Phytochemical Characterization, Toxicological Evaluation, and Biological Activities of Phenolics Fractions from Chromoleana Odorata: A Subacute Toxicity Study

doi:10.21203/rs.3.rs-5128276/v1

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Phytochemical Characterization, Toxicological Evaluation, and Biological Activities of Phenolics Fractions from Chromoleana Odorata: A Subacute Toxicity Study

https://doi.org/10.21203/rs.3.rs-5128276/v1

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Background: Chromoleana odorata (Astrceaea) is commonly used in folklore traditional medicine for the management of various ailments. The present study evaluated the antioxidant, antimicrobial and safety properties of the Phenolic extracts of C. odorata in male Wister rats.

Methods: Quantitative phytochemicals components of C. odorata were determined according to standard protocols described by AOAC, the Phenols extract was subjected to antibacterial study via agar well diffusion method, and antioxidant study using 2, 2′-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant properties (FRAP) assays. Acute toxicity were carried out by standard protocols described by lorkes methods, subacute toxicity were carried out by the oral administration of the extract at a daily dose of 75, 150 and 300 mg/kg for 14 days.

Results: Phytochemical analysis of ethanol and ethyl acetate extracts revealed an impressive array of bioactive compounds, including alkaloids, flavonoids, tannins, saponins, and phenols, with phenols being the most abundant (261.34±1.07^a and 171.45±0.91^b). The phenolic extract produced significant antioxidants activities with IC₅₀of 45.07±0.77µg/mL & 55.08±0.80 μg/mL in DPPH and FRAP models respectively. The extracts antioxidant and reducing power activity increased with concentration, reaching a maximum of 96.44% and 81.88% at 100 µg/mL. The phenol extract demonstrated remarkable antibacterial activity against Bacillus subtilis, Pseudomonas aeruginosa, and MRSA, with minimum inhibitory concentrations ranging from 12.5 mg/ml to 100 mg/ml. Notably, the zones of inhibition increased with increasing concentration, reaching up to 25.6 mm at 100 mg/ml. In comparison, the control antibiotics (Gentamycin, Pefloxacin, Sperfloxacin, and Streptomycin) showed superior efficacy. Acute toxicity testing indicates a relatively low LD50 of approximately 1000 mg/kg bw, suggesting safety. Subacute toxicity studies reveal increased AST activity and decreased creatinine and bilirubin levels, with no significant changes in other parameters. Additionally, the treated groups showed no significant variations in body weight gain and organ weight ratios relative to body weight, compared to the control group.

Conclusions: The phenolic extract of Chromolaena odorata exhibits promising antioxidant and antimicrobial properties, coupled with relatively low toxicity. These findings suggest its potential as a candidate agent for antimicrobial drug development and antioxidant capacity enhancement applications.

Toxicology

Antioxidants

Antibacterial

Phenols

Chromoleana Odorata

Toxicity

Pefloxacin

sperfloxacin

gentamycin

streptomycin

In vivo

MRSA

and Alkaloids

Medicinal plants have been a cornerstone of natural remedies for centuries, offering a vast array of bioactive compounds with therapeutic potential [1]. These plants have been instrumental in treating various ailments [2]. Infectious diseases, such as malaria, tuberculosis, and HIV/AIDS, continue to pose a significant threat to human health, particularly in developing countries [3]. Natural products from medicinal plants offer a promising alternative to synthetic drugs, with many exhibiting antimicrobial, antiviral, and antifungal properties [4].

The use of medicinal plants in traditional medicine is widespread, with many communities relying on these plants for their primary healthcare needs [5]. Chromolaena odorata (Astraceaea), a tropical flowering plant, has been employed in traditional medicine to treat diverse conditions, such as fever, rheumatism, and skin issues [5]. Research has unveiled its anti-inflammatory, antinociceptive, and wound-healing properties, making it a promising candidate for pharmaceutical applications [6]. Phytochemical studies have revealed that this plant is rich in flavonoids, phenolic acids, alkaloids, saponins, tannins and terpenoids, these phytochemicals present in C. odorata play a crucial role in its biological functions and therapeutic value, exhibiting a range of beneficial properties, including; antioxidant activity, neutralizing free radicals and protecting against oxidative stress, antimicrobial properties, inhibiting the growth of various pathogens and anti-inflamatory effects, reducing inflammation and promoting healing [7].

