xanthium strumarium research paper

Phytochemical investigation of the fruits of Xanthium strumarium and their cytotoxic activity

• Published: 07 January 2022
• Volume 76 , pages 468–475, ( 2022 )

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• Xiang-Wei Xu 1 ,
• Yi-Yuan Xi 2 ,
• Jun Chen 1 ,
• Feng Zhang 3 ,
• Ju-Jia Zheng 2 &
• Peng-Hai Zhang 3  

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Eight pentacyclic triterpenoids including two new ones ( 1 , 2 ) were isolated from the fruits of Xanthium strumarium . Their structures were elucidated by extensive spectroscopic analysis. All isolates were evaluated for in vitro cytotoxic activity on HepG2, A549, HCT116 and SW480 cancer cells. Among them, the new compound 2 was found to exhibit significant cytotoxic activity on A549, HCT116 and SW480 cancer cells with IC 50 values of 9.68, 4.27 and 7.58 μM, respectively. Further, 2 was selected for cell cycle analysis and results revealed that 2 could cause HCT116 cell cycle arrest in G1 phase. In addition, Annexin V-FITC/PI staining assay showed that 2 could induce the death of HCT116 cells.

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Acknowledgements

This work was financially sponsored by grant from the Chinese Medicine Science and Technology Program of Zhejiang Province (2021ZB324). In addition, we are grateful to the Department of Instrumental Analysis of Wenzhou Medical University for the measurement of the UV, IR, HREIMS and NMR, and the workers of Kunming Plant Biotechnology Co., Ltd.

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Xiang-Wei Xu & Jun Chen

School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, China

Yi-Yuan Xi & Ju-Jia Zheng

Department of Oncology, Yongkang Hospital of Traditional Chinese Medicine, Yongkang, 321300, People’s Republic of China

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Xu, XW., Xi, YY., Chen, J. et al. Phytochemical investigation of the fruits of Xanthium strumarium and their cytotoxic activity. J Nat Med 76 , 468–475 (2022). https://doi.org/10.1007/s11418-021-01588-w

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Received : 27 August 2021

Accepted : 24 November 2021

Published : 07 January 2022

Issue Date : March 2022

DOI : https://doi.org/10.1007/s11418-021-01588-w

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Traditional Uses, Botany, Phytochemistry, Pharmacology, Pharmacokinetics and Toxicology of Xanthium strumarium L.: A Review

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• 1 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. [email protected].
• 2 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. [email protected].
• 3 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. [email protected].
• 4 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. [email protected].
• 5 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. [email protected].
• 6 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. [email protected].
• 7 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. [email protected].
• 8 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. [email protected].
• 9 Sichuan Neautus Traditional Chinese Herb Limited Company, Chengdu 611731, China. [email protected].
• 10 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. [email protected].
• 11 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. [email protected].
• PMID: 30669496
• PMCID: PMC6359306
• DOI: 10.3390/molecules24020359

Xanthium strumarium L. (Asteraceae) is a common and well-known traditional Chinese herbal medicine usually named Cang-Er-Zi, and has been used for thousands of years in China. The purpose of this paper is to summarize the progress of modern research, and provide a systematic review on the traditional usages, botany, phytochemistry, pharmacology, pharmacokinetics, and toxicology of the X. strumarium . Moreover, an in-depth discussion of some valuable issues and possible development for future research on this plant is also given. X. strumarium , as a traditional herbal medicine, has been extensively applied to treat many diseases, such as rhinitis, nasal sinusitis, headache, gastric ulcer, urticaria, rheumatism bacterial, fungal infections and arthritis. Up to now, more than 170 chemical constituents have been isolated and identified from X. strumarium , including sesquiterpenoids, phenylpropenoids, lignanoids, coumarins, steroids, glycosides, flavonoids, thiazides, anthraquinones, naphthoquinones and other compounds. Modern research shows that the extracts and compounds from X. strumarium possess wide-ranging pharmacological effects, including anti- allergic rhinitis (AR) effects, anti-tumor effects, anti-inflammatory and analgesic effects, insecticide and antiparasitic effects, antioxidant effects, antibacterial and antifungal effects, antidiabetic effects, antilipidemic effects and antiviral effects. However, further research should focus on investigating bioactive compounds and demonstrate the mechanism of its detoxification, and more reasonable quality control standards for X. strumarium should also be established.

Keywords: Xanthium strumarium L.; botany; pharmacokinetics; pharmacology; phytochemistry; toxicology; traditional usages.

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Original research article, fractionation of xanthium strumarium l. foliage phenolics, in-vitro antioxidant activities, and in-vivo anti-diabetic potential.

• 1 Department of Chemistry, Government College University Lahore-Pakistan, Lahore, Pakistan
• 2 Division of Science and Technology, University of Education Lahore-Pakistan, Lahore, Pakistan
• 3 Department of Zoology, Lahore College for Women University Lahore-Pakistan, Lahore, Pakistan

Introduction: The present research aimed to fractionate Xanthium strumarium L. (XSL) foliage phenolics into a set of solvents and evaluate their antioxidant potential and in-vivo anti-diabetic activity against Alloxan monohydrate-induced diabetic mice.

Methodology: For this purpose, XSL foliage was fractionated into petroleum ether, ethyl acetate, ethanol, and water via orbital type shaking and tested for the presence of phenolics, and their antioxidant and antidiabetic potential.

Results and discussion: The results revealed that the ethyl acetate fraction of XSL foliage contained the highest amount of total phenolics 95.25 mg GAE/g of extract, followed by ethanol (65.14 mg GAE/g), petroleum ether (25.12 mg GAE/g), water (12.20 mg GAE/g), and XSL powder (69.13 mg GAE/g). At the end of treatment time (day 18 of oral administration of 400 mg/kg body weight of mice), the ethyl acetate fraction significantly ( p ≤ 0.05) lowered blood glucose level (353 ± 10.6 to 220 ± 25.5 mg/dL) which might due to the elevated level of phenolic compounds in this fraction.

Conclusion: Overall, it can be speculated that ethyl acetate and ethanol may work efficiently for the enrichment of XSL phenolic without compromising their antidiabetic potential.

1 Introduction

Despite the prevalence and widespread use of anti-diabetic medications like metformin, sulfonylureas, thiazolidinediones, and insulin, diabetes ranks as a major global health concern. International Diabetic Federation estimates indicate that the prevalence of diabetes has increased to 463 million in 2019 and figures may rise to 578 (10.2%) and 700 million (10.9%) by 2030 and 2045, respectively ( Akhtar et al., 2022 ). Additionally, it has been predicted that by 2030, diabetes will account for 3.3% of all fatalities worldwide, making it the seventh greatest cause of death ( Kokil et al., 2015 ). For centuries herbs have been used to meet healthcare needs through medical procedures and traditional remedies. Traditional medicine employs at least 1200 different plant species for their potential biological activities and almost half of these have been studied for their phytochemistry, and antioxidant activities ( Krupa et al., 2019 ; Mawoza et al., 2019 ).

