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Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control
SAJB-01357; No of Pages 7 South African Journal of Botany xxx (2015) xxx–xxx
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Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control Mohamed Abdel aziz Balah Plant Protection Department, Ecology and Dry Land Agriculture Division, Desert Research Center, 1-Mathaf El-Mataria St, EL-Mataria, Cairo, BOX: 11753, Egypt
a r t i c l e
i n f o
Article history: Received 28 June 2014 Received in revised form 28 February 2015 Accepted 29 July 2015 Available online xxxx Keywords: Conyza dioscoridis Pre-emergence Phytotoxicity activity Flavonoids Sesquiterpene Convolvulus arvensis
a b s t r a c t The potential herbicidal activity of certain chemicals isolated from Conyza dioscoridis (L.) desf. leaves was bioassayed on seed germination and growth parameters of Convolvulus arvensis (L.) and several weeds including Portulaca oleracea (L.), Phalaris paradoxa (L.), Corchorus olitorius (L.) and Echinochloa crus-galli (L.) Beauv. Bioassay guided isolation showed seven phytotoxic natural compounds including three sesquiterpene compounds. An inseparable mixture of the eudesmane derivatives (15-hydroxyisocostic acid and the corresponding aldehydic acid) was isolated from the leaves of C. dioscoridis. Relative to controls, this mixture reduced total biomass of C. arvensis by (86.81%) and methyl 15-oxo-eudesome-4, 11(13)-diene 12-oate as well as 1α, 9α-dihydroxy-αcyclocostunolide showed 86.28% and 76.19% reductions respectively, compared to its control. Also, four flavonoids caused the reductions in C. arvensis total biomass fresh weight by: isorhamnetin 3-sulfate (80.61%), isorhamnetin 3-O-rutinoside (80.39%), rhamanetin (76.25%) and epicatechin (70.58%) compared to its control. The activity of these chemicals may be nominating them to develop as natural herbicides in the future. © 2015 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Controlling bindweeds (Convolvulus arvensis L.) in agricultural fields is still extremely difficult, despite many efforts to minimize their impact using cultural, chemical and biological methods. Field bindweed reproduces by buds in their secondary roots that can develop into new plants and seeds with tough seed coats that can remain viable in the soil for years (Wiese and Phillips, 1976). The use of commercial herbicides is considered the most effective method for controlling weeds; however herbicides can be poisonous to mammals and many other forms of wild life, result in ground water contamination, and have other adverse impacts on the environment. This has led to intensified efforts in recent years to identify environmentally benign forms of chemical weed control particularly through increasing the allelopathic potential of promising plants (Mendes and Vermelho, 2013). Thus, much interest has been centered on the possible incorporation of natural products phytotoxin as safe, selective, and cost-effective herbicides. Natural products may offer novel molecular target sites and mechanisms of action for new herbicides (Duke et al., 2000). The environmental half-life of many natural compounds is shorter, and they are generally less toxic to the environment than many synthetic herbicides (Duke et al., 2000), thus a large number of natural products are being tested as possible bio herbicides. Conyza dioscoridis was chosen for investigation because it is widely used as a traditional medicine in Egypt and considered to be a rich source of
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the potentially bioactive compounds. C. dioscoridis is a richly branched hairy shrub grown commonly in the Nile and Mediterranean regions, desert oases, and tropical Africa (Boulos and El-Hadidi, 1984). Previous chemical analyses of C. dioscoridis and closely related species revealed the presence of typical acetylenes (Abou-Zid et al., 2008), di- and triterpenes (Ahmed, 1991; Galal et al., 1998; Lis et al., 2003; Yanfang et al., 2008), terpenoids (Maniruddin and Ahmed, 1990), flavones and saponins (Liu et al., 2001; Mukhtar et al., 2002; Tzakou et al., 2005), sulfated flavonoids (Ahmed et al., 1987) and coumarins (Boti et al., 2007). Eudesmanolides and two isocostic acids were isolated from C. dioscoridis (Dawidar and Metwally, 1985) and seven eudesmanes as well as the isocostic acids were later reported from the same plant (Mahmoud, 1997) and clerodanes (Bohlmann and Wegner, 1982; Jolad et al., 1988; Yang et al., 1989). A unusual sesquiterpene mixture of 2E, 11E-13hydroxy-10-sesquigeranic and 2Z, 11E-13-hydroxysesquigeranic acids previously isolated as methyl esters from Conyza hypoleuca A. Rich (Zdero et al., 1991). Oat growth (fresh and dry weights of above and underground parts) was inhibited from phytotoxic activity of upper leaves and inflorescence tissues of C. albida in pot studies (Economou et al., 2002). The objective of the present work was to identify chemicals from crude extracts of these plants that are responsible for phytotoxic effects against several economically important weeds such as C. arvensis and several weeds including Portulaca oleracea (L.), Phalaris paradoxa (L.), Corchorus olitorius (L.) and Echinochloa crus-galli (L.) Beauv. Dose– response relationships between phytotoxin concentrations and growth parameters of the weed species were evaluated. Purified active components were identified by spectroscopic methods.
http://dx.doi.org/10.1016/j.sajb.2015.07.006 0254-6299/© 2015 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006
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M.A. Balah / South African Journal of Botany xxx (2015) xxx–xxx
2. Materials and methods C. dioscoridis leaves were collected from Swia Oasis, 560 km from Cairo and preserved at the Desert Research Center, Cairo, Egypt. Seeds of onion (Allium cepa L.), alfalfa (Medicago sativa L.) and wheat (Triticum aestivum L.) were obtained from Agriculture Research Center, Egypt. Bindweeds (C. arvensis), purslane (P. oleracea) and little seed (P. paradoxa), molokhia (C. olitorius) and barnyard grass (E. crus-galli) seeds were collected from citrus and olive field North Sinai, Egypt. Plant specimen was identified by plant taxonomist (Tâckholm, 1974). 3. Aqueous extract preparation Ten grams of C. dioscoridis air dried plant parts was extracted with 100 ml double distilled deionized water using a rotary shaker for 5 h at 25 °C. The mixture was filtered through two layers of cheese cloth to remove debris, and centrifuged to precipitate small particles for 10 min at 3500 rpm and finally through whatman #4 paper. The filtrate was considered as 100 g dry wt./liter solution, and diluted to different concentration 0, 2, 4, 6, 8, 10 (g dry wt.100 ml−1) using distilled water.