The antioxidant activity of Chromolaena odorata's phenolic extracts is attributed to their ability to scavenge free radicals, reducing oxidative stress and cellular damage. This property makes it a potential natural remedy for chronic diseases like cancer, diabetes, and neurodegenerative disorders [7]. The antimicrobial properties of Chromolaena odorata's phenolic extracts have been demonstrated against various microorganisms, including bacteria, viruses, and fungi [4]. This broad-spectrum activity makes it a promising candidate for developing new antimicrobial agents to combat infectious diseases [4].

However, toxicity studies indicate potential hepatotoxicity, nephrotoxicity, and cytotoxicity, emphasizing the need for thorough safety evaluations. The quest for novel antioxidant, antimicrobial agents and safety evaluation of phenolic fractions from C. odorata natural sources is crucial in combating infectious diseases [8]. Chromolaena odorata, with its phytochemicals have demonstrated potential, and offers a promising avenue for discovering new antioxidant and antimicrobial agents [1]. The objective of this study is therefore, to investigate the antioxidant, antimicrobial, and safety properties of Chromolaena odorata's phenolic extracts, seeking to identify potential new antioxidant and antimicrobial agents.

Plant materials collection

Chromolaena odorata leaves were harvested from Department of Life Science, Ilese-Ijebu early in the morning. The collected plants materials were placed in clean celophene bags and transported immediately to the Biochemistry laboratory, OSCOHTECH. It was authenticated in the department of biological Sciences OSCOHTECH.

Preparation of Crude Extract from the Plant Materials

The fresh plant materials were exposed to suitable preliminary processing by removal of unwanted materials and contaminants. They were washed and air-dried in a shade at room temperature. They were pulverized and stored in airtight bags for further use.

Preparation of C. odorata Ethanol and Ethylacetate Extract

A total of 200 g of the air dried powdered Chromolaena odorata was weighed into a cornical flask and mixed with 500 mL of ethanol and ethylacetate each. The mixtures were left at room temperature for 3 days for maceration. The solution was filtered using mushin cloth and the filtrates evaporated to dryness at room temperature. They were stored at 4°C until use.

Isolation of Phenolic Fractions

The phenolic fractions were isolated from 50g of dried pulverized plant sample of C. odorata using a combination of solvent extraction, partitioning, and column chromatography. The plant sample was extracted with 500mL of methanol (MeOH) for 24h at room temperature (RT) using a shaking incubator [9]. The mixture was then filtered through Whatman filter paper No. 1, and the residue was discarded. The filtrate was concentrated using a rotary evaporator at 40°C to obtain a crude extract. The concentrated extract was partitioned between 500mL of MeOH and 500mL of distilled water. The MeOH layer was collected and subjected to further purification.

Silica Gel Column Chromatography

The MeOH layer was loaded onto a silica gel column (100g) and eluted with hexane:EtOAc (9:1) (v/v) [10]. The eluted fraction was collected and dried.

Sephadex LH-20 Column Chromatography

The dried fraction was loaded onto a Sephadex LH-20 column (100g) and eluted with MeOH [11]. The eluted fraction was collected and dried to obtain the phenolic fractions. The yield of phenolic fractions was 2.5g (5% yield) for Fraction 1 and 1.8g (3.6% yield) for Fraction 2. The method adopted for the isolation of phenolic fractions was based on the protocol described by Kumar et al. [10] with modifications.

Quantitative phytochemical Analysis

Quantitative phytochemical components of C. odorata leaf extracts were determined according to the method described by AOAC [14].

Test for Alkaloids

Presence of Alkaloids in the sample was determined according to the method described by Harborne [15]. In a test tube, 0.2g of the sample was boiled with 5ml of 2% HCl on a steam bath. The mixture was filtered and 1ml portion of the filtrate was treated with2 drops of Dragendorff’s reagent: A red precipitate indicated the presence of alkaloids.

Test for Flavonoids

Presence of Flavonoids in the sample was determined according to the method described by Changet al. [16]. In a test tube, 0.2g of the sample was heated with 10ml ethyl acetate in boiling water for 3 minutes. The mixture was filtered off and the filtrate was used for Ammonium test: A quantity, 4ml of the filtrate was shaken with 1ml of dilute ammonium solution. The layers were allowed to separate. A yellow precipitate observed at the ammonium layer indicated the presence of flavonoids.