Xanthium strumarium Linn (hereafter XSL) belongs to the Asteraceae family and is commonly identified as cocklebur, sheep bur, hedgehog bur weed, ditch bur, sea burdock, clot bur, and button bur by different civilizations in the world ( Ghahari et al., 2017 ). The plant has habitats worldwide, but temperate zones are the areas of high prevalence, especially India, Russia, Iran, Australia, North Korea, Japan, Pakistan, America, South Africa, China, Eastern Asia, and some parts of South Asia. It frequently grows along roadsides, and on plains, hills, and mountains. The flowering period starts during July/August whereas fruits are usually ripe in September/October ( Fan et al., 2019 ). The fruit as a whole clings to animal fur for its dispersal and is considered poisonous, however, it possesses cytotoxic, sedative diuretic, antitussive antimalarial, antispasmodic, anti-rheumatic antibacterial, and antifungal properties ( Clayton et al., 2020 ). Few researchers have evaluated the antidiabetic potential of X. strumarium L. (XSL), but the majority of these studies utilized aqueous or alcoholic extracts of XSL foliage ( Ahmad et al., 2016 ). An acute review of previously published research regarding phenolics reveals that the solubility of these compounds varies widely and similar can be speculated about the stability and biological functions of these bioactives. Keeping in view these facts, we have planned to fractionate XSL foliage phenolics into a range of solvents including petroleum ether, ethyl acetate, ethanol, and water and each fraction of XSL foliage was subsequently assessed for its total phenolic content (TPC), radical scavenging capacity, in-vitro antioxidant activity, and antidiabetic potential ( in-vivo ).

2 Materials and methods

The experimental work regarding the fractionation of XSL foliage was done in the Biochemistry laboratory of Government College University Lahore. The in-vivo trials associated with the present research were carried out at the animal house in the Lahore College for Women University’s Zoology Department. Formal approval was obtained from the institutional ethical committee (No. GCU-IIB-2596) dated 31 October 2022. Animals (Swiss albino mice) were supplied by F. Z traders, Lahore, Pakistan, and at the time of the experiment the average age and weight ranged from 4 to 8 weeks and 22–24 g, respectively. The chemicals and solvents used during the current study were of analytical grade and acquired from the companies Sigma Aldrich Chemical Co. (United States) and Merck (Germany).

2.1 Sample collection

The XSL foliage was collected during the month of June 2022 from a remote location in Pakistan’s District Narowal and was identified as rough cocklebur (voucher no. LCW -1016) by Dr. Shubnam Shaheen Associate Professor, Department of Botany, Lahore College for Women University Lahore.

2.2 Preparation of XSL fractions

The XSL foliage was air dried at 40°C after being cleaned with distilled water to remove dirt and contaminants. The dried XSL foliage was powdered prior to the fractionation with particle mesh sizes ranging from 24 to 60 sorted by electromagnetic vibrator for 15 min, packed in an opaque plastic bag, and stored. Petroleum ether, ethyl acetate, ethanol, and water were sequentially used as solvents during the fractionation process. A carefully-weighed 25 g of dried XSL foliage powder was placed in a 500 mL Erlenmeyer flask and shaken with 250 mL of petroleum ether for 12 h in an orbital shaker (Gallenkamp, UK) at 250 rpm under ambient conditions 25°C–30°C. The resultant aliquot was filtered using Whatman filter paper 1, and residues were mixed with the second extraction solvent, i.e. , ethyl acetate, and shaken under the above-mentioned conditions. The residues of step 2 and step 3 were subsequently subjected to ethanol- and water-based fractionation following the above-mentioned procedure. All four fractions were separately dried using a rotating evaporator (SB-651; EYELA, Tokyo, Japan) operated at 35°C and reduced pressure, weighed to calculate the percent yield of each fraction (g/100 g of XSL powder), and stored at −10°C until the required dosages were made. Finally, the amount of extractable bioactives (total extract) was calculated by adding the percent yield of each fraction.

2.3 Total phenolic content

The TPC in every fraction of XSL foliage powder was measured using Folin-Ciocalteu reagent as documented by ( Elagdi et al., 2023 ). Gallic acid was processed as a positive control to express the results as milligrams of gallic acid equivalent (mg GAE)/g) of extract. The five different concentrations of the gallic acid standard (50, 40, 30, 20, and 10 mg/mL) and 1.0 mg/mL of each XSL foliage powder fraction were introduced into the test tube containing 2.5 mL of 10% (v/v). To the Folin-Ciocalteu reagent was added 2 mL of 7.5% (w/v) Na 2 CO 3, and the mixture was allowed to stand in the dark at room temperature for half an hour. The absorbance was measured at 760 nm using a Shimadzu 160-UV spectrophotometer. For each extract, all the calculations were done in triplicates.

2.4 DPPH assay

The radical scavenging potential of each XSL foliage fraction was estimated by using the method described by Villano et al. with minor changes ( Villaño et al., 2007 ). In this assay, 0.1 mM solution of 1,1-diphenyl-2-picrylhydrazyl and 1.0 mg/mL of each XSL extract was prepared in HPLC grade methanol. The equal volumes 2.5 mL of DPPH solution and extract were incubated at room temperature for half an hour under dark, and at 517 nm absorbance was measured and compared with ascorbic acid acting as a standard. The free radical scavenging potential of the XSL fraction was expressed as %inhibition calculated by the following formula where As and Ac stand for the absorbance of the sample and control respectively. The IC 50 (Conc. providing 50% inhibition) value is used to express the DPPH assay results. IC 50 value can be measured by plotting a graph between the scavenging effect and the corresponding extract concentration. AAI (Antioxidant activity index) is used to measure the antioxidant potential and can be calculated by dividing the final DPPH concentration (µg/mL) by IC 50 (µg/mL) ( Scherer and Godoy, 2014 ).