(0.3% v/v) for 1–2 min and washed four times with sterile doubledistilled water, then ten seeds were placed on two filter papers Whatman #1 in a sterilized 9-cm petri-dish. Aliquot (10 ml) from each concentration was used for treatments. Sterile water was used as negative controls. C. arvensis seeds were exposed to scarification before the use to overcome its dormancy. Petri dishes were sealed with parafilm and incubated in the dark at 25 °C. Germination percentage (G %) was recorded after 7 days of incubation, the experiment was terminated then stem and root lengths of weed seedlings were measured and have been repeated independently at least twice. 5.1.2. Organic extracts Fifty milligrams of chloroform and ethyl acetate from each crude extract, dissolved in 10 ml of ethanol and diluted with water to a concentration series of 200, 400, 800, 1000 and 1200 μg ml−1 was tested in a similar way in the phytotoxicity test against C. arvensis, P. oleracea, C. olitorius and E. crus-galli. Seeds germination (G %), the shoot length and root lengths of plant seedlings were recorded after 7 days of incubation. 5.2. Bioassay by seedling
4. Organic extracts Air dried plant parts were ground and 300 g were extracted by soaking in 1500 ml of ethanol/water (2:1) for 14 h followed by shaking for 5 h. The filtrate was heated to 40 °C to evaporate ethanol solvent whereas, water solution extracted successively with petroleum ether, chloroform and ethyl acetate with partitioning three times to equal volume. Chloroform and ethyl acetate extracts were added to the top of the column; a glass column (30-cm long and 2 cm radius) was filled with 35-g silica gel for column chromatography (Merck) 60 mesh in methanol. Five grams of anhydrous sodium sulfate was put in the upper layer and eluted in a successive system with increasing polarity using 90 ml each of hexane, hexane: chloroform (1:1), chloroform, chloroform: ethyl acetate (1:1), ethyl acetate and methanol. Twelve fractions with 45 ml volume were collected from each solvent system, evaporated to dryness and re-dissolved in 5 ml ethanol 70% for the use in bioassays. Control treatment was treated with the same volume of ethanol 70% without extracts. Selected active fractions were further purified using TLC and a HPLC system equipped with an auto sampler and a photodiode array detector (Dionex). Samples were run on an analytical C18 column (5 μm, 4.6–150 mm) using gradient elution. Mobile phase consisted of 0.1% (v/v) acetic acid in water (Soln. A)–MeOH (Soln. B) using the following linear gradient: 10% to 90% Soln. B over 60 min. The flow rate of the mobile phase was 0.7 ml min−1 and the injection volume was 20 μl. 4.1. Chemical identification and structure elucidation Chemical structures of isolated compounds were deduced using 1H and 13C NMR spectra data obtained from Varian instrument, operating at 400 MHz. The spectra were run in CD3OD; chemical shifts (d) are given in ppm and the coupling constant (J) in hertz (Hz), using TMS as an internal standard. Chromatographically, pure materials were dissolved in pure methanol and subjected to ultraviolet spectrophotometric investigation using UV–VIS spectrophotometers thermo (Nicolet evolution 300). ESIMS was measured with a Thermo Finnigan LCQ instrument and a Fision VG Autospec apparatus. 5. Phytotoxicity bioassays 5.1. Bioassay by seeds 5.1.1. Water extracts Seeds of weeds (C. arvensis, P. oleracea and P. paradoxa) and crops (T. aestivum, A. cepa and M. sativa) were surface sterilized using NaOCl
Seeds of C. arvensis were surface sterilized using NaOCl (0.3% v/v) for 10–12 min and washed four times in sterile double distilled water. C. arvensis seeds were exposed to scarification before the use to overcome its dormancy. Seeds were placed on static Murashige and Skoog (MS) basal media and allowed to germinate for 7 days until roots and shoots emerged. Seven day old seedlings were transferred to tissue culture tube containing 5 ml of liquid MS media where the roots were submerged. Seedlings were treated with the concentrations of ethanolic extracts. Control treatment was treated with the same volume of ethanol without extracts. The pH distribution in a liquid media with and without extracts ranged from pH 5.5 to pH 6.0. Plant cultures were maintained at room temperature and shake every day for 10 min with an orbital platform shaker set at 90 rpm at 25 °C. Total biomass of each seedling was recorded 10 days after treatment. 5.3. Statistical analysis All experiments were designed in a randomized complete block design with four replicates and have been repeated independently at twice. The effective dose (ED50 values) (that provided 50% reduction in plant parameter) for each growth parameter was calculated by plotting concentration on a log scale (X) and the response (Y) on probit scale mathematically transformed, the data appear linear and sign the point in a semi-log graph paper. Data were statistically analyzed by ANOVA, according to Snedecor and Cochran (1990) and treatment means were compared by Duncan and LSD test at 5% level of probability. 6. Results and discussion 6.1. Aqueous leachate as a primary test guided for C. dioscoridis herbicidal activity against some weeds and crops Germination and seedling length of the tested species inhibited under different concentrations of C. dioscoridis aqueous extracts are presented in (Table 1). The highest applied concentration of 8 and 10 g DW 100 ml− 1 has strongest suppressive effect on seeds germination of C. arvensis, P. oleracea and P. paradoxa. Minimum inhibition concentration were recorded for 2 g DW 100 ml−1 which significantly reduced the root (42.3%) and shoot length (66.7%) and germination (24.0%) of C. arvensis (Table 1). The ED50 values of aqueous extracts were 2.0, 2.4 and 1.6 g DW 100 ml− 1 for shoot length, 2.25, 1.6 and 0.6 g DW 100 ml− 1 for root length, and 2.3, 2.4 and 1.6 g DW 100 ml− 1 for germination in C. arvensis, P. oleracea and P. paradoxa (Table 3). Extraction of C. dioscoridis by aqueous extracts showed inhibitory effects on a
Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006
M.A. Balah / South African Journal of Botany xxx (2015) xxx–xxx
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Table 1 Effect of Conyza dioscoridis aqueous extracts on some weed growth parameters. Concentration (g DW 100 ml−1)
Weeds species
0
1
2
4
6
8
10
LSD (0.05)
Germination % C. arvensis P. oleracea P. paradoxa
83.3 (0.00) 96.7 (0.00) 86.7 (0.00)
73.3 (12.0) 90.0 (6.92) 43.3 (50.05)
63.3 (24.0) 80.0 (17.3) 20.0 (76.9)
43.3 (48.0) 56.7 (41.4) 0.00 (100)
16.7 (79.9) 10.0 (89.6) 0.0 (100)
0.00 (100) 0.00 (100) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
17.03 15.80 13.90
Shoot length (cm) C. arvensis P. oleracea P. paradoxa
7.20 (0.00) 2.60 (0.00) 4.00 (0.00)
4.10 (43.1) 1.90 (26.9) 1.80 (55.0)
2.40 (66.7) 1.70 (34.6) 0.40 (90.0)
2.20 (69.4) 1.00 (61.5) 0.00 (100)
1.50 (79.2) 0.50 (80.7) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
1.63 0.82 2.07
Root length (cm) C. arvensis P. oleracea P. paradoxa
2.60 (0.00) 1.40 (0.00) 1.80 (0.00)
1.30 (50.0) 0.70 (50.0) 0.20 (88.9)
1.50 (42.3) 0.50 (64.3) 0.10 (94.4)
1.20 (53.8) 0.20 (85.7) 0.00 (100)
0.40 (84.6) 0.10 (92.8) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
0.68 0.63 0.45
Values between brackets are inhibition and activation (–) percents.
Economou and Nektarios (2003). Growth inhibition of Conyza albida extracts was found to be on Poa pratensis, Lolium perenne, Festuca arundinacea, Pennisetum clandestinum and Dichondra repens. The higher root growth inhibition might be due to permeability of allelopathic substances to root than shoot tissue (Nishida et al., 2005) and due to the direct contact between the root and phytotoxic compounds present in extract which might inhibit cell division of meristimatic tissue in the growing root tip (Rietjens and Alink, 2003).
wide range of weed plant species, including dicotyledons (C. arvensis and P. oleracea) and the monocotyledons plants (P. paradoxa) at the same concentration. The root and shoot growth of test plant species were more suppressed than the seed germination. The tested crops ED50 of T. aestivum, A. cepa and M. sativa values were 6.6, 4.4 and 6.7 g DW 100 ml− 1 for shoot length; 6.0, 3.0 and 4.9 g DW 100 ml−1 for root length, and 6.8, 4.4 and 6.8 g DW 100 ml−1 for germination (Table 3). The observed inhibition concentration was 6.0 g DW 100 ml−1 which significantly reduced the root length 56.06%, shoot length 57.6% and germination 83.5% of T. aestivum. Finally, the highest concentration of 10 g DW 100 ml−1, aqueous extracts decreased M. sativa germination significantly by 99.13%, root length by 95.05% and shoot length by 96.55% compared to the control (Table 2). Inhibitory activity was dependent on the aqueous extract concentrations and the higher extract concentration had the stronger inhibitory activity. Wandscheer and Pastorini (2008) analyzing the allelopathic effect of leaves and roots of Raphanus raphanistrum L. on seedlings of lettuce and tomato, found that the extracts were more active at higher concentrations. The inhibition effect was found to increase with increasing concentrations of different aqueous extracts (Sisodia and Siddiqui, 2009; Gulzar and Siddiqui, 2014). The effectiveness of the C. dioscoridis allelochemicals was much higher on the root growth than shoot growth of all tested plants. Comparing the extract concentrations required for 50% inhibition, germination and root and shoot growth of weeds were the most sensitive to C. dioscoridis allelochemicals than crop parameters. These results suggest that C. dioscoridis aqueous extracts may contain feasible growth inhibitory substances and may possess to further work that will be needed to identify allelopathic constituents. These results were supported by
7. Dose response curve of chloroform and ethyl acetate extracts against weed seedlings Successive use of solvent partitioning to ethanol 95% extracts by petroleum ether, chloroform and ethyl acetate leads to two active extracts. Growth inhibitory test of chloroform and ethyl acetate extracts tested on seedling development of C. arvensis and other economic weeds revealed that both extracts remarkably inhibited weeds total biomass as compared with controls. The ED50 values of both chloroform and ethyl acetate extracts were 430 μg ml− 1 and 550 μg ml−1, for C. arvensis, respectively (Fig. 1). The ED50 values of chloroform extracts were 445 μg ml−1 (P. oleracea), 925 μg ml− 1 (C. olitorius) and 1020 μg ml−1 (E. crus galli) (Fig. 2). A difference was found between the effect of chloroform and ethyl acetate extracts in depressing weed total biomass. However, the inhibitory effect of chloroform was clearly pronounced with C. arvensis weeds than ethyl acetate which was entirely affected. Comparing ED50 values reveals that total biomass of C. arvensis were the most sensitive to the C. dioscoridis extracts than C. olitorius and E. crus-galli. The inhibitory effect might be occurred through a variety of mechanisms like reduced mitotic activity in roots
Table 2 Effect of Conyza dioscoridis aqueous extracts on some crops growth parameters. Crops
Concentration (g DW 100 ml−1) 0
1
2
4
6
8
10
LSD (0.05)
83.3 (0.00) 80.0 (0.00) 96.6 (0.00)
83.3 (0.00) 73.3 (8.37) 90.0 (6.83)
70.0 (15.9) 46.6 (41.7) 73.3 (24.2)
60.0 (27.9) 10.0 (87.5) 66.6 (31.1)
36.6 (56.06) 0.00 (100) 46.6 (51.7)
0.00 (100) 0.00 (100) 3.30 (96.6)
0.00 (100) 0.00 (100) 0.00 (100)
12.04 14.31 16.52
Shoot length (cm) T. aestivum 13.70 (0.00) A. cepa 1.80 (0.00) M. sativa 3.40 (0.00)
12.00 (12.4) 1.40 (22.2) 3.50 (−2.94)
12.80 (6.57) 0.90 (50.0) 3.00 (11.7)
7.80 (43.0) 0.30 (83.33) 2.20 (35.3)
5.80 (57.6) 0.00 (100) 1.50 (55.8)
0.00 (100) 0.00 (100) 0.20 (94.1)
0.00 (100) 0.00 (100) 0.00 (100)
4.32 0.38 0.45
Root length (cm) T. aestivum A. cepa M. sativa
12.00 (−23.7) 0.40 (55.50) 3.20 (15.8)
9.70 (0.00) 0.30 (66.67) 1.80 (52.6)
3.20 (67.0) 0.10 (88.87) 1.20 (68.4)
1.60 (83.5) 0.00 (100.0) 0.80 (78.9)
0.00 (100) 0.00 (100) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
1.69 0.32 0.76
Germination % T. aestivum A. cepa M. sativa
9.70 (0.00) 0.90 (0.00) 3.80 (0.00)
Values between brackets are inhibition and activation (–) percents.
Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006
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M.A. Balah / South African Journal of Botany xxx (2015) xxx–xxx
Table 3 Quantitative bioassay of Conyza dioscoridis aqueous extracts on plants growth parameters. ED50 (g DW 100 ml−1)
Plant species
C. arvensis P. oleracea P. paradoxa T. aestivum A. cepa M. sativa
Root length
Shoot length
Germination
2.25 1.60 0.60 6.00 3.00 4.90
2.00 2.40 1.60 6.60 4.40 6.70
2.30 2.40 1.60 6.80 4.40 6.80
and shoots, reduced rate of ion uptake, inhibition of photosynthesis, respiration and enzyme action (Rice, 1974; Einhellig, 1995; Einhellig, et al., 2004). 8. Fractionation effect of chloroform and ethyl acetate bioassay on C. arvensis seedlings Chromatographic analysis of chloroform extract of C. dioscoridis on the column chromatography yielded six active fractions which eluted by hexane:chloroform (F4, F5 and F6) and chloroform:ethyl acetate (F7, F8 and F9). Fractions F4, F5, and F6 were found to be phytotoxic on the bioassay at 20 μg ml− 1 concentration, these fractions significantly reduced C. arvensis total biomass fresh weight by 69.32%,71.80% and 91.19%, respectively, also F7, F8, and F9 showed 92.19%, 93.79% and 69.50% reduction, than the controls respectively. Meanwhile, chromatographic analysis of ethyl acetate extracts yielded four active fractions (F4, F5, F6) and (F7) detected from bioassay guided fractionation which eluted by (hexane: chloroform and chloroform: ethyl acetate), respectively. These fractions at 20 μg ml−1 concentration significantly reduced C. arvensis total biomass fresh weight by 88.35%, 89.96%,77.42% and 79.39%, respectively, relative to the controls (Fig. 3). 9. Phytotoxicity bioassays of organic extracts and purified compounds Chloroform fractions F6, F7, and F8 rechromatographed by preparative TLC eluted with hexane-ethyl acetate (3:2 and 1:1, v/v) subsequently purified by HPLC to yield seven active compounds showed potent by causing mortality 10 days after application though decreasing total biomass fresh weight at 20 μg ml− 1. Bioassay guided isolation showed three sesquiterpene phytotoxic compounds that caused the reductions in C. arvensis total biomass fresh weight by 15-hydroxy isocostic acid plus isocostic acid 4-carboxaldehyde (86.81%), methyl 15-oxoeudesome-4, 11(13)-diene 12-oate (86.28%) and 1α, 9α-dihydroxy-
α-cyclocostunolide (76.19%), compared with the control. Also, four natural products (flavonoids) caused the reductions in C. arvensis total biomass fresh weight by: isorhamnetin 3-sulfate (80.61%), isorhamnetin 3O-rutinoside (80.39%), rhamnetin (76.25%) and epicatechin (70.58%) when compared to its control (Fig. 4). The primary herbicidal isolate from C. dioscoridis chloroform extracts was identified as an approximately 3:2 mixture of 15-hydroxyisocostic acid and the corresponding aldehyde acid by high resolution LCMS and extensive 2D NMR studies in comparison with the literature (Dawidar and Metwally, 1985; Mahmoud, 1997). 15-hydroxyisocostic acid: C15H22O3 (M+ − H 249.1494); 13C NMR: 39.7 (C1), 19.0 (C2), 27.2 (C3), 129.4 (C4), 140.3 (C5), 32.0 (C6), 41.1 (C7), 29.8 (C8), 41.2 (C9), 34.5 (C10), 143.7 (C11), 170.8 (C12), 124.7 (C13), 25.0 (C14), 63.2 (C15). H-NMR: 2.17, 2.10 (m, H-3),1.78 (dd, H-6, J = 14 Hz), 2.71 (d, H-6−, J = 14 Hz), 2.4 (d, H-7, J = 13 Hz), 6.0, 5.6 (s, H-13),1.0 (s, H-14), 4.09, 4.02 (H-15, J = 11.5 Hz), 3.7 (s, OMe), isocostic acid 4-carboxaldehyde: C15 H20O 3 (M + − H 247.1342); 13 C NMR: 17.6 (C2), 23.8 (C3), 25.3 (C14), 27.0 (C8), 30.2 (C6), 36.7 (C10), 39.7 (C7), 39.7 (C1), 41.6 (C9),125.4 (C13), 133.1 (C4), 140.2 (C11), 163.8 (C5), 170.8 (C12), 191.4 (C15). 1H-NMR: 1.05 (s, H-14), 1.46 (dddd, H-8 J = 12 Hz),1.53 (dd, H-6− J = 13 Hz),1.63 (d, H-7 J = 13 Hz), 1.79 (ddd, H-15), 1.95 (m, H-2−),1.96 (ddd, H-6 J = 12 Hz), 2.08 (m, H-2), 5.47 (s, H-3), 5.59 (s, H-13), 6.22 (s, H-13), 9.5 (s, H-4). UV spectral data of the compound with methanol exhibited a major band at λmax 237, and shoulders at λmax 260.5 and 279. The compound (2) was identified as methyl 15-oxo-eudesome-4, 11(13)-diene 12-oate which isolated from chloroform extract, UV spectral data of the compound with methanol exhibited a major band at λmax 232, and a shoulder at λmax 231, C16H22O3 with m/z 261.15 (M + 1), 231.2,181.3, 1H-NMR: 1.21 (s, H-14), 2.06 (m, H-7), 2.27 (m, H-3), 6.24, 5.63 (s, H-13), 10.17 (s, H-15), 3.77 (s, O Me). 13C-NMR: 18.64 (C-2), 20.89 (C-8), 20.21 (C-12), 20.66 (C-14), 22.07 (C-13), 28.26 (C-11), 30.56 (C-3), 34.27 (C-10), 42.60 (C-4), 44.23 (C-1), 44.51 (C-9), 50.29 (C-5), 51.18 (C-7), 74.45 (C-6),175.92 (C-15), OMe (51.64) (Dawidar and Metwally, 1985). The compound (3) was identified as 1α,9α-dihydroxy-αcyclocostunolide using UV spectral data of the compound with methanol exhibited a major band at λmax 227.5, and shoulder at λmax 225.8, molecular weight 232 and molecular formula C15H18O3 with m/z: 233.12 [M+ + 1], 187.3,151.2, 1H-NMR (CD3OD): 0.82 (s, H-14), 1.99 (s, H-15), 3.00 (d, H-5, J = 11 Hz), 3.28 (d, H-7, J = 11 Hz), 3.9 (d, H-1, J = 4 Hz), 3.97 (t, H-9, J = 2.5 Hz), 4.05 (H-6, J = 11 Hz), 5.32 (d, H-13), 5.52 (s, H-3), 6.07 (d, H-13, J = 3.5 Hz),13C-NMR: 16.11 (C-14),17.35 (C-15), 26.17 (C-8), 27.97 (C-2), 39.41 (C-9), 40.93 (C-3), 50.34, 81.89 (C-6), 119.73 (C-13), 127.00 (C-1), 127.18 (C-5), 136.97 (C-10), 139.99 (C-4), 141.53 (C-11), 170.51 (C-12) (Dawidar and Metwally, 1985).
Chloroform
Total biomass (g)
Ethyl acetate
Concentration (µg/ml) Fig. 1. Allelopathic potential of C. dioscorides extracts on C. arvensis total biomass fresh weight. The error bars means the standard deviations between replicates.
Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006
M.A. Balah / South African Journal of Botany xxx (2015) xxx–xxx
C. olitorius
E. crus galli
Total biomass (g)
P. oleracea
5
Concentration (µg/ml) Fig. 2. Allelopathic potential of chloroform extracts on the tested weeds total biomass fresh weight. The error bars means the standard deviations between replicates.
