Herbicidal activity of n-hexane, ethyl acetate and methanol purple nutsedge (Cyperus rotundus L.) extracts on Amaranthus spinosus L.

Environmentally friendly weed control is highly recommended to be implemented in agriculture. The objective of this research was to assess the herbicidal efficacy of n-hexane, ethyl acetate, and methanol extracts derived from the tuber of Cyperus rotundus L. on Amaranthus spinosus L. The tuber was consecutively extricated with n-hexane, ethyl acetate, and methanol. These extracts were given to A. spinosus in pot culture at concentrations of 5%, 10%, 15%, and 20%.The herbicide (2,4-D at 0.686 kg a.i. ha−1) and distilled water were acted as positive and negative controls. The extract had variable effects on weed control percentage, leaf area, root length, shoot and root dry weight of A. spinosus. Major compounds in GC-MS in n-hexane of C. rotundus extract were: 7-Isopropenyl-1, 4a- dimethy l-4,4a,5,6,7,8-hexahydro-3H-naphthalen-2-one (19.60%), phthalic acid (7%), dibutyl phthalate (3.97%); in ethyl acetat extract were propanetriol, 1-acetate (37,40%) and 7 Isopropenyl-1,4a-dimethyl-4,4a,5,6,7,8-hexahyro-3H-naphthalen-2-one (6.42%); in methanol extract were d-glycero-d-ido-heptose (21.31%) and octaethylene glycol monododecyl ether (39.15%). The 20% of those extracts causing 53.33%, 64.33% and 55.33 destruction of A. spinosus respectively at 14 Days After Aplication. Thus, ethyl acetate is the most potent herbicide extract to be developed as an agent against A. spinosus.


Introduction
Weeds are regarded as an eminent significant living impediment to crop productivity, resulting in harvest reductions that vary from 45-95%, contingent upon environmental factors and agronomic methodologies [1].Weeds cause yield losses greater than the losses caused by other agricultural pests [2].Herbicides have been employed for the purpose of weed control, however, a majority of them presents a substantial menace to both the environment and human health.Numerous studies have reported on the adverse effects of synthetic herbicides, including emergence of herbicide resistance in weeds [3,4], adverse consequence on soil [5], the toxicity of substances to non-target organisms, disruption of the overall ecology, and the persistence of chemical residues in the environment [6].It is imperative to investigate the allelopathic capacity of plants as a viable substitute for conventional herbicides in a natural context [7,8].It is environmentally safe, can converse the available resources and also may mitigate the problems raised by synthetic chemicals [9].The allelochemicals that are released into the environment have a stimulatory or inhibitory effect on neighboring weeds, plants, and microbes, depending on their concentration [4].Numerous pernicious annual and perennial weeds have been classified as species exhibiting allelopathic properties.Cyperus rotundus, commonly known 1297 (2024) 012032 IOP Publishing doi:10.1088/1755-1315/1297/1/012032 2 as purple nutsedge, is a perennial weed that is prevalent in cultivated fields and has the ability to infiltrate both field crops and landscapes (Figure1).This weed has been documented to possess allelopathic activity against various crops, including barley, radish, rice, sorghum, and soybean [10].C. rotundus tuber flour was able to suppress weed germination of Asystasia gangetica effectively on paper material [11].The utilization of tuber residues from C. rotundus lead to a serious minimization in the dry weight of jute (Chorchorus olitorius) and barnyard grass (Echinochloa crus-galli) by 85.96% and 58.28%, respectively, in comparison to the control group.This effect was observed with the application of 80 g of soil surface [12].The application of purple nutsedge extract can control Richardia brasiliensis weed which comes from the broadleaf weed group [13].It has been found that using purple nutsedge extract in concentrations below 14% can promote growth, but it may also impede the production of chlorophyll in upland rice [14].We screened C. rotundus [15,16] among five perennial plants; Australian acacias, merkus Pine, teak, Indian almond, jatropha, and five weed species which are cogon grass, billygoat weed, purple nut sedge, siam weed, and carpet grass against Amaranthus spinosus L., (Amaranthaceae).It showed that the extract of C. rotundus applied at concentration of 20 % gaved severe effect (85.83%) and concentration 30% (91.50%) completely suppressed at 7 days after its application.
It is imperative to employ specific solvents based on the characteristics of the intended compound, as variances in solvent polarity yield varying quantities and varieties of allelochemical compounds [17].The present research has selected spiny amaranth (Amaranthus spinosus L.) as the focal plant owing to its rapid and consistent germination, sensitivity, and competitive nature with crops [18,19].This work aimed to test the herbicidal activity of n-hexane, ethyl acetate and methanol tuber extracts of C. rotundus on growth of weed A. spinosus in pot assays.Additionally, a GC-MS analysis was performed on the extracts to identify the allelochemicals responsible for the observed effects.

