Animal Science

In vitro antimicrobial and antioxidant activity of leaves and aqueous extract of four medicinal plants with phytobiotic potential in animal production

0000-0002-3940-1026R. Aroche^{1  2}  , 0000-0002-8799-3572X. Jiang²  , 0000-0003-2641-1767R. Rodríguez³  , 0000-0003-2047-9840X. Li²  , 0000-0001-9759-1078Carolina Avellaneda⁴  , 0000-0003-2167-4904Y. Martínez^{4  *} 

¹Departamento de Zootecnia, Facultad de Ciencias Agropecuarias, Universidad de Granma, Cuba.

²Key Laboratory of Feed Biotechnology of Ministry of Agriculture and Rural Affairs, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.

³Centro de Estudio de Producción Animal, Facultad de Ciencias Agropecuarias, Universidad de Granma, Cuba.

⁴Departamento de Ciencia y Producción Agropecuaria, Escuela Agrícola Panamericana, Zamorano, Honduras.

ABSTRACT

The aim of this study was to evaluate the in vitro antimicrobial and antioxidant properties of leaves and aqueous extracts from four medicinal plants (Anacardium occidentale, Psidium guajava, Morinda citrifolia, and Moringa oleifera). Six bacterial strains were used in the study, and the minimum bactericidal concentration (MBC) was determined for leaves, while the minimum inhibitory concentration (MIC) and MBC were evaluated for aqueous extracts. Additionally, the in vitro antioxidant activity was measured using 1,1-diphenyl-2-picryl-hydrazyl, and secondary metabolites were identified and quantified by ultra-high-performance liquid chromatography. Results showed that A. occidentale and P. guajava had the highest antimicrobial activity against all bacterial strains, with A. occidentale and P. guajava also demonstrating the highest antioxidant activity. A. occidentale had a high concentration of quercetin 3-O-glucoside-7-O-rhamnoside, kaempeferol-7-O-glucoside, quercetin, caffeic acid, and cinnamic acid in its leaves. In conclusion, A. occidentale and P. guajava are the most effective plants in terms of in vitro antimicrobial and antioxidant activity in their leaves and aqueous extracts, while Moringa oleifera has good antioxidant activity but no bactericidal effect, and Morinda citrifolia has no antimicrobial or antioxidant effect.

Key words: antimicrobial activity; bactericidal; fine powder; leaves; secondary metabolite; antioxidant

Currently, there is a growing interest in researching natural alternatives to subtherapeutic antibiotics, particularly medicinal plants that possess beneficial phytochemical compounds and exhibit antimicrobial, anti-inflammatory, and antioxidant properties (^{Oanh et al. 2023}). Various studies have demonstrated that phytogenic compounds can enhance the genetic expression of farm animals in different production schemes without the use of preventive antibiotics in their diet (^{Skoufos et al. 2020}). Moreover, ^{Karásková et al. (2015)} reported that phytobiotics can reduce oxidative rancidity in foods/feeds, promote productivity naturally, and be used as an adjuvant in the treatment of various animal diseases. In this context, medicinal plants such as A. occidentale, P. guajava, M. citrifolia, and M. oleifera have been frequently employed worldwide to alleviate or eliminate different disease symptoms in humans and animals (^{Aroche et al. 2018}).

Low concentrations of A. occidentale (Anacardiaceae family) have been found to increase egg production and quality and decrease pig diarrheal syndrome (^{Martínez et al. 2013} and ^{Aroche et al. 2017}). In animal production, P. guajava promote egg production, eggshell thickness, and reduce liquid feces in pigs after weaning (^{Ceballos-Francisco et al. 2020}). The inclusion of M. citrifolia in animal diets has also been found to promote egg production and weight gain in pigs (^{Salazar et al. 2017} and ^{Aroche et al. 2018}). Likewise, M. oleifera possess anti-inflammatory, antioxidant, and antimicrobial activity (^{Dhakad et al. 2019}) and has been recommended as a protein source in animal diets (^{Valdivié et al. 2020}).

These four plants have garnered significant global interest in animal production due to their nutraceutical properties that enhance productive indicators, intestinal health, and the quality of the final product (^{Ramírez et al. 2020}). Despite the productive results of their nutraceutical use in animals, few investigations have identified the secondary metabolites responsible for the possible comparative antibacterial and antioxidant effect in vitro. This information would allow elucidating the medicinal benefits reported in animals of zootechnical interest. Therefore, the objective of this research was to evaluate the in vitro antimicrobial and antioxidant activity of leaves and aqueous extracts from four medicinal plants (A. occidentale, P. guajava, M. citrifolia, and M. oleifera).

