- MS system.

5 AUTOTOXIC COMPOUND FROM ALFALFA LEAF 319 Quantification of Detected Compounds by HPLC. Analyses of the compounds were based on the method of Banwart et al. (1985). Chlorogenic and salicylic acids were identified by retention times or standard addition, and contents were calculated by comparing peak areas with those of standards. Bioassay. Activity of suspected bioactive materials was evaluated in germination tests with alfalfa seeds. Analytical grade chlorogenic acid (10 3 to 10 4 M in water) was prepared with some modifications by the method of Williams and Hoagland (1982). Chlorogenic acid had a similar retention time to one of the peaks of the purified extract analyzed by HPLC. Solutions were stored at 50 C until used. Salicylic acid was detected by HPLC analysis, but exhibited no activity in an assay according to Hedge and Miller (1992). Alfalfa seed cv. Pioneer 5472 (5 g) were surface sterilized with a solution of distilled water Clorox (90 : 10) for 5 min, and then rinsed with distilled water. One hundred alfalfa seeds were evenly placed on filter paper (Whatmam No. 1) in sterilized 9-cm glass Petri dishes. Test solution (10 ml) was added to each Petri dish and dishes placed in a lighted chamber at 24 C. Percent germination was defined as radicle protrusion through the seed coat. Seedling growth was recorded on the fourth and fifth day after seeding. After measuring seedling growth, the seedling leaves (cotyledons), stems, and roots were separated, and dried at 85 C for 6 hr to obtain dry weight. Each treatment was replicated five times in a randomized design, and bioassays were repeated twice. Analysis of variance for the data was carried out by the general linear model procedure of the SAS program (SAS Institute, 1988). Means were separated on basis of least significant difference (LSD) at the 0.05 probability level. RESULTS Low germination percentages occurred in seed bioassays with fractions from the microcrystalline cellulose thin-layer chromatography (bands 5 9) and from Sephadex LH-20 column chromatography (fractions 4 and 5) (Tables 1 and 2). Results of HPLC analyses indicated the presence of a large number of unknown peaks and considerable overlapping of peaks (Figures 1 and 2). Table 3 shows peak area, area percent, and height percent data on chlorogenic and salicylic acids chromatograms from HPLC analysis of fresh alfalfa fraction 4 from Sephadex LH-20 column chromatography. HPLC identification of chlorogenic and salicylic acids was confirmed by GC-MS (Figures 3 and 4). The UV spectrum of collected portions of eluant was obtained following freeze-drying and subsequent dissolving of the freeze-dried solid material in methanol. The solid material was analyzed by GC-MS. The mass spectrum of chlorogenic and salicylic acids showed molecular ions at m/ z , respectively, and major fragmentation ions at 163 and 180, and m/ z

6 320 CHUNG, SEIGLER, MILLER, AND KYUNG TABLE 1. ALFALFA SEED GERMINATION AFTER TREATMENT WITH AQUEOUS EXTRACT FRACTIONS OF FRESH ALFALFA LEAVES SEPARATED BY MICROCRYSTALLINE CELLULOSE TLC PLATES Band a Range (cm) Recovery (g) b Germination (%) a After visualizing under UV light, the separated fractions were scraped from the TLC plate. b Recovery amount means recrystalized amount. TABLE 2. ALFALFA SEED GERMINATION AFTER TREATMENT WITH FRACTIONS OF AQUEOUS EXTRACT OF FRESH ALFALFA LEAVES SEPARATED BY SEPHADEX LH-20 COLUMN CHROMATOGRAPHY Recovery amount Germination Fraction (g) a (%) a Recovery amount means recrystalized amount. and at 92 and 120, respectively. These spectra were essentially identical to those obtained for authentic standards (reference data not shown). Alfalfa seed germination, seedling length, and weight of alfalfa were inhibited by chlorogenic acid, with a greater degree of inhibition caused by higher concentration (10 3 M) (Table 4). The concentration of chlorogenic acid (0.39 mg/ g) was 13 times higher than that of salicylic acid (0.03 mg/ g) in fresh alfalfa leaves.

7 AUTOTOXIC COMPOUND FROM ALFALFA LEAF 321 FIG. 1. Chromatogram of fraction 4 from Sephadex LH-20 column chromatography by high-performance liquid chromatography. FIG. 2. Chromatogram of fraction 5 from Sephadex LH-20 column chromatography by high-performance liquid chromatography.