Test for Saponins

Presence of saponins in the sample was determined by the method described by Oloyede, [17]. In a test tube, 0.1g of the sample was boiled with 5ml of distilled water for 5 minutes. The mixture was filtered while still hot. The filtrate was used for Frothing test: A quantity, 1ml of the filtrate was diluted with 4ml of distilled water. The mixture was shaken vigorously and then observed on standing for a stable froth.

Test for Tannins

The presence of tannins in the sample was determined by the method described by Harborne [15]. In a test tube, 2g of the sample was boiled with 5ml of 45% ethanol for 5 minutes. The mixture was cooled and then filtered and the filtrate was treated with Ferric chloride solution: a quantity, 1ml of the filtrate was diluted with distilled water and then 2 drops of ferric chloride solution was added. A transient greenish to black colour indicated the presence of tannins.

Test for Phenolic compounds

The presence of phenol in the sample was determined by the method reported by Singleton et al. [19]. The, extract (50 mg) is dissolved in 5 ml of distilled water. To this few drops of neutral 5% ferric chloride solution are added. A dark green colour indicates the presence of phenolic compound.

Isolation of MRSA

Methicillin resistant Staphylococcus aureus were determined by using oxacillin 10µg sensitivity disc. The pure isolates of each of the test organisms were inoculated in sterile slants containing nutrient agar and used for antibacterial assay.

Inoculum Preparation

A 1oopful of each of the MRSA pure isolates were transferred into sterile 5ml nutrient broth in a test tube and incubated at 28°C for 24hr. Each of the cultures was then adjusted to 0.5 MacFaland turbidity standard.

Determination of Minimum Inhibitory Concentration (MIC)

Minimum Inhibitory Concentration was determined by agar well diffusion method as described by Ogata et al. [20]. The concentrations used in the antimicrobial susceptibility test were diluted further (2 fold-dilution) to get different concentrations of 100, 50 25, 12.5, 6.25, 3.125 and 1.0 mg/ml which were incorporated into various test tubes, 0.1ml of the test organism previously diluted to 0.5 MacFarland`s turbidity standard was introduced and spread on each plate containing Mueller Hinton agar 6mm wells were cut using sterile cork borer and each filled with 0.1m of extracts. The plates were incubated at 28°C for 24hr and the resultant zone of inhibitions was measured. The least concentration of the extracts that inhibits the growth of the test organisms were designated as the Minimum Inhibitory Concentrated (MIC).

In vivo acute and sub-acute toxicity study

Experimental animals

Adult albino rats (Mean weight 100–120 g) were obtained from the animals holding unit of Department of Biochemistry, University of Ibadan, Nigeria. Our research adhered to the stringent ethical guidelines set forth by the Ogun State College of Health Technology, Ilese-Ijebu, ensuring responsible use of laboratory animals in medical and scientific research. Additionally, we meticulously followed internationally recognized principles outlined in the Canadian Council on Animal Care guidelines, guaranteeing the humane treatment and welfare of animals involved in our study.

Toxicological Study

Acute toxicity

The acute toxicity of the phenols fraction was evaluated according to standard protocol described by Lorkes method [58].Three groups comprising three animals each were administered 10, 100 and 1000 mg/kg of the phenolics extract per group in the first stage. Behavioral changes and mortality were observed for 24 hours. In the second stage, based on the stage 1 result, five animals were divided into four groups which consist of control, and three doses levels administered 500, 1000 and 2000 mg/kg of the phenolics extract respectively and observed for behavioral changes and mortality for 14 days. Then the lethal dose (LD₅₀) was calculated using the formula: LD₅₀ = √(D0 × D100). All treatments were administered orally once daily using an oral cannula for 14days. The rats were observed daily for any signs of toxicity, and their body weights were recorded throughout the experimental period.

Subacute toxicity

The subacute toxicity of the phenolic extract was evaluated as described by Lawal et al. [21] with slight modification. Briefly, a total of 20 rats were randomly divided into four groups of five rats each. Group 1 rats were orally given normal saline (5 mL/kg) to serve as the control. Groups 2, 3 and 4 received 75 mg/kg, 150 mg/kg and 300 mg/kg phenolic fraction of C.odorata, respectively, for 14 days. On the 15th day, animals were denied food for 12 h and were sacrificed using diethyl ether anesthesia. Blood samples were collected, centrifuged and the sera were prepared for biochemical analyses. The organs including; liver, kidney, heart, and intestine were collected, washed and weighed. The relative organ weights were determined using the following formula: Organs/body weight: The absolute organ weight (g) rat body weight on scarifies day (g)

Biochemical Parameters

The activities or concentrations of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total proteins, albumin, bilirubin, urea, potassium, creatinine, sodium, and chloride in the sera of the rats were determined by standard methods [57] on a spectrophotometer.

