Introduction:

Infections from wounds and subsequent death peaked in the 19th century [1]. The discovery of harmful microorganisms in the 20th   century opened up a new area of study for the phenomenon of wound infection [2].  Exogenous   or   endogenous   infections   of   the   wound   might   occur [3].   Staphylococcus   aureus   is   one   of   the   germs   that   has   been identified from wound infections in adults most frequently, and it is also the most common   cause   of   wound   infections   in   newborns, the   majority   of   which   are resistant   to   treatments [4]. Staphylococcus aureus, Streptococcus pyogenes, Pneumococcus, and   coliform   bacteria   such   Escherichia   coli, Proteus species, and Pseudomonas aeruginosa are the most prevalent pyogenic bacteria. It would be assumed that the etiologic agents of the infection would be present in the pus and exudates from an infected wound or open abscess [5].

The   creation   of   numerous   pharmaceuticals   and   chemotherapeutic   agents   from traditional plants that are abundant in the tropics is a result of the rising demand for medications   derived   from   natural   sources [6].  

Originally from North and Central   America, the flowering shrub Chromolaena odorata was later brought to portions of Asia, Africa, and Australia. Armstrong’sweed, baby   tea, bitter   bush, butterfly   weed, Christmas   bush, devil   weed, eupatorium, Jack in the bush, king weed, paraffin bush, paraffin weed, Siam weed, turpentine weed, and   triffid weed are some of the other names for C.odorata [7].

Traditional medical practitioners have treated human burns,soft tissue wounds, ulcerated wounds, burn wounds, postnatal wounds, leech bites, dyspepsia, and skin infections with the fresh leaves of C. odorata or the decoction [8]. Other pharmacological effects of this plant include anti-helminthic, analgesic, anti-inflammatory, antipyretic, antispasmodic [9], antimycobacterial, insecticidal, antioxidant [10], anti- gonorrheal, fungicidal, diuretic [11], blood coagulating, and antimicrobial effects. However, there is currently very little knowledge about the plant's capacity for healing wounds [12].

It   is   well   recognized   and   established   that   some   wound   microorganisms   are antibiotic   resistant.   Given   that   some   of   these   species, such   as   the   extremely refractory   bacterium   Pseudomonas   aeruginosa, are   resistant   to   most   synthetic antibiotics, this study was carried out to determine whether a natural extract from a plant would be useful in treating illnesses brought on by these bacteria. The herb Chromolaena   odorata   was chosen   because   herbalists commonly utilized it in traditional medicine. They are inexpensive, simple to obtain, and may have significant therapeutic value.

Materials And Methods:
Study Area:

In Owerri, Nigeria's Federal Medical Center (FMC), this study was carried out. Along   Orlu   road   in   Owerri, there   is   a   tertiary   medical   center   called   Federal Medical Centre. It is a sizable hospital with many fully functional arms. In addition to   providing   patient   treatment, the   hospital   serves   as   a   training   ground   for numerous health care professions. It has received accreditation from the Nigerian Medical   and   Dental   Council   for   the   training   of   house  officers, the   Nigerian Pharmacy Council for   the   training   of   pharmacy   interns, the Nigerian Medical Laboratory Science Council for the training of laboratory scientists/interns, among other bodies.

Plant Material:

Chromolaena odorata leaves were bought in bulk from vendors at the Owerri relief market and authenticated at Imo State University's Department   of   Plant   Science   and   Biotechnology.   The   bulbs   and   leaves   were cleaned and air-dried for 21 days at room temperature.

Collection and Identification of Bacteria:

The   Federal   Medical   Centre (F.M.C.)   at   Owerri's   Medical   Microbiology Laboratory provided the cultures of the wound-isolate isolates used in this study. 

Re-identified bacteria isolates were preserved on nutrient agar and kept at 4°C for a short period of time.

Control Samples 

Broad spectrum antibiotic ciprofloxacin was standardised and utilized as a positive control, while   ethanol   and   distilled   sterile   water   were   employed   as   negative controls for ethanolic extracts. 

