Allelopathic activity of Acacia concinna pod extracts

*Corresponding author: Sutjaritpan Boonmee, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan. E-mail: sutjaritpanbm@gmail.com Received: 02 September 2016; Revised: 13 February 2017; Accepted: 16 February 2017; Published Online: 27 February 2017 Boonmee and Kato-Noguchi: Allelopathic activity of Acacia concinna Emir. J. Food Agric ● Vol 29 ● Issue 4 ● 2017 251 Sivaraj, 2012). In addition, antioxidant, anti-coagulant, anti-platelet, anti-thrombotic, antidermatophytic and immune adjuvant activities are some of the valuable pharmacological properties of the plant (Kapoor et al., 1969, Kukhetpitakwong et al., 2006; Todkar et al., 2010; Rao et al., 2014; Sharma et al., 2014). Nonetheless, no study has been conducted regarding the allelopathic properties of this plant species. Therefore, the allelopathic activity and the allelopathic substances of A. concinna pods were investigated. MATERIALS AND METHODS Plants material A. concinna (Willd.) DC. pods were collected from an area of Chiang Rai Province, Thailand in September 2014. Tiny particles soil or other contaminants were removed from the mature pods by washing thoroughly with tap water. The mature pods were then dried in the shade and ground into powder. Dicotyledonous plants; cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), alfalfa (Medicago sativa L.), and rapeseed (Brassica napus), and monocotyledonous plants; barnyard grass (Echinochloa crus-gallis L.), Italian ryegrass (Lolium multiflorum Lam.), foxtail fescue (Vulpia myuros (L.) C. C. Gmel.) and timothy (Phleum pretense L.) were selected for the bioassay. Extraction Powder of pods (100 g) of A. concinna was extracted with 70% (v/v) aqueous methanol (500 mL) and kept in a sealed container for 48 h. The extract was filtered through a sheet of filter paper (No.2, Toyo Roshi Kaisha Ltd., Japan). The residue was extracted again with 500 mL of cold methanol for 24 h and filtrated. The two filtrates were mixed and the solvent was removed until dry by using a rotary evaporator with water bath at 40°C. Bioassay The residue of the pod extracts was dissolved with cold methanol and an aliquot of pod extracts at final concentrations of 1, 3, 10, 30, 100 and 300 mg dry weight equivalent extract/mL was added to a sheet of filter paper (No.2) in a 28 mm Petri dish. The solvent on the filter paper was evaporated in a draft chamber. Subsequently, 0.6 mL of 0.05% (v/v) aqueous solution of polyoxyethylene sorbitan monolaurate (Tween 20; Nacalai, Kyoto, Japan), a mild surfactant and has no toxicity was added to the filter paper. Ten seeds of cress, alfalfa, lettuce and rapeseed were placed on filter paper in the Petri dishes. Ten germinated seeds of barnyard grass, Italian ryegrass, foxtail fescue and timothy were placed on filter paper in the Petri dishes after incubation with distilled water in dark at 25°C for 60, 72, 48 and 120 h, respectively. Control seeds or germinated seedlings were placed on a filter paper soaked with the aqueous solution of Tween 20 without the extract. After incubation for 48 h in the dark at 25°C, the length of shoots and roots of all test plant seedlings was measured. The percentage length of seedlings was determined by reference to the length of control seedlings. The bioassay was conducted in two independent experiments with three replications (10 seedlings/replication) for each experiment (n=60). The concentrations required for 50% inhibition (I50 value) of the test plants were calculated from the regression equation of the concentration response curves. Isolation and purification of an allelopathic substance The pod extracts of A. concinna pods were evaporated to produce an aqueous residue using a rotary evaporator with water bath at 40°C (Fig. 1). Subsequently, the aqueous residue was then adjusted to pH 7.0 with 1 M phosphate buffer and partitioned three times with an identical volume of ethyl acetate. The ethyl acetate fraction was dehydrated over anhydrous Na2SO4, filtrated and evaporated until dry. The residue was separated by a column of silica gel (60 g; silica gel 60, 70-230 mesh, Nacalai Tesque, Kyoto, Japan), eluted stepwise with increasing amounts of ethyl acetate in n-hexane (10% per step, v/v, 150 mL per step) and methanol (300 mL). Cress bioassay was used to determine the inhibitory activity of all separated fractions with three replications (10 seedlings/replication). The growth inhibitory activity was found in the fraction eluted by 70% ethyl acetate in n-hexane. The active fraction was evaporated until dry and the residue was separated by a column of Sephadex LH-20 (100 g, GE Healthcare, Uppsala, Sweden), eluted with increasing amounts of methanol in water (20% per step, v/v, 150 mL) and methanol (300 mL). The most active fraction was eluted by 40% aqueous methanol and evaporated