2.5 FRAP assay

The ferric-reducing ability of plasma (FRAP) comprising XSL foliage fractions was determined by using the strain and Benzie method with minor changes ( Benzie and Strain, 1996 ). In this method, 300 µL distilled water was mixed in 100 µL of plant extract followed by the addition of 3 mL of frap reagent. The frap reagent was prepared by mixing 25 mL acetate buffer (0.3 M) at PH = 3.6 and 2.5 mL (10 mM) of TPTZ (2, 4, 6- tripyridyl-s-triazine) in hydrochloric acid (40 mM) and 2.5 mL of FeCl 3 (20 mM). The mixture was incubated under ambient conditions (30°C) and absorbance was measured at 593 nm by a Shimadzu 160-UV spectrophotometer. Frap assay evaluates the antioxidant activity of plant extract by reducing metal ions using electron donation. Ascorbic acid was used as the standard and the FRAP value was measured by the following equation, Where A O and A S are the absorbance of the standard and sample respectively.

2.6 Induction of diabetes in mice

For the animal study, 45 male albino mice were selected and were kept for 1 week of acclimatization. With a maximum of six animals per cage, the animals were housed in sterile polypropylene cages and kept at room temperature. The bedding material was sterile rice husk. These animals were housed in a controlled setting with respect to temperature (25°C), humidity (45%–57% range), and light period (12: 12-h dark-light cycle). Alloxan monohydrate was intraperitoneally injected at a dosage of 120 mg/kg to cause hyperglycemia in male albino mice that were more than 8 weeks old and weighed 22–24 g. The blood glucose levels were assessed by tail clip sampling 48 h following alloxan administration. When the blood sugar level exceeded 185 mg/dL, mice were considered diabetic ( Nagappa et al., 2003 ). The animals were divided into seven groups each having six mice and the body weight and blood glucose (using glucometer) level of control and treated groups were monitored on days 1, 3, 6, 9, 12, 15, and 18 of treatment.

C: Mice who had not been given any drug or alloxan monohydrate (Normal control).

DC: Mice were given alloxan monohydrate (Diabetic control).

XSCP: Diabetic mice were administered with crude powder of XSL.

XSPE: Diabetic mice were administered a petroleum ether fraction of XSL.

XSEA: Diabetic mice were administered with ethyl acetate fraction of XSL.

XSETH: Diabetic mice were administered an ethanolic fraction of XSL.

XSW: Diabetic mice were administered with an aqueous fraction of XSL.

2.7 Acute toxicity studies

The process was completed in accordance with OECD (Organization for Economic Co-operation and Development) guidelines to check the toxicity ( Ahmad et al., 2016 ). To investigate the acute toxicity, different doses of XSL foliage fractions were administered to male Swiss albino mice. The control group received the vehicle (normal saline only), whereas the treatment groups orally received plant fractions in dosages of 2 and 5 g/kg. The adverse effects were thoroughly monitored for 2 days after the dose was introduced. The body weight of the mice before and after administration, any sign of toxicity such as changes in the fur, skin, and eyes, as well as changes in the respiratory, circulatory, and central nervous systems, behavior patterns, signs of tumors, salivation, diarrhea, sleep, and coma were also noted.

2.8 Blood collection and determination of blood glucose

To determine the blood glucose level blood samples were collected from fasted mice on days 0, 1, 3, 6, 9, 12, 15, and 18 of the treatment. Blood was collected 1 from the mouse tails by snipping with a sharp razor, and glucose level was measured by glucometer (Accu-Chek Active, Roche Limited, Pakistan) 2 as described by Arya et al. (2012) .

2.9 Statistical analysis

All the parameters including extract yield, TPC, and DPPH radical scavenging activity were measured in triplicate and the estimates were compared 3 for significant difference p ≤ 0.05 by using Duncan’s multiple range 4 test (MRT) ( Tallarida and Murray, 1987 ).

3 Results and discussion

Table 1 shows the percentage yield 5 of fractions of XSL foliage depending on the solvent polarity that significantly affected the fraction yield in the order of petroleum ether < ethyl acetate < ethanol < water. Fraction yield depends upon the solvent polarity and the difference in fraction yield in different solvents was due to the solubility of phytochemicals in the respective solvent ( Dirar et al., 2019 ). TPC of different fractions of XSL in ethyl acetate was 95.25 ± 7.41 mg GAE/g, followed by ethanol 65.14 ± 7.06 mg GAE/g, petroleum ether 25.12 ± 7.06 mg GAE/g, and water 12.2 ± 5.80 mg GAE/g. The TPC in crude powder of XSL foliage was found to be 69.13 ± 6.01 mg GAE/g. In the previous studies TPC value of ethyl acetate and aqueous leave extract of XSL extracted by maceration and hot extraction method was 59.98 and 35.92 mg of GAE/g DW, respectively ( Pillai and Thebe, 2023 ). The TPC value of ethanolic extract prepared from the aerial parts of XSL using the cold percolation method was determined 84.86 ± 5.13 mg of GAE/g DW ( Ly et al., 2021 ). Similarly, aqueous and ethyl acetate fractions were obtained from 80% methanolic extract of XSL dried leaves using the cold percolation method, and their TPC values were determined as 75.24 ± 13.31 and 166.26 ± 27.98 mg of GAE/g DW ( Guemmaz et al., 2018 ; Pillai and Thebe, 2023 ). In other studies ethyl acetate and 80% ethanol extract were extracted from XSL dried leaves by dynamic maceration, static maceration, and soxhlet method, and their TPC values were determined as 70.07 ± 1.6, 64.51 ± 1.0, 69.38 ± 1.3 mg GAE/g DW (Ethanol), 27.19 ± 1.0, 21.98 ± 3.6 and 23.19 ± 0.3 mg GAE/g DW (Ethyl acetate) respectively ( Scherer and Godoy, 2014 ). No literature reported on petroleum ether fraction. The discrepancy in TPC value is due to factors like sample variety, sample quantity, type of extraction techniques, seasonal variation, extracted bioactive components, and geographic location.

TABLE 1 . The XSL foliage fraction yield in various solvents and their Total Phenolic Content (TPC).