Preparation of Ciprofloxacin 15ug/ml:

One milliliter of   distilled water was   used to dissolve about   400 milligrams of ciprofloxacin. To make 1ml (15ul/dl), 985ul of distilled water was added after around 15ul had been pipetted out

Preparation of Plant Extract:

Chromolaena odorata leaves were air dried and powdered. A 100g sample of the paste was soaked for 48 hours in 1000ml of ethanol and water. Utilizing Whatman No. 1 filter paper and the Soxhlet apparatus to prepare the homogenized plant extracts, the filtrate was then evaporated   using   a   rotary   evaporator.   According   to   Nagesh   and Samreen, the extracts were collected in sterile screw-cap bottles and kept in the refrigerator at 4°C (2016).

Preparation of Bacterial Inoculum:

On nutritional agar, test organism cultures were kept alive. Using an inoculating look, four to six colonies were selected quickly, suspended in 5 ml of broth, and incubated at 37 oC. The broth culture's turbidity was then adjusted to conform to 0.5 Macfarlands norms. This gives the pathogenic organism needed for the test, which ranges from 1 x 106 to 5 x 106 (colony forming unit/ml).

Preparation of the Extract's Serial Dilution:

The formula is 100g = 100,000mg = 100mg/ml (stock) 1000ml 1000ml 

3.6 Preparation of Serial Dilution of the Extract

  100g     = 100,000mg = 100mg/ml (stock)

1000ml          1000ml

100mg/ml           50mg/ml            25mg/ml           12.5mg/ml               6.25mg/ml

3.125mg/ml.

The extract was serially diluted by two-fold from the stock (100 mg/ml), yielding 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, and 3.125 mg/ml.

Antimicrobial Activity of the Crude Extract:

Using   the   agar   well   diffusion   method, the   extract's   antibacterial   activity   was assessed. Satdive and other (2012). On the surface of a sterile Mueller-Hinton agar plate, a tenth of a milliliter of each standard test organism was streaked before being let to stand for 15 minutes. On the solidified Hinton agar plate, 0.1ml of the extracts (both stock and serially diluted) were cultivated into each well using a sterile   6mm   cork-borer.   Distilled   water   served   as   the   negative   control, and ciprofloxacin (15ug) as the positive control. The plate was incubated for 18 to 24 hours at 37°C. Using a transparent meter ruler, clear zones surrounding the wells were counted as signs of inhibition and measured in millimeters (ml).

Determination of Minimum Inhibitory Concentration (M.I.C):

Allium sativum and Chromolaena odorata ethanol extracts were tested for their minimum bacterial concentration (M.B.C.). The Minimum Inhibitory Concentration (M.I.C.) was established using the broth dilution method with ethanol as the M.I.C. and M.B.C. [13]. Test tubes were combined with an equal volume of extracts from Chromolaena odorata, and nutritional broth in various concentrations.

Test   tubes   were   filled   with   0.5   milliliters   of   the   various   ethanolic   extract concentrations (100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, and 3.125 mg/ml). The equal volume (about 0.5ml) of various bacterial suspensions were injected into the appropriate test tubes for each pair. These test tubes, together with the positive and negative control test tubes, were infected at 37°C for 18 hours.   The   M.I.C   was   chosen   as   the   lowest   extract/antibiotic   concentration   at which the organism showed no signs of growth after 14 hours of incubation. The tubes that exhibited no signs of growth were plated out onto Mueller Hinton agar plates   devoid   of   antibiotics   to   perform   the   M.B.C   assay.   After   culturing   on Mueller-Hinton   agar   plates   without   antibiotics, the   M.B.C.   was   chosen   as   the lowest concentration of antibiotic/extract that completely stopped bacterial growth.

Phytochemical Analysis

According to Yadav and Agarwala, the ethanol extract underwent a phytochemical examination   utilizing   standardized   methods   to   determine   the   phytochemical ingredients.

Qualitative Analysis:

Determination of saponins

A test tube containing 0.5g of the material was separately agitated; foaming that persisted after warming was considered proof of the presence of saponins.