The second herbicidal isolate chemical group from C. dioscoridis was identified by comparing UV spectrum MS, H NMR and C NMR spectra with authentic samples and published data as: rhamanetin compound (4) with UV spectral data of the compound with methanol exhibited a major band at λmax 320.5, and with molecular weight 316, with m/z: 315 [M+ − 1], 261.3, 203.2, 147.2 and 1H-NMR (CD3OD) δ ppm: 3.8 (3H, s, 7-O CH3), 6.20 (1H, d, 6-H), 6.48 (1H, d, 8-H), 6.94 (1H, d, 5′-H), 7.6 (1H, m, 6′-H), 7.75 (1H, d, 2′-H), 9.40 (1H, s, OH), 9.73 (1H, s, OH), 10.76 (1H, s, OH), 12.06 (1H, s, OH). 13C-NMR δ ppm: 93.5 (C-8), 98.0 (C-6), 102.7 (C-10), 111.6 (C-2′), 115.3 (C-5′), 121.5 (C-6′), 121.8 (C-1′), 135.6 (C-3), 146. 3 (C-3′), 147.2 (C-4′), 148.7 (C-2), 160.3 (C-9), 156.0 (C-5), 163.6 (C-7), 175.72 (C-4), 55.4 (7-OCH3) (Ahmed et al., 1987). Isorhamnetin 3-sulfate compound (5) with UV spectral data of the compound with methanol exhibited a major band at λmax 320, and shoulder at λmax 340 (30) with molecular weight 394.3 with m/z: 395.3, 349.3, 317 (100%), 277, 229. 1H-NMR: (CD3OD): 3.83 s (3H, 3′OCH3), 6.17 d (1H, H-6), 6.43 d (1H, H-8), 6.86 d (1H, H-5′), 7.61 dd (1H, H-6′), 8.04 d (1H, H-2′), 9.70 (4′-OH), 10.80 (7-OH), 12.85 (3-OSO3H) and 12.84 (5-OH). 13C-NMR: C-8 (93.4), C-3 (132.5), C-6 (98.3), C-10 (104.1), C-5′ (115.0), C-6′ (121.9), C-2′ (113.6), C-1′ (121.4), (146.9), C-4′ (149.3), C-2 (155.9), C-9 (156.0), C-5 (161.3), C-7 (163.8), C-4 (177.6), C-3′ OCH3 (55.6) (Ahmed et al., 1987). Isorhamnetin 3-O-rutinoside compound (6) with UV spectral data of the compound with methanol exhibited a major band at λmax 319.8, and shoulder at λmax 340, with molecular weight 624 and m/z: 625 [M + H]+, 523.4, 455.5, 345.1, 273.3, 233.3, 187.3, 151.2 and 1H-NMR
(CD3OD): δ 1.2 (1H, d, J = 6.0 Hz, H-6′′′), 3.86 (1H, s, OCH3), sugar moiety: 5.5 (1H, d, J = 8 Hz, H-1′′′ rhamnose), 5.31 (1H, d, J = 7.4 Hz, H-1′′′ glucose), 6. 3 (1H, s, H-6), 6.3 (1H, s, H-8), 6.83 (1H, d, J = 8.0 Hz, H-5′), 7.74 (1H, d, J = 8.0 Hz, H-6′), 7.84 (1H, s, H-2′); 13C-NMR (CD3OD): δ 17.7 (C-6′′′), 56.6 (C-3′-OCH3), 67.6 (C-6′′), 67.6 (C-5′′′), 70.6 (C-4′′), 70.7 (C-2′′′), 70.9 (C-3′′′), 72.8 (C-4′′′), 74.9 (C-2′′), 76.4 (C-5′′), 77.0 (C-3′′), 93.6 (C-8), 98.7 (C-6), 101.2 (C-1′′′), 103.0 (C-1′′), 104.4 (C-10), 113.3 (C-2′), 114.8 (C-5′), 121.6 (C-6′), 122.5 (C-1′), 134.0 (C-3), 147.1 (C-3′), 149.7 (C-4′), 157.3 (C-2), 157.6 (C-9), 161.8 (C-5), 165.0 (C-7) (Ahmed et al., 1987). Epicatechin compound (7) with UV spectral data of the compound with methanol exhibited a major band at λmax 231.5, and shoulder at λ max 325, with molecular weight 290, with m/z: 289 [M − H+], 231.3, 183.3 1H-NMR (CD3OD): 2.41 (dd, J = 2.8, H-4), 2.56 (dd, J = 4.5 Hz, H-4), 3.8 (s, H-3), 4.4 (s, H-2), 5.5 (d, J = 2.08 Hz, H-8), 5.6 (d, J = 2.08 Hz, H-6), 6.4 (d, J = 8.12 Hz, H-5′), 6.48 (d, J = 9.77 Hz, H-6′), 6.6 (s, H-2′), 13C-NMR: 29.1 (C-4), 67.4 (C-3), 79.7 (C-2), 95.6 (C-8), 96.5 (C-6), 100.1 (C-10), 115.4 (C-2′), 116.0 (C-5′), 119.4 (C6′),132.2 (C-1′), 145.6 (C-3′), 145.7 (C-4′), 157.2 (C-9), 157.4 (C5),157.7 (C-7), from the above result it could be identified as epicatechin, these data were compared with UV spectrum MS, 1H-NMR and 13C-NMR spectra with authentic sample and published data. As a result, the C. dioscoridis crude extract have a broad spectrum activity since they are effective against both mono and di-cotyledon, this is probably due to the plant which is widely used as antifungal and antibacterial activity (Fatope et al., 2004; Katuura et al., 2007), corresponding to bioassay, the toxicity of aqueous and organic extracts increased
Chloroform
Total biomass (g)
Ethyl acetate
Fractions (20 µg/ml) Fig. 3. Bioassay of column chromatography fractions on C. arvensis total biomass fresh weights. The error bars means the standard deviations between replicates.
Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006
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M.A. Balah / South African Journal of Botany xxx (2015) xxx–xxx
A= (Isorhamnetin 3 sulfate),
Total fresh weight reduction %
B=(Isorhamnetin 3-O- rutinoside), C=(Rhamnetin) , D=(Epicatechin), E=(15-Hydroxyisocostic cid + Isocostic acid 4- carboxaldehyde), F= (Isocostic acid 4carboxaldehyde), G= (1α,9α-dihydroxy-αcyclocostunolide). Concentration (50 µg/ml) Fig. 4. Bioassay of allelopathic constituents on C. arvensis total biomass fresh weights. The same letters within columns means not significant differences between treatments at P = 0.05. The error bars means the standard deviations between replicates.
with increasing concentration. Our results showed that chloroform extracts have a remarkable herbicidal activity against field bindweeds total biomass fresh weight. The activity of C. dioscoridis crude extracts due to several phytotoxic compounds responsible for their effect on weeds growth parameters and germination. However, no clear post emergence activity was recorded against C. arvensis (data not shown) 5 leaves stage. Few studies have mentioned allelopathic activity and identified compounds isolated from the genus Conyza. Economou et al. (2002) reported allelopathic activity of fleabane (Conyza albida Willd. ex Spreng) on oat (Avena sativa L.) growth fresh and dry weight. Previously, many sesquiterpenes have been isolated from Conyza sp. (Dawidar and Metwally, 1985; Ahmed et al., 1987; Mahmoud, 1997; Fatope et al., 2004). The present work adds the evidence supporting potential herbicidal activity of Conyza spp sesquiterpene compounds, which has been identified as a mixture of the eudesmane derivatives 15-hydroxyisocostic acid from C. dioscoridis. The chemical structure of the isolated compounds was established by extensive analysis of 1 H-NMR, 13CNMR and mass spectrometry data and confirmed by comparison of the spectroscopic data with those reported in the literature (Dawidar and Metwally, 1985; Ahmed et al., 1987). The highest phytotoxic activity appeared in the sesquiterpene mixture of the eudesmane derivatives 15-hydroxyisocostic acid, isolated from C. dioscoridis followed with methyl 15-oxo-eudesome-4, 11(13)-diene 12-oate and the lower toxicity was observed in the flavonoid compounds. The phytotoxic effect of the previous sesqueterpene compounds was reported for the first time by the authors in this pepper. The allelopathic potentials of the isolated compounds were previously recorded by Al-Watban and Salama (2012) (isorhamnetin 3-O-rutinoside), Weidenhamer and Romeo (2004) (rhamanetin), Weir and Vivanco (2008) (epicatechin) and others. However, the herbicidal activity of isorhamnetin 3-sulfate and isorhamnetin 3-O-rutinoside was not recorded formerly. To our knowledge, this is the first information on the herbicidal activity of extracts and sesquiterpene constituents of C. dioscoridis against field bindweeds (C. arvensis). Finally, one can conclude that weeds control using these compounds might be feasible and further work is needed to recommend this as an herbicide substitute or structurally leading to new synthetic herbicides.