Materials and Methods

Plant material

Around 20 kg per plant of fresh leaves of A. occidentale, P. guajava, M. oleifera and M. citrifolia were collected in Granma province, Cuba, during the low rainy season of February/2019. This zone is characterized by a flat topography and brown soil with carbonates (^{Hernandez et al. 2019}), authenticated by specialists from the Faculty of Agricultural Sciences of the University of Granma. The plants were more than one year old and without any pathology. The leaves were dried in the shade for five (A. occidentale, P. guajava and M. oleifera) and ten days (M. citrifolia), with free air circulation to constant weight and then dried in a stove (WSU 400, German) with air recirculation for 1 hour at 60 °C. Subsequently, the leaves were crushed in a hammer mill with parallel blades, at 1 mm of size. The samples were stored at room temperature of 26 °C, in fully airtight plastic bags until further use. In vitro experiments were performed at the Feed Research Institute of the Chinese Academy of Agricultural Sciences to determine the antibacterial and antioxidant activity of the leaves and their aqueous extracts.

Preparation of the fine powder and aqueous extract

To obtain the fine powder, 5 kg per plant leaves were ground in a commercial grain crushing machine (Zhejiang Horus Industry and Trade Co., Ltd., Zhejiang, China) through a 40 mesh (0.45 mm) sieve (Yoston, China) and stored in completely airtight bags until use for microbiological tests. Also, 16.67 g of the leaves of each plant were weighed and mixed with 500 mL of water for aqueous extraction. The aqueous extract was obtained by the sonication method, using an ultrasonic extractor (model SY-1000E, China) for 50 minutes at 50 °C, allowed to stand for 1 hour, and filtered through Whatman filter paper No. 1. It was subsequently condensed through a rotary evaporator (model RE-2000, China), under reduced pressure at 45 °C at 60 rpm to reach an amount less than 10 mL of extract (^{Fieser 2004}). The extract was frozen at -80 °C for at least 4 hours, and finally dried in a lyophilizing machine (model LGJ-18, China).

Minimum bactericidal concentration of fine powder

The MBC of the fine powder from leaves of the four plants was determined for dilution method (^{Rios et al. 1988}) in triplicate. For this, bacterial culture was prepared in culture medium at a concentration of approximately 15 x 10⁷ CFU/mL compared to theoretical optical density (550 nm absorbance) that defines the level of 0.5 in the McFarland turbidimetric scale, and was inoculated and incubated for 12 hours, later, 90 mm diameter Petri dishes were prepared with Mueller-Hinton Agar (MHA) at different concentrations of the fine powder. The concentration of the bacterial suspensions was adjusted to 0.5 (optical density) by using a spectrophotometer. Each bacterial culture of 100 µL was inoculated, which consisted in strains of enterotoxigenic Escherichia coli (ETEC) K88+ and ATCC 1515, Staphylococcus aureus ATCC 43300 and ATCC 25923, Salmonella enteritidis: ATCC 3377, and Salmonella typhimurium ATCC 14028. In the first period, concentrations of 5, 15 and 30 mg/mL of fine powder in the culture medium were tested to identify the minimum concentration of each plant in that range for each bacterium tested. Then, concentrations less than 5.0 mg/mL (0.125, 0.25, 0.50, 1.0, 2.0, 3.0 and 4.0 mg/mL) were used for fine powder of leaves of A. occidentale against the six bacteria strains and same concentrations of fine powder of leaves of P. guajava against E. coli ATCC 1515, S. aureus ATCC 25923, S. enteritidis: ATCC 3377, and S. typhimurium ATCC 14028; concentrations of 5.0 to 15.0 mg/mL (5.0, 6.0, 7.0, 8.0, 11.0, 12.0, 13.0, 14.0 and 15.0 mg/mL) of fine powder in culture medium for P. guajava against E. coli K88+ and S. aureus ATCC 43300 and for M. citrifolia against S. aureus ATCC 43300 and 25923; finally, 15 to 30 mg/mL of fine powder of culture medium with M. oleifera against S. aureus ATCC 43300 (^{Rios et al. 1988}).

