1Department of Biological Safety Science, National Institute for Agro-Environmental Sciences, 3-1-3 Kan-nondai, Tsukuba, Ibaraki 305-8604, Japan. E-mail; email@example.com
2TA Stevia Co. Ltd., Shibuya, Tokyo, Japan.
Allelopathic studies on the aerial parts of Stevia rebaudiana Hemsl. were carried out following a specific protocol derived from the definition of allelopathy. Bioactivity guided extraction and chromatographic techniques were used to isolate and purify the allelochemicals from the aqueous methanolic extract of stevia. The pure compounds are being studied by extensive mass and NMR spectroscopic methods in order to determine their structures. Their biological activity and threshold levels for activity were tested and optimized through the total activity guided bioassay procedures. These compounds promoted the growth of target species (lettuce and cucumber) significantly. This is the first report of isolation of allelochemicals as plant growth promoters from Stevia rebaudiana.
Plant growth promoting activity of Stevia rebaudiana Hemsl. was investigated using bioassay guided total activity approach.
Allelopathy; plant growth promoters, Stevia rebaudiana Hemsl., total activity.
Allelopathy is an important mechanism of plant interference caused by the addition of plant- manufactured secondary metabolites in the soil environment. A large number of plants produce inhibitory or stimulatory effects on the germination and growth of neighbouring or successional plants by releasing allelopathic substances into soil, either as exudates from living plant tissue or by decomposition of plant residues (Inderjit 1996; Narwal 1999; Putnam and Tang 1986; Rice 1984). When the plant-produced substances are beneficial, such association can stimulate plant growth, increase disease resistance, and improves the plant’s ability to withstand environmental stress. Allelopathic studies on the aerial parts of Stevia rebaudiana were carried out following a specific protocol derived from the definition of allelopathy.
S. rebaudiana is a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America (Paraguay and Brazil). It is often referred to as "the sweet herb of Paraguay". Stevia plant, its extract and stevioside have been used since long as a sweetener in South America, Asia and in different countries of Europe (Kinghorn 2002; Swanson, Mahady et al. 1992). In Brazil, Japan and Korea, stevia leaves and its highly refined extracts are officially used as low calorie sweetener (Kim and Kinghorn 2002; Mizutani and Tanaka 2002).
Residues and extracts of S. rebaudiana exhibited plant growth promoting activity on lettuce and cucumber and other crop plants. The purpose of this study was to isolate and identify the growth-promoting substances from the leaves of S. rebaudiana.
1H NMR spectra were recorded at 600 MHz, and 13C NMR spectra at 150 MHz on JEOL JNM α 600 spectrometer in CD3OD. Tetramethylsilane (TMS) was used as an internal reference. All the reagents were commercially available. Silica gel GF254, analytical chromatoplates, charcoal, and silica gel grade 60, 70-230 mesh for column chromatography were purchased from Wako, Japan and Merck, Germany.
Isolation and identification of growth promoting substances
Dried leaf powder of S. rebaudiana was soaked in 80 % methanol for 2 weeks. The extract was filtered and evaporated under vacuum to obtain a dark brown gummy material. A portion of the gummy material was partitioned with various organic solvents. All these fractions, along with the crude extract, were subjected to bioassays using lettuce and cucumber seedlings as test species. Aqueous fraction and the hexane fraction of crude extract were found to be active (Figure-1 a&b and Figure-2 a&b). The aqueous fraction (Fr-3) was subjected to charcoal column chromatography and eluted with water (Fr-3-1), water-methanol (1:1) (Fr-3-2), methanol (Fr-3-3), acetone (Fr-3-4) and finally with ethyl acetate (Fr-3-5). The bioassay results indicate that the major activity was separated into the acetone-eluted fraction. Fraction 3-4 was further subjected to bioassay directed silica gel chromatography to obtain pure compound-C (500 mg, 0.3 % of the dried leaves). The structure of compound-C is being elucidated by detailed spectroscopic analysis of EIMS, FABMS, 1H-NMR, COSY, 13C-NMR, DEPT, HMQC and HMBC etc. The second active fraction (hexane fraction) is also being purified.
Measurement of phytotoxic activity
A filter paper (27 mm °, Type 1, Toyo Roshi Kaisha, Ltd, Tokyo) was placed in a glass Petri dish (27 mm °). Different concentrations of test solutions (0.5 ml) were added in the Petri dish. Six pre-germinated (16 h at 20á░C in the dark) seedlings of lettuce (Lactuca sativa cv. Great Lakes 366) and cucumber were placed on the filter paper, and incubated for 48 h at 20á░C in the dark. The growth promoting activity on root and shoot elongation was detected by measuring the length of the root and comparing the data obtained with that of untreated controls.
Bioactivity guided extraction and chromatographic procedures were used to isolate and purify the allelochemicals from the aqueous methanolic extract of stevia. The pure compounds are being identified by extensive mass and NMR spectroscopic methods. Comparison of the growth promoting activity of extracts on cucumber/lettuce root/shoot growth revealed a higher activity of the original extract fractionated into the hexane and water fractions (Figure-1 a&b and Figure-2 a&b).
Figure-1. Effect of stevia extracts on root (a) and shoot (b) growth of cucumber at 3 different concentrations. Error bar representsá▒ standard deviation (N=15).
Figure-2. Effect of stevia extracts on root (a) and shoot (b) growth of lettuce at 3 different concentrations. Error bar representsá▒ standard deviation (N=15).
Chromatographic purifications of the most active aqueous fraction gave compound-C. The growth promoting activity of the original water fraction was not greatly lost, and compound-C accounted for almost all of the activity observed in the aqueous fraction. Compound-C enhanced the growth of cucumber and lettuce shoots up to 147 % and 140 %, respectively (Figure-3 a&b). Growth of radish and tomato seedlings also increased to a considerable extent when exposed to an appropriate concentration of the isolated compound (Figure-4 a&b). These results suggest that compound-C is the cause of the growth promoting activity of the water fraction. Studies are in progress for finalization of the structure of compound-C.
Figure-3. Effect of compound-C on the root and shoot growth of cucumber (a) and lettuce (b) seedlings. Error bar representsá▒ standard deviation (N=15).
Figure-4. Effect of compound-C on the root and shoot growth of tomato (a) and radish (b) seedlings. Error bar representsá▒ standard deviation (N=15).
Identification of plants, which release beneficial allelochemicals, is very important. Through exudates and leachates these plants release wide array of secondary metabolites which can have a positive influence on plant growth; enhancing the availability of minerals and nutrients, improving nitrogen fixation ability, improving plant health through the biocontrol of phytopathogens, inducing systemic plant disease resistance, and facilitating plant establishment, growth and development. These beneficial substances by virtue of their metabolic versatility are the key agents of change in soil agro-ecosystems. Interactions between plant root systems and these growth promotory substances will have a profound effect on crop health and yield.
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