Acknowledgment The author would like to thank the natural product and phytochemistry staff of the National Research Center and Ain Shams University
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Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006
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Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control Mohamed Abdel aziz Balah Plant Protection Department, Ecology and Dry Land Agriculture Division, Desert Research Center, 1-Mathaf El-Mataria St, EL-Mataria, Cairo, BOX: 11753, Egypt
a r t i c l e
i n f o
Article history: Received 28 June 2014 Received in revised form 28 February 2015 Accepted 29 July 2015 Available online xxxx Keywords: Conyza dioscoridis Pre-emergence Phytotoxicity activity Flavonoids Sesquiterpene Convolvulus arvensis
a b s t r a c t The potential herbicidal activity of certain chemicals isolated from Conyza dioscoridis (L.) desf. leaves was bioassayed on seed germination and growth parameters of Convolvulus arvensis (L.) and several weeds including Portulaca oleracea (L.), Phalaris paradoxa (L.), Corchorus olitorius (L.) and Echinochloa crus-galli (L.) Beauv. Bioassay guided isolation showed seven phytotoxic natural compounds including three sesquiterpene compounds. An inseparable mixture of the eudesmane derivatives (15-hydroxyisocostic acid and the corresponding aldehydic acid) was isolated from the leaves of C. dioscoridis. Relative to controls, this mixture reduced total biomass of C. arvensis by (86.81%) and methyl 15-oxo-eudesome-4, 11(13)-diene 12-oate as well as 1α, 9α-dihydroxy-αcyclocostunolide showed 86.28% and 76.19% reductions respectively, compared to its control. Also, four flavonoids caused the reductions in C. arvensis total biomass fresh weight by: isorhamnetin 3-sulfate (80.61%), isorhamnetin 3-O-rutinoside (80.39%), rhamanetin (76.25%) and epicatechin (70.58%) compared to its control. The activity of these chemicals may be nominating them to develop as natural herbicides in the future. © 2015 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Controlling bindweeds (Convolvulus arvensis L.) in agricultural fields is still extremely difficult, despite many efforts to minimize their impact using cultural, chemical and biological methods. Field bindweed reproduces by buds in their secondary roots that can develop into new plants and seeds with tough seed coats that can remain viable in the soil for years (Wiese and Phillips, 1976). The use of commercial herbicides is considered the most effective method for controlling weeds; however herbicides can be poisonous to mammals and many other forms of wild life, result in ground water contamination, and have other adverse impacts on the environment. This has led to intensified efforts in recent years to identify environmentally benign forms of chemical weed control particularly through increasing the allelopathic potential of promising plants (Mendes and Vermelho, 2013). Thus, much interest has been centered on the possible incorporation of natural products phytotoxin as safe, selective, and cost-effective herbicides. Natural products may offer novel molecular target sites and mechanisms of action for new herbicides (Duke et al., 2000). The environmental half-life of many natural compounds is shorter, and they are generally less toxic to the environment than many synthetic herbicides (Duke et al., 2000), thus a large number of natural products are being tested as possible bio herbicides. Conyza dioscoridis was chosen for investigation because it is widely used as a traditional medicine in Egypt and considered to be a rich source of
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the potentially bioactive compounds. C. dioscoridis is a richly branched hairy shrub grown commonly in the Nile and Mediterranean regions, desert oases, and tropical Africa (Boulos and El-Hadidi, 1984). Previous chemical analyses of C. dioscoridis and closely related species revealed the presence of typical acetylenes (Abou-Zid et al., 2008), di- and triterpenes (Ahmed, 1991; Galal et al., 1998; Lis et al., 2003; Yanfang et al., 2008), terpenoids (Maniruddin and Ahmed, 1990), flavones and saponins (Liu et al., 2001; Mukhtar et al., 2002; Tzakou et al., 2005), sulfated flavonoids (Ahmed et al., 1987) and coumarins (Boti et al., 2007). Eudesmanolides and two isocostic acids were isolated from C. dioscoridis (Dawidar and Metwally, 1985) and seven eudesmanes as well as the isocostic acids were later reported from the same plant (Mahmoud, 1997) and clerodanes (Bohlmann and Wegner, 1982; Jolad et al., 1988; Yang et al., 1989). A unusual sesquiterpene mixture of 2E, 11E-13hydroxy-10-sesquigeranic and 2Z, 11E-13-hydroxysesquigeranic acids previously isolated as methyl esters from Conyza hypoleuca A. Rich (Zdero et al., 1991). Oat growth (fresh and dry weights of above and underground parts) was inhibited from phytotoxic activity of upper leaves and inflorescence tissues of C. albida in pot studies (Economou et al., 2002). The objective of the present work was to identify chemicals from crude extracts of these plants that are responsible for phytotoxic effects against several economically important weeds such as C. arvensis and several weeds including Portulaca oleracea (L.), Phalaris paradoxa (L.), Corchorus olitorius (L.) and Echinochloa crus-galli (L.) Beauv. Dose– response relationships between phytotoxin concentrations and growth parameters of the weed species were evaluated. Purified active components were identified by spectroscopic methods.
http://dx.doi.org/10.1016/j.sajb.2015.07.006 0254-6299/© 2015 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006
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2. Materials and methods C. dioscoridis leaves were collected from Swia Oasis, 560 km from Cairo and preserved at the Desert Research Center, Cairo, Egypt. Seeds of onion (Allium cepa L.), alfalfa (Medicago sativa L.) and wheat (Triticum aestivum L.) were obtained from Agriculture Research Center, Egypt. Bindweeds (C. arvensis), purslane (P. oleracea) and little seed (P. paradoxa), molokhia (C. olitorius) and barnyard grass (E. crus-galli) seeds were collected from citrus and olive field North Sinai, Egypt. Plant specimen was identified by plant taxonomist (Tâckholm, 1974). 3. Aqueous extract preparation Ten grams of C. dioscoridis air dried plant parts was extracted with 100 ml double distilled deionized water using a rotary shaker for 5 h at 25 °C. The mixture was filtered through two layers of cheese cloth to remove debris, and centrifuged to precipitate small particles for 10 min at 3500 rpm and finally through whatman #4 paper. The filtrate was considered as 100 g dry wt./liter solution, and diluted to different concentration 0, 2, 4, 6, 8, 10 (g dry wt.100 ml−1) using distilled water.
(0.3% v/v) for 1–2 min and washed four times with sterile doubledistilled water, then ten seeds were placed on two filter papers Whatman #1 in a sterilized 9-cm petri-dish. Aliquot (10 ml) from each concentration was used for treatments. Sterile water was used as negative controls. C. arvensis seeds were exposed to scarification before the use to overcome its dormancy. Petri dishes were sealed with parafilm and incubated in the dark at 25 °C. Germination percentage (G %) was recorded after 7 days of incubation, the experiment was terminated then stem and root lengths of weed seedlings were measured and have been repeated independently at least twice. 5.1.2. Organic extracts Fifty milligrams of chloroform and ethyl acetate from each crude extract, dissolved in 10 ml of ethanol and diluted with water to a concentration series of 200, 400, 800, 1000 and 1200 μg ml−1 was tested in a similar way in the phytotoxicity test against C. arvensis, P. oleracea, C. olitorius and E. crus-galli. Seeds germination (G %), the shoot length and root lengths of plant seedlings were recorded after 7 days of incubation. 5.2. Bioassay by seedling
4. Organic extracts Air dried plant parts were ground and 300 g were extracted by soaking in 1500 ml of ethanol/water (2:1) for 14 h followed by shaking for 5 h. The filtrate was heated to 40 °C to evaporate ethanol solvent whereas, water solution extracted successively with petroleum ether, chloroform and ethyl acetate with partitioning three times to equal volume. Chloroform and ethyl acetate extracts were added to the top of the column; a glass column (30-cm long and 2 cm radius) was filled with 35-g silica gel for column chromatography (Merck) 60 mesh in methanol. Five grams of anhydrous sodium sulfate was put in the upper layer and eluted in a successive system with increasing polarity using 90 ml each of hexane, hexane: chloroform (1:1), chloroform, chloroform: ethyl acetate (1:1), ethyl acetate and methanol. Twelve fractions with 45 ml volume were collected from each solvent system, evaporated to dryness and re-dissolved in 5 ml ethanol 70% for the use in bioassays. Control treatment was treated with the same volume of ethanol 70% without extracts. Selected active fractions were further purified using TLC and a HPLC system equipped with an auto sampler and a photodiode array detector (Dionex). Samples were run on an analytical C18 column (5 μm, 4.6–150 mm) using gradient elution. Mobile phase consisted of 0.1% (v/v) acetic acid in water (Soln. A)–MeOH (Soln. B) using the following linear gradient: 10% to 90% Soln. B over 60 min. The flow rate of the mobile phase was 0.7 ml min−1 and the injection volume was 20 μl. 4.1. Chemical identification and structure elucidation Chemical structures of isolated compounds were deduced using 1H and 13C NMR spectra data obtained from Varian instrument, operating at 400 MHz. The spectra were run in CD3OD; chemical shifts (d) are given in ppm and the coupling constant (J) in hertz (Hz), using TMS as an internal standard. Chromatographically, pure materials were dissolved in pure methanol and subjected to ultraviolet spectrophotometric investigation using UV–VIS spectrophotometers thermo (Nicolet evolution 300). ESIMS was measured with a Thermo Finnigan LCQ instrument and a Fision VG Autospec apparatus. 5. Phytotoxicity bioassays 5.1. Bioassay by seeds 5.1.1. Water extracts Seeds of weeds (C. arvensis, P. oleracea and P. paradoxa) and crops (T. aestivum, A. cepa and M. sativa) were surface sterilized using NaOCl