Minimum inhibitory concentration and minimum bactericidal of aqueous extract

For this study, microdilution method was used for MIC and dilution method for MBC of the aqueous extract (^{Rios et al. 1988}). Stock solution of 13.0 mg/mL was prepared, which was used to prepare in serial dilutions of 13.0, 6.5, 3.25, 1.63, 0.81, 0.41, 0.2, 0.1, 0.05, 0.03, and 0.01 mg/mL. The inoculum of E. coli (ETEC) K88+, S. aureus ATCC 43300, and S. typhimurium ATCC 14028 were prepared in culture medium (Mueller Hinton Broth) at a concentration of approximately 15 x 10⁷ CFU/mL compared to theoretical optical density (550 nm absorbance) that defines the level of 0.50 in the McFarland turbidimetric scale. Then, 200 µL/well of each dilution was placed in 96-well microplates and 2 µL of each bacterial culture was inoculated for triplicates, incubated for 12 hours at 37 °C to determine its absorbance in a plate reader (ELISA, BIO-TEK, Synergy HT). Determination of the MBC was carried out for triplicated, 100 µL of supernatant from those wells where bacterial growth was inhibited and seeded with a sterile glass triangular spatula in 90 mm diameter Petri dishes with Mueller-Hinton Agar (MHA), and incubated for 12 hours at 37 °C.

Antioxidant activity of aqueous extract

For this study, leaves from the four plants were evaluated with 1,1-diphenyl-2-picryl-hydrazyl (DPPH−) (^{Shen et al. 2010}), where a solution of 0.1 mM of DPPH− in methanol was prepared. Later, 1 mL of this solution was taken and vigorously mixed in a vortex with 3 mL of the different concentrations (10.0, 5.0, 2.5, 1.25, 0.625, 0.313, 1,156, 0.078, 0.039, 0.020 and 0.010 mg/mL) of the extract, and 200 µL of each concentration were placed in a 96-well microplate. The solutions were left to stand at RT in the dark for 30 min and then, the absorbance at 517 nm was measured with the use of a plate reader (ELISA brand, BIO-TEK, Synergy HT). Butylated hydroxytoluene (BHT) was used as reference. Low absorbance values indicate high free radical scavenging capacity, or high antioxidant capacity, which was calculated using the following formula: Antioxidant effect of DPPH−(% inhibition) = [(A0 -A1) / A0 * 100], where A0 is the absorbance of the control reaction, and A1 is the absorbance in the presence of the extracts and the reference. Then, % of inhibition was plotted against concentration and the calibration curve for BTH was: y = 139.34x + 10.42, r² = 0.8672. All samples were evaluated in triplicate and the results were averaged and shown as IC₅₀ values (mg/mL).

Identification and quantification of major compounds from leaves of the four plants. pretreatment method

The sample leaves (around 40 mg) from A. occidentale, P. guajava, M. citrifolia and M. oleifera were added to 4 mL of extractant consisting in 0.8 mL of EDTA buffer solution + 3.2 mL of methanol, and were shaken under ultrasonic for 30 min. Then, centrifugation for 5 min to take the supernatant and the membrane was done. EDTA buffer solution consisted in 7.10 g of anhydrous sodium hydrogen phosphate, 1.95 g of disodium EDTA and 8.40 g of citric acid, dissolved in 650 mL of water.

Chromatographic method. ultra-high-performance liquid chromatography-MS/MS conditions

chromatographic analysis was performed on a waters acquity ultrahigh-performance liquid chromatography system, using an Agilent Zorbax Eclipse Plus C18 column (3.0 x 150 mm, 1.8 μm). Mobile phase A: water (0.1% formic acid and 0.2 mmol/L ammonium acetate), mobile phase B: methanol (0.1% formic acid and 0.2 mmol/L ammonium acetate). Separation gradient (0-1 min: 10 % B, 1-2 min: 10 % B-60 % B, 2-7.5 min: 60 % B-90 % B, 7.5-8.0 min: 90 % B-100 % B, 8.0-8.1 min: 10 % B). The injection volume was 2 μL and the flow rate 0.30 mL/min (^{Fang et al. 2007}). MS was performed on a Sciex Triple Quad 4500 MS/MS, and electrospray ionization coupled with multiple reaction monitoring (MRM) model. The resulting optimized values were as follows: source temperature 450 °C, ion spray voltage 4500 V, collision gas: 9 psi, curtain gas 10 psi, ion source gas (GS 1) 18 psi, and ion source gas (GS 2) 0 psi. The corresponding declustering potential (DP) and collision energy (CE) are presented in table 1.

