Research Article | Open Access
Yumnam Asha Devi1 , Prathiba Gnanasekaran2 and Haorongbam Joldy Devi1
1Department of Microbiology, Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu, India.
2Department of Microbiology, Sathyabama Dental College and Hospital, Chennai, Tamil Nadu, India.
Article Number: 9755 | © The Author(s). 2024
J Pure Appl Microbiol. 2024;18(4):2528-2538. https://doi.org/10.22207/JPAM.18.4.24
Received: 23 July 2024 | Accepted: 01 October 2024 | Published online: 16 November 2024
Issue online: December 2024
Abstract

The goal of the present investigation was to demonstrate the antibacterial activity of different solvent extracts (methanol, ethanol, cold aqueous and hot aqueous) of Crassocephalum crepidioides against ATCC bacterial cultures of Staphylococcus aureus, Escherichia coli, Methicillin resistant Staphylococcus aureus, Pseudomonas aeruginosa and its antioxidant potential. Furthermore, the chemical constituents present in the extract was perused by Gas chromatography-mass spectroscopy (GC-MS), along with in vitro cytotoxicity assessment. All the extracts were shown to be sensitive against S. aureus, MRSA and P. aeruginosa except for the ethanolic extract which was resistant to P. aeruginosa. Of all the extracts, hot aqueous extract found to be the most effective. It was found that Minimum inhibitory concentration (MIC) value of hot aqueous extract against S. aureus, MRSA and P. aeruginosa were 5 mg/mL, 5 mg/mL and 40 mg/mL, respectively. DPPH results showed that C. crepidioides leaf extract has potent antioxidant activity with IC50 value of 57.9 µg/mL. 22 compounds were detected in hot aqueous extract through Gas chromatography-mass spectroscopy. The results of the cytotoxicity evaluation displayed that the IC50 value of the hot aqueous extract of C. crepidioides on Vero cell lines was 292 µg/mL. This study concludes that C. crepidioides leaf extract is non-toxic, has various bioactive components and strong antibacterial and antioxidant activities, thus making it a promising therapeutic agent for various biomedical applications.

Keywords

Antibacterial Activity, Antioxidant Potential, Crassocephalum crepidioides, Phytochemicals, GC-MS, Cytotoxicity, Vero Cell Lines

Introduction

Antibiotics are widely used for treating bacterial infections in human as well as animal. However, there has been a steady increase in the antibiotic resistant bacteria and decrease in the potential antibacterial drugs to treat various infections.1 Over-use, improper, unsystematic uses of antibacterial drugs and genetic mutations led to the development of drug resistance where the pathogens become ineffective to the available antibiotics.2 Antibiotic-resistant bacterial infections typically result in significant disease and mortality as well as economic problem on medical management globally.3 The Global Research on Antimicrobial Resistance study showed that almost 5 million human deaths were linked with drug-resistant bacterial infections.4 The diminishing efficacy of antibiotics and the rise of antibiotic resistance in pathogenic bacteria have led to a sustained exploration of medicinal plants for their potential to combat resistant strains.5

A free radical is a molecule that possesses an unpaired electron in one of its atomic orbitals and is capable of existing independently. They can function as oxidants or reductants by donating or accepting electrons from other molecules.6 In addition to the body’s normal, crucial metabolic developments, external causes such as contact to X-rays, ozone, tobacco smoke, air pollution and manufacturing chemicals can also produce free radicals and other reactive oxygen species (ROS).7 Overproduction of free radicals causes secondary damage through the cytotoxic and mutagenic effects of liberated metabolites, in addition to direct damage by the oxidation of DNA, proteins, lipids, and carbohydrates.8 Antioxidants are crucial in preventing cancers, cardiovascular diseases and neurological illnesses as well as aging and inflammation. Natural plant products are believed to have antioxidant properties which protect cells from the harmful effects caused by free radicals.9,10

For millennia, plants have been used to cure a variety of bacterial illnesses. A potential strategy to combat antibiotic-resistant pathogens involves harnessing the therapeutic properties of medicinal plants, which offer a wide range of promising remedies through their secondary compounds.11 Phytochemicals present in the plants include the phenolic compounds, alkaloids, flavonoids, saponin, tannin, quinones and coumarins, etc.12 Phenolic compounds are considered as important phytochemicals because of its antioxidant and antimicrobial properties.13 Flavonoids possess various medicinal benefits including antimicrobial, free radical scavenging, anti-inflammatory and antihistamines properties.14 Tannins are a class of polyphenols which contain antioxidant and antibacterial properties.15 Alkaloids has anticancer, antibacterial and anti-inflammatory properties.16,17

Crassocephalum crepidioides is a species belonging to Asteraceae Family which is an upright, annual and succulent herb growing in many tropical and subtropical regions. It is commonly known as Thickhead, Redflower rag leaf, Fireweed and locally as Terapaibi in Manipur, India.18 It has been utilized as food constituents and traditional medicine.19 The plant is found abundantly in the yards, roadsides, gardens and rice fields. It is traditionally used by local people of Manipur as a remedy for many ailments. The leaves are practised for the healing of stomach ulcer, hypertension and boosting the immune system.20 Leafy paste has been employed to treat minor wounds by the local people of Manipur.18 Researchers reported that it has antibacterial,21 antioxidant,22 anti-inflammatory,23 antidiabetic,22 antitumor24 and wound healing activity.25 Therefore, this study assesses the antibacterial, antioxidant potentials, along with the chemical constituents of Crassocephalum crepidioides and its toxicity nature was investigated on Vero cell lines to ensure the safe use of the plant.