8 322 CHUNG, SEIGLER, MILLER, AND KYUNG TABLE 3. PEAK AREA, AREA PERCENT, AND PEAK HEIGHT OF CHLOROGENIC AND SALICYLIC ACIDS OBTAINED BY HPLC ANALYSIS OF ALFALFA LEAF FRACTIONS FROM SEPHADEX LH-20 COLUMN CHROMATOGRAPHY Fraction Compound Chlorogenic acid Peak area (mm 2 ) Peak area (% of total) Peak height (% of total) Salicylic acid Peak area (mm 2 ) Peak area (% of total) Peak height (% of total) DISCUSSION Bioactive compounds separated from fresh alfalfa leaves are responsible for some of the autotoxic activity of alfalfa. Although the compounds from fractions 2 7 had no exact match with standards, two peaks in these fractions had similar HPLC retention times with chlorogenic acid (14.78 min) and salicylic acid (29.74 min). Fractions 4 and 5 from Sephadex LH-20 column chromatography inhibited alfalfa seed germination in bioassays. Chlorogenic acid is biologically active against alfalfa seed germination, seedling growth, and weight. Flavonoids are reportedly involved in alfalfa autotoxicity and allelopathy (Miller et al., 1988; Dornbos et al., 1990), and peaks corresponding to chlorogenic and salicylic acids on HPLC analysis remained after fractionation. This suggests that compounds in these extracts are polar. Preliminary identification based upon microcrystalline cellulose TLC, Sephadex LH-20 column chromatography, and HPLC analysis suggests that chlorogenic acid contributes to alfalfa autotoxicity. Similar results were found by Abdul-Rahman and Habib (1989), who isolated phenolic acids, including chlorogenic acid from alfalfa root exudates and residues, and found biological activity on bladygrass (Imperata cylindrica) germination and root length. Chlorogenic acid shows phytotoxic activity in several crop residues (Rice, 1984), and analytical grade phenolic compounds tested at 10 3 and 10 5 M indicate that chlorogenic acid is an active compound on germination of nine crop and weed species (Williams and Hoagland, 1982). We conclude that the autotoxic and/ or allelopathic effect of alfalfa may be due in part to the presence of chlorogenic acid. Purification of phytotoxic compounds by microcrystalline cellulose TLC and column chromatography with activated charcoal treatment may be useful for isolation of specific compounds that inhibit seed germination and seedling

9 FIG. 3. Mass spectrum of chlorogenic acid from high-performance liquid chromatography. AUTOTOXIC COMPOUND FROM ALFALFA LEAF 323

10 324 FIG. 4. Mass spectrum of salicylic acid from high-performance liquid chromatography. CHUNG, SEIGLER, MILLER, AND KYUNG

11 (sample/ TABLE 4. GERMINATION AND SEEDLING LENGTH AND WEIGHTS OF ALFALFA SEEDLINGS IN RESPONSE TO CHLOROGENIC ACID CONCENTRATIONS Length [cm (%)] a Dry weight [mg (%)] a Concentration Germination (M) (%) a Root Shoot Leaf Root Stem Control 88.6 (100%) 4.3 (100%) 3.7 (100%) 0.72 (100%) 0.32 (100%) 0.78 (100%) (81.0%) 3.6 (84.0%) 2.5 (67.6%) 0.65 (90.3%) 0.25 (78.1%) 0.67 (85.9%) (61.6%) 3.0 (70.0%) 3.2 (86.5%) 0.53 (73.6%) 0.17 (53.1%) 0.56 (71.8%) LSD (0.05) CV (%) DRLW, Dry lead weight; DRRW, Dry root weight. a Values in parenthesis are relative ratio, where relative ratio control) 100. AUTOTOXIC COMPOUND FROM ALFALFA LEAF 325

