Indian Journal of Experimental Biology Vol. 46, December 2008, pp. 831-835
Isolation and characterization of bioactive and antibacterial compound from Helianthus annuus linn. S Sankaranarayanan1, P Bama1, M Deccaraman1, M Vijayalakshimi1 K Murugesan2, PT Kalaichelvan2 & P Arumugam3 1 Department of Industrial Biotechnology, Dr MGR Educational and Research Institute University, Mathuravoyal, Chennai 600095, India 2 Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, India 3 Gloris BioMed Research Centre (P) Ltd, No. 171 First Street Vijayaragavapuram, Saligramam, Chennai 600 093, India
Received 12 May 2008; revised 22 October 2008 A bioactive compound with antibacterial activity was isolated and purified from the extract of leaves of sunflower, Helianthus annuus. The bioactive compound was characterized using 1H and 13C NMR. The compound induced auxin, gibberellins and cytokinin in Oryza sativa and Phaseolus mungo. It also showed activity against Xanthomonas oryzae pv. oryzae. Keywords: Antibacterial activity, Auxin, Bioactive compound, Cytokinin, Gibberellin, Helianthus annuus, Oryza sativa, Phaseolus mungo.
Helianthus annuus Linn. an oilseed crop is well known for its allelopathic compounds, several phenol and terpene compounds, including sesquiterpene, lactones, heliespirones A, annuionones, helibisabonols and heliannuols1. Heliannols A, D and E have special relevance due to high phytotoxic activity2 Annuione A and its relative compound show existence of six germacranolides and their biological activity has been determined by inhibition in Avena colaptile test and antimicrobial test3. Sesquiterpenoids have a wide spectrum of biological activities4 particularly Heliangine. A sesquiterpene lactone from leaves of H. tuberosus inhibits the elongation of Avena coleoptiles5, but the same compound promoted the root formation in phaseolus cuttings6. Cultivated sunflower has great allelopathic potential and inhibits weed seedling growth of velvel leaf, thorn apple, morning glory, wild mustards7. Leaves of H. annuus possess antimicrobial activity against many bacteria8. Leaf extract of sunflower having iso-chlorogenic acid and chlorogenic acid inhibits the growth of nitrogen fixing and nitrifying bacteria9. The seed-borne Xanthomonas oryzae pv. __________ Telephone: +9144-23721767, +9144-24724044; Fax: +9144-22352498 E-mail:
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oryzae severely affects the production of rice cultivates in Asia, Australia, Latin, America and Africa10-12. Several broad spectrum bactericides, recommended for control of bacterial blight of rice, however the chemical methods of disease management are expensive and may affect beneficial microbial population in the ecosystem. In the present investigations, attempt has been made to study the growth promoting and inhibiting activity of a bioactive compound from H. annuus leaves on Phaseolus mungo (dicotyledons) and Oryza sativa (monocotyledon). Antibacterial activity of the isolated compound has been evaluated against Xanthomonas oryzae pv. oryzae. Materials and Methods Plant materials and pathogen — Seeds of H. annuus L. (cv CO3) were purchased at Saravana Agro Service, Villupuram, Tamilnadu State. Plants were raised and maintained at the Field Research Laboratory, Centre for Advanced Studies in Botany, University of Madras, Maduravoyal, Chennai. Two months old leaves were collected from two month old plants and used for isolation of the bioactive compound. Seeds of paddy (O. sativa L.cv ADT 45) and Phaseolus mungo were obtained from a certified commercial seed vendor (Saravana Agro Service,
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Villupuram, and Tamilnadu State). The bacterial culture of Xanthomonas oryzae pv. oryzae was procured from bacterial culture collection of Centre for Advance Studies in Botany, University of Madras, Chennai, India and was maintained on nutrient agar medium at 37 °C. Preparation of plant extract — Fresh leaves (~2 kg) collected as above were washed thoroughly with deionized water and rinsed with distilled water. The leaves were ground in a mortar and pestle and the leaf homogenate was allowed to stand in distilled water overnight at 27º± 2ºC in dark. The homogenate was filtered through cheese cloth and the filtrate was initially extracted with five different solvents such as water, acetone, ethyl acetate, methanol and dichloromethane. Among the different solvents tested, dichloromethane resulted in relatively higher amount of bioactive compound and hence only this solvent was used in the subsequent experiments. The water phase of the extract was removed using a separating funnel and concentrated under vacuum with reduced pressure (Rotary evaporator R201B2 Shaanxi Taikang Biotechnology Co Ltd). Isolation and identification of bioactive compound — The concentrated dichloromethane extract (~2.5 g) was eluted through a column of silica gel (mesh size 60-120; 3 cm dia × 60 cm length) with an eluent comprising petroleum ether and ethyl acetate (9:1 ratio). Three ml fractions were collected and individual fractions were tested for the presence of the active compound by developing the fractions on pre-coated TLC plates (60F254) with hexane and acetone in the ratio of 9:1 as solvent system. There were six different spots on the TLC plate when illuminated with UV light with Rf value of 0.7, 0.6, 0.54, 0.47, 0.42, and 0.40, respectively from the point of origin of the sample. The compound with Rf value of 0.7 showed relatively higher bioactive and antibacterial activity and therefore, this compound was chosen for identification and further characterization. The bioactive compound was identified after analyzing the spectra obtained from Two Dimensional Correlated Proton Nuclear Magnetic Resonance (1H NMR) spectra recorded at 400 MHz on JEOL GsX 400 Spectrophotometer and 13C NMR spectra recorded at 100 MHz on JEOL GsX400 spectrophotometer. Chemical shifts were reported in ppm (δ) using tetramethylenesilane as internal
standard and coupling constants were expressed in Hertz.