Determination of tannins

Separately, 0.5g of the extract was mixed with 10ml of distilled water in a test tube before being filtered. 5% Iron III Chloride was added in two drips. The presence of tannins was indicated by the blue-black coloring.

Determination of alkaloids:

The material, which weighed 0.5g, was dissolved in 5ml of 1% hydrochloric acid (HCl). Dragendroff's   reagent   was   used   to   process   the   filtrate.   Alkaloids   were present as evidenced by the red precipitate's formation.

Determination of glycosides:

One beaker received 1g of the material, 5ml of sulphuric acid was added, and the other beaker received 5ml of water. The contents of the two beakers were filtered into test tubes with labels after being heated for three minutes. After adding 0.5ml of sodium hydroxide to the filtrate to make it alkaline, the mixture was let to stand for three minutes. A good reaction for glycosides was shown by the reddish-brown precipitate that was present in the filtrate.

Determination of flavonoids:

1ml of the extract was combined with a piece of magnesium ribbon and 5ml of strong hydrochloric acid. Flavones were denoted by colors ranging from orange to red, flavonols from red to crimson, and flavonones from crimson to magenta.

Determination of phenols:

One milliliter of the extract was put to a test tube along with four drops of ferric chloride   solution.   Blueish   black   coloration   formed   suggested   the   presence   of phenols.

Determination of carbohydrates:

5 ml of Benedict's reagent were applied to 1 ml of the filtrate. When the mixture was heated, a crimson precipitate formed, which indicates the presence of reducing sugar [14].

Chromatographic analysis:

For this analysis, 50 liters of Datura stramonium ethanolic extract were aspirated and   injected   into   the   sample   inlet, which   transports   the   sample   to   all   other compartments.   The   spectra   were   analyzed, peak   by   peak   and   compound   by compound, on the linked computer after a turnaround time of 27 minutes.

Statistical Analysis:

After data analysis, the results were displayed as means and standard deviations.

The   zones   of   inhibition   were   compared   using   analysis   of   variance (ANOVA), which was used to identify the significant differences. When the P value is less than 0.05, statistical significance has been determined. Tables were used to display the results.

Results:

Test organisms		Concentrations
	Raw	100mg/ml (Stock)	50mg/ml	25mg/ml	12.5mg/ml	6.25mg/ml	Ciprofloxacin (15ug/ml)	Negative Control
S. aureus	16.05 ±0.04	11.10 ±0.05	8.15 ±0.26	6.10 ±0.10	0.00	0.00	25.67±0.15	0.00
E. coli	18.20 ±0.12	12.50 ±0.15	9.40 ±0.05	7.20 ±0.00	0.00	0.00	30.33±0.00	0.00
P. aeruginosa	22.00 ±0.20	14.00 ±0.00	11.10 ±0.11	8.50 ±0.10	0.00	0.00	30.33±0.50	0.00

Table 4.1 Mean ± standard deviation values of zones of inhibition (in mm) of ethanolic extracts of Chromolaena odorata leaves on the test organisms.
Key: ± Standard Deviation

Bacterial Isolates	MIC (mg/ml)	MBC (mg/ml)
Staphylococcus aureus	25	100
Escherichia coli	50	100
Pseudomonas aeruginosa	50	100

Table 4.2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of ethanolic extract of Chromolaena odorata on test organisms

Compound	Quantity (%)
Tannin	3.854
Phenol	5.329
Flavonoid	1.246
Alkaloid	9.602
Phytate	3.171
HCN	3.517