12 326 CHUNG, SEIGLER, MILLER, AND KYUNG growth. In the present study, however, it was not possible to determine specifically which toxic compounds were present in the extracts. A method for purification and isolation of bioactive compounds from alfalfa leaves has been developed to bioassay compounds related to alfalfa autotoxicity. Tests for phytotoxicity of compounds from alfalfa on other crops and potential use in weed control are warranted. REFERENCES ABDUL-RAHMAN, A. A., and HABIB, S. A Allelopathic effect of alfalfa (Medicago sativa L.) on bladygrass (Imperata cylindrica). J. Chem. Ecol. 15: BANWART, W. L., PORTER, P. M., GRANATO, T. C., and HASSETT, J. J HPLC separation and wavelength area ratios of more than 50 phenolic acids and flavonoids. J. Chem. Ecol. 11: BOHNENBLUST, K. E Effect of allelopathy or autotoxicity on alfalfa seedling establishment, p. 25, in Report 28th Alfalfa Improvement Conference, Davis, California. CHUNG, ILL-MIN, and MILLER, D. A. 1995a. Effect of alfalfa plant and soil extracts on germination and growth of alfalfa. Agron. J. 87: CHUNG, ILL-MIN, and MILLER, D. A. 1995b. Natural herbicide potential of alfalfa residue on selected week species. Agron. J. 87: DORNBOS, D. L., JR., SPENCER, G. F., and MILLER, R. W Medicarpin delays alfalfa seed germination and seedling growth. Crop Sci. 30: GOPLEN, B. P., and WEBSTER, G. R Selection in Medicago sativa L. for tolerance to alfalfa sick soils in central Alberta. Agron. J. 61: GUENZI, W. D., KEHR, W. R., and MCCALLA, T. M Water soluble phytotoxic substances in alfalfa forage: Variation with variety, cutting, year, and stage of growth. Agron. J. 56: HALL, M. H., and HENDERLONG, P. R Alfalfa autotoxic fraction characterization and initial separation. Crop Sci. 29: HEDGE, R. S., and MILLER, D. A Concentration dependency and stage of crop growth in alfalfa autotoxicity. Agron. J. 84: KEHR, W. R., WATKINS, J. E., and OGDEN, R. L Alfalfa establishment and production with continuous alfalfa and following soybeans. Agron. J. 75: KLEIN, R. R., and MILLER, D. A Allelopathy and its role in agriculture. Commun. Soil Plant Anal. 11: MABRY, T. J., MARKHAM, K. R., and THOMAS, M. B The Systematic Identification of Flavonoids. Springer-Verlag, New York, 354 pp. MCELGUNN, J. D. and HEINRICHS, D. H Effects of root temperature and a suspected phytotoxic substance on the growth of alfalfa. Can. J. Plant Sci. 50: MILLER, D. A Allelopathic effects of alfalfa. J. Chem. Ecol. 9: MILLER, R. W., KLEIMAN, R., POWELL, R. W., and PUTNAM, A. R Germination and growth inhibitors of alfalfa. J. Nat. Prod. 51: NIELSEN, K. F., CUDDY, T., and WOODS, W The influence of the extract of some crops and soil residues on germination and growth. Can. J. Plant Sci. 40: PORTER, P. M., BANWART, W. L., and HASSETT, J. J HPLC isolation and GC-MS identification of genistein, daidzein, and coumestrol from unhydrolyzed soybean root extracts. Environ. Exp. Bot. 25: PUTNAM, A. R Allelopathic research in agriculture: Past highlights and potential, pp. 1 8,

13 AUTOTOXIC COMPOUND FROM ALFALFA LEAF 327 in A. C. Thompson (ed.). The Chemistry of Allelopathy. ACS Symposium Series. American Chemical Society, Washington, D.C. RICE, E. L Allelopathy. Academic Press, Orlando, Florida. 422 pp. SAS Institute SAS/ STAT User s Guide, 6.03 ed. SAS Institute, Cary, North Carolina. 108 pp. WALLER, G. R., JURYSTA, M., and THORNE, R. L. Z Allelopathic activity of root saponins from alfalfa (Medicago sativa L.) on weeds and wheat. Bot. Bull. Acad. Sin. 34:1 10. WILLIAMS, R. D., and HOAGLAND, R. E The effects of naturally occurring phenolic compounds on seed germination. Weed Sci. 30:

Preliminary phytoconstituents screening of some weeds and their potential toxicity on rice variety- Tarom via decomposition bioassay

2011 International Conference on Environmental, Biomedical and Biotechnology IPCBEE vol.16 (2011) (2011)IACSIT Press, Singapoore Preliminary phytoconstituents screening of some weeds and their potential