Effect of bioactive compound on seed germination — Twenty five healthy seeds of O. sativa and P. mungo were washed with distilled water and blotted with sterile filter paper. The seeds were placed in a Petri dish containing 5 ml of test solution with different concentrations of purified compound and allowed for germination at 25ºC for 10 days. The pH of the test solution was adjusted to 6.0 with 1M, NaoH before treatment of the seeds. Five replicates were maintained for each concentration. The test solutions (10-4 M or 10-5 M) were prepared using H2O and MES (2-[Nmorpholino] ethanesulfonic acid, 10 mM). Parallel controls without bioactive compound were maintained along with the test concentrations. The root and shoot length values were recorded and tested by Duncan multiple ranges test. Difference between experimented and controls were significant at P = 0.05.
Extraction and estimation of growth hormones — The growth regulator hormones namely auxin, gibberellin and cytokinin were extracted and estimated following the methods as describers earlier13-15.
Anti bacterial activity of the bioactive compound — The bioactive compound was tested against the culture of xanthomonas oryzae pv. oryzae on an agar medium at 37 °C incubated for 24 hr using the disc diffusion method16. Results Seeds of O. sativa and P. mungo treated with bioactive compounds at 25-100 μg/ml. O. sativa showed increased shoot length (3.05-4.30 cm) and root length (3.10-4.57 cm) when compared with the bioactive compound. Whereas, P. mungo showed decreased shoot length (11.79-7.84 cm) and decreased root length (10.12-7.30 cm) with increase in bioactive compound concentration. Statistical data was obtained by ANOVA analysis (Table 1).
NMR spectrum — The proton NMR spectrum of the purified compound having Rf value 0.7 revealed the presence of an aldehyde functional group whose peak corresponds to value data 9.53 and 9.40 ppm; olyphynic functional groups at 6 to 4 ppm; OCH2 at
SANKARANARAYANAN et al.: ISOLATION & CHARACTERIZATION OF BIOACTIVE COMPOUND
around 3.8, 3.7, 3.4 ppm; and methylenes groups around 1.8 ppm. This revealed that it might be an unsaturated aliphatic aldehyde with OCH2 groups (Fig. 1).
Effect of bioactive compound on germination of O. sativa and P. mungo seeds — Seeds of O. sativa and P. mungo were treated with the bioactive compound at 25-100 µg/ml and its efficacy was measured in terms of elongation of root and shoot length. There was an increase in the length of shoot and root after the bioactive compound treatment as compared to control. Increase in shoot length in O. sativa ranged from 3.05-4.30 cm, while root length increased from 3.10-4.57 cm. Increase in shoot length was directly proportional to concentration of bioactive
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compound used. In contrast, the shoot and root length of P. mungo decreased with increasing concentrations of bioactive compound (shoot length, 11.79-7.84 cm; root length 10.12-7.30 cm). ANOVA followed by POST-HOC tests performed with shoot and root lengths of O. sativa for the treatments range 25-100 µg/ml of bioactive compound showed no significant difference between them. However, there was significant differences at 0.5% level between treated and control ones. There was significant difference at P < 0.05 level in shoot and root lengths of O. sativa and P. mungo between control and treated ones. However, there was positive effect (increase in shoot and root length) in O. sativa and the negative effect (decrease in shoot and root length) in P. mungo. (Fig. 2).