Materials and Methods

Collection and extraction of the plant leaves
The green leaves of C. crepidioides (Benth.) S. Moore were collected from different areas of Kakching District of Manipur, India and was authenticated by Dr. P. Palani, Centre for Advanced Studies in Botany, University of Madras, Chennai. After being shade-dried for 10 days, the leaves were powdered and extracted using various solvents including methanol, ethanol, and water. In each Erlenmeyer flask, 25 g of the powdered medicinal plant leaves were mixed with 250 mL of the corresponding solvent and incubated in a shaking incubator at 28°C for 3 days.26 For the hot aqueous extract, 25 g of the powder was mixed with water and heated at 60°C for 2 hrs, then it was cooled and left undisturbed for 24 hrs.27 Subsequently, all the solutions were filtered, dried and stored for future use.

Qualitative screening of Phytochemicals
Each extract was screened for the investigation of six different phytochemicals such as tannin, saponin, phenols, flavonoids, terpenoids and alkaloids conferring to the following protocols.

Tannin test
The extract (0.5 g) was mixed with 10 mL of distilled water, then a few drops of 5% ferric chloride were added to the extract. Black or blue-green coloration was the indication of tannin presence.28

Saponin test
The foam formation that persists for 5 min after shaking the extract (0.5 g) with the distilled water (10 mL) denotes the positive result for saponins.29

Flavonoids test
Few drops of 10% NaOH solution was added to 1 mL of the extract. Initially, bright yellow color formed but it gradually turned colorless after the addition of dilute hydrochloric acid which confirms the positive results of flavonoids.29

Phenol test
The change of the aqueous extract to dark green or bluish-black after adding 10% ferric chloride solution indicates the presence of phenol.28

Terpenoids test
Salkowski test: Conc. H2SO4 (3 mL) was added slowly along the side of the test tube containing the extract (5 mL) and chloroform (2 mL). Terpenoids presence was confirmed by reddish-brown coloration at the junction of two layers.28

Alkaloids test
Mayer’s test: Cream colored precipitate formation after adding 1 mL of Mayer’s reagent (Potassium mercuric iodide solution) to the extract in a test tube was the indication of the positive result for alkaloids.29

Bacterial cultures
Four ATCC cultures such as Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were obtained from Centre for Drug Discovery and Development, Sathyabama University, Chennai and Methicillin resistant Staphylococcus aureus ATCC 43300 was purchased from HiMedia, Maharashtra, India. The obtained cultures were subculture onto the Nutrient agar medium and incubated for 24 hrs at 37°C. After the incubation, one colony was taken and inoculated into the Brain Heart Infusion broth, then, incubated for 3 hrs.

Antibacterial Activity Assay
The antibacterial potential of each extract was evaluated against the test organisms using the agar well diffusion method.30 Lawn culture of the test organisms was made onto the sterile Muller Hinton agar plates. Wells of 8 mm diameter were made using sterile micropipette tip. A stock solution of 500 mg/mL concentration of various solvent extracts was prepared and 100 µl from the stock solution was loaded onto the wells and the final concentration is 50 mg/mL, incubated at 37°C for 24 hrs. Ciprofloxacin (5 µg), distilled water and 20% DMSO (Dimethyl sulfoxide) were used as controls. After the incubation, inhibition zone diameter was measured. The experiment was conducted in triplicate.

Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC)
By employing agar well diffusion method, MIC of the solvent extract with strong antibacterial activity was determined against the test organisms. Two-fold dilutions were made to make the concentrations ranging from 3.125 to 400 mg/mL. Wells were created on Muller Hinton agar plates and lawn culture was made using sterile cotton swab. Following the addition of 100 µl of various conc. to each well, the plates were incubated.31 Distilled water and ciprofloxacin (5 µg) were utilized as negative and positive controls respectively. Zone of inhibition was noted after 24 hrs of incubation. The lowest concentration which produces zone of inhibition was considered to be the MIC of the extract.