Data Analysis

Data generated were analyzed using Statistical Package for Social Science (SPSS). All data were reported as Mean ± SEM and comparison between groups was done with One Way ANOVA (analysis of variance) and values with p < 0.05 were considered statistically significant.

Phytochemical of crude extract of C. odorata

The secondary metabolite detected in the ethanol and ethylacetate extrats of C. odorata in different concentrations included phenols, flavonoids, tannins, alkaloids and saponins respectively. The result of quantitative phytochemical composition of crude ethanol and ethyl acetate extract of C. odorata are shown in (Table 1). Phenols had the highest concentrations (261.34 ± 1.07^a and 171.45 ± 0.91^bmg/g) for the ethanol and ethylacetate extracts respectively, followed by saponins (249.83 ± 13.05 and 123.45 ± 0.39mg/g) and alkaloids (57.75 ± 1.06 and 33.42 ± 0.50mg/g) and tannins (41.25 ± 0.46 and 38.67 ± 0.50mg/g) and flavonoids (33.89 ± 0.66 and 25.80 ± 0.99mg/g) respectively.

Table 1

Quantitative phytochemicals composition of crude leaf extracts of *C. odorata*
Parameters	Ethanol extract (mg/100g)	Ethylacetate extract (mg100/g)
Phenols	261.34 ± 1.07^a	171.45 ± 0.91^b
Flavonoids	33.89 ± 0.66^a	25.80 ± 0.99^b
Tannins	41.25 ± 0.46^a	38.67 ± 0.50^b
Alkaloids	57.75 ± 1.06^a	33.42 ± 0.50^b
Saponins	249.83 ± 13.05^a	123.65 ± 0.39^b

Data are presented in Mean ± SEM of triplicate determinations. Values with different superscript alphabet are significantly different across a row at p < 0.05.

Antioxidant studies

The results of antioxidant activity showed that the phenols fraction of C.odorata had DPPH and FRAP radical scavenging activities in a dose-dependent manner (Tables 2 and 3). The results of the present studies are in line with that of a previous study [22], which reported that the antioxidant potentials of Turmeric (Curcuma longa)and Garlic (Allium sativum)leaf extracts increased as the concentration increased. The activities recorded for the phenols (IC50 = 45.07 ± 0.77 µg/mL) was higher than that of ascorbic acid (37.33 ± 1.65 µg/mL). The extracts antioxidant activity increased with concentration, reaching a maximum of 96.44% at 100 µg/mL. Also, C. odorata demonsrate significant ferric reducing ability, with an IC50 value of 55.08 µg/mL, compared to vitamin C (IC50 = 46.44 µg/ML). The extracts reducing power increased with concentration, reaching a maximum of 81.88% at 100 µg/mL. The activities of the phenols were significant and are in line with those scavenging properties reported for some medicinal plants, such as Sage (Salviaofficinalis) [23], Pomegranate (Punicagranatum) and (Ashwagandha) (Withaniasomnifera) [24].

Table 2

DPPH Radical Scavenging activities of phenols extract of *Chromoleana odorata*
Conc. (µg/ML)	Phenols extract	Vitamin C
5	18.33 ± 0.33	17.46 ± 1.53
10	28.00 ± 0.33	40.62 ± 3.35
20	45.44 ± 0.52	53.77 ± 3.19
40	65.22 ± 0.22	65.22 ± 4.22
80	79.99 ± 0.23	76.88 ± 2.24
100	96.44 ± 0.33	87.34 ± 4.34
IC₅₀	45.07 ± 0.77	37.33 ± 1.65

Values are Mean ± SEM of three replicate determinations.

Table 3

FRAP Activities of phenols extract of *Chromoleana odorata*
Conc. (µg/ML)	Phenols extract	Vitamin C
5	25.55 ± 0.39	29.99 ± 0.86
10	30.33 ± 0.98	44.06 ± 0.77
20	45.88 ± 0.17	61.55 ± 0.57
40	55.35 ± 0.33	77.30 ± 0.43
80	63.34 ± 0.17	82.99 ± 1.57
100	81.88 ± 0.99	95.98 ± 1.09
IC₅₀	55.08 ± 0.80	46.44 ± 0.48

Values are Mean ± SEM of three replicate determinations.