3.1 Antioxidant activity

The DPPH assay evaluates the antioxidant’s scavenging capacity towards the DPPH radical. DPPH is a stable free radical having a blue color in alcoholic solution, while in the presence of antioxidants its reduction changes color to yellow. DPPH assay depends upon the antioxidant’s potential to donate hydrogen or electrons and is spectrophotometrically analyzed. Absorbance is inversely proportional to the antioxidant potential which is directly proportional to % inhibition. Table 2 shows the antioxidant activity of petroleum ether (XSPE), ethyl acetate (XSEA), ethanol (XSETH), and water (XSW) fractions of XSL. The IC 50 value decreased in the order of 101.20 > 88.02 > 54.60 > 46.11 and the antioxidant index value (AAI) increased in the order of 1.87 > 0.90 > 0.28 > 0.05 respectively. In the present study the ethyl acetate fraction has the lowest IC 50 value followed by the ethanol fraction, while in the previous literature the IC 50 value of different solvent extracts of Xanthium strumarium aerial parts has been determined. IC 50 values of aqueous and ethyl acetate extracts (obtained by maceration and hot percolation method) have been determined at 2465.21 and 1856.02 μg/mL, respectively ( Kim et al., 2005 ; Guemmaz et al., 2018 ; Pillai and Thebe, 2023 ). Similarly Aqueous and ethyl acetate fractions of 80% methanol extract of dried XSL leaves obtained by cold percolation method have demonstrated IC 50 values 46.00 ± 0.0006, 17.00 ± 0.0004 μg/mL respectively ( Pillai and Thebe, 2023 ). In another study, 80% ethanol and ethyl acetate extract prepared from XSL dried leaves by dynamic maceration, static maceration, and soxhlet method and their IC 50 values were determined as 53.01 ± 1.20, 47.83 ± 1.40, 53.34 ± 1.52 μg/mL (Ethanol), 369.83 ± 13.58, 346.35 ± 16.50, and 423.97 ± 22.27 μg/mL (Ethyl acetate) in the DPPH assay ( Scherer and Godoy, 2014 ). There was no literature reported on petroleum ether fraction. The variation in the IC 50 in the present study and previous reports might be due to the above-mentioned factors as discussed before.

TABLE 2 . DPPH Radical scavenging activity of different fractions of XSL foliage powder.

FRAP assay evaluates the antioxidant potential of antioxidants in terms of the reduction of ferric to ferrous ions. Ascorbic acid is used as standard and the values of reducing potential of antioxidants are compared to it. With the increase of antioxidant concentration, absorbance increases, and the resulting reducing power also increases. The frap value will be greater the higher the antioxidant potential. Table 3 depicts the Frap values and absorbance of different fractions of XSL foliage. Ethyl acetate has greater absorbance with FRAP value of 0.238 ± 0.06 (µm ascorbate/g) followed by ethanol (0.212 ± 0.07), petroleum ether (0.194 ± 0.04) and water (0.179 ± 0.01) µm ascorbate/g. In the literature cited the ferric reducing capacity of ethyl acetate and water extracts of XSL have been determined to be 0.996 ± 0.101 and 0.412 ± 0.009 µm ascorbate/g at a concentration of 100 μg/mL ( Kim et al., 2005 ; Pillai and Thebe, 2023 ). While the FRAP value in this research work was measured at 30 μg/mL. The FRAP values of ethanol and petroleum ether fractions were not reported in the literature.

TABLE 3 . FRAP Assay of different fractions of XSL foliage powder.

3.2 Body weight

Table 4 elaborates on the difference in body weight between the experimental and control group animals after treatment with different fractions of XSL foliage. The mice administered alloxan monohydrate (120 mg/kg) lost body weight from 1% to 3% which was averted by the treatment of different fractions and powdered samples of the selected plant. The control group animals (C) which use only the vehicle shows an increase in body weight from 21.5 ± 2.12 to 24.75 ± 1.77 (14%) while diabetic control (DC) loss in body weight from 23.50 ± 0.71 to 21.05 ± 0.78 (10%) on day 18 days.

TABLE 4 . Effect of different fractions of XSL on body weight in alloxan-induced diabetic mice.

The animals in the XSCP sample lost body weight by 23 ± 2.83 to 22.40 ± 2.69 (3%) on the first day and after receiving with powdered sample increased by 24 ± 2.82 (7%) on the last day of the trial. The XSPE and XSEA animals reduced their body weight from day 1 23.50 ± 0.71 to 23.15 ± 0.50 (1%) and 21.50 ± 0.71 to 21.15 ± 0.64 (2%) respectively and after being treated with petroleum ether and ethyl acetate fractions, the weight of XSPE and XSEA animals increased up to 23 ± 0.57, 24 ± 0.71 (1% and 13%) of their initial body weight. The animals treated with the ethanol and water fractions showed body weight reduction of 23.5 ± 0.71 to 23.05 ± 0.64 (2%), 22.75 ± 1.06 to 22.55 ± 1.06 (1%), and after treatment increased up to 24.15 ± 0.64, 22.70 ± 0.71 (5% and 1%) respectively. The results shown are based on the mean of six mice and the percentage change indicates the change from day 1 to the end of the study (day 18).

3.3 Antidiabetic activity

The blood glucose level of diabetic mice (CD) was significantly ( p ≤ 0.05) higher as compared to normal mice (C) and the effect of powdered sample and different fractions of XSL on diabetic mice was studied. Data assembled in Table 5 shows the blood glucose levels of the control and treatment groups on days 1, 3, 6, 9, 12, 15, and 18 of treatment. The blood glucose level of C (control group), who simply used a vehicle, did not vary significantly ( p ≤ 0.05) during the treatment time (as shown by subscript and superscript letters). However, the blood glucose level of diabetic mice (DC) was significantly ( p ≤ 0.05) higher as compared to the control group. Fasting mean blood glucose level of diabetic control DC and XSCP on day one after being diabetic was 345 ± 7.07 mg/dL, and 355 ± 7.07 mg/dL, respectively. On day 18 the blood glucose level of DC (diabetic control) increased up to 364 ± 2.12 mg/dL, while oral administration of powdered sample to XSCP significantly ( p ≤ 0.05) decreased the blood glucose level to 278 ± 10.6 mg/dL (22%). On day one, blood glucose levels of diabetic-induced mice of XSPE, XSEA, XSETH, and XSW were measured as 325 ± 21.2 mg/dL, 353 ± 10.6 mg/dL, 327 ± 9.2 mg/dL, and 316 ± 8.5 mg/dL, respectively. There was no substantial decrease in the blood glucose level of the XSPE group (treated with petroleum ether) observed and two mice out of six died on days 8 and 13 of the treatment, while on day 18 the mean blood glucose level of the remaining mice decreased upto 297 ± 26.2 mg/dL (9%). Animals treated with ethyl acetate (XSEA) showed a substantial decrease in the blood glucose level from day 1 353 ± 10.6 mg/dL to 220 ± 25.5 mg/dL (38%) on day 18. The blood glucose levels of animals treated with ethanol (XSETH) and water (XSW) fractions on the day 18 were 266 ± 15.6 mg/dL and 306 ± 14.1 mg/dL respectively.

TABLE 5 . Blood Glucose Level of alloxan-induced diabetic mice at different intervals after XSL fractions administration.