MBC was performed by touching inhibition zone of MIC plate of 4 lowest concentration of plant extract showing invisible growth and subculture onto the Nutrient agar plates. Following a 24 hrs incubation period at 37°C, the bacterial growth on these plates was monitored. The concentration of the plant extract that did not produce any bacterial growth on freshly inoculated plates was considered as the MBC.32

Antioxidant activity
The antioxidant capacity of hot aqueous extract of C. crepidioides was assessed by measuring its 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Hot aqueous extract of C. crepidioides at varying concentrations (20, 40, 60, 80 and 100 µg/mL) was combined with 0.004% (w/v) methanol containing DPPH (0.1 mM). Ascorbic acid was employed as a standard. The solution was shaken vigorously and placed in the dark condition at room temperature for 30 mins. At 517 nm, the absorbance of the reaction mixtures was measured. The negative control was used as the blank sample containing distilled water and DPPH solution. The percentage of DPPH scavenging effect was evaluated.33

Inhibition % = (Absorbance of control-Absorbance of test sample)/(Absorbance of control) × 100

GC-MS
The chemical components of hot aqueous extract of C. crepidioides were analyzed by GCMS QP 2010 Ultra (Shimadzu, Japan) which was equipped with a Rtx-5 MS fused silica column (5% diphenyl 95% dimethyl polysiloxane 30 m x 250 µm, film thickness of 0.25 µm. At 250°C, the injector was operated. With a split ratio of 1:50, the samples were injected in the split mode and the injection volume was 1 µl. Helium gas as a carrier gas was used at a steady flow rate of 1 mL/min. The oven temperature was as follows: 80°C was the starting temperature, which was maintained for 4 mins. The temperature then increased to 100°C at a rate of 2°C/min and then to 280°C at a rate of 5°C/min, which was maintained for 5 mins. The total elution was 54 mins. The compounds identification was based on matching the obtained mass spectra with the available mass spectral records (Willey 8, NIST 11 and 17 libraries).

In vitro Cytotoxicity analysis
The toxicity of the hot aqueous extract of C. crepidioides was evaluated using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) reduction assay34 on Vero cell lines. Vero cells were deposited onto a 96-well microtiter plate that contained DMEM medium supplemented with 10% FBS, penicillin G and streptomycin at a density of 1×104 cells/well and incubated for 24 hrs at 37°C with 5% CO2. Following this, the medium was substituted with new medium, then, the various concentrations (1-500 µg/mL) of hot water extract of C. crepidioides were added and incubated for 24 hrs. After incubation, MTT reagent was added and incubated for 4 hrs. After removing the medium containing the MTT reagent, DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was measured to determine the cell viability (%) and calculated according to the formula35:

 Cell viability (%) = (Absorbance of Sample)/(Absorbance of Control)×100

Statistical study
The data were displayed as mean±SD. Using SPSS version 25.0 software, the obtained data of antibacterial activity were studied by one-way analysis of Variance (ANOVA) and Duncan test with p-value of ˂0.05 being considered statistically significant.

RESULTS AND DISCUSSION

Phytochemicals screening
Medicinal herbs have been discovered to be the rich source of phytochemicals with various pharmacological activities that can be utilized as a promising counteragent to treat a variety of illness. Phytochemicals present in the plant have been explored for their pharmacological action and potency including antibacterial activities.21 Qualitative phytochemicals screening was performed for each extract for the presence and absence of tannin, saponin, phenol, alkaloids, flavonoids and terpenoids. The results (Table 1) revealed that Tannin and Phenols were detected in methanolic, cold and hot aqueous except ethanolic extract. Saponin was present in cold and hot aqueous extract but absent in methanolic and ethanolic extracts. Methanolic and ethanolic extracts showed positive results for alkaloids. Flavonoids were present in all the extracts. Terpenoids was found only in hot aqueous extract. The presence of these phytochemicals in the plant extract can deliver a first description for their antibacterial properties. The variations in the antimicrobial properties of the extract may result from variations in both their chemical makeup and the way that their bioactive components work.36

Table (1):
Phytochemicals screening of methanolic, ethanolic, cold and hot aqueous extracts

Phytochemicals
Methanol
Ethanol
Cold aqueous
Hot aqueous
Tannin
+
+
+
Saponin
­-
+
+
Phenols
+
+
+
Alkaloids
+
+
Flavonoids
+
+
+
+
Terpenoids
+

‘+’ denotes the presence of the phytochemical and ‘-’ denotes the absence of the phytochemical

The findings of our analysis of phytochemical screening of hot aqueous extract C. crepidioides were consistent with previously published research; yet, alkaloids were not present.21 Adjatin et al. revealed that flavonoids, gallic tannin, and cathartic tannin were detected in the C. crepidioides aqueous extract.37 Alkaloids and terpenoids were absent from the cold aqueous extract, while tannin, saponin, flavonoids, and phenol were detected. There were alkaloids and flavonoids in the ethanolic extract. In the methanolic extract, tannin, alkaloids, flavonoids, phenol were present whereas saponin, terpenoids were absent.