Location and Time
The beginning of the research was in early June to the end of October 2021.The study was carried out at Syiah Kuala University (USK); The Faculty of Mathematics and Science-Biology laboratory and Chemistry research.Pot studies were completed in the screen house of Experimental Farm, Faculty of Agriculture, Aceh Province, Indonesia.The geographical coordinates of the location are defined by a latitude of 5°34ˈ3,44°U and a longitude of 95°22ˈ34, 49°T accompanied by an altitude of 3 meters above sea level.

Experimental Design
This study employed a Completely Random design.The treatments were carried out consisting of C. rotundus extract at 5%, 10%, 15%, and 20% concentration of n-hexane, ethyl acetate, and methanol (12 treatments altogether), the positive control was carried out using (2,4-D at 0.686 kg a.i.ha -1 ) while the negative control used distilled water.The experiment conducted on three separate occasions.

The preparation of botanical extract and seed of A. spinosus
The C. rotundus tubers came from our agricultural faculty's experimental farm, Syiah Kuala University.The seeds of A. spinosus were gathered in Meunasah Gle, Sigli, Pidie.Suwarno, a botanist, identified both plants.The C. rotundus tubers with the same size and colour were collected and cleaned for uniformity.After a 14-day period at room temperature, the substance underwent a grinding process to ensure uniformity.The grinded tubers (6 kg) were placed in 1 L of ammonia and left for 1 hour.Subsequently, the samples underwent sequential extraction six times with methanol, seven times with ethyl acetate, and eight times with n-hexane.Every single extraction necessitated the use of 5 L of solvent and spanned duration of three days.The organic fractions obtained following each solvent extraction were subjected to filterring, stirring, and drying via a rotary evaporator.In order to obtain concentrations of 5%, 10%, 15%, and 20%, the dried remains of the three mentioned extractions were mixed in a solution consisting solely of distilled water.

Pot culture
Soil samples were obtained from Lampakuk Village, Aceh Besar, up by the deepness of 200 mm.The collected soil was subjected to a drying process for a period of seven days and subsequently sieved to eliminate any plant remains.Each plastic pot was filled with 1000 gram of soil.The seeds of A. spinosus were immersed in water for a duration of two hours.Subsequently, five seeds were planted in each pot by the deepness of 20 mm.After a period of seven days following the sowing process, the act of thinning was executed with the intention of preserving solely one robust plant per individual pot.On day 21, plants were treated with either water or plant extract via foliar spraying (15 ml per pot) as per the designated treatments.The pots were subjected to a daily irrigation regimen, receiving a precise quantity of 200 ml of tap water on two separate occasions.
The growth parameters of A. spinosus were documented 28 days post-application.as well as weed control at 7, 14, 21, and 28 post-application The leaf area was measured and the percentage of A. spinosus weed control was evaluated referring to evaluation table using a 0-100 rating scale (Table 1).The dry weights of the shoots and roots were documented after being baked at 60°C for 48 hours or until a consistent dry weight was attained.The measurement of the root's length was conducted after it had been thoroughly rinsed with tap water.

Analysis of GC-MS
The instrument employed for the analysis is a single Quadrupole GC-MS of unit size, possessing a mass range of 1.2 TB 1100 U with unit mass resolution.It is equipped with a triple off-axis Thermo Scientific Dynamax XR detector featuring an electronic dynamic range exceeding 109.The mass filter owns double filter for silent sound.This instrument is capable of ionizing sample molecules using electron impact ionization, which involves bombarding the sample with high-energy electrons to produce ions.The scanning capability allows detection of all ions produced during the ionization process, providing comprehensive sample analysis.Additionally, chemical ionization is employed, with both positive and negative ions.The scanning capability of the instrument extends up to 20000 U/s.

Analysis of data
The complete dataset was subjected to an analysis of variance using the F test, followed by Duncan's new multiple range test at a significance level of 5%.The aforementioned analyses were conducted utilizing SPSS version 16 (SPSS Inc., Chicago-Illinois, United States).