Ethanol fraction lowers the blood glucose level by up to 19% while water fraction decreases it by only 3%. Overall, it can be claimed that the ethyl acetate fraction of XSL (XSEA) contains substantial amounts of phenolic bioactive antioxidants which decreased the blood glucose level of diabetic mice. However, more comprehensive studies with a larger number of mice might be helpful to generalize these findings.

In the previous studies, the aqueous leaf extract of XSL was also used as an antidiabetic agent at a dose of 500 mg/kg and 250 mg/kg body weight respectively in alloxan-induced diabetic mice for 10 days and a significant reduction in blood glucose level was observed at the high dose of XSL aqueous extract ( Mouhamad, 2022 ). Similarly, methanolic extract of XSL leaves prepared by the soxhlet apparatus was used to determine the antioxidant potential ( in-vitro ) and antidiabetic potential ( in-vivo ) in albino rats by oral administration at a dose of 200 mg/kg and 400 mg/kg body weight for 9 days ( Umer et al., 2016 ). In another study hypoglycemic effect of ethanolic extract of XSL and its isolated compound were evaluated in alloxan-induced diabetic mice for 14 days and compared with Glibenclamide 10 mg/kg b. w; an antidiabetic drug ( Ahmad et al., 2016 ). Leaf and fruit methanolic extract of X. strumarium obtained by the soxhlet method was used to measure the antidiabetic effect in diabetic mice by oral administration of 400 mg/kg b. w of extract for a period of 24 days ( Harikumar et al., 2012 ). Methanolic extract of XSL stem was used to evaluate the hypoglycemic effect at doses of 100 mg/kg and 200 mg/kg body weight and antidiabetic potential was compared with Glibenclamide 0.6 mg/kg in Streptazocin-induced diabetic rats ( Narendiran et al., 2011 ). By surveying the literature, it can be observed that there is no research work published about crude foliage fractionation and comparison of their antioxidant, antidiabetic potential, and variation in body weight. In the present research work, four fractions of XSL were obtained and their total phenolic content (TPC), antioxidant, and antidiabetic potential were studied. The antidiabetic potential of ethyl acetate fraction (XSEA) fractions was comparable with standard Glibenclamide. By comparing results observed by all fractions, it has been observed that the XSEA fraction showed high TPC value, antioxidant potential and antidiabetic effect followed by the ethanol, petroleum ether, and aqueous fractions.

4 Conclusion

The aim of the current research was to assess the antidiabetic potential of different fractions of XSL foliage on diabetic mice. After 18 days of constant administration of XSL fractions, the blood glucose level of mice significantly decreased. These verdicts reinforced the traditional usage of the XSL foliage as an antidiabetic mediator as well as for the treatment of a number of ailments ( Rahman et al., 2022 ). In order to boost insulin release from pancreatic cells, the conventional medication Glibenclamide has been used to treat diabetes for several decades. Several plant extracts were reported to exhibit hypoglycemic effects through stimulatory actions on insulin release, which is a possible pathway by which plant extracts lower blood glucose levels. This mechanism involves elevating the pancreatic insulin secretion from islets of Langerhans β cells or through its breakdown from the confined form ( IfedibaluChukwu et al., 2020 ). According to some reports, other plants may also influence blood glucose levels by stimulating insulin release. Some wild herbs have chemical components that have hypoglycemic effects. There is evidence that certain types of plant-derived compounds, including carboxyatractyloside, chlorogenic acid, caffeic acid, and other phenolic compounds lower blood glucose levels. It has been found that plant’s extracts and metabolites have pharmacological effects. The results obtained supported the use of XSL in conventional medical system to cure diabetes. To determine the precise pathway of the anti-diabetic activity of the XSL fractions, a more comprehensive pharmacological and chemical research with large number of subjects is required.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

Ethics statement

The animal study was approved by Institute of industrial biotechnology Government College University, Lahore. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

AS conducted experiments and prepared the draft, MM conceptualized and supervised this research, and SS, SA, AR, and AA reviewed the draft. All the authors endorsed the final version of the article.

The authors declare financial support was received for the research, authorship, and/or publication of this article. The authors with affiliation are thankful to the Higher Education Commission of Pakistan for financial support of this research via NRPU project # 17549.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

1 Petroleum ether fraction of X. strumarium L. Foliage.

2 Ethyl acetate fraction of X. strumarium L. Foliage.

3 Ethanol fraction of X. strumarium L. Foliage.

4 Water fraction of X. strumarium L. Foliage.

5 Crude powder of X. strumarium L. Foliage.

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Keywords: Xanthium strumarium , L. Foliage, fractionations, phenolics, antioxidants, antidiabetics

Citation: Shaheen A, Akram S, Sharif S, Rashid A, Adnan A and Mushtaq M (2023) Fractionation of Xanthium strumarium L. foliage phenolics, in-vitro antioxidant activities, and in-vivo anti-diabetic potential. Front. Chem. 11:1279729. doi: 10.3389/fchem.2023.1279729

Received: 18 August 2023; Accepted: 30 October 2023; Published: 20 November 2023.

Reviewed by:

Copyright © 2023 Shaheen, Akram, Sharif, Rashid, Adnan and Mushtaq. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Muhammad Mushtaq, [email protected] ; Saima Sharif, [email protected]

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The influence of environmental factors on seed germination of xanthium strumarium l.: implications for management.

1 Government Reclamation Research Station, Mian Channu, Pakistan

Ansar Hussain

2 Department of Plant Breeding and Genetics, Ghazi University, D. G. Khan, Pakistan

Muhammad Ifnan Khan

Muhammad arif.

3 Department of Plant Protection, Ghazi University, D. G. Khan, Pakistan

Muhammad Mudassar Maqbool

4 Department of Agronomy, Ghazi University, D. G. Khan, Pakistan

Hassan Mehmood

5 Department of Soil Science, Bahauddin Zakariya University, Multan, Pakistan

Muhammad Iqbal

6 Cotton Research Institute, Multan, Pakistan

Jawaher Alkahtani

7 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia

Mohamed Soliman Elshikh

Associated data.

All relevant data are within the paper.