until dry. The residue was dissolved with 20% (v/v) aqueous methanol and loaded onto a reverse phase C18 cartridge (1.2 × 6.5 cm; YMC, Kyoto, Japan). The cartridge was eluted with increasing amounts of methanol in water (20% per step, v/v, 30 mL) and methanol (60 mL). The most active fraction was eluted by 40% aqueous methanol and evaporated until dry. Finally, the residue was purified by using reverse phase HPLC (4.6 mm × 250 i.d., Inertsil ODS-3; GL Science, Tokyo, Japan) eluted at a flow rate of 0.8 mL/min with 35% (v/v) aqueous methanol and detected at 220 nm. Statistical analysis The bioassay experiment was conducted in a completely randomized design. Data processing were analyzed by ANOVA and subsequent post hoc analysis with Dunnett’s test. Two-tailed Pearson Correlation test was used to analyze the correlation of coefficient (R) between the extract concentration and the growth of test plants. All statistical analyses were performed using SPSS version 16.0. The concentrations required for 50% inhibition of each test plants were analyzed by GraphPad Prism 5.04. Boonmee and Kato-Noguchi: Allelopathic activity of Acacia concinna 252 Emir. J. Food Agric ● Vol 29 ● Issue 4 ● 2017 RESULTS Effects of pod extracts of A. concinna on shoot length of test plants Shoot length of eight test plants was significantly inhibited by A. concinna pod extracts at concentrations ≥3 mg dry weight equivalent extract/mL (P<0.001) (Fig. 2). At 30 mg dry weight equivalent extract/mL, shoots of lettuce and rapeseed seedlings were completely inhibited and Italian ryegrass, timothy, foxtail fescue, alfalfa, cress, and barnyard grass were significantly inhibited by 0.4, 2.1, 3.2, 7.1, 14.1 and 25.6% of control length, respectively. The extract at 300 mg dry weight equivalent extract/mL, shoots of cress and barnyard grass seedlings were significantly inhibited by 0.7 and 2.3% of control length and the other test plants were completely inhibited. Regression analysis between pod extract concentration and shoot length percentage showed negative correlation coefficient (R) in all test plants (Table 1). The shoot length of barnyard grass and cress had a strong negative correlation with pod extracts and giving the correlation coefficients of -0.669 and -0.743, respectively (P<0.01). The I50 value on the shoot length of all test plants ranged between 1.00 to 5.17 mg dry weight equivalent extract/mL (Table 2). Shoot length of foxtail fescue was the most susceptible to the pod extracts, followed by lettuce, Italian ryegrass, timothy, rapeseed, alfalfa, cress, and barnyard grass. Effects of pod extracts of A. concinna on root length of test plants Root length of eight test plants was significantly inhibited by A. concinna pod extracts at concentrations ≥1 mg dry weight equivalent extract/mL (P<0.001) (Fig. 2). At 10 mg dry weight equivalent extract/mL, roots of barnyard grass, Fig 1. Procedure for extraction and isolation of substance ACP-1. Boonmee and Kato-Noguchi: Allelopathic activity of Acacia concinna Emir. J. Food Agric ● Vol 29 ● Issue 4 ● 2017 253 foxtail fescue and timothy seedlings were completely inhibited, and Italian ryegrass, lettuce, cress, rapeseed and alfalfa were significantly inhibited by 0.7, 2.3, 9.2, 11.3 and 14.8% of control length, respectively. The extract at 300 mg dry weight equivalent extract/mL, the root of cress was significantly inhibited by 0.4% of control length, and the other test plants were completely inhibited. There was a negative correlation coefficient (R) between pod extract concentration and root length percentage in all test plants (Table 1). The root length of cress, alfalfa and barnyard grass had a significant negative correlation with the pod extracts and giving the correlation coefficients of -0.543, -0.521 and -0.428, respectively (P<0.01). The I50 value on the root length of all test plants ranged between 0.02 to 2.59 mg dry weight equivalent extract/mL (Table 2). Root growth of barnyard grass was the most susceptible to the extract, followed by cress, lettuce, foxtail fescue, timothy, Italian ryegrass, rapeseed and alfalfa. Isolation of an allelopathic substance from pod extracts of A. concinna The pod extracts of A. concinna were purified as described in Fig. 1. The biological activity of the separated fractions through a silica gel column is shown in Fig. 3. Only fraction 6 (F6) showed significant inhibitory activity. Shoots and roots of cress were inhibited by 23.5 and 33.2% of control length, respectively. Fraction 6 was further purified by Sephadex LH-20 and reverse phase C18 cartridge, an active substance (ACP-1) was separated by reverse phase HPLC. The active substance was found at the retention time of 40-55 min. The residue was colorless. Shoots and roots of cress were inhibited by the substance at 27.1 and 14.4% of control length, respectively.