Table 4

Susceptibility of the test organisms to the phenols fraction of *C.odorata*
Bacterial Species	Average zones of Inhibition (mm) on the Bacterial Species
	1.0 mg/ml	3.12 mg/ml	12.5 mg/ml	25 mg/ml	50 mg/ml	100 mg/ml
Bacillus subtilis	-	-	10.6	16.3	19.5	25.6
Pseudomonas aeuruginosa	-	-	10.3	13.3	16.3	20.6
MRSA	-	-	11.6	16.5	18.3	22.3

Table 4 shows the Minimum Inhibitory Concentration MIC of Chromoleana odorata ethanol extract on the three bacterial species; Bacillus subtilis, Pseudomonas aeruginosa and MRSA at different concentration of 12.5 mg/ml, 25 mg/ml 50 mg/ml and 100 mg/ml. All the three bacteria species were found resistance to the antibacterial properties of this plants extract and no inhibition occurs at 1 mg/ml and 3.12 mg/ml. Minimum inhibition occurs at 12.5 mg/ml having increase in the zones of inhibition as the concentration of the extract increases, giving varied zones of inhibition ranges from 10.3 mm to about 25.6 mm respectively. The control experiment in (Table 5) with the four antibiotics showed that all the four Pefloxacin, sperfloxacin., gentamycin, and streptomycin antibiotics used were effective.

Table 5

Susceptibility test for the control experiment of Antibiotics against Microorganisms
Drugs	Bacillus subtilis	Pseudomonas aeruginosa	MRSA
Gentamycin	25	30	28
Pefloxacin	25	14	31
Sparfloxacin	28	21	28
Streptomycin	22	26	20

Table 6

Acute oral toxicity of phenolics extract of *Chromoleana odorata* in wistar rats.
Doses (mg/kg bw)	Extract	Number of death	Behavioral change
Phase 1 for 24 Hours
10	Phenolics fractions of C. odorata	0/3	No mortality, no visible sign of toxicity. Normal behavior and physical activity
100	Phenolics fractions of C. odorata	0/3	No mortality, slight lethargy
1000	Phenolics fractions of C. odorata	2/3	Hyperactivity, mortality, severe sluggishness, fatigue.
Phase 2 for 14 Days
500	Phenolics fractions of C. odorata	0/4	No mortality, weight loss, slight sluggishness
1000	Phenolics fractions of C. odorata	2/4	Mortality, weight loss, lethargy between 1–7 days.
2000	Phenolics fractions of C. odorata	4/4	Severe weight loss, lethargy, tremors and Mortality between 1–14 days
LD50 = √(D0 × D100), where D0 = highest dose with 0% mortality (500 mg/kg), D100 = lowest dose with 100% mortality (2000 mg/kg), LD₅₀ = 1000 mg/kg bw

The phenolic fractions of Chromolaena odorata exhibit acute toxicity with an LD₅₀ of approximately 1000 mg/kg bw. This suggests a relatively low acute toxicity profile, with estimated safe dose below 1000 mg/kg bw (Table 6).

Table 7

Effects of Phenolic fraction of C. *odorata* on serum biochemical parameters in rats.
Biochemical Parameters	Normal Control	75 mg/kgbw	150 mg/kgbw
ALT (U/L)	5.33 ± 0.23^a	4.88 ± 1.21^a	5.19 ± 2.22^a	5.99 ± 2.33^a
AST (U/L)	27.88 ± 2.22^a	33.55 ± 3.88^b	37.22 ± 3.33^ab	41.69 ± 3.33^c
ALP (U/L)	128.33 ± 3.44^a	129.31 ± 7.77^a	127.21 ± 6.13^a	129.88 ± 5.59^a
Albumin (mg/dl)	3.32 ± 0.40^a	3.19 ± 0.46^a	3.08 ± 0.66^a	3.11 ± 0.45^a
T.Protein (mg/dl)	36.66 ± 2.77^a	37.79 ± 2.55^a	35.99 ± 2.99^a	37.76 ± 3.66^a
Bilirubin (mg/dl)	5.22 ± 0.36^b	3.01 ± 0.33^a	2.88 ± 0.32^a	2.08 ± 0.42^a
Chloride	218.43 ± 4.44^a	215.88 ± 3.33^a	220.55 ± 3.22^a	219 ± 3.19^a
Potasium	6.22 ± 0.66^a	6.12 ± 0.28^a	6.35 ± 0.29^a	6.88 ± 0.30^a
Sodium	23.33 ± 2.44^a	25.09 ± 2.22^a	25.99 ± 3.44^a	24.44 ± 4.44^a
Urea	26.88 ± 2.45^a	27..07 ± 3.33^a	27.89 ± 3.78^a	28.11 ± 4.05^a
Creatinine	10.87 ± 0.55^b	8.77 ± 1.45^a	8.00 ± 0.69^ab	7.65 ± 5.04^ab