Table 1 MRM conditions, retention time parameters for the analytes. 

Number	CAS#	Compound	Ret Time (min)	Precursor Ion (m/z)	Product Ion (m/z)	DP (V)	CE (V)	Polarity
1	18016-58-5	Quercetin 3-O-glucoside-7-O-rhamnoside	4.66	611.0	303.0	187	30	Positive
					129.0	192	35
2	28338-59-2	Cyanidin 3-O-rutinoside	4.08	594.9	286.7	111	90	Positive
					449.3	128	30
3	520-27-4	3',5,7-Trihydroxy-4'-methoxyflavone 7-rutinoside	4.80	607.2	339.0	32	36	Negative
					299.0	153	38
4	70831-56-0	Cichoric acid	3.97	473.1	314.9	79	40	Negative
					200.9	44	53
5	16290-07-6	Kaempferol-7-O-glucoside	4.51	447.0	284.0	233	51	Negative
					151.0	240	63
6	522-12-3	Quercitrin	4.98	447.0	271.0	273	56	Negative
					300.0	273	37
7	331-39-5	Caffeic acid	4.17	181.0	89.0	72	42	Positive
					117.0	68	30
8	104-46-1	cis-Anethol	6.10	147.1	77.0	54	28	Negative
					62.1	17	42
9	140-67-0	4-Allylanisole	6.10	146.8	77.1	68	30	Negative
					116.9	54	45
10	87-66-1	Pyrogallol	2.34	127.0	81.0	53	27	Positive
					53.0	48	33
11	621-82-9	Cinnamic Acid	6.07	146.8	77.0	66	28	Negative
					103.0	71	16

Statistical analysis

All analyzes are performed in triplicate. For the quantification of secondary metabolites, descriptive statistics were performed. Also, for the antioxidant test, data were processed by simple classification ANOVA in a completely randomized design. Before this, the normality of the data was verified using the ^{Kolmogorov (1941)} and ^{Smirnov (1948)} test, and for uniformity of variance, the ^{Bartlett test (1939)}. In necessary cases, means using ^{Duncan (1955)} test at the significance level of P<0.05 were separated. All analyzes were carried out in accordance with the IBM®SPSS® Statistics, version 22.0 (2013) (SPSS Inc., Chicago, IL, USA).

Results and Discussion

Principal compounds from A. occidentale and P. guajava leaves

The content of principal compounds from A. occidentale and P. guajava leaves are shown on table 2, where it is observed the higher concentration of quercetin 3-O-glucoside-7-O-rhamnoside, kaempferol-7-O-glucoside, quercetin, caffeic acid and cinnamic acid from A. occidentale leaves compared to P. guajava.

Table 2 Quantification of majority compounds from leaves of A. occidentale and P. guajava 

Compounds	A. occidentale (µg/g)	P. guajava (µg/g)
Quercetin 3-O-glucoside-7-O-rhamnoside	0.54±0.03	0.12±0.01
Chicoric acid	0.62±0.04	1.3±0.08
Kaempeferol-7-O-glucoside	1.95±0.12	<0
Quercetin	10.25±0.9	<0
Caffeic acid	0.22±0.01	<0
Cinnamic acid	0.25±0.02	0.07±0.00

Data expressed as mean (n=3) ± standard deviation.

Polyphenols are the major secondary metabolites distributed in all plants, with higher emphasis on isoflavonoids, anthocyanins, flavonols, and flavones in A. occidentale and P. guajava. The quantification of the main secondary metabolites in these two plants such as quercetin 3-O-glucoside-7-O-rhamnoside, chicoric acid, kaempeferol-7-O-glucoside, caffeic acid, and cinnamic acid could support the antibacterial and antioxidant effects found in this study. Theoretically, authors such as ^{Roepke and Bozzo (2013)} have mentioned that 3-O-glucoside-7-O-rhamnoside is a rare secondary metabolite in plants with proven antioxidant and antimicrobial properties against E. coli. Furthermore, caffeic and chicoric acids have potential as antidiabetic agents, demonstrated by ^{Mukhtar et al. (2004)} who found a reduction in glucose concentration in laboratory mice when they used extracts of A. occidentale and P. guajava, respectively. In addition, the flavonoid kaempferol-7-O-glucoside was identified and quantified in the leaves of A. occidentale, which is a phytochemical widely studied for its antimicrobial properties (^{Singh et al. 2011}).