Table 4.3: Quantitative Phytochemical Analysis of Chromolaena odorata Results

Peak number	Retention time(minute)	Name	Percentage
1	1.511	Benzene-ethanamine	3.448
1	1.511	Bactolin	3.448
2	1.448	Methyl hydrogen disulfide	46.240
2	1.448	Hydrazine	46.240
3	2.010	Cyclohexane	1.587
3	2.010	Hexane	1.587
4	3.280	Thiazole	0.051
4	3.280	Butanoic acid	0.051
5	3.400	Cyclohexyl-ethylamine	0.085
5	3.400	Benzene-ethanamine	0.085
5	3.400	Dextroamphetamine	0.085
5	3.400	dl-phenylephrine	0.085
5	3.400	Phenylephrine	0.085
5	3.400	Thiophene-3-ol	0.085
6	7.890	Benzene methanol	0.041
6	7.890	2-iodohistidine	0.041
7	8.728	Ethanamine	0.037
7	8.728	Amphetamine	0.037
8	10.650	6,9,12-octadecatrienoic acid	0.061
8	10.650	Allyl(dimethyl)benzyl oxysilone	0.061
9	11.114	1-hydroxy-4-dimethylhydrazonomethyl	0.033
9	11.114	3-(E)-octen-2-one	0.033
10	12.826	Diisopropyl(ethoxy)silane	0.051
11	13.658	2,5-methylene-1-thamnitol	0.041
12	14.275	1-ethoxy-1-methyl-1-silacyclohexane	0.051
12	14.275	Adipic acid	0.051
13	15.113	3-ethyl-4-hydroxy-4	0.034
14	15.359	2,3-O-Benzal-d-mannosan	0.051
14	15.359	2,3-O-Benzal-d-mannosan	0.051
15	16.553	Artemiseole	0.048
16	17.212	Amphetamine	0.051
17	17.258	9,10-secochola	0.051
18	17.649	Benzenamine	0.080
18	17.639	Benzene propanoic acid	0.069
19	20.700	4-hydroxy-4-(2-methylcychonex-3-enyl)	0.102
19	20.700	Hydrocinnamic acid	0.102
20	21.803	5,7-dodecadiyn-1,2-diol	0.488
20	21.803	3-cyclohexen-1-ol	0.488
21	22.590	3,7,11,15-tetramethyl-2-hexadeen 1-ol	0.035
21	22.590	Phytol, acetate	15.821
22	27.914	7-methyl-Z-tetradecen-1-ol acetate	15.821
23	28.596	Octadecanoic acid	1.400

Table 4.4: Chromatographic Analysis of the Ethanolic Extract of Chromolaena Using Gas Chromatography-Mass Spectrometry

Discussion:

According to the study's findings, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus   aureus   are   all   susceptible   to   the   antibacterial   effects   of Chromolaena odorata extracts.  This is consistent with results from other scholars [14, 16], which also demonstrated the bactericidal efficacy of ethanolic extracts of Chromolaena odorata. It was foud that S. aureus was more vulnerable to the active compounds   found   in   Chromolaena   odorata   in   this investigation. S. aureus had an 8.30 mm for Chromolaena odorata extract zone of growth inhibition diameter. With diameters of 7.8 mm for Chromolaena odorata extract, P. aeruginosa is less sensitive. Of all the test microorganisms tested, E. coli was the least   susceptible, with   growth   inhibition   diameters   for    Chromolaena odorata extract of 7.5 mm, The production of enzymes by E. coli and P. aeruginosa may be the cause of these behaviors.These enzymes may degrade or inactivate some of the bioactive phytoconstituents in Chromolaena odorata. Additionally, it has been noted that the gram-negative bacteria's complex cell envelope slows or obstructs the passage of numerous antimicrobial drugs through the cell wall [17]. All test microorganisms had their development generally inhibited to varied degrees, which is consistent with the findings [18]. S. aureus was more susceptible than E. coli, according to the findings [19], and the same finding was made in our investigation. It is common practice to assess a variety of   compounds, including   antibiotics, antiseptics, disinfectants, and chemotherapeutic agents, using the MIC and MBC assay techniques [20, 21]. High MIC and MBC values are typically given by antimicrobial agents with low activity against an organism, while low MIC and MBC values are typically given by antimicrobial agents with high activity. Chromolaena odorata ethanol extract have different M.I.C. and M.B.C. values in this investigation, with M.I.C. values ranging from 25 to 50 mg/ml and M.B.C values of 100 mg/ml and higher. This indicates that the concentration or quantity of extract supplied to the medium has a significant impact on how well microorganisms are inhibited.