Antibacterial activity assay
The antibacterial potential of methanolic, ethanolic, cold and hot aqueous extracts of C. crepidioides were performed by agar well diffusion method against the test organisms. S. aureus, MRSA and P. aeruginosa were found to be sensitive to the methanolic extract of C. crepidiodes. The ethanolic extract was found to be sensitive to S. aureus and MRSA. Both the cold and hot water produced significant inhibition zone against S. aureus, MRSA and P. aeruginosa. Among the extracts, hot aqueous extract was found to produce higher zone of inhibition when compared to other extracts which was shown in Table 2.
S. aureus and MRSA was the most sensitive to all of the solvent extracts while P. aeruginosa was the least sensitive to methanol, cold and hot aqueous, but resistant to ethanolic extract. It was found that E. coli was resistant to all the extracts.

Table (2):
Inhibition zone in diameter of different solvent extracts of C. crepidioides, Ciprofloxacin, Distilled water and DMSO against the test organisms

Extracts
S. aureus
P. aeruginosa
E. coli
MRSA
Ethanol
10.83 ± 0.28e
11.84 ± 0.76d
Methanol
12.5 ± 0.5d
11.16 ± 0.28b
12 ± 0.5d
Cold water
16 ± 0.5c
11 ± 0.5b
15.83 ± 0.29c
Hot water
17.33 ± 0.57b
12 ± 1b
18.16 ± 0.76b
Ciprofloxacin (Positive control)
22.16 ± 0.76a
28 ± 0.5a
31.16 ± 0.76a
22.5 ± 0.5a
Distilled water (Negative control)
DMSO (Negative control)

Results expressed as mean ± SD (mM). Different letters in the same column denotes significantly different (p ˂ 0.05)

Among the extracts, hot aqueous extract was shown to be the most efficient with the zone of inhibition of 17.33 ± 0.57 mm and 12 ± 1 mm against S. aureus and P. aeruginosa respectively. Omotayo et al. revealed that the hot aqueous extract of C. crepidioides were sensitive to Staphylococcus aureus, Klebsiella pneumonia and Escherichia coli.21 The essentials oils of C. crepidioides was effective against pathogenic organisms.38 The variation in these findings might be due to the plant extraction process, concentration and type of cultures used. As previously reported, the methanolic extract of C. crepidioides was found to be sensitive against S. aureus and P. aeruginosa, which is consistent with the present study.39

MIC and MBC
Since the hot aqueous extract of C. crepidioides revealed highest inhibition zone, the hot aqueous extract of C. crepidioides was used to check the MIC. The MIC value of hot aqueous extract was found to be 5 mg/mL against S. aureus (13.66 ± 0.28 mM), MRSA (12.16 ± 0.28 mM) and is not determined for P. aeruginosa (Figure 1). According to Omotayo et al., the hot aqueous extract of C. crepidioides was sensitive against S. aureus with MIC value of 45 mg/mL.21

Figure 1. MIC of hot aqueous extract of C. crepidiodes against S. aureus and MRSA

The concentration showing no bacterial growth after subculturing the MIC onto the nutrient agar plate was considered as the MBC. Hot aqueous extract of C. crepidioides was able to kill S. aureus and MRSA having MBC of
10 mg/mL respectively.

Antioxidant activity
The DPPH radical scavenging activity of hot aqueous extract of C. crepidioides was tested to check its antioxidant potential by DPPH assay. The scavenging ability of the tested extract showed significance in a concentration-dependent manner at low concentration. Scavenging activity increased as concentration increased. It was found that the scavenging activity of hot aqueous extract of C. crepidioides was 78 ± 1.9% (100 µg/mL), 67.5 ± 1.7% (80 µg/mL), 53.6 ± 1.7% (60 µg/mL), 33 ± 1.8% (40 µg/mL) and 26 ± 1.7% (20 µg/mL) which is shown in Figure 2. The IC50 value of hot aqueous extract of C. crepidioides and ascorbic acid was found to be 57.9 µg/mL and 40.9 µg/mL, respectively. Antioxidant activity increases with decreasing IC50 value and vice versa. IC50 is the concentration needed in a system to scavenge 50% of DPPH radicals.23 The current findings demonstrated that C. crepidioides is a potent antioxidant which may be helpful in the management of pathological harm caused by free radicals.

Figure 2. DPPH radical scavenging activity of hot aqueous extract of C. crepidioides and Ascorbic acid

According to Akinpelu et al., hot water extract of C. crepidioides exhibits antioxidant activity with an IC50 value of 0.29 mg/mL.23 The flavonoids and phenolics present in the extract may help reduce oxidative stress in cells and hinder the activities of a-amylase and a-glucosidase, acetylcholinesterase and butyrylcholinesterase.40 Many phenolic compounds produced from plants such as flavonoids, anthocyanins, catechins, etc., have demonstrated strong antioxidant activity.41

GC-MS
The GC-MS chromatogram of hot aqueous extract of C. crepidioides was shown in Figure 3.