. In addition, antioxidant, anti-coagulant, anti-platelet, anti-thrombotic, antidermatophytic and immune adjuvant activities are some of the valuable pharmacological properties of the plant (Kapoor et al., 1969, Kukhetpitakwong et al., 2006;Todkar et al., 2010;Rao et al., 2014;Sharma et al., 2014).Nonetheless, no study has been conducted regarding the allelopathic properties of this plant species.Therefore, the allelopathic activity and the allelopathic substances of A. concinna pods were investigated.

Extraction
Powder of pods (100 g) of A. concinna was extracted with 70% (v/v) aqueous methanol (500 mL) and kept in a sealed container for 48 h.The extract was filtered through a sheet of filter paper (No.2, Toyo Roshi Kaisha Ltd., Japan).The residue was extracted again with 500 mL of cold methanol for 24 h and filtrated.The two filtrates were mixed and the solvent was removed until dry by using a rotary evaporator with water bath at 40°C.

Bioassay
The residue of the pod extracts was dissolved with cold methanol and an aliquot of pod extracts at final concentrations of 1, 3, 10, 30, 100 and 300 mg dry weight equivalent extract/mL was added to a sheet of filter paper (No.2) in a 28 mm Petri dish.The solvent on the filter paper was evaporated in a draft chamber.Subsequently, 0.6 mL of 0.05% (v/v) aqueous solution of polyoxyethylene sorbitan monolaurate (Tween 20; Nacalai, Kyoto, Japan), a mild surfactant and has no toxicity was added to the filter paper.Ten seeds of cress, alfalfa, lettuce and rapeseed were placed on filter paper in the Petri dishes.Ten germinated seeds of barnyard grass, Italian ryegrass, foxtail fescue and timothy were placed on filter paper in the Petri dishes after incubation with distilled water in dark at 25°C for 60, 72, 48 and 120 h, respectively.Control seeds or germinated seedlings were placed on a filter paper soaked with the aqueous solution of Tween 20 without the extract.After incubation for 48 h in the dark at 25°C, the length of shoots and roots of all test plant seedlings was measured.The percentage length of seedlings was determined by reference to the length of control seedlings.The bioassay was conducted in two independent experiments with three replications (10 seedlings/replication) for each experiment (n=60).The concentrations required for 50% inhibition (I 50 value) of the test plants were calculated from the regression equation of the concentration response curves.