Table 1 — Effect of bioactive compound on phytohormones of O. sativa and P. mungo Bioactive compound conc. (µl)
Auxilin
O.sativa Gibberlin
Cytokinin
Auxin
P. mungo Gibberlin
Cytokinin
Control
0.005±0.001a
0.031±0.001a
1.004±2.234a
0.018±0.000a
0.018±0.000a
0.019±0.001a
25
0.007±0.000b
0.046±0.001b
0.006±0.001a
0.018±0.001a
0.016±0.001b
0.018±0.000b
50
0.009±0.001c
0.049±0.000c
0.007±0.000a
0.014±0.000b
0.015±0.000c
0.016±0.001c
75
0.011±0.001d
0.055±0.001d
0.009±0.000a
0.015±0.000c
0.012±0.000d
0.014±0.000d
100
0.016±0.000e
0.059±0.000e
0.011±0.000a
0.012±0.000d
0.010±0.001e
0.011±0.000e
Anova followed by Duncan's Multiple Range's test. Different superscripts in the same column of the mean values are significant different at P < 0.05 level
Fig. 1 — Proton NMR spectrum of the bioactive compound
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Effect of bioactive compound on the production of growth regulator — Seeds of O. sativa (monocotyledon) treated with bioactive compound showed increase in growth regulators, auxin (0.007-0.016); giberellin (0.046-0.059); and cytokinin (0.006-0.011). In P. mungo (dicotyledon) treated seeds with bioactive compound showed a decrease in growth regulators, auxin (0.018-0.012); giberellin (0.016-0.010); and cytokinin (0.018-0.011). The statistical data was obtained by ANOVA for O. sativa and P. mungo (Table 1).
Effect of bioactive compound against Xanthomonas oryzae pv. oryzae — The bioactive compound inhibited the growth of Xanthomonas oryzae pv. oryzae. The percentage of inhibition at different concentrations was 33, 44, 57 and 70% at 25, 50, 75 and 100 μg/ml, respectively. It showed highest inhibition at 100μg/ml when compared to the respective control (Table 2). Discussion Sunflower (H. annuus), an important oil crop, contains six bioactive norsesquiterpenes. Of which, three non ionone type bisnorsesquiterpenes and a new norbisabolene are potential allelopathic agents. These compounds exhibit selective inhibiting effect in shoot and root length in monocotyledons with an average inhibition of 45% on seed germination of Allium cepa and an average stimulation of 50 % on the root growth of A. cepa and Hordeum vulgare16. In the present study, isolated bioactive compound, an unsaturated aliphatic aldehyde showed growth
promoting activity in O. sativa, (monocotyledons) and suppressive activity in P. mungo (dicotyledons). The purified aliphatic aldehyde compound in O. sativa with increase in concentration increased the elongation of shoot and root as well as phytohormone concentration. Two types of allelochemicals such as bisnorsesquiterpenes (+) – dehydro vomifoliol and annuoionone D from sunflower leaves showed similar stimulatory effect in monocotyledons species (Allium cepa and Hordeum vulgare)17. In P. mungo, the bioactive compound showed decrease in shoot, root length and decrease in phytohormone concentration with increased concentration of bioactive compound from H. annuus. Comparatively Annuionone, a bioactive compound from sunflower leaves is a potent growth inhibitor used for development of a herbicide model against Coronopisdidymus (L.) sm., Medicago polymorpha L., Rumex dentatus L. and Phalaris minor Retz18. The purified unsaturated aliphatic aldehyde compound was efficient in controlling growth and multiplication of X. oryzae pv. oryzae. Similar observation has already been reported with the leaf extracts of Datura metel which significantly reduced the in vitro growth of Rhizoctonia solani and X. oryzae pv. oryzae. Methanolic extract of D. metel contains antibacterial active compound having RF value of 0.70519. The bioactive compound purified from the sunflower leaves showed antibacterial activity and effectively inhibited the growth of X. oryzae pv. oryzae with the maximum inhibition of 70% when treated at 100 µg/ml. A new germacronolide with a α-methylene-γ-lactone moiety from H. annuus Table 2 — Antibacterial activity of bioactive compound from H. annuus leaves tested against Xanthomonas oryzae pv. oryzae [Values are mean ± SD of 5 replications] Purified comp. Conc. (µg/ml) 25 50 75 100
Inhibition (mm) 10 12 15 18
Chloramphenicol Fig. 2 — Effect of bioactive compound on shoot and root length of O. sativa and P. mungo [Anova followed by Duncan’s Multiple Range’s test. Different superscripts in the same plant of the parameters are significantly different at P