Figure 3. GC-MS chromatogram of C. crepidioides hot aqueous extract

By comparing the observed spectra with the available standards, 22 compounds were found (Table 3) from the hot aqueous extract of C. crepidioides.

Table (3):
Compounds detected through GC-MS analysis, along with their retention time, area % and chemical formula in the hot aqueous extract of C. crepidioides

Peak
Retention Time (RT)
Area (%)
Name
 Formula
1
4.493
0.50
Silicic acid, diethyl bis(trimethylsilyl) ester
C10H28O4Si3
2
5.466
2.19
Cyclotetrasiloxane, octamethyl-
C8H24O4Si4
3
6.258
1.01
Penta siloxane, dodecamethyl-
C12H36O4Si5
4
32.510
2.50
Hexadecanoic acid, methyl ester
C17H34O2
5
32.611
2.39
Pentadecanoic acid, methyl ester
C16H32O2
6
34.380
0.84
Benzene propanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, ethyl
C19H30O3
7
34.466
2.63
4-(3,5-Di-tert-butyl-4-hydroxyphenyl) butyl acrylate
C21H32O3
8
34.550
1.78
Hexadecanoic acid, ethyl ester
C18H36O2
9
34.620
2.88
Hexadecanoic acid, ethyl ester
C18H36O2
10
36.126
14.89
13-Hexyloxacyclotridec-10-en-2-one
C18H32O2
11
36.195
3.13
Bicyclo [8.2.0] dodecan-11-one, 12-chloro-
C12H19C1O
12
37.018
5.30
9,12-Octadecadienoic acid (Z, Z)-, methyl ester
C19H34O2
13
37.198
14.00
9-Octadecenoic acid, methyl ester, (E)-
C19H36O2
14
37.830
5.17
Methyl stearate
C19H38O2
15
39.189
1.21
Octadecanoic acid, ethyl ester
C20H40O2
16
40.881
0.78
9-Octadecenoic acid, 12-hydroxy-, methyl ester, (Z)-
C19H36O3
17
41.975
8.65
Ricinoleic acid
C18H34O3
18
43.764
1.38
9-Octadecenoic acid (Z)-, oxiranylmethyl ester
C21H38O3
19
45.765
1.53
13-Hexyloxacyclotridec-10-en-2-one
C18H32O2
20
46.445
16.48
Glycidyl (Z)-9-nonadecenoate
C22H40O3
21
49.077
1.34
Hydroxycitronellal, trimethylsilyl ether
C13H28O2Si
22
49.892
9.44
13-Hexyloxacyclotridec-10-en-2-one
C18H32O2

Some of the abundantly found compounds were Glycidyl (Z)-9-nonadecenoate (16.48%), 9-Octadecenoic acid, methyl ester, (E)- (14.00%), 13-Hexyloxacyclotridec-10-en-2-one (9.44%), Ricinoleic acid (8.65%), 9,12-Octadecadienoic acid (Z, Z)-, methyl ester (5.30%), Methyl stearate (5.17%), Hexadecanoic acid, ethyl ester (2.88), Hexadecanoic acid, methyl ester (2.50%), Cyclotetrasiloxane, octamethyl- (2.19%), etc. Among the identified compounds, Hexadecenoic acid, ethyl ester has antioxidant, pesticide and nematicide properties as previous literature reported.42 9-octadecenoic acid methyl ester has antioxidant activity,43 antibacterial activity as previously reported.44 Cyclotetrasiloxane, octamethyl- have been reported to possess antimicrobial property.42 Hexadecenoic acid, methyl ester has antimicrobial, anti-inflammatory, anti-cancer, hepatoprotective, hypocholesterolemic, antihistaminic, antiarthritic properties.45,46 Persson et al. reported that ricinoleic acid has antibacterial effect.47 The majority of the obtained compounds have mainly antimicrobial, antioxidant and anti-inflammatory properties. These phytoconstituents also referred to as ‘phytoprotectants’ which are of ecological relevance in biomedical study.48

Cytotoxicity analysis
The cytotoxicity analysis was done on Vero cell lines to ensure the safe use of hot aqueous extract of C. crepidioides. IC50 value of less than 20 µg/mL was considered to be toxic as previously reported.49,50 In the current study, IC50 value of hot aqueous extract of C. crepidioides was 292 µg/mL, hence indicating the non-toxicity of the extract to Vero cell lines as shown in Figure 4.