Isolation and purification of an allelopathic substance
The pod extracts of A. concinna pods were evaporated to produce an aqueous residue using a rotary evaporator with water bath at 40°C (Fig. 1).Subsequently, the aqueous residue was then adjusted to pH 7.0 with 1 M phosphate buffer and partitioned three times with an identical volume of ethyl acetate.The ethyl acetate fraction was dehydrated over anhydrous Na 2 SO 4 , filtrated and evaporated until dry.The residue was separated by a column of silica gel (60 g; silica gel 60, 70-230 mesh, Nacalai Tesque, Kyoto, Japan), eluted stepwise with increasing amounts of ethyl acetate in n-hexane (10% per step, v/v, 150 mL per step) and methanol (300 mL).Cress bioassay was used to determine the inhibitory activity of all separated fractions with three replications (10 seedlings/replication).The growth inhibitory activity was found in the fraction eluted by 70% ethyl acetate in n-hexane.The active fraction was evaporated until dry and the residue was separated by a column of Sephadex LH-20 (100 g, GE Healthcare, Uppsala, Sweden), eluted with increasing amounts of methanol in water (20% per step, v/v, 150 mL) and methanol (300 mL).The most active fraction was eluted by 40% aqueous methanol and evaporated until dry.The residue was dissolved with 20% (v/v) aqueous methanol and loaded onto a reverse phase C 18 cartridge (1.2 × 6.5 cm; YMC, Kyoto, Japan).The cartridge was eluted with increasing amounts of methanol in water (20% per step, v/v, 30 mL) and methanol (60 mL).The most active fraction was eluted by 40% aqueous methanol and evaporated until dry.Finally, the residue was purified by using reverse phase HPLC (4.6 mm × 250 i.d., Inertsil ODS-3; GL Science, Tokyo, Japan) eluted at a flow rate of 0.8 mL/min with 35% (v/v) aqueous methanol and detected at 220 nm.

Statistical analysis
The bioassay experiment was conducted in a completely randomized design.Data processing were analyzed by ANOVA and subsequent post hoc analysis with Dunnett's test.Two-tailed Pearson Correlation test was used to analyze the correlation of coefficient (R) between the extract concentration and the growth of test plants.All statistical analyses were performed using SPSS version 16.0.The concentrations required for 50% inhibition of each test plants were analyzed by GraphPad Prism 5.04.

Effects of pod extracts of A. concinna on shoot length of test plants
Shoot length of eight test plants was significantly inhibited by A. concinna pod extracts at concentrations ≥3 mg dry weight equivalent extract/mL (P<0.001) (Fig. 2).At 30 mg dry weight equivalent extract/mL, shoots of lettuce and rapeseed seedlings were completely inhibited and Italian ryegrass, timothy, foxtail fescue, alfalfa, cress, and barnyard grass were significantly inhibited by 0.4, 2.1, 3.2, 7.1, 14.1 and 25.6% of control length, respectively.The extract at 300 mg dry weight equivalent extract/mL, shoots of cress and barnyard grass seedlings were significantly inhibited by 0.7 and 2.3% of control length and the other test plants were completely inhibited.Regression analysis between pod extract concentration and shoot length percentage showed negative correlation coefficient (R) in all test plants (Table 1).The shoot length of barnyard grass and cress had a strong negative correlation with pod extracts and giving the correlation coefficients of -0.669 and -0.743, respectively (P<0.01).The I 50 value on the shoot length of all test plants ranged between 1.00 to 5.17 mg dry weight equivalent extract/mL (Table 2).Shoot length of foxtail fescue was the most susceptible to the pod extracts, followed by lettuce, Italian timothy, rapeseed, alfalfa, cress, and barnyard grass.

Effects of pod extracts of A. concinna on root length of test plants
Root length of eight test plants was significantly inhibited by A. concinna pod extracts at concentrations ≥1 mg dry weight equivalent extract/mL (P<0.001) (Fig. 2).At 10 mg dry weight equivalent extract/mL, roots of barnyard grass, foxtail fescue and timothy seedlings were completely inhibited, and Italian ryegrass, lettuce, cress, rapeseed and alfalfa were significantly inhibited by 0.7, 2.3, 9.2, 11.3 and 14.8% of control length, respectively.The extract at 300 mg dry weight equivalent extract/mL, the root of cress was significantly inhibited by 0.4% of control length, and the other test plants were completely inhibited.There was a negative correlation (R) between pod extract concentration and root length percentage in all test plants (Table 1).The root length of cress, alfalfa and barnyard grass had a significant negative correlation with the pod extracts and giving the correlation coefficients of -0.543, -0.521 and -0.428, respectively (P<0.01).The I 50 value on the root length of all test plants ranged between 0.02 to 2.59 mg dry weight equivalent extract/mL (Table 2).Root growth of barnyard grass was the most susceptible to the extract, followed by cress, lettuce, foxtail fescue, timothy, Italian ryegrass, rapeseed and alfalfa.