Xanthium strumarium L. (Common cocklebur) is a noxious weed prevailing in different ecosystems around the world. It incurs significant yield and economic losses in different cropping systems globally. Successful management of any weed species depends on sound knowledge of seed germination biology. However, detailed knowledge on seed germination biology of the species is missing. Therefore, we investigated the impact of different environmental factors on seed germination and seed burial depths on seedling emergence of two X . strumarium populations. The impact of different sorghum mulch doses (0–10 t ha -1 ) on seedling emergence of the tested populations was also explored. Seed germination was evaluated under different photoperiods (0, 12 and 24), constant temperatures (0–50°C with 5°C stepwise rise), and different levels of pH (3–12), salinity (0–600 mM) and osmotic potential (0 to -1.6 MPa). Seedling emergence was observed for seeds buried at different depths (0–15 cm). Seeds of both populations proved non-photoblastic; however, higher germination was recorded under 12-hour photoperiod. The seeds germinated under a wide range of constant temperatures (10–45°C), pH (4–10), osmotic potentials (0 to -0.8 MPa) and salinity levels (0–400 mM NaCl). However, the highest germination was observed under 30–31°C temperature and neutral pH (7.51–7.52). Seeds were able to withstand 400 mM salinity and -1.00 MPa osmotic potential. Seedling emergence was initially improved with increasing burial depth and then a sharp decline was noted for the seeds buried >3 cm depth. Most of the seeds of both populations did not emerge from >8 cm depth. Different sorghum mulch doses linearly suppressed seedling emergence of tested populations, and 5.83–5.89 t ha -1 mulch application suppressed 50% of seedling emergence. Seedling emergence was completely retarded with 8 t ha -1 sorghum mulch. The tested populations germinated under diverse environmental circumstances indicating that the species can become troublesome in marginal habitats and cropped lands. Deep burial of seeds and application of sorghum mulches suppressed seedling emergence. Thus, deep burial followed by shallow tillage and application of sorghum mulches could be used as a successful strategy to manage the species in agricultural fields. Nonetheless, management strategies must be developed to control the species in other habitats.

Introduction

Xanthium strumarium L. (Common cocklebur), a member of the Asteraceae, is an annual weed species propagated by seeds [ 1 , 2 ]. It is native to North America and Argentina [ 3 ], and regarded as a noxious weed species of corn and soybean crops throughout the world [ 4 – 8 ]. Moreover, it produce large amounts of allergenic pollens due to close relatedness of Xanthium and Ambrosia genus [ 9 – 11 ]. The contact with glandular hairs of the plant causes dermatitis in allergenic individuals [ 12 ]. Thus, the species exerts allergenic impact on human population and causes yield and quality losses in different crops.

The infestation of X . strumarium in cotton caused 5% yield losses in Mississippi, the USA [ 13 ]. Similarly, 6–27% cotton yield is lost in North Carolina [ 14 ] due to its infestation and critical period of competition is 2–10 weeks after crop emergence [ 15 ]. Similarly, groundnut yield is reduced by 31–39% with 0.5 plants m -2 density and yield losses may reach ~88% if the density increases to 4 plants m -2 [ 16 , 17 ]. Xanthium strumarium infestation in maize causes lower yield losses than in soybean, cotton and groundnut. A 10% reduction in maize yield occurs at 1 plants m -2 density and reaches to 27% with 4.7 plants m -2 [ 18 ]. Nonetheless, it also reduces the yield of horticultural crops [ 19 ]. A yield reduction of 5–50% is recorded in snap bean with 0.5 to 8 plants m -2 density [ 20 ].

Xanthium strumarium detrimentally influences livestock production as the animals eating the young plants of species may be poisoned. The young plants of the species are attractive and eaten by the pasture animals. Carboxyatractyloside, a poisoning compound is present in the leaves of young plants of the species, whereas it is not found in older plants [ 21 , 22 ]. The detrimental impacts of the species on grazing animals have been reported from Australia, where it is frequently noted in pastures [ 21 , 23 ]. Nonetheless, ‘burrs’ of the species are attached to legs, tails and manes; thus, causing discomfort to the animals.

Xanthium strumarium is distributed in several geographic regions of the world and started to exert negative impacts on crop yields, biodiversity and economy [ 24 – 27 ]. Therefore, management strategies are inevitable for the species. The successful management of any weed species depends on the sound knowledge of seed germination biology [ 28 – 30 ]. Seed germination is the first transition step from ‘seed’ to ‘seedling’ in the life cycle of plant species, and readily affected by various environmental factors [ 31 – 33 ]. Nonetheless, seed dormancy level of the seed at different times strongly regulates seed germination of different plant species [ 34 , 35 ]. Seed germination traits of weed species greatly vary within the same population [ 36 ] and among different populations of the same species [ 28 – 30 ]. Seed germination under wide range of environmental conditions guarantees successful establishment and dispersal. Similarly, retarding seed germination or making seeds dormant is the most successful weed management strategy [ 37 ]. However, sound knowledge of seed germination biology is essential to manage/suppress weed seed germination.

The seeds of X . strumarium are rarely dormant, although they possess impermeable seed coat, which becomes permeable soon after seed dispersal. The mature ‘burr’ contains two seed, of which lower one germinates immediately after dispersal (after dry storage for some time), whereas the upper seed remains dormant until the testa is intact [ 38 – 42 ]. More than 80% of the seeds produced by X . strumarium are viable and exhibit high germination potential [ 19 ]. Several studies have investigated the impact of individual environmental factors on seed germination of the species [ 43 , 44 ]. The seeds are non-photoblastic and do not have strict light requirement for germination [ 45 ]. The seeds rarely emerge if buried >15 cm depth [ 45 ]. The species requires high moisture for seed germination and rarely emerge if field capacity is <75% [ 46 ]. However, the seeds can absorb moisture under increased negative osmotic potential. The seeds lose their viability after few years of dispersal [ 47 ]. Although, some information is available on seed germination, the complete knowledge relating to the impact of different environmental factors on seed germination of X . strumarium is missing.

The use of different mulches has gained increased importance for moisture conservation, weed management and improve soil nutrients [ 48 – 52 ]. The use of plant-based mulches not only lowers cost incurred on crop production, but also solves residue management issue. Nonetheless, mulches did not pose negative impact to crop production; therefore, can be successfully used for weed management [ 53 , 54 ]. Sorghum [ Sorghum bicolor (L.) Moench] is an important allelopathic crop and have been reported to suppress seed germination and seedling emergence of several weed species in different crops [ 55 – 60 ]. However, the impact of sorghum mulches on seedling emergence of X . strumarium has merely been tested.

The current study was conducted to determine the impact of different environmental factors on seed germination of X . strumarium populations collected from agricultural and ruderal habitats. We were interested to know; i) whether there are differences among seed germination potential of populations stemming from different habitats, ii) are seeds able to germinate under diverse environmental conditions, iii) what is the optimum seed burial depth to retard seedling emergence and iv) what is the optimum dose of sorghum mulch to suppress the seedling emergence of the species. The results of the study would help to develop suitable and effective management strategies against the species.