Figure 4. Cytotoxicity analysis of hot aqueous extract of C. crepidioides on Vero cell lines

Inhibitory concentration is the concentration that kills 50% of viable cells and is written as IC50. Nguyen reported that C. crepidioides hydroethanolic leaf extract showed no toxicity to the cells at the concentrations of 125, 62.5, and 31.25 µg/mL.25

CONCLUSION

The results of the current study concludes that the hot aqueous extract of C. crepidioides leaf exhibits better antibacterial activity than the methanolic, ethanolic and cold aqueous extract. S. aureus was the most sensitive to hot water extract of C. crepidioides. The antioxidant potential of C. crepidioides was confirmed by DPPH assay. Phytochemicals such as tannin, saponin, flavonoids, phenol and terpenoids were found in the extract which are accountable for their antibacterial and antioxidant activities. GC-MS study gives an insight into bioactive compounds present in the extract which possess many biological properties. Hence, C. crepidioides leaf extract can be utilized as an alternative therapeutic agent for various biomedical applications. Further investigation on the isolation of bioactive compounds is needed.

Declarations

ACKNOWLEDGMENTS
None.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

FUNDING
None.

DATA AVAILABILITY
All datasets generated or analyzed during this study are included in the manuscript.

ETHICS STATEMENT
Not Applicable.