Isolation of an allelopathic substance from pod extracts of A. concinna
The pod extracts of A. concinna were purified as described in Fig. 1.The biological activity of the separated fractions through a silica gel column is shown in Fig. 3.Only fraction 6 (F6) showed significant inhibitory activity.Shoots and roots of cress were inhibited by 23.5 and 33.2% of control length, respectively.Fraction 6 was further purified by Sephadex LH-20 and reverse phase C 18 cartridge, an active substance (ACP-1) was separated by reverse phase HPLC.The active substance was found at the retention time of 40-55 min.The residue was colorless.Shoots and roots of cress were inhibited by the substance at 27.1 and 14.4% of control length, respectively.

DISCUSSION
A. concinna pod extracts inhibited the shoot and root length of selected test plants with different growth inhibition percentages.The effectiveness of the extracts varied with their concentration and test plants.These results are supported by others studies which noted that the inhibitory activity depended on extract concentration (Parvez et al., 2004, Shunjie et al., 2008, Moosavi et al., 2011;Mousavi et al., 2013).Differences in the biochemical and physiological nature of test plants may be responsible for inhibitory effects of the extracts (Sodaeizadeh et al., 2009).The I 50 values of A. concinna pod extracts indicated that the extract had higher inhibitory effects on roots than their shoots.A similar result was reported by Arowosegbe and Afolayan (2012) that Aloe ferox extract had inhibition on the root growth of test plants more than on the shoot growth.This may be because the root is the first exposed to the extracts for absorbance of allelochemicals (Salam and Kato-Noguchi, 2010).In this study, all test plants were germinated and/or grown in a single Petri dish which contained only aqueous methanol extracts of A. concinna pods.During the initial growth of the plants, light is unnecessary and the plants use nutrients from their seeds and there was no intra-species competition for nutrients (Fuerst and Putnam, 1983).Therefore, the inhibitory activity on the growth of test plants resulted from the presence of allelochemicals in the pod extracts rather than extraneous competition factors.Bioassay-directed fractionations of the extract resulted in the isolation of an allelopathic active substance ACP-1 with growth inhibitory activity.This research is the first report of the presence of allelopathic active substances in A. concinna pods.

CONCLUSIONS
A. concinna pod extracts showed the growth inhibitory effect on selected test plants.The allelopathic active substance ACP-1 was isolated from the pod extracts and is likely to be responsible for the inhibitory effects of the extracts.The pods and the extracts could, therefore, be utilized as a weed management options in sustainable agriculture.Further research under the field condition is necessary to substantiate these results.

Fig 1 .
Fig 1. Procedure for extraction and isolation of substance ACP-1.

Fig 2 .
Fig 2. The growth inhibitory effects of Acacia concinna pod extracts on the shoot and root length of cress, lettuce, alfalfa, rapeseed, barnyard grass, Italian ryegrass, foxtail fescue and timothy.All test plants were treated with the concentrations corresponding to the extracts obtained from 1, 3, 10, 30, 100 and 300 mg dry weight equivalent extract/mL (mg/mL).Mean ± SE from two independent experiments with three replications (10 seedlings/replication) for each experiment (n=60) are shown.Significant differences between control and treatments are represented by asterisks: *P<0.05,**P<0.01,***P<0.001(One-way ANOVA, Post hoc by Dunnett's test).

Fig 3 .
Fig 3.The effects of separated fraction through silica gel column on the growth of cress.The pod extract was used for the experiment in the concentration of 100 mg dry weight equivalent extract/mL.Mean ± SE from three replications with 10 seedlings/replication (n=30) are shown.Significant differences between control and treatments are represented by asterisks: **P<0.01,***P<0.001(ANOVA, Post hoc by Dunnett's test).