Moreover, cinnamic acid is an organic acid that occurs naturally in many medicinal plants and quantified in both A. occidentale and P. guajava. This acid has low toxicity and a wide spectrum of functional activities, such as antibacterial, antiviral and antifungal properties (^{Sova 2012}), which supports the antimicrobial effect found in the leaves of the plant in study (tables 3 and 4). Although positive results have been found for the secondary metabolites quantified in the leaves of the plant under study (mainly A. occidentale), in farm animals, they are not conclusive. Thus, these results could contribute to understand how medicinal plants (mainly leaves of A. occidentale and P. guajava and their extracts), due to their antimicrobial and antioxidant function, can completely replace growth-promoting antibiotics in farm animals, as demonstrated by ^{Martínez et al. (2013)}, ^{Más et al. (2016)}, ^{Aroche et al. (2017)}, ^{Salazar et al. (2017)} and ^{Aroche et al. (2018)} in poultry and pigs.

Antimicrobial activity of leaves powder and aqueous extract

The MBC of the leaf powder from the plants against six strains of pathogenic bacteria is showed in table 3. Leaf powder of A. occidentale showed the greatest bactericidal effect in the study, mainly against S. aureus ATCC 25923 and 43300 and S. typhimurium ATCC 14028, however, against E. coli K88+ concentration of 4 mg/mL was needed to inhibit the growth. Likewise, the leaf powder of P. guajava showed a bactericidal effect by reducing the growth of Gram-negative and Gram-positive bacteria, with the lowest concentration (1.0 mg/mL) for S. aureus ATCC 43300 and the highest concentration (11 mg/mL) for E. coli K88+. Also, the leaves of M. citrifolia and M. oleifera only showed bactericidal activity against the strains of S. aureus ATCC 43300 and ATCC 25923 although with higher doses (8 and 16 mg/mL respectively) than the inhibitory effects of the leaves of A. occidentale and P. guajava.

Table 3 MBC of the leaf powder of four plants against six bacterial strains (mg/mL). 

Bacteria	AO ^1	PG ^2	MC ^3	MO ^4
E. coli K88+	4.0	11.0	NI ^5	NI
E. coli ATCC 1515	4.0	5.0	NI	NI
S. aureus ATCC 43300	1.0	1.0	8.0	16.0
S. aureus ATCC 25923	0.5	5.0	15.0	NI
S. enteritidis ATCC 3377	4.0	4.0	NI	NI
S. typhimurium ATCC 14028	2.0	2.0	NI	NI

¹ A. occidentale. ² P. guajava. ³ M. citrifolia. ⁴ M. oleifera. ⁵ No inhibition. Data were obtained by triplicated (n = 3).

MIC and MBC of the aqueous extract of the leaves of the four plants in study are shown in table 4. Similar to the fine powder of the leaves, the aqueous extracts of A. occidentale and P. guajava had the highest bactericidal activity. It should be noted that MIC and MBC to inhibit the growth of E. coli K88+ is the same in both medicinal plants (6.5 mg/mL).

Table 4 MIC and MBC of the aqueous extract of the leaves of the plants (mg/mL). 

Extracts	E. coli K88+		S. aureus 43300		S. typhimurium 14028
	MIC	MBC	MIC	MBC	MIC	MBC
AO^1	6.5	6.5	0.81	0.81	3.25	3.25
PG^2	6.5	6.5	0.81	1.63	6.5	6.5
MC^3	NI^5	NI	6.5	NI	NI	NI
MO^4	NI	NI	NI	NI	NI	NI

¹ Anacardium occidentale. ² Psidium guajava. ³ Morinda citrifolia. ⁴ Moringa oleifera. ⁵ No Inhibition. Data were obtained by triplicated (n = 3).