References
  1. Famuyide IM, Aro AO, Fasina FO, Eloff JN, McGaw LJ. Antibacterial and antibiofilm activity of acetone leaf extracts of nine under-investigated south African Eugenia and Syzygium (Myrtaceae) species and their selectivity indices. BMC Complement Altern Med. 2019;19:(1)141.
    Crossref
  2. Vaou N, Stavropoulou E, Voidarou C, Tsigalou C, Bezirtzoglou E. Towards Advances in Medicinal Plant Antimicrobial Activity: A Review Study on Challenges and Future Perspectives. Microorganisms. 2021;9(10):2041.
    Crossref
  3. CDC. Antibiotic Resistance Threats in the United States, 2019. Atlanta, GA: U.S. 2019 Antibiotic Resistance Threats Report; Antimicrobial Resistance | CDC. Accessed 20 March, 2024.
  4. Food and Agriculture Organisation of the United Nations. Antimicrobial Resistance. https://www.fao.org/antimicrobial-resistance/background/what-is-it/en/. Accessed 2 December, 2023.
  5. Al-Abboud MA, Ismail KS, Mashraqi A, Albishi S, Al-Namazi AA, Masrahi YS. GC-MS analysis and antibacterial activities of some plants belonging to the genus Euphorbia on selected bacterial isolates. Open Chemistry. 2023;21(1):20220325.
    Crossref
  6. Cheeseman KH, Slater TF. An introduction to free radical biochemistry. British Medical Bulletin. 1993;49(3):481-493.
    Crossref
  7. Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacogn Rev. 2010;4(8):118-26.
    Crossref
  8. Favier A. Oxidative stress in Human diseases. Ann Pharm Fr. 2006;64(6): 390-396.
    Crossref
  9. Willcox JK, Ash SL, Catignani GL. Antioxidants and prevention of chronic disease. Crit Rev Food Sci Nutr. 2004;44(4):275-295.
    Crossref
  10. Zouirech O, Alyousef AA, EI Barnossi A, et al. Phytochemical Anaiysis and Antioxidant, Antibacterial, and Antifungal Effects of Essential Oil of Black Caraway (Nigella sativa L.) seeds against Drug-Resistant Clinically Pathogenic Microorganisms. Biomed Res Int. 2022;2022(1):5218950.
    Crossref
  11. Anand U, Nandy S, Mundhra A, Das N, Pandey DK, Dey A. A review on Antimicrobial Botanicals, Phytochemicals and Natural Resistance Modifying Agents from Apocynaceae Family:Possible Therapeutic Approaches against Multidrug Resistance in Pathogenic Microorganisms. Drug Resistance Updates. 2020;51:100695.
    Crossref
  12. Mohammed MJ, Anand U, Altemimi AB, Tripathi V, Guo Y, Pratap-Singh A. Phenolic Composition, Antioxidant Capacity and Antibacterial Activity of White Wormwood (Artemisia Herbia-alba). Plants. 2021;10(1):164.
    Crossref
  13. Naczk M, Shahidi F. Extraction and analysis of phenolics in food. J Chromatogr A. 2004;1054(1-2):95-111.
    Crossref
  14. Montoro P, Braca A, Pizza C, De-Tommasi N. Structure-antioxidant activity relationships of flavonoids isolated from different plant species. Food Chem. 2005;92(2):349-355.
    Crossref
  15. Fraga-Corral M, Otero P, Cassani L, et al. Traditional Applications of Tannin Rich Extracts Supported by Scientific Data: Chemical Composition, Bioavailability and Bio accessibility. Foods. 2021;10(2):251.
    Crossref
  16. Nithya TG, Jayanthi J, Ragunathan MG. Antioxidant activity, Total phenol, Flavonoid, Alkaloid, Tannin, and Saponin contents of Leaf Extracts of Salvinia molesta D. S. Mitchell (1972). Asian J Pharm Clin Res. 2016;9(1):200-203.
  17. Asha Y, Prathiba G, Joldy H, Balakumar V. Countermeasure for bacteria causing wound infection. IJBPAS. 2023;12(4):1632-1644.
    Crossref
  18. Das A. Evaluation of the effect of Crassocephalum crepidioides on the wound healing in Albino rats. Int J Adv Res. 2022;10(10):402-411.
    Crossref
  19. Silalahi M. Crassocephalum crepidioides (Bioactivity and Utilization). International Conference of Education and Science. 2022.
    Crossref
  20. Sujata W, Laitonjam WS. Constituents of the leaf’s extracts of Crassocephalum crepidioides (Benth.) Moore. Indian J Chem. 2015;54(3):439-443
  21. Omotayo MA, Avungbeto O, Sokefun OO and Eleyowo OO. Antibacterial activity of Crassocephalum crepidioides (Fireweed) and Chromolaena odorata (Siam weed) hot aqueous leaf extract. Int J Pharm Bio Sci. 2015; 5(2):114-122. https://ijpbs.com/ijpbsadmin/upload/ijpbs_55942e15ef74e.pdf
  22. Adedayo BC, Oyeleye SI, Ejakpovi II, Oboh G. Effects of hot water treatment on the radicals scavenging, lipid peroxidation, and a-amylase and a-glucosidase inhibitory abilities of Crassocephalum crepidioides leaves. Nutrafoods. 2015;14(4):217-225.
    Crossref
  23. Akinpelu BA, Godwin A, Gbadegesin T, et al. Comparative Studies on Anti-Inflammatory, Antioxidant and Antimutagenic Activities of Crassocephalum crepidioides (Bent) Leaf Cold and Hot Water Extracts. Asian Food Science Journal. 2019;9(1):1-12.
    Crossref
  24. Tomimori K, Nakama S, Kimura R, Tamaki K, Ishikawa C, Mori N. Antitumor activity and macrophage nitric oxide producing action of medicinal herb, Crassocephalum crepidioides. BMC Complement Altern Med. 2012;12:78.
    Crossref
  25. Can NM, Thao DTP. Wound healing Activity of Crassocephalum crepidioides (Benth) S. Moore. Leaf Hydroethanolic Extract. Oxid Med Cell Longev. 2020;2020:2483187.
    Crossref
  26. Begashaw B, Mishra B, Tsegaw A, Shewamene Z. Methanol leaves extract Hibiscus micranthus Linn exhibited antibacterial and wound healing activities. BMC Complement Altern Med. 2017;17:337.
    Crossref
  27. Abdelgadir AE, Karamallah AA, Abualhassan AM, Hamid AA, Sabahelkhier MK. Antimicrobial activities of Syzygium cumini leave extracts against selected microorganisms. Nova Journal of Medical and Biological Sciences. 2015;4(2):1-10.
    Crossref