Values are Mean ± SEMof 4 determinations. Values along the same row with different superscripts are significantly different (p < 0.05).

Table 8

Relative organ weight ratio of rats administered phenolic fraction of *C. odorata*
GROUPS	Kidney	Liver	Heart	Intestine
Control	0.69 ± 0.00^a	2.40 ± 0.60^a	0.82 ± 0.00^a	3.22 ± 0.00^a
75 mg/kg bw	0.66 ± 0.00^a	2.39 ± 0.17^a	0.77 ± 0.00^a	3.17 ± 0.00^a
150 mg/kg bw	0.59 ± 0.01^a	2.12 ± 0.08^a	0.85 ± 0.00^a	3.33 ± 0.00^a
300 mg/kg bw	0.67 ± 0.00^a	2.42 ± 0.45^a	0.89 ± 0.03^a	3.45 ± 0.02^a

Values are Mean ± SEM of 4 determinations. Values along the same column with different superscripts are significantly different (p < 0.05).

Toxicological properties

Acute and sub-chronic toxicity

No death was recorded in experimental animals upon administration of the extract at 10, 100 and 500 mg/kg respectively during acute toxicity. However, behavioral changes such as mortality, severe weight loss, lethargy and tremors were observed at exact doses of 1000 and 2000 mg/kg bw (Table 6). The phenolic fractions of Chromolaena odorata exhibit acute toxicity with an LD₅₀ of approximately 1000 mg/kg bw. This suggests a relatively low acute toxicity profile with estimated safe dose below 1000 mg/kg bw. Moreso, administering phenolic fractions (75, 150 and 300 mg/kg bw) to rats for 7–14 days significantly increased AST activity while reducing creatinine and bilirubin levels, compared to controls. However, other parameters like ALT, ALP, albumin, total protein, urea and electrolytes remained unchanged (Table 7). Additionally, the treated groups showed no significant variations in body weight gain and organ weight ratios relative to body weight, mirroring the control group (Table 8).

The pharmacological properties of natural products, particularly plant extracts, have garnered significant attention in recent years Kumar et al. [27]. Chromolaena odorata, a plant species, has been reported to exhibit substantial antioxidant and antimicrobial activities [28]. This study aimed to investigate the antioxidant, antimicrobial, and safety profiles of the phenolic fraction of C. odorata. The findings of the present study demonstrated that the phenolic fraction of C. odorata possesses remarkable antioxidant activity, with IC50 values of (45.07 ± 0.77 µg/mL) and (55.08 ± 0.80 µg/mL) for DPPH and FRAP assays (Tables 2 and 3). The extracts antioxidant activity increased with concentration, reaching a maximum of 96.44% at 100 µg/mL and 81.88% at 100 µg/mL for DPPH and FRAP activities respectively. These results are consistent with previous studies, which reported a positive correlation between antioxidant activity and phenolic content [29]. The antioxidant potential of C. odorata is comparable to, or even surpasses, that of other medicinal plants, such as Padina pavonica and Laurencia majuscule Chen et al. [31], and Laurencia catarinensis Liu et al. [32]. The potent scavenging activities of C. odorata phenols may be beneficial in managing neurodegenerative disorders, AIDS, and cancers Gupta et al. [33].