Likewise, a higher concentration of the aqueous extract of P. guajava (compared to the aqueous extract of A. occidentale leaves) is necessary to inhibit and eliminate S. typhimurium ATCC 14028, similar occurred for the bactericidal effect of this product against S. aureus ATCC 43300. The aqueous extract of M. citrifolia only inhibited the growth of S. aureus ATCC 43300 at doses of 6.5 mg/mL, however, it did not show bactericidal activity at the concentrations studied (maximum concentration of 13 mg/mL). Also, the M. oleifera extract did not show inhibitory or bactericidal activity.

A. occidentale is known for its antibacterial properties, mainly in its flowers, bark and leaves (^{da Silva et al. 2016}). In addition, it has been used in the prevention and treatment of oral diseases (being the first contact of the digestive system with the food) by inhibiting the bacteria in this cavity and therefore the formation of biofilm (^{Anand et al. 2015}). Also, ^{Melo Menezes et al. (2014)} found that both crude extract and isolated tannins of A. occidentale have inhibitory activity against microorganisms that are part of the composition of oral biofilm. Therefore, they hypothesized that the mechanisms of the antimicrobial action of tannins, the enzymatic inhibition, the modification of cellular metabolism by its action on the membranes and binding with metal ions, decrease the access to metabolism to the microorganisms that are outside the biofilm. The present study results showed a potent antimicrobial and antioxidant activity, which is related to the high content of polyphenols and flavonoids contained in its leaves, in addition to other medicinal compounds (^{Siracusa et al. 2020}).

^{Souza et al. (2017)} observed the antioxidant and anti-inflammatory activity in vitro in A. occidentale leaves extract when used in RAW 264.7 macrophage cells due to the lower oxidative damage of these cells and the decrease in inflammatory parameters induced by lipopolysaccharides stimulation. Additionally, ^{Brito et al. (2020)}, pointed out that pentagalloil hexoside, a precursor to the formation of hydrolyzed tannins such as ellagitannins and gallotanins, was found in all the organs of A. occidentale, these chemical compounds are responsible for several functional properties, with higher emphasis on the antimicrobial activity. Thus, the present study showed that A. occidentale was the plant with the highest antimicrobial and antioxidant capacity compared to the other three plants studied.

Regarding the effect of A. occidentale in animal production, specifically in poultry and pig production, ^{Aroche et al. (2017)} found that dietary supplementation with 1.0 % of a mixed powder made from 40 % of A. occidentale leaves powder increased growth performance and decreased the diarrhea incidence in weaned piglets. Furthermore, ^{Más et al. (2016)} showed that the dietary inclusion in low concentrations of A. occidentale and P. guajava leaves powder promoted growth and reduced dehydration in pigs before and after weaning. In this sense, ^{Aroche et al. (2018)} showed positive results in feed efficiency and IgG production when they added 0.5 % of a mixture of plants representing 60 % of A. occidentale in broiler diets.

P. guajava has also shown strong bactericidal activity on its leaves and aqueous extract, as it requires a small amount to eliminate bacteria such as E. coli, S. aureus, and Salmonella. Similarly, ^{Salihu Abdallah et al. (2019)} verified that the aqueous and methanolic extracts of P. guajava leaves have antimicrobial activity against S. aureus and S. typhi. The aqueous extract was effective with MIC of 12.5 mg/mL for both bacteria and MBC between 25 and 50 mg/mL for S. aureus and S. typhimurium respectively. In this study, the concentrations of aqueous extract were necessary to obtain the MIC and MBC against these bacteria, and were lower than those, which may be due to the variety of the plant used, the origin, the extraction methods, among other factors. Also, ^{Chero-Nepo and Ruiz-Barrueto (2016)} determined that the alcoholic extract of P. guajava inhibits the growth of Streptococcus mutans due to its bactericidal power.

Antioxidant activity of aqueous extract

Table 5 shows the IC₅₀ of the aqueous extract of the leaves of the four plants. A. occidentale plant with the highest free radical trapping activity compared to the other three plants, as it reflects the lower IC₅₀, being even lower (P<0.001) than the positive control BHT. Furthermore, P. guajava did not show (P>0.05) statistical differences with A. occidentale and BHT. However, M. oleifera and M. citrifolia had the lowest results in antioxidant activity, as they require the highest concentration to inhibit the DPPH− reaction.