  28. Iqbal E, Salim KA, Lim LBL. Phytochemical screening, total phenolics and antioxidant activities of bark and leaf extracts of Goniothalamus velutinus (Airy Shaw) from Brunei Darussalam. J King Saud Univer Sci. 2015;27(3):224-232.
    Crossref
  29. Devi HJ, Gnanasekaran P, Devi YA. Selection of effective plant extract as a disinfecting agent using hot and cold-water extraction. Eco Env Con. 2021;28(4):1874-1881.
    Crossref
  30. Alarfaj K, Almatroudi A, Alrumaihi F, et al. Evaluation of the White Garden Snail (Theba pisana) Mucus Slime for its Efficacy as an Antimicrobial Agent. J Pure Appl Microbiol. 2024;18(2):900-906.
    Crossref
  31. Venkateswarulu TC, Srirama K, Mikkili I, et al. Estimation of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of antimicrobial peptides of Saccharomyces boulardii against selected pathogenic strains. Karbala International Journal of Modern Science. 2019;5(4).
    Crossref
  32. Mostafa AA, Al-Askar AA, Almaary KS, Dawoud TM, Sholkamv EN, Bakri MM. Antimicrobial activity of some plant extracts against bacterial strains causing food poisoning diseases. Bakri Saudi J Biol Sci. 2018;25(2):361-366.
    Crossref
  33. Boppudi HB, Rao YS, Kuchi C et al. Zinc oxide nanoparticles as an efficient antioxidant, photocatalyst, and heterogeneous catalyst in C-P bond synthesis. Results in Chemistry. 2023;6:101227.
    Crossref
  34. Njeru SN, Obonyo MA, Nyambati SO, Ngari SM. Antimicrobial and Cytotoxicity properties of the crude extracts and fractions of Premna resinosa (Hochst.) Schauer (Compositae): Kenyan traditional medicinal plant. BMC Complement Altern Med. 2015;15:295.
    Crossref
  35. Fouda A, Al-Otaibi WA, Saber T, et al. Antimicrobial, Antiviral and In-Vitro Cytotoxicity and Mosquitocidal Activities of Portulaca oleracea- Based Green Synthesis of Selenium Nanoparticles. J Funct Biomater. 2022;13(3):157.
    Crossref
  36. Hemeg HA, Moussa IM, Ibrahim S, et al. Antimicrobial effect of different herbal plant extracts against different microbial population. Saudi J Biol Sci. 2020;27(12):3221-3227.
    Crossref
  37. Adjatin A, Dansi A, Badoussi E, et al. Phytochemical screening and toxicity studies of Crassocephalum rubens (Juss. Ex Jacq) S. Moore and Crassocephalum crepidioides (Benth.) S. Moore consumed as vegetable in Benin. Int. J. Curr. Microbiol. App. Sci. 2013; 2(8): 1-13. https://www.ijcmas.com/vol-2-8/A.Adjatin,%20et%20al.pdf
  38. Owokotomo IA, Owokotomo EP. Antibacterial and brine shrimps’ lethality studies of the essential oils of Crassocephalum crepidioides (Benth S. More) grown in south west Nigeria. Afr J Pure Appl Chem. 2018;12(1):1-7.
    Crossref
  39. Baral R, Karki A, Karki S, et al. Phytochemical Screening, Free Radical Scavenging, and In vitro Anti-bacterial Activity Studies of various extracts of selected medicinal plants of Nepal. Current Perspectives on Medicinal and Aromatic Plants. 2021;4(1):22-35.
    Crossref
  40. Adedayo BC, Oboh G, Oyeleye SI, Ejakpovi II, Boligon AA, Athayde ML. Blanching alters the phenolic constituents and in vitro antioxidant and anticholinesterases properties of fireweed (Crassocephalum crepidioides). Journal of Taibah University Medical Sciences. 2015;10(4):419-426.
    Crossref
  41. Zahin M, Bokhari NA, Ahmad I, et al. Antioxidantss, antibacterial, and antimutagenic activity of Piper nigrum seeds extracts, Saudi J Biol Sci. 2021;28(9):5094-5105.
    Crossref
  42. Mishra V, Tomar S, Yadav P, Vishwakarma S, Singh MP. Elemental Analysis, Phytochemical Screening and Evaluation of Antioxidant, Antibacterial and Anticancer Activity of Pleurotus ostreatus through In Vitro and In Silico Approaches. Metabolites. 2022;12(9):821.
    Crossref
  43. Shaheed KA, AlGaraawi NI, Alsultany AK, Abbas ZH, Khshayyish IK, Al Khazali MT. Analysis of Bioactive phytochemical compound of (Cyperus iria L.) By using gas chromatography-mass spectrometry, International Conference on Agricultural Sciences. IOP Conf Ser Earth Environ Sci. 2019;388.
    Crossref
  44. Zahara K, Bibi Y, Arshad M, Kaukab G, Al Ayoubi S, Qayyum A. In-vitro examination and isolation of antidiarrheal compounds using five bacterial strains from invasive species Bidens bipinnata L. Saudi J Bio Sci. 2022;29(1):472-479.
    Crossref
  45. Shaaban MT, Ghaly MF, Fahmi SM. Antibacterial activities of hexadecenoic acid methyl ester and green-synthesized silver nanoparticles against multidrug-resistant bacteria. J Basic Microbio. 2021;61(6):557-568.
    Crossref
  46. Krishnamoorthy K, Subramaniam P. Phytochemical profiling of Leaf, Stem and Tuber Parts of Solena amplexicaulis (Lam.) Gandhi using GC-MS. Int Sch Res Notices. 2014;2014:567409.
    Crossref
  47. Persson C, Robert E, Carlsson E, et al. The effect of unsaturated fatty acid and triglyceride oil addition on the mechanical and antibacterial properties of acrylic bone cements. J Biomater Appl. 2015;30(3):279-289.
    Crossref
  48. Hossain MA, Alsabari KM, Weli AM, Al-Riyai Q. Gas chromatography-mass spectrometry analysis and total phenolic contents of various crude extract from the fruits of Datura metel L. J Taibah Univ Sci. 2013;7(4):209-215.
    Crossref
  49. Zirihi GN, Mambu L, Guede-Guina F, Bodo B, Grellier P. In vitro antiplasmodial activity and cytotoxicity of 33 West African plants used for treatment of malaria. J Ethnopharmacol. 2005;98(3):281-285.
    Crossref
  50. Afagnigni AD, Nyegue MA, Djova SV, Etoa F-X. LC-MS analysis, 15-lipoxygenase inhibition, cytotoxicity, and genotoxicity of Dissotis multiflora (Sm) Triana (melastomataceae) and Paullinia pinnata Linn (Sapindaceae). J Trop Med. 2020;2020(1):8.
    Crossref

Article Metrics

Article View: 387

Share This Article

© The Author(s) 2024. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.