Seeds of C. arvensis were surface sterilized using NaOCl (0.3% v/v) for 10–12 min and washed four times in sterile double distilled water. C. arvensis seeds were exposed to scarification before the use to overcome its dormancy. Seeds were placed on static Murashige and Skoog (MS) basal media and allowed to germinate for 7 days until roots and shoots emerged. Seven day old seedlings were transferred to tissue culture tube containing 5 ml of liquid MS media where the roots were submerged. Seedlings were treated with the concentrations of ethanolic extracts. Control treatment was treated with the same volume of ethanol without extracts. The pH distribution in a liquid media with and without extracts ranged from pH 5.5 to pH 6.0. Plant cultures were maintained at room temperature and shake every day for 10 min with an orbital platform shaker set at 90 rpm at 25 °C. Total biomass of each seedling was recorded 10 days after treatment. 5.3. Statistical analysis All experiments were designed in a randomized complete block design with four replicates and have been repeated independently at twice. The effective dose (ED50 values) (that provided 50% reduction in plant parameter) for each growth parameter was calculated by plotting concentration on a log scale (X) and the response (Y) on probit scale mathematically transformed, the data appear linear and sign the point in a semi-log graph paper. Data were statistically analyzed by ANOVA, according to Snedecor and Cochran (1990) and treatment means were compared by Duncan and LSD test at 5% level of probability. 6. Results and discussion 6.1. Aqueous leachate as a primary test guided for C. dioscoridis herbicidal activity against some weeds and crops Germination and seedling length of the tested species inhibited under different concentrations of C. dioscoridis aqueous extracts are presented in (Table 1). The highest applied concentration of 8 and 10 g DW 100 ml− 1 has strongest suppressive effect on seeds germination of C. arvensis, P. oleracea and P. paradoxa. Minimum inhibition concentration were recorded for 2 g DW 100 ml−1 which significantly reduced the root (42.3%) and shoot length (66.7%) and germination (24.0%) of C. arvensis (Table 1). The ED50 values of aqueous extracts were 2.0, 2.4 and 1.6 g DW 100 ml− 1 for shoot length, 2.25, 1.6 and 0.6 g DW 100 ml− 1 for root length, and 2.3, 2.4 and 1.6 g DW 100 ml− 1 for germination in C. arvensis, P. oleracea and P. paradoxa (Table 3). Extraction of C. dioscoridis by aqueous extracts showed inhibitory effects on a
Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006
M.A. Balah / South African Journal of Botany xxx (2015) xxx–xxx
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Table 1 Effect of Conyza dioscoridis aqueous extracts on some weed growth parameters. Concentration (g DW 100 ml−1)
Weeds species
0
1
2
4
6
8
10
LSD (0.05)
Germination % C. arvensis P. oleracea P. paradoxa
83.3 (0.00) 96.7 (0.00) 86.7 (0.00)
73.3 (12.0) 90.0 (6.92) 43.3 (50.05)
63.3 (24.0) 80.0 (17.3) 20.0 (76.9)
43.3 (48.0) 56.7 (41.4) 0.00 (100)
16.7 (79.9) 10.0 (89.6) 0.0 (100)
0.00 (100) 0.00 (100) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
17.03 15.80 13.90
Shoot length (cm) C. arvensis P. oleracea P. paradoxa
7.20 (0.00) 2.60 (0.00) 4.00 (0.00)
4.10 (43.1) 1.90 (26.9) 1.80 (55.0)
2.40 (66.7) 1.70 (34.6) 0.40 (90.0)
2.20 (69.4) 1.00 (61.5) 0.00 (100)
1.50 (79.2) 0.50 (80.7) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
1.63 0.82 2.07
Root length (cm) C. arvensis P. oleracea P. paradoxa
2.60 (0.00) 1.40 (0.00) 1.80 (0.00)
1.30 (50.0) 0.70 (50.0) 0.20 (88.9)
1.50 (42.3) 0.50 (64.3) 0.10 (94.4)
1.20 (53.8) 0.20 (85.7) 0.00 (100)
0.40 (84.6) 0.10 (92.8) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
0.68 0.63 0.45
Values between brackets are inhibition and activation (–) percents.
Economou and Nektarios (2003). Growth inhibition of Conyza albida extracts was found to be on Poa pratensis, Lolium perenne, Festuca arundinacea, Pennisetum clandestinum and Dichondra repens. The higher root growth inhibition might be due to permeability of allelopathic substances to root than shoot tissue (Nishida et al., 2005) and due to the direct contact between the root and phytotoxic compounds present in extract which might inhibit cell division of meristimatic tissue in the growing root tip (Rietjens and Alink, 2003).
wide range of weed plant species, including dicotyledons (C. arvensis and P. oleracea) and the monocotyledons plants (P. paradoxa) at the same concentration. The root and shoot growth of test plant species were more suppressed than the seed germination. The tested crops ED50 of T. aestivum, A. cepa and M. sativa values were 6.6, 4.4 and 6.7 g DW 100 ml− 1 for shoot length; 6.0, 3.0 and 4.9 g DW 100 ml−1 for root length, and 6.8, 4.4 and 6.8 g DW 100 ml−1 for germination (Table 3). The observed inhibition concentration was 6.0 g DW 100 ml−1 which significantly reduced the root length 56.06%, shoot length 57.6% and germination 83.5% of T. aestivum. Finally, the highest concentration of 10 g DW 100 ml−1, aqueous extracts decreased M. sativa germination significantly by 99.13%, root length by 95.05% and shoot length by 96.55% compared to the control (Table 2). Inhibitory activity was dependent on the aqueous extract concentrations and the higher extract concentration had the stronger inhibitory activity. Wandscheer and Pastorini (2008) analyzing the allelopathic effect of leaves and roots of Raphanus raphanistrum L. on seedlings of lettuce and tomato, found that the extracts were more active at higher concentrations. The inhibition effect was found to increase with increasing concentrations of different aqueous extracts (Sisodia and Siddiqui, 2009; Gulzar and Siddiqui, 2014). The effectiveness of the C. dioscoridis allelochemicals was much higher on the root growth than shoot growth of all tested plants. Comparing the extract concentrations required for 50% inhibition, germination and root and shoot growth of weeds were the most sensitive to C. dioscoridis allelochemicals than crop parameters. These results suggest that C. dioscoridis aqueous extracts may contain feasible growth inhibitory substances and may possess to further work that will be needed to identify allelopathic constituents. These results were supported by
7. Dose response curve of chloroform and ethyl acetate extracts against weed seedlings Successive use of solvent partitioning to ethanol 95% extracts by petroleum ether, chloroform and ethyl acetate leads to two active extracts. Growth inhibitory test of chloroform and ethyl acetate extracts tested on seedling development of C. arvensis and other economic weeds revealed that both extracts remarkably inhibited weeds total biomass as compared with controls. The ED50 values of both chloroform and ethyl acetate extracts were 430 μg ml− 1 and 550 μg ml−1, for C. arvensis, respectively (Fig. 1). The ED50 values of chloroform extracts were 445 μg ml−1 (P. oleracea), 925 μg ml− 1 (C. olitorius) and 1020 μg ml−1 (E. crus galli) (Fig. 2). A difference was found between the effect of chloroform and ethyl acetate extracts in depressing weed total biomass. However, the inhibitory effect of chloroform was clearly pronounced with C. arvensis weeds than ethyl acetate which was entirely affected. Comparing ED50 values reveals that total biomass of C. arvensis were the most sensitive to the C. dioscoridis extracts than C. olitorius and E. crus-galli. The inhibitory effect might be occurred through a variety of mechanisms like reduced mitotic activity in roots
Table 2 Effect of Conyza dioscoridis aqueous extracts on some crops growth parameters. Crops
Concentration (g DW 100 ml−1) 0
1
2
4
6
8
10
LSD (0.05)
83.3 (0.00) 80.0 (0.00) 96.6 (0.00)
83.3 (0.00) 73.3 (8.37) 90.0 (6.83)
70.0 (15.9) 46.6 (41.7) 73.3 (24.2)
60.0 (27.9) 10.0 (87.5) 66.6 (31.1)
36.6 (56.06) 0.00 (100) 46.6 (51.7)
0.00 (100) 0.00 (100) 3.30 (96.6)
0.00 (100) 0.00 (100) 0.00 (100)
12.04 14.31 16.52
Shoot length (cm) T. aestivum 13.70 (0.00) A. cepa 1.80 (0.00) M. sativa 3.40 (0.00)
12.00 (12.4) 1.40 (22.2) 3.50 (−2.94)
12.80 (6.57) 0.90 (50.0) 3.00 (11.7)
7.80 (43.0) 0.30 (83.33) 2.20 (35.3)
5.80 (57.6) 0.00 (100) 1.50 (55.8)
0.00 (100) 0.00 (100) 0.20 (94.1)
0.00 (100) 0.00 (100) 0.00 (100)
4.32 0.38 0.45
Root length (cm) T. aestivum A. cepa M. sativa
12.00 (−23.7) 0.40 (55.50) 3.20 (15.8)
9.70 (0.00) 0.30 (66.67) 1.80 (52.6)
3.20 (67.0) 0.10 (88.87) 1.20 (68.4)
1.60 (83.5) 0.00 (100.0) 0.80 (78.9)
0.00 (100) 0.00 (100) 0.00 (100)
0.00 (100) 0.00 (100) 0.00 (100)
1.69 0.32 0.76
Germination % T. aestivum A. cepa M. sativa
9.70 (0.00) 0.90 (0.00) 3.80 (0.00)
Values between brackets are inhibition and activation (–) percents.
Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006
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M.A. Balah / South African Journal of Botany xxx (2015) xxx–xxx
Table 3 Quantitative bioassay of Conyza dioscoridis aqueous extracts on plants growth parameters. ED50 (g DW 100 ml−1)
Plant species
C. arvensis P. oleracea P. paradoxa T. aestivum A. cepa M. sativa
Root length
Shoot length
Germination
2.25 1.60 0.60 6.00 3.00 4.90
2.00 2.40 1.60 6.60 4.40 6.70
2.30 2.40 1.60 6.80 4.40 6.80
and shoots, reduced rate of ion uptake, inhibition of photosynthesis, respiration and enzyme action (Rice, 1974; Einhellig, 1995; Einhellig, et al., 2004). 8. Fractionation effect of chloroform and ethyl acetate bioassay on C. arvensis seedlings Chromatographic analysis of chloroform extract of C. dioscoridis on the column chromatography yielded six active fractions which eluted by hexane:chloroform (F4, F5 and F6) and chloroform:ethyl acetate (F7, F8 and F9). Fractions F4, F5, and F6 were found to be phytotoxic on the bioassay at 20 μg ml− 1 concentration, these fractions significantly reduced C. arvensis total biomass fresh weight by 69.32%,71.80% and 91.19%, respectively, also F7, F8, and F9 showed 92.19%, 93.79% and 69.50% reduction, than the controls respectively. Meanwhile, chromatographic analysis of ethyl acetate extracts yielded four active fractions (F4, F5, F6) and (F7) detected from bioassay guided fractionation which eluted by (hexane: chloroform and chloroform: ethyl acetate), respectively. These fractions at 20 μg ml−1 concentration significantly reduced C. arvensis total biomass fresh weight by 88.35%, 89.96%,77.42% and 79.39%, respectively, relative to the controls (Fig. 3). 9. Phytotoxicity bioassays of organic extracts and purified compounds Chloroform fractions F6, F7, and F8 rechromatographed by preparative TLC eluted with hexane-ethyl acetate (3:2 and 1:1, v/v) subsequently purified by HPLC to yield seven active compounds showed potent by causing mortality 10 days after application though decreasing total biomass fresh weight at 20 μg ml− 1. Bioassay guided isolation showed three sesquiterpene phytotoxic compounds that caused the reductions in C. arvensis total biomass fresh weight by 15-hydroxy isocostic acid plus isocostic acid 4-carboxaldehyde (86.81%), methyl 15-oxoeudesome-4, 11(13)-diene 12-oate (86.28%) and 1α, 9α-dihydroxy-
α-cyclocostunolide (76.19%), compared with the control. Also, four natural products (flavonoids) caused the reductions in C. arvensis total biomass fresh weight by: isorhamnetin 3-sulfate (80.61%), isorhamnetin 3O-rutinoside (80.39%), rhamnetin (76.25%) and epicatechin (70.58%) when compared to its control (Fig. 4). The primary herbicidal isolate from C. dioscoridis chloroform extracts was identified as an approximately 3:2 mixture of 15-hydroxyisocostic acid and the corresponding aldehyde acid by high resolution LCMS and extensive 2D NMR studies in comparison with the literature (Dawidar and Metwally, 1985; Mahmoud, 1997). 15-hydroxyisocostic acid: C15H22O3 (M+ − H 249.1494); 13C NMR: 39.7 (C1), 19.0 (C2), 27.2 (C3), 129.4 (C4), 140.3 (C5), 32.0 (C6), 41.1 (C7), 29.8 (C8), 41.2 (C9), 34.5 (C10), 143.7 (C11), 170.8 (C12), 124.7 (C13), 25.0 (C14), 63.2 (C15). H-NMR: 2.17, 2.10 (m, H-3),1.78 (dd, H-6, J = 14 Hz), 2.71 (d, H-6−, J = 14 Hz), 2.4 (d, H-7, J = 13 Hz), 6.0, 5.6 (s, H-13),1.0 (s, H-14), 4.09, 4.02 (H-15, J = 11.5 Hz), 3.7 (s, OMe), isocostic acid 4-carboxaldehyde: C15 H20O 3 (M + − H 247.1342); 13 C NMR: 17.6 (C2), 23.8 (C3), 25.3 (C14), 27.0 (C8), 30.2 (C6), 36.7 (C10), 39.7 (C7), 39.7 (C1), 41.6 (C9),125.4 (C13), 133.1 (C4), 140.2 (C11), 163.8 (C5), 170.8 (C12), 191.4 (C15). 1H-NMR: 1.05 (s, H-14), 1.46 (dddd, H-8 J = 12 Hz),1.53 (dd, H-6− J = 13 Hz),1.63 (d, H-7 J = 13 Hz), 1.79 (ddd, H-15), 1.95 (m, H-2−),1.96 (ddd, H-6 J = 12 Hz), 2.08 (m, H-2), 5.47 (s, H-3), 5.59 (s, H-13), 6.22 (s, H-13), 9.5 (s, H-4). UV spectral data of the compound with methanol exhibited a major band at λmax 237, and shoulders at λmax 260.5 and 279. The compound (2) was identified as methyl 15-oxo-eudesome-4, 11(13)-diene 12-oate which isolated from chloroform extract, UV spectral data of the compound with methanol exhibited a major band at λmax 232, and a shoulder at λmax 231, C16H22O3 with m/z 261.15 (M + 1), 231.2,181.3, 1H-NMR: 1.21 (s, H-14), 2.06 (m, H-7), 2.27 (m, H-3), 6.24, 5.63 (s, H-13), 10.17 (s, H-15), 3.77 (s, O Me). 13C-NMR: 18.64 (C-2), 20.89 (C-8), 20.21 (C-12), 20.66 (C-14), 22.07 (C-13), 28.26 (C-11), 30.56 (C-3), 34.27 (C-10), 42.60 (C-4), 44.23 (C-1), 44.51 (C-9), 50.29 (C-5), 51.18 (C-7), 74.45 (C-6),175.92 (C-15), OMe (51.64) (Dawidar and Metwally, 1985). The compound (3) was identified as 1α,9α-dihydroxy-αcyclocostunolide using UV spectral data of the compound with methanol exhibited a major band at λmax 227.5, and shoulder at λmax 225.8, molecular weight 232 and molecular formula C15H18O3 with m/z: 233.12 [M+ + 1], 187.3,151.2, 1H-NMR (CD3OD): 0.82 (s, H-14), 1.99 (s, H-15), 3.00 (d, H-5, J = 11 Hz), 3.28 (d, H-7, J = 11 Hz), 3.9 (d, H-1, J = 4 Hz), 3.97 (t, H-9, J = 2.5 Hz), 4.05 (H-6, J = 11 Hz), 5.32 (d, H-13), 5.52 (s, H-3), 6.07 (d, H-13, J = 3.5 Hz),13C-NMR: 16.11 (C-14),17.35 (C-15), 26.17 (C-8), 27.97 (C-2), 39.41 (C-9), 40.93 (C-3), 50.34, 81.89 (C-6), 119.73 (C-13), 127.00 (C-1), 127.18 (C-5), 136.97 (C-10), 139.99 (C-4), 141.53 (C-11), 170.51 (C-12) (Dawidar and Metwally, 1985).
Chloroform
Total biomass (g)
Ethyl acetate
Concentration (µg/ml) Fig. 1. Allelopathic potential of C. dioscorides extracts on C. arvensis total biomass fresh weight. The error bars means the standard deviations between replicates.
Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006
M.A. Balah / South African Journal of Botany xxx (2015) xxx–xxx
C. olitorius
E. crus galli
Total biomass (g)
P. oleracea
5
Concentration (µg/ml) Fig. 2. Allelopathic potential of chloroform extracts on the tested weeds total biomass fresh weight. The error bars means the standard deviations between replicates.