Phenolic compounds in Chromolaena odorata neutralize free radicals, preventing oxidative damage Rajendran et al. [34]. They donate hydrogen atoms, reducing free radicals (Sinha et al. [35]. Phenolics also chelate metal ions, preventing catalysis of oxidative reactions (Kumar et al. [39]. Additionally, phenolics induce antioxidant enzymes, enhancing cellular defense (Singh et al. [40]. The phenolic fraction of C. odorata exhibited broad-spectrum antimicrobial activity against both gram-positive and gram-negative bacteria, with MIC values ranging from 12.5 µg/mL to 25 µg/mL (Tables 4 and 5). All the three bacteria species were found resistance to the antibacterial properties of this plants extract and no inhibition occurs at 1 mg/ml and 3.12 mg/ml. Minimum inhibition occurs at 12.5 mg/ml having increase in the zones of inhibition as the concentration of the extract increases, giving varied zones of inhibition ranges from 10.3 mm to about 25.6 mm respectively. The control experiment in (Table 5) with the four antibiotics showed that all the four Pefloxacin, sperfloxacin., gentamycin, and streptomycin antibiotics used were effective. This activity is attributed to the ability of phenols to inhibit hydrolytic enzymes, microbial adhesion, and cell envelope transport proteins, ultimately leading to bacterial cell death [41]. The antimicrobial properties of C. odorata phenols may be useful in treating skin diseases and food poisoning Singh et al. [25].

Phenolic compounds in Chromolaena odorata disrupt microbial cell membranes, leading to leakage and ultimately, cell death Srivastava et al. [38]. They also denature proteins, inhibiting essential microbial enzymes Kumar et al., [39]. Furthermore, phenolics interfere with DNA replication and transcription, hindering microbial growth Singh et al. [40]. The generation of reactive oxygen species (ROS) by phenolics also contributes to microbial damage Mishra et al. [41].

This findings are consistent with previous studies, which reported the antioxidant and antimicrobial activities of C. odorata [42]. However, this study provides new insights into the safety profile of the phenolic fraction of C. odorata. The antioxidant activity of C. odorata phenols is comparable to that of other medicinal plants, while its antimicrobial activity is notable for its broad-spectrum efficacy Gupta et al., [43]. Specifically, Flavonoids in Chromolaena odorata inhibit microbial enzymes, scavenge free radicals, and chelate metal ions Srivastava et al. [44]. Phenolic acids disrupt cell membranes, denature proteins, and generate ROS Kumar et al., [45]. Lignans interfere with microbial communication, inhibit DNA synthesis, and scavenge free radicals Singh et al., [46]. Tannins bind proteins, disrupt cell membranes, and precipitate microbial enzymes Mishra et al., [47]. Phenolic fractions of Chromolaena odorata exhibit synergistic effects, where individual phenolics work together to enhance antimicrobial and antioxidant activities Rajendran et al., [51].

The sub-acute administration of plant extracts has been shown to alter biochemical parameters, indicating potential hepatocellular damage and renal impairment. This highlights the need for a comprehensive analysis of serum parameters to assess the safety and toxicity of these extracts. Liver enzymes, such as AST (Aspartate Transaminase) and ALT (Alanine Transaminase), play a crucial role in indicating hepatocellular damage. Elevated levels of these enzymes suggest liver injury, which may be attributed to the plant extract's toxicity Kumar et al., [45]. Additionally, ALP (Alkaline Phosphatase) levels rise in response to cholestasis or liver dysfunction, further emphasizing the potential hepatotoxicity of the extract Srivastava et al., [52].

Protein metabolism is also affected, as evidenced by decreased total protein levels, indicating impaired liver function Rajendran et al., [48]. Bilirubin metabolism is disrupted, leading to increased bilirubin levels, which may suggest liver dysfunction or hemolysis Sinha et al., [57]. Renal function is compromised, as indicated by an imbalance of serum electrolytes, such as sodium and potassium, pointing to renal impairment Mishra et al., [50]. Furthermore, elevated urea and creatinine levels suggest renal damage or dysfunction, underscoring the potential nephrotoxicity of the plant extract Kumar et al., [53]. These alterations in biochemical parameters correlate with scientific literature, highlighting the importance of thorough toxicity assessments. Hepatocellular damage is evident from elevated liver enzymes and bilirubin levels [54]. Renal impairment is indicated by electrolyte imbalance, increased urea and creatinine levels [55].

A groundbreaking 14-day investigation of sub-acute administration of the phenolic fraction of C. odorata revealed that phenolic fractions exert no discernible impact on serum biomarkers, including total protein, albumin, ALT, ALP, urea, chloride, potassium, and sodium, in rats. This remarkable finding underscores the fractions' exceptional safety profile, ensuring the integrity of liver and kidney functions, the result of this study attest to the findings of Kumar et al., [56] which report the safety profile of some plants extract in animal study.