Materials and methods

Site selection and seed collection.

The seeds of Xanthium strumarium L. populations were collected from Mian Channu. The seeds were collected at maturity, brought to lab, dried under shade (to meet after ripening requirements) and stored at 25°C until use. The seeds were collected from agricultural (30.419203, 72.300121) and ruderal (30.415984, 72.301836) population. Mature ‘burrs’ were collected from 50–60 mother plants. There are no specific permissions required for seed collection and the study did not involve any endangered species. Five laboratory and two greenhouse experiments were conducted to determine the seed germination biology of both populations.

General experimental procedure

The ‘burrs’ rapidly loose seed dormancy and dormancy release treatments are not required [ 38 , 42 ]. Therefore, ‘burrs’ of the species were used in the experiments and seeds were not taken off from the ‘burrs’. The 90×15 mm Petri dishes were used to observe seed germination of ‘burrs’ (seeds hereafter). The dishes contained two layers of Whatman no. 1 filter paper, which was moistened with 5 ml deionized water or treatment solution. Paraffin film was used to seal the Petri dishes in order to prevent moisture loss. The dishes were kept at respective environmental conditions for 21 days and then seed germination was observed. There were 20 seeds in one Petri dish and each treatment had five replications. Two Petri dishes were considered as a single replication; thus, each treatment had 10 dishes and 200 seeds. All germination experiments were conducted at 30°C and 12 hours photoperiod with an exceptions for temperature and photoperiod experiments. Seed germination was recorded 21 days after the initiation of the experiments. The non-germinating seeds were tested for viability according to Onen et al. [ 30 ] and germination was adjusted for viability. All experiments were terminated after 21 days and repeated over time (two experimental runs for each treatment). The experiments were laid out according to randomized complete block design with split plot arrangement. Populations were regarded as main plot, whereas experimental treatments were randomized in sub-plots.

Experiment 1: Photoperiod

Seeds were incubated under three different photoperiods (0, 12 and 24 hours) to observe seed germination. The incubators were illuminated with cool, white fluorescent lamps at 380 μ Em −2 s −1 intensity. The dishes of 0-hour photoperiod were wrapped in four layers of aluminum foil for excluding the effects of light.

Experiment 2: Constant temperatures

Seed germination of both populations was recorded under 10 different constant temperatures (5–50°C with 5°C stepwise increase).

Experiment 3: pH

Seed germination was noted under 3–11 pH levels. Thus, 10 pH levels were included in the experiment representing, acidic, neutral and alkaline medium. The method of Chauhan et al. [ 61 ] was used to prepare solutions of different pH levels.

Experiment 4: Salinity

Seed germination was recorded under eight different NaCl concentrations (50, 100, 150, 200, 300, 400, 500 and 600 mM). The control treatment (only distilled water) was also included in the experiment for comparison. Sodium chloride (NaCl) was dissolved in distilled water to make the solutions of respective concentrations [ 28 ].

Experiment 5: Osmotic potential

Seed germination of populations stemming from agricultural and ruderal habitats was observed under eight different osmotic potentials (-0.2 to -1.6 MPa), with -0.2 MPa difference among the treatments. The 0 MPa osmotic potential, regarded as control was included in the study for comparison. Polyethylene glycol 6000 was dissolved in distilled water to prepare solutions of respective osmotic potentials [ 62 ].

Experiment 6: Seed burial

The emergence of seeds arising from ruderal and agricultural populations was noted in a pot experiment conducted in greenhouse under controlled conditions. Ten different (0, 0.5, 1, 2, 4, 6, 8, 10, 12 and 15 cm) burial depths were included in the experiment. A total 20 seeds were buried at desired depth. Seedling emergence was recorded 21 days after the initiation of experiment. The pots were irrigated daily to exclude the danger of moisture stress. A mist sprinkler was used to irrigate the pots. The greenhouse was maintained at 30°C and 12-hour photoperiod throughout the experiment.

Experiment 7: Sorghum mulch

The impact of different doses of sorghum mulch on seedling emergence was recorded in pot experiment. The respective doses were applied at the soil surface after seed sowing. A total 20 seeds were sown in the pots and mulches were applied according to the treatments on soil surface. The greenhouse was maintained at 30°C and 12-hour photoperiod throughout the experiment. The pots were irrigated daily to exclude the danger of moisture stress. A mist sprinkler was used for irrigation. Sorghum plants (above ground parts) were dried to prepare mulches. The dried plants were chopped in a grinding mil to prepare mulches. The resultant powder was regarded as mulch and used according to the treatments.

Statistical analysis

The final germination percentage data collected were modelled using two different models (sigmoid and Gaussian). The final germination percentage data of osmotic potential, salinity and mulch experiments were modelled by three-parameter sigmd model. The model was

Here; G = seed germination percentage, G max = maximum germination percentage, T 50 = respective environmental condition for retarding 50% of maximum germination, and G rate = slope.

Similarly, final germination percentage data of temperature, pH and seed burial experiments were analyzed by a three-parameter Gaussian model. The model was:

Here, “a” = the highest seed germination or seedling emergence, “b” = respective environmental condition to achieve the highest germination or seedling emergence and “c” = width of the “bell”. The data of photoperiod experiment were analyzed by two-way analysis of variance (ANOVA) [ 63 ]. The homogeneity of variance and normality were tested prior to ANOVA [ 64 ]. Least significant difference at 5% probability was used to separate the means. SPSS version 21.0 [ 65 ], and SigmaPlot version 13.0 were used for ANOVA and models, respectively.

Seed germination was influenced by different photoperiods. The seeds of both populations had no strict light requirement for germination, i.e., non-photoblastic. However, higher seed germination percentage was recorded for 12-hour photoperiod compared to 0 and 24-hour photoperiods ( Fig 1 ).

Different constant temperatures strongly mediated the germination of both populations stemming from different habitats. An increase in seed germination percentage was recorded with rise in temperature up to 35°C and then a sharp decline was noted. Agricultural population had higher seed germination (92.66%) compared to ruderal population (81.74%). The highest germination was recorded at 30.62 and 31.08°C for agricultural and ruderal populations, respectively ( Fig 2 ).

Different pH levels included in the study altered seed germination of populations arising from different habitats. An increase in seed germination was recorded with increasing pH up to 8 and then seed germination sharply declined. Like temperature experiment, agricultural population exhibited higher seed germination (90.64%) compared to ruderal population (85.94%). The highest germination was recorded under 7.51 and 7.52 pH for agricultural and ruderal populations, respectively ( Fig 3 ).