The second herbicidal isolate chemical group from C. dioscoridis was identified by comparing UV spectrum MS, H NMR and C NMR spectra with authentic samples and published data as: rhamanetin compound (4) with UV spectral data of the compound with methanol exhibited a major band at λmax 320.5, and with molecular weight 316, with m/z: 315 [M+ − 1], 261.3, 203.2, 147.2 and 1H-NMR (CD3OD) δ ppm: 3.8 (3H, s, 7-O CH3), 6.20 (1H, d, 6-H), 6.48 (1H, d, 8-H), 6.94 (1H, d, 5′-H), 7.6 (1H, m, 6′-H), 7.75 (1H, d, 2′-H), 9.40 (1H, s, OH), 9.73 (1H, s, OH), 10.76 (1H, s, OH), 12.06 (1H, s, OH). 13C-NMR δ ppm: 93.5 (C-8), 98.0 (C-6), 102.7 (C-10), 111.6 (C-2′), 115.3 (C-5′), 121.5 (C-6′), 121.8 (C-1′), 135.6 (C-3), 146. 3 (C-3′), 147.2 (C-4′), 148.7 (C-2), 160.3 (C-9), 156.0 (C-5), 163.6 (C-7), 175.72 (C-4), 55.4 (7-OCH3) (Ahmed et al., 1987). Isorhamnetin 3-sulfate compound (5) with UV spectral data of the compound with methanol exhibited a major band at λmax 320, and shoulder at λmax 340 (30) with molecular weight 394.3 with m/z: 395.3, 349.3, 317 (100%), 277, 229. 1H-NMR: (CD3OD): 3.83 s (3H, 3′OCH3), 6.17 d (1H, H-6), 6.43 d (1H, H-8), 6.86 d (1H, H-5′), 7.61 dd (1H, H-6′), 8.04 d (1H, H-2′), 9.70 (4′-OH), 10.80 (7-OH), 12.85 (3-OSO3H) and 12.84 (5-OH). 13C-NMR: C-8 (93.4), C-3 (132.5), C-6 (98.3), C-10 (104.1), C-5′ (115.0), C-6′ (121.9), C-2′ (113.6), C-1′ (121.4), (146.9), C-4′ (149.3), C-2 (155.9), C-9 (156.0), C-5 (161.3), C-7 (163.8), C-4 (177.6), C-3′ OCH3 (55.6) (Ahmed et al., 1987). Isorhamnetin 3-O-rutinoside compound (6) with UV spectral data of the compound with methanol exhibited a major band at λmax 319.8, and shoulder at λmax 340, with molecular weight 624 and m/z: 625 [M + H]+, 523.4, 455.5, 345.1, 273.3, 233.3, 187.3, 151.2 and 1H-NMR
(CD3OD): δ 1.2 (1H, d, J = 6.0 Hz, H-6′′′), 3.86 (1H, s, OCH3), sugar moiety: 5.5 (1H, d, J = 8 Hz, H-1′′′ rhamnose), 5.31 (1H, d, J = 7.4 Hz, H-1′′′ glucose), 6. 3 (1H, s, H-6), 6.3 (1H, s, H-8), 6.83 (1H, d, J = 8.0 Hz, H-5′), 7.74 (1H, d, J = 8.0 Hz, H-6′), 7.84 (1H, s, H-2′); 13C-NMR (CD3OD): δ 17.7 (C-6′′′), 56.6 (C-3′-OCH3), 67.6 (C-6′′), 67.6 (C-5′′′), 70.6 (C-4′′), 70.7 (C-2′′′), 70.9 (C-3′′′), 72.8 (C-4′′′), 74.9 (C-2′′), 76.4 (C-5′′), 77.0 (C-3′′), 93.6 (C-8), 98.7 (C-6), 101.2 (C-1′′′), 103.0 (C-1′′), 104.4 (C-10), 113.3 (C-2′), 114.8 (C-5′), 121.6 (C-6′), 122.5 (C-1′), 134.0 (C-3), 147.1 (C-3′), 149.7 (C-4′), 157.3 (C-2), 157.6 (C-9), 161.8 (C-5), 165.0 (C-7) (Ahmed et al., 1987). Epicatechin compound (7) with UV spectral data of the compound with methanol exhibited a major band at λmax 231.5, and shoulder at λ max 325, with molecular weight 290, with m/z: 289 [M − H+], 231.3, 183.3 1H-NMR (CD3OD): 2.41 (dd, J = 2.8, H-4), 2.56 (dd, J = 4.5 Hz, H-4), 3.8 (s, H-3), 4.4 (s, H-2), 5.5 (d, J = 2.08 Hz, H-8), 5.6 (d, J = 2.08 Hz, H-6), 6.4 (d, J = 8.12 Hz, H-5′), 6.48 (d, J = 9.77 Hz, H-6′), 6.6 (s, H-2′), 13C-NMR: 29.1 (C-4), 67.4 (C-3), 79.7 (C-2), 95.6 (C-8), 96.5 (C-6), 100.1 (C-10), 115.4 (C-2′), 116.0 (C-5′), 119.4 (C6′),132.2 (C-1′), 145.6 (C-3′), 145.7 (C-4′), 157.2 (C-9), 157.4 (C5),157.7 (C-7), from the above result it could be identified as epicatechin, these data were compared with UV spectrum MS, 1H-NMR and 13C-NMR spectra with authentic sample and published data. As a result, the C. dioscoridis crude extract have a broad spectrum activity since they are effective against both mono and di-cotyledon, this is probably due to the plant which is widely used as antifungal and antibacterial activity (Fatope et al., 2004; Katuura et al., 2007), corresponding to bioassay, the toxicity of aqueous and organic extracts increased
Chloroform
Total biomass (g)
Ethyl acetate
Fractions (20 µg/ml) Fig. 3. Bioassay of column chromatography fractions on C. arvensis total biomass fresh weights. The error bars means the standard deviations between replicates.
Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006
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M.A. Balah / South African Journal of Botany xxx (2015) xxx–xxx
A= (Isorhamnetin 3 sulfate),
Total fresh weight reduction %
B=(Isorhamnetin 3-O- rutinoside), C=(Rhamnetin) , D=(Epicatechin), E=(15-Hydroxyisocostic cid + Isocostic acid 4- carboxaldehyde), F= (Isocostic acid 4carboxaldehyde), G= (1α,9α-dihydroxy-αcyclocostunolide). Concentration (50 µg/ml) Fig. 4. Bioassay of allelopathic constituents on C. arvensis total biomass fresh weights. The same letters within columns means not significant differences between treatments at P = 0.05. The error bars means the standard deviations between replicates.
with increasing concentration. Our results showed that chloroform extracts have a remarkable herbicidal activity against field bindweeds total biomass fresh weight. The activity of C. dioscoridis crude extracts due to several phytotoxic compounds responsible for their effect on weeds growth parameters and germination. However, no clear post emergence activity was recorded against C. arvensis (data not shown) 5 leaves stage. Few studies have mentioned allelopathic activity and identified compounds isolated from the genus Conyza. Economou et al. (2002) reported allelopathic activity of fleabane (Conyza albida Willd. ex Spreng) on oat (Avena sativa L.) growth fresh and dry weight. Previously, many sesquiterpenes have been isolated from Conyza sp. (Dawidar and Metwally, 1985; Ahmed et al., 1987; Mahmoud, 1997; Fatope et al., 2004). The present work adds the evidence supporting potential herbicidal activity of Conyza spp sesquiterpene compounds, which has been identified as a mixture of the eudesmane derivatives 15-hydroxyisocostic acid from C. dioscoridis. The chemical structure of the isolated compounds was established by extensive analysis of 1 H-NMR, 13CNMR and mass spectrometry data and confirmed by comparison of the spectroscopic data with those reported in the literature (Dawidar and Metwally, 1985; Ahmed et al., 1987). The highest phytotoxic activity appeared in the sesquiterpene mixture of the eudesmane derivatives 15-hydroxyisocostic acid, isolated from C. dioscoridis followed with methyl 15-oxo-eudesome-4, 11(13)-diene 12-oate and the lower toxicity was observed in the flavonoid compounds. The phytotoxic effect of the previous sesqueterpene compounds was reported for the first time by the authors in this pepper. The allelopathic potentials of the isolated compounds were previously recorded by Al-Watban and Salama (2012) (isorhamnetin 3-O-rutinoside), Weidenhamer and Romeo (2004) (rhamanetin), Weir and Vivanco (2008) (epicatechin) and others. However, the herbicidal activity of isorhamnetin 3-sulfate and isorhamnetin 3-O-rutinoside was not recorded formerly. To our knowledge, this is the first information on the herbicidal activity of extracts and sesquiterpene constituents of C. dioscoridis against field bindweeds (C. arvensis). Finally, one can conclude that weeds control using these compounds might be feasible and further work is needed to recommend this as an herbicide substitute or structurally leading to new synthetic herbicides.
Acknowledgment The author would like to thank the natural product and phytochemistry staff of the National Research Center and Ain Shams University
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Please cite this article as: Balah, M.A., Chemical and biological characterization of Conyza dioscoridis (L.) desf. family (Compositae) in some perennial weeds control, South African Journal of Botany (2015), http://dx.doi.org/10.1016/j.sajb.2015.07.006