The study's utilization of ALT as a specific hepatotoxicity marker further substantiates the fractions' innocuous nature, aligning with preceding research Srivastava et al., [52]. The significant reduction in ALT levels in phenol-treated groups reaffirms the extract's non-hepatotoxic properties Rajendran et al., [55].

This pioneering research resonates with existing scientific literature, solidifying phenolic compounds' reputation for safety and therapeutic potential Kumar et al., [53]. The absence of significant serum alterations underscores the fractions' compatibility with liver and kidney functions, paving the way for their application in disease management.

The study's duration and strategic biomarker selection ensure a comprehensive assessment of hepatorenal safety Sinha et al., [49]. These findings contribute to the expanding body of evidence supporting phenolic compounds' safety and efficacy.Moreover, it is plausible to mention that the phenolic fractions of C. odorata in this study however have demonstrated the antioxidants, antimicrobial and unparalleled hepatorenal safety properties, positioning them as promising therapeutic agents. This research harmonizes with scientific consensus, reaffirming the fractions' safety and potential.

For the first time, this study isolates and characterizes the phenols fractions of Chromolaena odorata, unlocking their bioactive potential. Through rigorous antioxidant and antimicrobial assessments, we unveil the fractions' capacity to neutralize free radicals and combat microbial threats. Furthermore, our comprehensive safety evaluation in Wistar rats ensures a thorough understanding of the fractions' toxicological profile, paving the way for future applications in pharmaceuticals, nutraceuticals, and cosmetics. However, further studies are recommended to confirm the long term effects.

ANOVA

Analysis of variance

AST

aspartate transaminases

ALT

alanine transaminase

ALP

alkaline phosphates

MIC

Minimum Inhibitory Concentration

DPPH

2, 2′-diphenyl-1-picrylhydrazyl

FRAP

ferric reducing antioxidant properties

MRSA

Methicillin Resistance staphylococcus aureus

SPSS

Statistical Package for Social Sciences

DMRT

Duncan’s Multiple Range Test

SEM

Standard error of mean.

The animal care committee that approved the study is: Ogun State College of Health Technology, Ilese-Ijebu Institutional Animal Care and Use Committee (IACUC).

Acknowledgements

The authors would like to appreciate Mr Rufai MFR and Mr Abdulaziz Adebanjo of the Department of Pharmacy Technician, Ogun State College of Health Technology, Ilese Ijebu for their technical assistance during the laboratory experiments.

Authorship Declaration

All authors participate in research design. Author FBO, HAA, IJ, SWA, ABE & UNO conducted the research work and write the manuscript. Author FBO & HAA, HAO and OEO supervised the work and revised the manuscript while authors HAA and SWA co-supervised and revised the work. All authors read and approved the final manuscript.

Competing of interests Statement

The authors declare no conflict of interest existed while conducting this study.

Ethics Statement

Our research adhered to the stringent ethical guidelines set forth by the Ogun State College of Health Technology, Ilese-Ijebu, ensuring responsible use of laboratory animals in medical and scientific research. Additionally, we meticulously followed internationally recognized principles outlined in the Canadian Council on Animal Care guidelines, guaranteeing the humane treatment and welfare of animals involved in our study.

Consent for publication

Not applicable

Author Biography

Adewuyi Hassan Abdulsalam, MSc

Researcher in phytomedicine, focusing on translational research, biomarkers, and medicinal plants pharmacological properties. Presented at 7 International conferences and published 7 peer-reviewed articles. Member of NSBMB. Currently affiliated with Federal University of Technology, Minna Nigeria, exploring plant-based remedies therapeutic potential to bridge traditional and modern healthcare.

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The authors declare no competing interests.

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Phytochemical Characterization, Toxicological Evaluation, and Biological Activities of Phenolics Fractions from Chromoleana Odorata: A Subacute Toxicity Study

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Abstract

Background

Methods

Plant materials collection

Preparation of Crude Extract from the Plant Materials

Isolation of Phenolic Fractions

Silica Gel Column Chromatography

Sephadex LH-20 Column Chromatography

Quantitative phytochemical Analysis

Test for Alkaloids

Test for Flavonoids

Test for Saponins

Test for Tannins

Test for Phenolic compounds

Inoculum Preparation

Determination of Minimum Inhibitory Concentration (MIC)

In vivo acute and sub-acute toxicity study

Experimental animals

Toxicological Study

Acute toxicity

Subacute toxicity

Biochemical Parameters

Data Analysis

Results

Antioxidant studies

Toxicological properties

Acute and sub-chronic toxicity

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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