Table 5 IC₅₀ of the aqueous extract of the leaves of the four plants 

	AO^1	PG^2	MC^3	MO^4	BTH	SEM±	P-value
IC50 (mg/mL)	0.028^a	0.069^ab	6.269^d	0.603^c	0.093^b	0.65	<0.001

¹ A. occidentale. ² P. guajava. ³ M. citrifolia. ⁴ M. oleifera. IC₅₀: Extract concentration required to inhibit the DPPH− reaction by 50 %. Data are mean (n = 3). Values followed by different letters within a row are significantly different (P<0.05) according to the ^{Duncan (1955}). BHT used as a positive control.

Regarding the antioxidant activity, ^{Flores et al. (2015)} identified the chemical composition of seven cultivars of P. guajava and founded a high content of flavonoids, in addition of anthocyanins, proanthocyanins, triterpenes and other compounds. Likewise, ^{Feng et al. (2015)} and ^{Flores et al. (2015)} showed that there is high correlation between flavonoid content and the antioxidant capacity of the plant, which agree with our findings, where P. guajava was the second plant to show a high antioxidant power.

On the other hand, M. oleifera is a multipurpose plant with multiple nutritional benefits, but also has been studied for its antimicrobial and antioxidant effects, since its use in human and animal nutrition is increasingly popular (^{Wang et al. 2018}). Likewise, M. citrifolia has innumerable health benefits, however, when these two plants are compared with A. occidentale and P. guajava, they may be at a disadvantage due to the lower content of secondary metabolites responsible for the aforementioned activity. This research demonstrated the marked difference for antimicrobial and antioxidant effect of both the leaves and the aqueous extract of A. occidentale and P. guajava compared to M. citrifolia and M. oleifera.

However, in the case of M. oleifera, researchers such as ^{Siddhuraju and Becker (2003)}, determined that this plant presents high antioxidant power in its ethanolic and methanolic extracts, which was related to abundant flavonoid content, especially quercetin and kaempferol. ^{Shih et al. (2011)} found high antioxidant activity in the ethanolic extract of various parts of this plant, where the leaves showed the highest activity, with an IC₅₀ of 0.287 mg/mL, which is less than that found in this study (0.603 mg/mL). This difference could be due to the difference on the extraction (aqueous) method used this study. In relation to animal production, authors such as ^{Zhang et al. (2018)} found positive effects of M. oleifera on performance of fattening pigs, with a marked effect due to increased activity of the enzyme superoxide dismutase and decreased serum malondialdehyde concretion.

M. citrifolia only inhibited the growth of staphylococcal strains, in both forms, as fine powder and as aqueous extract, however, did not show any antimicrobial effect with the other bacterial strains. These results agree with ^{Almeida et al. (2019)}, who reported several studies that probe the antimicrobial and antioxidant properties of M. citrifolia based on its chemical compounds in the plant parts. Also, antibacterial activity was found by ^{Pandiselvi et al. (2019)}, specifically with Staphylococcus aureus. The difference in terms of the least antimicrobial effect in this study could be due to the use of methanolic extract. The antioxidant activity of the leaves of M. citrifolia was the lowest of among the four plants. Very little literature has been published about the antioxidant capacity of the leaves of this plant. Besides, there are several investigations that show this quality in its fruits (^{Senthilkumar et al. 2016} and ^{Thorat et al. 2017}). ^{Sunder et al. (2016)} demonstrated the multiple uses of M. citrifolia in livestock and poultry as a natural growth promoter due to its immunomodulatory, antioxidant, and hypocholesterolemic properties.

Conclusions

It is concluded that A. occidentale and P. guajava are the plants with the highest antimicrobial and antioxidant activity in their leaves and aqueous extract. M. oleifera has good antioxidant in vitro activity, although it does not have high antimicrobial power, and M. citrifolia is the plant that has the least antioxidant activity in its aqueous extract.

Acknowledgments

We would like to thank Dr. Yan Zhao from Institute of Quality Standard and Testing Technology for Agro-Products of CAAS for the assistance of analysis of major compounds from leaves.

References

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Received: February 08, 2023; Accepted: May 29, 2023

^*Email: [e-mail]

Conflict of interests: The authors declare that there is no conflict of interest between them.

Authors contribution: R. Aroche: Data curation, Investigation, Formal analysis, Writing - original draft. X. Jiang: Data curation, Investigation, Formal analysis, Writing - original draft. R. Rodríguez: X. Li: Investigation, Formal analysis. Carolina Avellaneda: Writing - original draft. Y. Martínez: Conceptualization, Data curation, Investigation, Formal analysis, Writing - original draft