Weed Control
According to Figure 2, it was observed that the concentration of 20% of the ethyl acetate extract of C. rotundus showed an increased level of inhibition and a minor effect (64.34% inhibition) on the growth of A. spinosus at 14 days post application, which was no notable disparity was detected from the concentration of 20% of the methanol extract (55.33%).The results of the field research indicated that the treatment with the ethyl acetate fraction caused a rapid reaction, which commenced with the development of dense surface of leaves, chlorosis of stem, and there is a pale color overall.(52.34% inhibition) at 7 DAA.Over the course of the following days, a portion of the leaves of A. spinosus underwent burning and loss, resulting in a 64.34% reduction in control at 14 days after application.However, by 21 days, the weed had undergone regeneration and was leading with 58.67% control (as depicted in Figure 2).The application of a 20% methanol C. rotundus extract impeded the growth of A. spinosus, providing 54.00% control at 7 days, 50.33% at 14 days, 55.33% at 28 days after application, and maintained a steady level thereafter.The study revealed that the utilization of the methanol extract of C. rotundus at a concentration of 20% resulted in the onset of leaf surface tightening, stem yellowing and paleness, and some leaf necrosis.Furthermore, the n-hexane extract of C. rotundus at a concentration of 20% exhibited a yield of 31.33% at 7 DAA, 53.33% at 14 DAA, and a recovery rate of 38.83% at 28 DAA.The observed effect is akin to that of ethyl acetate and methanol.Additionally, Figure 3 illustrated a positive correlation between the concentration of the diverse extracts administered to the target plant and the inhibition value.This phenomenon is attributed to the allelopathy impact on target plants, which elicit various responses at higher concentrations [18].Our research findings have revealed that complete inhibition was documented seven days after application, similar to the effects of the herbicide 2,4-D procedure,.The phytotoxic manifestations caused by 2,4-D resulted in leaf dying then led to the death of the stems.

Leaf area, Root Length, Shoot Dry Weight, and Root Dry Weight
The concentration of ethyl acetate extract derived from C. rotundus at 15% was found to be the most effective treatment in inhibiting leaf area, exhibiting an 89% inhibition rate over the control.This treatment was not significantly different from the treatments involving all n-hexane and ethyl acetate extracts at varying concentrations.However, it was significantly different from the methanol treatment at varying concentrations at 28 days after application.
Nonetheless, the outcome of the leaf area assessment exhibited irregularity, as evidenced by the concentration tested, which yielded an inhibition rate of 88% at a concentration of 10% and 91% at a concentration of 5% after 28 days post-application when compared to the control group (distilled water).This incongruity may have been initiated by unanticipated modifications in environmental variables, such as illumination, carbon dioxide levels, warmth, land dampness, humidness, precipitation, or whirlwind throughout the administering of the extract [21].The utilization of plant extracts post-emergence may be significantly influenced by environmental factors, which can modify the processes of penetration and translocation.Furthermore, environmental conditions may exert a secondary impact by modifying the physiological phase of the weed [22].
Regarding root length (as depicted in Figure 4), both inhibitory and stimulatory growth of plants were observed.The lowest level of root inhibition was attributed to the application of the n-hexane extract of C. rotundus at a concentration of 5% (resulting in 76% inhibition), which did not exhibit a substantial deviation when compared to the utilization of 10%.15%, and 20% n-hexane extracts.However, it was significantly different from all ethyl acetate and methanol treatments at various concentrations.
The ethyl acetate C. rotundus extracts concentration 5 and 10 % extracts stimulated the root length (1 % and 6 % respectively), while the 15 % and 20% extracts inhibited the root length 15% and 4%, respectively.This phenomenon bears resemblance to the effects of allelochemicals on plant growth, which may either promote or hinder development depending on the concentration and specific plant species being affected Furthermore, allelochemicals may demonstrate additive, synergistic, or antagonistic interactions [23].There was an inconsistency in root length, where the root length in the ethyl acetate C. rotundus concentration of 15% causes 15% inhibition and 20% concentration causes 4% inhibition.The inconsistency was due to the measurements were taken 28 days after application, while recovery occurred at 14 days after application.
According to Figure 4, the application of 5% n-hexane extract resulted in the least amount of inhibition of shoot weight, which was not significantly different from n-hexane extract concentrations of 10%, 15% and 20% and synthetic herbicide 2,4-D, but significantly different from all ethyl acetate and methanol treatment at various concentrations (5%, 10%, 15% and 20%).Figure 4 also showed inconsistency of shoot weight inhibition where the ethyl acetate extract of C. rotundus with a concentration of 20% has a larger shoot weight (7.21 grams) compared to the inhibition caused by the ethyl acetate extract of C. rotundus with a lower concentration (5%, 10% and 15%).There is an increase (stimulation of 60%) as in the 20% ethyl acetate treatment (7.21 gram over control).The allelochemicals emanating from the donor plants can disrupt the processes of the recipient plant, including photosynthesis, respiration, water and hormonal balance, through the inhibition of enzyme activity [4].
Figure 4 illustrated the presence of plants that possess the ability to both inhibit and stimulate root length growth.Notably, the application of n hexane extract of C. rotundus at a concentration of 20% resulted in the highest inhibition of root dry weight, with a recorded inhibition rate of 64%.This rate did not exhibit a substantial deviation due to the application of n hexane extract at concentrations of 5%, 10%, and 15%.However, it was substantially dissimilar from the treatment with ethyl acetate extract and C. rotundus methanol at varying concentrations.All concentration of methanol extract of C. rotundus at 28 DAA stimulated root dry weight (20 %, 22 %, 9% and 5 %, respectively) over control.Figure 4 also showed that there was no consistent reduction in root dry weight where root weight in the ethyl acetate C. rotundus concentration treatment was 10% (22% inhibition) and 15% concentration experienced an inhibition of 7%.

GC-MS Analysis of Extracts
The primary components detected in the extracts of C. rotundus have been conferred in Table 2.The results of the GC-MS analysis revealed that the n-hexane extract predominantly comprised of 7-Isopropenyl-1,4a-dimethyl-4,4a,5,6,7, 8-hexahydro-3H-naphthalen-2-one (19.60%), followed by phthalic acid, hept-4-yl isobutyl ester (7.00%), and dibutyl phthalate (3.97 %).These compounds contributed to 30.57% of the overall composition as determined by GC-MS analysis.It is probable that these compounds played a role in the moderate phytotoxicity observed in A. spinosus when exposed to n-hexane, either independently or through a synergistic interaction.The compound 7-isopropenyl-1, 4a-dimethyl 4,4a,5,6,7,8-hexahydro-3H-naphthalen-2-one is registered under other names, namely (+)4,11-eudesmadien-3-one and (+) α-cyperone.[24].cyperone is the most active chemical αcomponent in puzzle grass.This compound belongs to the class of ketone sesquiterpenoids produced from the mevalonate acid pathway which has activity as an anti-feedant, hormone, antimicrobial, antibiotic and toxin as well as a plant growth regulator [25].α-cyperone is also found in the rhizome and tuber oils of nutgrass from South Africa and Saudi Arabia and in the essential oil of the nutgrass chemotypes (types H, K, M, and O) in Asia [26,27].This compound has good antioxidant activity, has a protective effect against DNA damage, a cytotoxic effect on human SH-SY5Y neuroblastoma cells, and also an antibacterial effect against foodborne pathogens [28].Phthalic acid has the capability to stimulate the antioxidant system by generating reactive oxygen species, leading to cellular harm and reduced growth of Mallus prunifolia [29].Phthalic acid and its secondary are recognized as major allelopathic autotoxic agents that create ongoing challenges for economically important crops such as tobacco, soybeans, and maize.They also show a critical part in the development of soil-borne diseases and pests, resulting in significant financial losses [30] [31].Dibutyl phthalate (DBP) has the capacity to impede seed germination and plant growth, hinder photosynthesis, and trigger oxidative harm [32].It is reported that dibutyl phthalate at low concentrations could increase nitrogen absorption but reduce phosphorus and potassium absorption, but at high concentrations it could inhibit N, P, and K uptake [33] According to the GC-MS analysis, the ethyl acetate extract was determined to primarily consist of 1,2,3-Propanetriol, 1-acetate (23.05 %) followed by 7-Isopropenyl-1,4a-dimethyl-4,4a,5,6,7,8hexahydro-3H-naphthalen-2-one (6.42%).The compounds mentioned above were found to constitute 29.47% of the total composition, ascertained through GC-MS analysis.In particular, 1,2,3-Propanetriol monoacetate, which is acknowledged for its antibacterial, anti-fungal, and antioxidant characteristics, was detected.The methanol extract contained mainly d-Glycero-d-ido-heptose (21.31%) followed by Octaethylene glycol monododecyl ether (39.15%).The aforementioned compounds contributed to 60.46% of the overall composition as determined by GC-MS analysis.

Conclusions
Upon conducting an analysis of leaf area, root length, dry shoot weight, dry root weight, and weed control, it was observed that all fractions of C. rotundus extract concentration at 20% exhibited bioherbicidal activity at the 28 days after application.The one with the highest potential is ethyl acetate extract concentration 20% with 64.33% inhibition, followed by methanol with 55.33% control and nhexane with 53.33% control.Henceforth, it can be concluded that ethyl acetate is the most efficacious extract for the formulation of a herbicidal agent targeting A. spinosus.

Figure 4 .
Figure 4. Effect of Cyperus rotundus L extract in the 3 extract at 28 DAA on A. spinosus leaf area, root length, shoot and root dry weight (%)

Table 1 .
The category system employed for evaluating the efficacy of weed control

Table 2 .
GC-MS analysis of the 3 extracts recovered from the aerial parts of Ageratum conyzoides.