Different salinity levels altered the seed germination of both populations. Seed germination was linearly decreased with increasing salinity level. Overall agricultural population had higher germination ability (95.71%) compared to ruderal population (93.28%). The 50% of the final germination of agricultural population was retarded by 248.55 mM NaCl salinity, whereas 240.56 mM was sufficient to retard 50% of the final seed germination of ruderal population ( Fig 4 ).

Different osmotic potentials strongly mediated the seed germination of tested populations. A linear reduction in seed germination of both populations was noted with increasing negative osmotic potential. The seeds of agricultural population exhibited higher germination (94.11%) compared to the seeds of ruderal population (87.87%). The osmotic potential required to retard the 50% of the final germination of agricultural population was -0.86 Mpa, whereas -0.87 osmotic potential retarded 50% of the final seed germination of ruderal population ( Fig 5 ).

Different burial depths strongly suppressed the seedling emergence of populations stemming from two distinct. An initial increase was witnessed in seedling emergence up to 3 cm and then a linear reduction was noted. Higher seedling emergence was recorded for agricultural population (82.44%) compared to ruderal population (71.54%). Peak seedling emergence was recorded at 2.88 and 2.87 cm seed burial depths for agricultural and ruderal populations, respectively ( Fig 6 ). Seedling emergence was <20 for the seeds buried at 8 cm depth. Similarly, rare seedling emergences was recorded for the seeds buried >12 cm burial depth.

The application of different doses of sorghum mulches strongly mediated the seedling emergence of the tested populations. A constant reduction in seedling emergence of both populations was observed with increasing dose of sorghum mulches. Higher seedling emergence was recorded for agricultural population (84.17%) compared to ruderal population (75.78%). The sorghum mulches required to stop 50% of the final seedling emergence percentage were 5.83 and 5.89 t ha -1 for agricultural and ruderal populations, respectively ( Fig 7 ). No seedling emergence was noted for >8 t ha -1 application of sorghum mulches in both populations.

Xanthium strumarium populations stemming from different habitats had higher seed germination potential under benign and adverse environmental conditions (Figs ​ (Figs2 2 – 5 ). High germination ability of both populations explain their successful naturalization in different habitats. Furthermore, seed germination ability further warrants range expansion ability to the areas facing different types of abiotic stress, including salinity and drought [ 28 , 30 ]. Seed dormancy and germination ability are strongly mediated by environmental conditions prevailing during seed set [ 36 , 66 ]. Seeds of different populations of the same species or even seeds of the same population significantly vary for their seed germination ability [ 28 – 30 , 36 ]. These inter and intra-population variations are explained by the adaptive ability and climate prevailing during seed development [ 36 ]. Several earlier studies have identified significant variations between different population of the same species for seed germination, growth and fecundity [ 28 – 30 , 34 , 67 ]. We report that tested populations stemming from different habitats differed in their seed germination ability (Figs ​ (Figs1 1 – 5 ), which is owed to their adaptive ability and environmental conditions faced by maternal plants. The genetic diversity within and among populations might be responsible for these differences; however, this claim needs thorough molecular investigations. The tested populations were able to germinate under diverse environments (Figs ​ (Figs1 1 – 5 ), indicating that both populations have a wide seed germination niche. There are several earlier studies indicating that weed species possess a broad seed germination niche, which help them to adapt stressful and benign environments [ 28 – 30 , 68 ].

Photoperiod had a slight impact on seed germination of tested populations. The seeds proved non-photoblastic; however, higher seed germination was noted for 12-hour photoperiod. The results are in line with Weaver and Lechowicz [ 19 ] who reported that seed germination of X . strumarium did not depend on light availability. Overall, ruderal population exhibited low germination than agricultural population, which can be attributed to the disturbance faced by agricultural population. The seeds of ruderal population did not face frequent disturbance; thus, are less tolerant to adverse environmental conditions.

Seed germination is strongly dependent on soil temperature since numerous enzyme activities are regulated by temperature [ 31 , 69 ]. The tested populations exhibited seed germination under almost all tested temperatures with little differences. The optimum temperature ranged from 31 to 32°C for agricultural and ruderal populations. The earlier findings have also indicated that optimum temperature for seed germination of X . strumarium is 30–35°C [ 43 , 44 ]. There were slight differences among tested populations for optimum temperature requirement. The differences in seed traits of different species due to selection in different environments have been explained earlier [ 70 , 71 ].

The establishment of plant species is strongly obstructed by salinity, pH and water stress. Germination ability under diverse pH, salinity and water stress levels guarantees the persistence and establishment of weed species [ 72 ]. The tested populations were able to germinate under diverse pH, salinity and osmotic potential levels. The germination of the tested populations under elevated pH, salinity and osmotic potential levels indicate that species could establish and persist in marginal habitats.

Seedling emergence increased up to 3 cm and followed by a sharp decline under deeper seed burials. The low emergence of the surface placed seeds can be linked to poor soil-seed contact and less water imbibition [ 33 ]. Seeds were able to emerge even form 8 cm seed burial depth. The emergence from deeper soil layers helps the species to persist in soil seed bank for longer time [ 73 ]. Several studies have reported that deep burial of the seeds of various weed species significantly decreased or even halted their seedling emergence [ 28 – 30 , 32 ]. It seems that burying seeds to maximum depth of emergence could combat the species agricultural habitats. However, topsoil is turned over by moldboard plow (conventional tillage) which help the seeds to included in the seed bank again. The weed management strategy must focus on decreasing seedling emergence, seed production and addition of seeds to soil seed bank. This will progressively reduce soil seed bank. Shallow tillage and management of the emerging seedlings through integrated weed management approach seems a viable option for the management of the species.

The use of different mulches is becoming popular in wake of sustainable agriculture [ 50 , 51 , 58 ]. Sorghum mulches have been successfully used to suppress the germination and growth of several weed species [ 56 , 57 , 60 ]. The applied mulched significantly reduced seedling emergence of the populations included in the study. Thus, deep burial, shallow tillage and application of sorghum mulches could be successfully used to manage the species in agricultural habitats.

The tested populations germinated under diverse environmental conditions, which indicates that the species can become noxious in marginal and cropped lands. The deep burial of seeds and application of mulches suppressed seedling emergence of the species. Thus, deep burial, shallow tillage and application of sorghum mulches could be successfully used to manage the species in agricultural habitats.

Acknowledgments

The authors extend their appreciation to the Researchers supporting project number (RSP-2020/193) King Saud University, Riyadh, Saudi Arabia.

Funding Statement

The current study was partially supported by Directorate of Land Reclamation, Government of Punjab, Pakistan. There was no additional external funding received for the study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability